| Publications on this gene: |
1.
Int J Gynecol Cancer ; 2(18):241-8.
P53 and bcl-2 assessment in serous ovarian carcinoma.
Palmer JE ,Sant Cassia LJ ,Irwin CJ ,Morris AG ,Rollason TP ,
Department of Gynaecological Oncology, University Hospitals Coventry & Warwickshire NHS Trust, Coventry, United Kingdom. jupalmer@supanet.com
The study objective was to determine the prognostic value of assessment of staining of p53 and bcl-2 in a well-selected group of serous ovarian carcinomas. Immunohistochemical detection was used to identify both p53 and bcl-2 positive tumors. One hundred thirty-two tumors were analyzed for positivity of staining, grade of staining intensity, and for p53 alone, percent expression rates. These were analyzed alongside traditional clinicopathologic parameters for their ability to predict overall survival (OS), disease-free survival (DFS), and response to chemotherapy (CR). Univariate COX analysis revealed percent p53 expression (P = 0.012) and p53 grade (P = 0.01) to be significant predictors of DFS. Neither the p53 nor bcl-2 measurement parameters were found significant for OS or prediction of CR. On multivariate analysis, incorporating clinicopathologic parameters, p53 parameters did not retain independent significance for any outcome measure. As in primary reported studies, bcl-2 was not found to be of clear independent prognostic value in this group of ovarian tumors. If mutation of p53 and its consequent overexpression is an early event in ovarian tumorigenesis, then p53 assessment may prove useful prognostically in the assessment of either low-grade ovarian carcinomas, as a possible indicator for progression, or in early-stage ovarian tumors, as a marker of tumor aggression or likelihood of recurrence. p53 analysis of a larger group of stage I ovarian tumors would be desirable to further explain the potential association with DFS.
Publication Type: Research Support, Non-U.S. Gov't;
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2.
Cancer Res 2008 Jan 15; 2(68):395-403.
t-Darpp promotes cancer cell survival by up-regulation of Bcl2 through Akt-dependent mechanism.
Belkhiri A ,Dar AA ,Zaika A ,Kelley M ,El-Rifai W ,
Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
t-Darpp is a cancer-related truncated isoform of Darpp-32 (dopamine and cyclic-AMP-regulated phosphoprotein of M(r) 32,000). We detected overexpression of t-Darpp mRNA in two thirds of gastric cancers compared with normal samples (P = 0.004). Using 20 micromol/L ceramide treatment as a model for induction of apoptosis in AGS cancer cells, we found that expression of t-Darpp led to an increase in Bcl2 protein levels and blocked the activation of caspase-3 and caspase-9. The MitoCapture mitochondrial apoptosis and cytochrome c release assays indicated that t-Darpp expression enforces the mitochondrial transmembrane potential and protects against ceramide-induced apoptosis. Interestingly, the expression of t-Darpp in AGS cells led to >or=2-fold increase in Akt kinase activity with an increase in protein levels of p-Ser(473) Akt and p-Ser(9) GSK3 beta. These findings were further confirmed using tetracycline-inducible AGS cells stably expressing t-Darpp. We also showed transcriptional up-regulation of Bcl2 using the luciferase assay with Bcl2 reporter containing P1 full promoter, quantitative reverse transcription-PCR, and t-Darpp small interfering RNA. The Bcl2 promoter contains binding sites for cyclic AMP-responsive element binding protein CREB/ATF1 transcription factors and using the electrophoretic mobility shift assay with a CREB response element, we detected a stronger binding in t-Darpp-expressing cells. The t-Darpp expression led to an increase in expression and phosphorylation of CREB and ATF-1 transcription factors that were required for up-regulating Bcl2 levels. Indeed, knockdown of Akt, CREB, or ATF1 in t-Darpp-expressing cells reduced Bcl2 protein levels. In conclusion, the t-Darpp/Akt axis underscores a novel oncogenic potential of t-Darpp in gastric carcinogenesis and resistance to drug-induced apoptosis.
Publication Type: Research Support, N.I.H., Extramural;
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3.
Mol Cells 2007 Dec 31; 3(24):378-87.
Mitochondrially targeted Bcl-2 and Bcl-X(L) chimeras elicit different apoptotic responses.
Liu S ,Pereira NA ,Teo JJ ,Miller P ,Shah P ,Song Z ,
Bioprocessing Technology Institute, 20 Biopolis Way, 06-01 Centros, 138668, Singapore.
The Bcl-2 family of proteins interacts at the mitochondria to regulate apoptosis. However, the anti-apoptotic Bcl-2 and Bcl-X(L) are not completely localized to the mitochondria. In an attempt to generate Bcl-2 and Bcl-X(L) chimeras that are constitutively localized to the mitochondria, we substituted their C-terminal transmembrane tail or both the C-terminal transmembrane tail and the adjacent loop with the equivalent regions from Bak or Bax mutant (BaxS184V) as these regions determine the mitochondrial localization of Bak and Bax. The effects of these substitutions on subcellular localization and their activities were assessed following expression in HeLa and CHO K1 cells. The substitution of the C-terminal tail or the C-terminal tail and the adjacent loop of Bcl-2 with the equivalent regions from Bak or the Bax mutant resulted in its association with the mitochondria. This change in subcellular localization of Bcl-2 chimeras triggered cells to undergo apoptotic-like cell death. The localization of this Bcl-2 chimera to the mitochondria may be associated with the disruption of mitochondrial membrane potential. Unlike Bcl-2, the loop structure adjacent to the C-terminal tail in Bcl-X(L) is crucial for its localization. To localize the Bcl-X(L) chimeras to the mitochondria, the loop structure next to the C-terminal tail in Bcl-X(L) protein must remain intact and cannot be substituted by the loop from Bax or Bak. The chimeric Bcl-X(L) with both its C-terminal tail and the loop structure replaced by the equivalent regions of Bak or Bax mutant localized throughout the entire cytosol. The Bcl-X(L) chimeras that are targeted to the mitochondria and the wild type Bcl-X(L) provided same protection against cell death under several death inducing conditions.
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4.
Rheumatology (Oxford) 2008 Feb ; 2(47):158-64.
Association of polymorphisms in complement component C3 gene with susceptibility to systemic lupus erythematosus.
Miyagawa H ,Yamai M ,Sakaguchi D ,Kiyohara C ,Tsukamoto H ,Kimoto Y ,Nakamura T ,Lee JH ,Tsai CY ,Chiang BL ,Shimoda T ,Harada M ,Tahira T ,Hayashi K ,Horiuchi T ,
Department of Medicine and Biosystemic Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
OBJECTIVE: Identification of the genes responsible for systemic lupus erythematosus (SLE). METHODS: All the exons and putative promoter regions of 53 candidate genes (TNFRSF6/Fas, TNFSF6/FasL, Fli1, TNFSF10/TRAIL, TNFSF12/TWEAK, Bcl-2, PTEN, FADD, TRADD, CDKN1A, TNFRSF1A/TNFR1, TNFRSF4/OX40, TNFSF4/OX40L, TNFSF5/CD40L, TNFSF13B/BAFF, ICOS, CTLA4, CD28, FYN, G2A, CR2, PTPRC/CD45, CD22, CD19, Lyn, PDCD1, PTPN6, TGFB1, TGFB2, TGFB3, TGFBR1, TGFBR2, TGFBR3, CD3Z, DNASE1, APCS, MERTK, C3, C1QA, C1QB, C1QG, C2, MBL2, IGHM, IL-2, IL-4, IL-10, IFNG, TNFA, MAN2A1, TNFRSF11A/RANK, TNFRSF11B/OPG, TNFSF11/OPGL) were screened for single nucleotide polymorphisms (SNPs) and their association with SLE was assessed by case-control studies. A total of 509 cases and 964 controls of Japanese descent were enrolled. RESULTS: A total of 316 SNPs was identified. When analysed in the Japanese population, the allele frequencies of T at rs7951 and G at rs2230201 of the C3 gene were 0.110 and 0.626, respectively, in SLE patients; significantly higher than the frequencies of 0.081 and 0.584, respectively, in controls [odds ratio (OR) = 1.40, 95% confidence interval (CI) = 1.05-1.86, P = 0.016 and OR=1.19, 95% CI = 1.01-1.41, P = 0.038, respectively]. The mean serum C3 level of carriers of the rs7951 T allele was significantly lower than that of non-carriers of the T allele in 87 SLE patients whose medical records were available (P = 0.0018). CONCLUSION: rs7951 T allele of the C3 gene was significantly associated with SLE, and decreased serum level of C3 seems to be correlated with this allele.
Publication Type: Multicenter Study; Research Support, Non-U.S. Gov't;
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5.
Eur J Histochem ; 4(51):291-300.
Expression and distribution of S-100 protein, CD83 and apoptosis-related proteins (Fas, FasL and Bcl-2) in thyroid tissues of autoimmune thyroid diseases.
Xu WC ,Chen SR ,Huang JX ,Zheng ZC ,Chen LX ,Lin JJ ,Li YG ,
The First Hospital of Shantou University Medical School, Guangdong, Shantou, China.
Previous studies have shown that dendritic cells (DCs) and apoptosis-related proteins play a critical role in the pathogenesis of autoimmune thyroid diseases (ATD).This study was designed to investigate the expression and distribution of S-100 protein, CD83 and apoptosis-related proteins (Fas, FasL and Bcl-2) in the thyroid tissues of ATD and their role in ATD pathogenesis as determined by immunochemical staing techniques and other methods. Pathological tissues of 30 patients with Hashimoto's thyroiditis (HT), 30 patients with Graves' disease (GD) and 30 cases of thyroid follicular adenoma (TFA, as control) were used for this study. A higher expression of S-100 in HT (4.2+/-3.1%) and GD (3.9+/-2.8%) vs TFA (0.95+/-0.64%) (p<0.001). was observed as well as a higher expression of CD83 in HT (22.58+/-13.96% and GD (29.92+/-14.43%) vs TFA (5.19+/-8.08%) (p<0.001). HT thyrocytes adjacent to thyroid infiltrating lymphocytes (TILs) showed greater increases in the levels of Fas and FasL than did the GD thyrocytes while HT TILs exhibited lower expression of Fas and FasL than did the GD TILs. GD thyrocytes expressed increased levels of the antiapoptotic protein Bcl-2 as compared to the low levels detected in HT thyrocytes. An opposite pattern was observed in the TILs in GD (low expression of Bcl-2) and HT (high expression of Bcl-2). The findings suggest that the high expression of DC markers is related to the pathogenesis of HT and GD. Up-regulation of both the number and matured functions of DCs may lead to the presentation of more antigens to lymphocytes which are related to the development of autoimmune thyroid diseases. The regulation of Fas/FasL/Bcl-2 in GD favors apoptosis of infiltrating lymphocytes and thyrocyte survival. The regulation of Fas/FasL/Bcl-2 in HT may promote thyrocyte apoptosis leading to hypothyroidism.
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6.
Blood 2008 Jan 1; 1(111):466-8.
Prognostic assessment of BCL2-938C>A polymorphism in chronic lymphocytic leukemia.
Rossi D ,Rasi S ,Capello D ,Gaidano G ,
Publication Type: Comment; Letter; Research Support, Non-U.S. Gov't;
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7.
Appl Immunohistochem Mol Morphol 2008 Jan ; 1(16):44-7.
Pancreatic endocrine tumors-c-erb B2 (Her-2/neu), bcl-2, and p-53 immunohistochemical testing and their value in assessing prognosis.
Proca DM ,Frankel WL ,
Department of Pathology, The Ohio State University, Marysville, OH 43040, USA. proca-1@medctr.osu.edu.
INTRODUCTION: In an attempt to identify better prognostic factors, and the genetic basis of pancreatic endocrine tumors (PETs), we evaluated immunohistochemical reactivity for c-erb B2 (Her-2/neu), bcl-2, and p53. METHODS AND MATERIALS: Two pathologists reviewed hematoxylin and eosin slides and immunohistochemical stains from 28 cases, 27 tumors and 1 nesidioblastosis. Using WHO criteria for malignancy (presence of local or lymphovascular invasion and/or metastases), cases were divided into malignant (20 cases) and benign or uncertain (8 cases). Nuclear staining in >1% of cells was considered positive for p53 and bcl-2, whereas membranous staining was considered positive for c-erb B2 (Her-2/neu). RESULTS: All cases were nonimmunoreactive with anti-c-erb B2 (Her-2/neu), but focal granular or diffuse cytoplasmic staining was seen in occasional neoplasms. One malignant PET showed reactivity with anti-p53, whereas all others were negative. bcl-2 reactivity was identified in 15/28 cases: 9/20 malignant PETs and 6/8 others were positive. CONCLUSIONS: c-erb B2 (Her-2/neu) and p53 are not useful prognostic factors in PET. c-erb B2 (Her-2/neu) staining must be carefully evaluated to avoid the misinterpretation of artifactual/background staining. bcl-2 is occasionally expressed in PET, but the significance of this finding is still to be determined.
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8.
Biochem Cell Biol 2007 Dec ; 6(85):741-50.
A new apoptosis inhibitor, CIAPIN1 (cytokine-induced apoptosis inhibitor 1), mediates multidrug resistance in leukemia cells by regulating MDR-1, Bcl-2, and Bax.
Li X ,Hong L ,Zhao Y ,Jin H ,Fan R ,Du R ,Xia L ,Luo G ,Fan D ,
State Key Laboratory of Cancer Biology & Institute of Digestive Diseases, Xijing Hospital, the Fourth Military Medical University, 17 Changle Western Road, Xi'an 710032, China.
We investigated the role of cytokine-induced apoptosis inhibitor 1 (CIAPIN1), a newly identified apoptosis inhibitor, in leukemia cell multidrug resistance (MDR) and its possible underlying mechanisms. CIAPIN1 was found to be overexpressed at the mRNA and protein levels in the vincristine-induced multidrug-resistant leukemia cell line HL-60/VCR, compared with HL-60, its parental cell line. In this study, we transfected HL-60 with a eukaryotic expression vector of CIAPIN1. In vitro drug sensitivity assays suggested that HL-60-CIAPIN1 cells conferred resistance to both P-glycoprotein (P-gp)-related and -unrelated drugs. Blocking CIAPIN1 expression in HL-60/VCR cells by CIAPIN1-specific small interfering RNA increased the cells' sensitivity to various chemotherapeutic drugs. Flow cytometry results suggested that CIAPIN1 expression could suppress adriamycin-induced apoptosis, accompanied by a decreased accumulation and increased release of adriamycin. Semiquantitative RT-PCR, Western blot analysis, and luciferase reporter assays suggested that CIAPIN1 could significantly upregulate the expression of MDR-1 and Bcl-2, the transcription of the MDR-1 gene, as well as downregulate the expression of Bax. Additionally, the inhibition of CIAPIN1 expression by RNA interference or P-gp inhibitor could partially reverse CIAPIN1-mediated MDR. Taken together, our findings suggest that downregulating CIAPIN1 could sensitize leukemia cells to chemotherapeutic drugs by downregulating MDR-1 and Bcl-2 and by upregulating Bax, yet not altering either glutathione-S-transferase activity or intracellular glutathione content in leukemia cells. Further study of CIAPIN1's function may reveal more of the mechanisms of leukemia MDR and result in the development of strategies to treat leukemia.
Publication Type: Research Support, Non-U.S. Gov't;
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9.
Int J Hematol 2007 Nov ; 4(86):352-7.
High frequency of BCL2 translocation in Thai patients with follicular lymphomas.
Rojnuckarin P ,Assanasen T ,Chotipuech A ,Ruangvejvorachai P ,Tansatit M ,Wannakrairot P ,Intragumtornchai T ,
Department of Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
Follicular lymphoma is characterized by chromosomal translocation involving BCL2 and immunoglobulin heavy chain genes (IgH). That the incidence of follicular lymphoma and the previously reported frequency of BCL2 translocation are lower in Asians than in Caucasians implies a different molecular pathology. The study of BCL2 rearrangement will yield deeper insights into the pathogenesis of follicular lymphomas and into clinical applications of molecular diagnosis for Asian follicular lymphoma patients. BCL2 /IgH translocation was analyzed in paraffin-embedded tissues from follicular lymphoma patients by using polymerase chain reaction (PCR) analysis of the major breakpoint region (MBR), the intermediate cluster region (ICR), and the minor cluster region. In addition, fluorescence in situ hybridization (FISH) analysis with split-signal BCL2 probes was performed. PCR analysis revealed BCL2 rearrangement in 12 (23.5%) of 51 cases (10 MBR and 2 ICR breakpoints). This frequency is lower than the frequencies reported from Western countries (40%-60%). DNA sequencing of the breakpoints revealed nucleotide insertions suggesting V(D)J recombination-mediated mechanisms. On the other hand, FISH analysis revealed 11 (84.6%) of 13 cases with positive signals for BCL2 translocation. Our results suggest that BCL2 translocation is essential for the pathogenesis of follicular lymphoma in Thai patients. In addition, the data demonstrate the low sensitivity of the PCR for diagnostic testing and suggest that split-signal FISH is the method of choice.
Publication Type: Research Support, Non-U.S. Gov't;
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10.
Leukemia 2008 Feb ; 2(22):370-7.
The BCL2 rheostat in glucocorticoid-induced apoptosis of acute lymphoblastic leukemia.
Ploner C ,Rainer J ,Niederegger H ,Eduardoff M ,Villunger A ,Geley S ,Kofler R ,
Division Molecular Pathophysiology, Department Biocenter, Medical University of Innsbruck, Innsbruck, Austria.
Glucocorticoid (GC)-induced apoptosis is essential in the treatment of acute lymphoblastic leukemia (ALL) and related malignancies. Pro- and anti-apoptotic members of the BCL2 family control many forms of apoptotic cell death, but the extent to which this survival 'rheostat' is involved in the beneficial effects of GC therapy is not understood. We performed systematic analyses of expression, GC regulation and function of BCL2 molecules in primary ALL lymphoblasts and a corresponding in vitro model. Affymetrix-based expression profiling revealed that the response included regulations of pro-apoptotic and, surprisingly, anti-apoptotic BCL2 family members, and varied among patients, but was dominated by induction of the BH3-only molecules BMF and BCL2L11/Bim and repression of PMAIP1/Noxa. Conditional lentiviral gene overexpression and knock-down by RNA interference in the CCRF-CEM model revealed that induction of Bim, and to a lesser extent that of BMF, was required and sufficient for apoptosis. Although anti-apoptotic BCL2 members were not regulated consistently by GC in the various systems, their overexpression delayed, whereas their knock-down accelerated, GC-induced cell death. Thus, the combined clinical and experimental data suggest that GCs induce both pro- and anti-apoptotic BCL2 family member-dependent pathways, with the outcome depending on cellular context and additional signals feeding into the BCL2 rheostat.
Publication Type: Research Support, Non-U.S. Gov't;
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11.
Leukemia 2008 Feb ; 2(22):339-43.
The BCL-2 promoter (-938C>A) polymorphism does not predict clinical outcome in chronic lymphocytic leukemia.
Kaderi MA ,Norberg M ,Murray F ,Merup M ,Sundström C ,Roos G ,Aleskog A ,Karlsson K ,Axelsson T ,Tobin G ,Rosenquist R ,
Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden.
The (-938C>A) polymorphism in the promoter region of the BCL-2 gene was recently associated with inferior time to treatment and overall survival in B-cell chronic lymphocytic leukemia (CLL) patients displaying the -938A/A genotype and may thus serve as an unfavorable genetic marker in CLL. Furthermore, the -938A/A genotype was associated with increased expression of Bcl-2. To investigate this further, we analyzed the -938 genotypes of the BCL-2 gene in 268 CLL patients and correlated data with treatment status, overall survival and known prognostic factors, for example, Binet stage, immunoglobulin heavy-chain variable (IGHV) mutational status and CD38 expression. In contrast to the recent report, the current cohort of CLL patients showed no differences either in time to treatment or overall survival in relation to usage of a particular genotype. In addition, no correlation was evident between the (-938C>A) genotypes and IGHV mutational status, Binet stage or CD38. Furthermore, the polymorphism did not appear to affect the Bcl-2 expression at the RNA level. Taken together, our data do not support the use of the (-938C>A) BCL-2 polymorphism as a prognostic marker in CLL and argue against its postulated role in modulating Bcl-2 levels.
Publication Type: Research Support, Non-U.S. Gov't;
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12.
Southeast Asian J Trop Med Public Health 2007 Sep ; 5(38):904-10.
Correlations in survivin expression with the expression of p53 and bcl-2 in invasive ductal carcinoma of the breast.
Al-Joudi FS ,Iskandar ZA ,Imran AK ,
School of Dental Sciences, University of Science of Malaysia, Kota Bharu. fajoudi@hotmail.com
This work studied the correlations between survivin, bcl-2 and p53 in infiltrating ductal carcinoma of the breast. A total number of 382 cases were collected from 3 hospitals in northeastern Malaysia. Survivin, bcl-2 and p53 were detected by immunohistochemistry on samples prepared from tissue blocks. Significant correlations were found between tumor histological grades and tumor size and lymph node involvement. Highly significant statistical correlations (p<0.001) were found in expression of the markers under study. It is concluded that such significant correlations may imply that the alterations in the expression take place in a concerted fashion, implying that many of these cases may share common abnormalities.
Publication Type: Research Support, Non-U.S. Gov't;
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13.
Cancer Lett 2008 Feb 8; 2(259):198-208.
sHA 14-1, a stable and ROS-free antagonist against anti-apoptotic Bcl-2 proteins, bypasses drug resistances and synergizes cancer therapies in human leukemia cell.
Tian D ,Das SG ,Doshi JM ,Peng J ,Lin J ,Xing C ,
Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
HA 14-1, a small-molecule antagonist against anti-apoptotic Bcl-2 proteins, was demonstrated to induce selective cytotoxicity toward malignant cells and to overcome drug resistance. Due to its poor stability and the reactive oxygen species (ROS) generated by its decomposition, chemical modification of HA 14-1 is needed for its future development. We have synthesized a stabilized analog of HA 14-1--sHA 14-1, which did not induce the formation of ROS. As expected from a putative antagonist against anti-apoptotic Bcl-2 proteins like HA 14-1, sHA 14-1 disrupted the binding interaction of a Bak BH3 peptide with Bcl-2 or Bcl-X(L) protein, inhibited the growth of tumor cells through the induction of apoptosis, and circumvented the drug resistance induced by the over-expression of anti-apoptotic Bcl-2 and Bcl-X(L) proteins. Interestingly, the impairment of extrinsic apoptotic pathway induced moderate resistance to sHA 14-1. The moderate resistance suggested that sHA 14-1 generated part of its apoptotic stress through the intrinsic pathway, possibly through its antagonism against anti-apoptotic Bcl-2 proteins. The resistance indicated that sHA 14-1 generated apoptotic stress through the extrinsic apoptotic pathway as well. The ability of sHA 14-1 to induce apoptotic stress through both pathways was further supported by the synergism of sHA 14-1 towards the cytotoxicities of Fas ligand and dexamethasone in Jurkat cells. Taken together, these findings suggest that sHA 14-1 may represent a promising candidate for the treatment of drug-resistant cancers either as a monotherapy or in combination with current cancer therapies.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't;
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14.
Cytokine 2007 Dec ; 3(40):226-34.
Interleukin-10 modulates pro-apoptotic effects of TNF-alpha in human articular chondrocytes in vitro.
John T ,Müller RD ,Oberholzer A ,Zreiqat H ,Kohl B ,Ertel W ,Hostmann A ,Tschoeke SK ,Schulze-Tanzil G ,
Department for Trauma and Reconstructive Surgery, Charité-University of Medicine, Campus Benjamin Franklin, FEM, Krahmerstrasse 6-10, 12207 Berlin, Germany.
The aim of this study is to determine if there is an antagonistic effect between tumour necrosis factor (TNF)-alpha and the immunoregulatory interleukin (IL)-10 on chondrocytes survival. Serum-starved primary human articular chondrocytes were stimulated with either 10 ng/ml recombinant TNF-alpha, IL-10 or a combination of both (at 10 ng/ml each). Chondrocyte apoptosis was determined by measuring caspase-3/7, -8 and -9 activities using caspase assays. Mitochondrial apoptotic inducer bax, and the suppressor bcl-2 were evaluated using western blotting at 48 h. Results indicated that TNF-alpha increased caspase activities and resulted in a significant (p = 0.001) increase in bax/bcl-2 ratio. Stimulation with IL-10 did not alter caspase activities, while co-treatment with IL-10 and TNF-alpha inhibited TNF-alpha induced caspase activities and significantly (p > 0.004) impaired bax/bcl-2 ratio. At 24 h, mRNA levels for collagen type II, TNF-alpha and IL-10 were determined using real-time RT-PCR. Stimulation with TNF-alpha or TNF-alpha and IL-10 significantly inhibited collagen type II and increased IL-10 and TNF-alpha mRNA expression. IL-10 modulated the pro-apoptotic capacity of TNF-alpha in chondrocytes as shown by the decrease in caspase activities and bax/bcl-2 ratio compared to TNF-alpha stimulated chondrocytes, suggesting a mostly antagonistic interplay of IL-10 and TNF-alpha on mitochondrial apoptotic pathways.
Publication Type: Research Support, Non-U.S. Gov't;
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15.
Transplant Proc 2007 Nov ; 9(39):2841-5.
Transplanted heart cardiomyocytes reveal continuous expression of antiapoptotic Bcl-2 protein.
Nozynski J ,Zakliczynski M ,Zembala-Nozynska E ,Konecka-Mrowka D ,Nikiel B ,Przybylski R ,Lange D ,Maruszewski M ,Zembala M ,
Department of Cardiac Surgery & Transplantation, Silesian Center for Heart Disease, Zabrze, Poland.
BACKGROUND: Apoptotic mechanisms take place in cardiocyte death during acute heart graft rejection and remodeling by the mitochondrial pathway. This process is suppressed by Bcl-2 protein. Besides that, knowledge about cardiocyte antiapoptotic responses after heart transplantation is scanty. We sought to estimate Bcl-2 expression in the absence of rejection. MATERIAL: The study group included endomyocardial biopsies taken at 1 week, 1 month, 1 through to 10 years after heart transplant, which showed rejection grade "0"; the control group were donor heart fragments. METHOD: Bcl-2 expression was determined immunohistochemically by NP030 antibody (DAKO) and Envision-DAB. The intensity of staining was assessed semiquantitatively. RESULTS: No Bcl-2 expression was seen in the controls; in the posttransplant groups, the significantly strongest sarcoplasmic reaction was observed at 1 week after heart transplant. Thereafter, the reaction decreased, and was weakest in the 3- and 5-year groups. From this time, Bcl-2 expression increased albeit without statistical significance. The intensity of the reaction showed no correlation with the time elapsed from heart transplant (Spearman r = 0.05; P > .05). CONCLUSION: The expression of antiapoptotic Bcl-2 protein occurs during the entire posttransplant period, being probably a preservative and adaptative response.
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16.
Cancer Res 2007 Nov 15; 22(67):10910-9.
Silencing of prion protein sensitizes breast adriamycin-resistant carcinoma cells to TRAIL-mediated cell death.
Meslin F ,Hamaï A ,Gao P ,Jalil A ,Cahuzac N ,Chouaib S ,Mehrpour M ,
INSERM U753, Laboratoire d'Immunologie des Tumeurs Humaines, Interaction Effecteurs Cytotoxiques-Système Tumoral, Institut Gustave Roussy PR1 and IFR 54, Villejuif, France.
We investigated the relationship between the resistance to the proapoptotic action of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and cellular prion protein (PrPc) function, using the TRAIL-sensitive MCF-7 human breast adenocarcinoma cell line and two TRAIL-resistant sublines: 2101 and MCF-7/ADR. All of the cell lines tested expressed TRAIL-R1 and TRAIL-R2. TRAIL decoy receptors were not detected, suggesting that the resistance of 2101 and MCF-7/ADR cells, strongly expressing PrPc, to TRAIL-mediated cell death was independent from the expression of TRAIL receptors and death-inducing signaling complex formation. Down-regulation of PrPc by small interfering RNA increased the sensitivity of Adriamycin- and TRAIL-resistant cells to TRAIL, but not to epirubicin/Adriamycin. TRAIL-mediated apoptosis in PrPc knocked-down cells was associated with caspase processing, Bid cleavage, and Mcl-1 degradation. In addition, an increased sensitivity of apoptosis-resistant cells to TRAIL after PrPc silencing was not associated with the increased recruitment of receptors and intracellular signaling molecule to the death-inducing signaling complex. Bcl-2 expression was substantially decreased after PrPc knock-down but the levels of Bcl-X(L) and Mcl-1 were not affected. The down-regulation of Bcl-2 was concomitant with Bax delocalization. Our findings support the notion that silencing of PrPc facilitates the activation of proapoptotic Bax by down-regulation of Bcl-2 expression, thereby abolishing the resistance of breast cancer cells to TRAIL-induced apoptosis.
Publication Type: Research Support, Non-U.S. Gov't;
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17.
Acta Biochim Biophys Sin (Shanghai) 2007 Nov ; 11(39):835-43.
Bcl-2 small interfering RNA sensitizes cisplatin-resistant human lung adenocarcinoma A549/DDP cell to cisplatin and diallyl disulfide.
Huang Z ,Lei X ,Zhong M ,Zhu B ,Tang S ,Liao D ,
Institute of Pharmacy and Pharmacology, University of South China, Hengyang 421001, China.
Bcl-2 is overexpressed in a variety of human tumors and is involved in tumorigenesis and chemoresistance. In this study, we investigated the inhibitory effect of the hairpin Bcl-2 small interfering (si)RNA on the expression of the Bcl-2 gene in the cisplatin (DDP)-resistant human lung adenocarcinoma cell line A549/DDP, and the effect of Bcl-2 siRNA on drug sensitization in A549/DDP cells. Bcl-2 siRNA and negative siRNA plasmids were constructed and stably transfected into A549/DDP cells. Reverse transcription-polymerase chain reaction, immunofluorescence microscopy and Western blot analysis were used to detect the target gene expression. Spontaneous cell apoptosis was detected by acridine orange and ethidium bromide staining. Drug sensitivity of the cells to DDP and diallyl disulfide (DADS) was analyzed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Expression levels of Bcl-2 mRNA and protein in siRNA stable transfectants were clearly reduced compared with negative siRNA transfectants and untreated cells. MTT results indicated that Bcl-2 transfectants had a higher cell inhibition rate after treatment with 0.2-200 microg/ml DDP or 50-200 microM DADS. Flow cytometry revealed increased apoptosis in Bcl-2 siRNA cells. After the addition of 20 microg/ml DDP or 100 microM DADS, siRNA targeting of the Bcl-2 gene specifically down-regulated gene expression in A549/DDP cells, increased spontaneous apoptosis, and sensitized cells to DDP and DADS.
Publication Type: Research Support, Non-U.S. Gov't;
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18.
Front Biosci 2008 ; (13):561-8.
Shikonin analogue (SA) 93/637 induces apoptosis by activation of caspase-3 in U937 cells.
Thangapazham RL ,Singh AK ,Seth P ,Misra N ,Mathad VT ,Raj K ,Maheshwari RK ,
Center for Combat Casualty and Life Sustainment Research, Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA.
Beta,beta-dimethyl acryl shikonin is an extract from the root of plant Arnebia nobilis which has been shown to possess anti-cancer activity. However, its toxicity limited further development of shikonin as a therapeutic agent. Subsequently, several analogues of beta,beta-dimethyl acryl shikonin were synthesized. One of these analogues, shikonin 93/637 was found to be significantly less toxic compared to shikonin. This study is aimed to determine the cell cycle associated differences in the susceptibility of U937 cells to apoptosis induced by shikonin analogue 93/637 (SA). Lower concentrations of SA (approximately 100 nM) showed no significant changes in cell growth. However, higher concentrations (approximately 500 nM) resulted in growth inhibition of U937 cells after 48 h of treatment with SA as measured by MTT assay. Flow cytometric analysis showed that SA treatment resulted in blocking of cell cycle progression in G1 phase. Decreased expression of Cyclin D, CDK 4 and PCNA was observed with SA treatment corroborating the G1 block. DNA gel electrophoresis showed an oligonucleotide ladder pattern, a distinct characteristic of DNA fragmentation associated with programmed cell death. Ribonuclease protection assay revealed inhibition of bcl2 expression at transcriptional level. SA treatment also resulted in induction of caspase-3 activity. The results suggest the involvement of bcl2 and Caspase-3 in SA induced apoptosis of human U937 cells.
Publication Type: Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.;
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19.
J Fr Ophtalmol 2007 Oct ; 8(30):799-806.
[Expression of Bax and Bcl-2 apoptotic factors in the conjunctiva of diabetic patients: a preliminary study]
Khalfaoui T ,Beltaief O ,Meddeb Amel O ,
Service d'Ophtalmologie, EPS Charles Nicolle, Tunis, Tunisie. khalfaouit@webmails.com
INTRODUCTION: Apoptosis, or programmed cell death, is necessary to multicellular organism survival, in contrast to involuntary necrosis that is devastating for tissue. It is positively or negatively regulated by proteins of the Bcl-2 family. The aim of our study was to analyze the expression of apoptotic factors Bax and Bcl-2 in the bulbar conjunctiva of diabetic patients without retinopathy and to compare it to the expression of these factors in nondiabetic patients. MATERIAL AND METHODS: Twenty-five conjunctival biopsies were obtained from diabetic patients without retinopathy. The ocular fundus and retinal fluorescein angiography results were normal. Normal human conjunctiva was taken from 15 patients undergoing senile cataract surgery. Immunohistochemical analysis was performed using indirect immunoperoxidase with antibodies against Bax and Bcl-2. RESULTS: In the human diabetic conjunctiva, The Bax protein was highly expressed in all specimens (100%). It was distributed in epithelial cells, vascular endothelial cells, fibroblasts, and inflammatory cells. The Bcl-2 protein was always at a low level or absent. In normal conjunctiva, Bax showed a significant level, whereas Bcl-2 showed no trace of positivity. CONCLUSION: Bax is often localized in tissues characterized by an elevated rate of apoptosis; in contrast, Bcl-2 is absent in these places. Our results suggest that diabetic human conjunctiva, with its inflammatory and cicatricial phenomena, is a privileged target for apoptotic cell death.
Publication Type: English Abstract;
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20.
J Cell Biochem 2007 Dec 1; 5(102):1171-9.
BCL-2 functions as an activator of the AKT signaling pathway in pancreatic cancer.
Mortenson MM ,Galante JG ,Gilad O ,Schlieman MG ,Virudachalam S ,Kung HJ ,Bold RJ ,
Department of Surgery, University of California Davis School of Medicine, Sacramento, California, USA.
BCL-2 is the prototypic anti-apoptotic protein involved in the regulation of apoptosis. Overexpression of BCL-2 is common in pancreatic cancer and confers resistance to the apoptotic effect of chemo- and radiotherapy. Although these cellular effects of BCL-2 are traditionally related to pathways involving the mitochondrial membrane, we sought to investigate whether BCL-2 is involved in other signaling pathways regulating cell survival and focused on AKT. We examined the effect of overexpression of BCL-2 in the MIA-PaCa-2 human pancreatic cancer cell line on the function and subcellular location of AKT. We observed that the stable subclones of MIA-PaCa-2 overexpressing BCL-2 demonstrated increased activity of AKT as well as IKK (a downstream target of AKT), increasing the transcriptional activity of NF-kappaB. Using immunoprecipitation techniques, we observed co-immunoprecipitation of AKT and BCL-2. Immunocytochemistry demonstrated co-localization of BCL-2 and AKT, which was abrogated by treatment with HA14-1, a small molecule inhibitor of BH-3-mediated protein interaction by BCL-2. Furthermore, treatment with HA14-1 decreased phosphorylation of AKT and increased sensitivity to the apoptotic effect of the chemotherapeutic agent, paclitaxel. These results demonstrate an additional mechanism of regulation of cell survival mediated by BCL-2, namely through AKT activation, in the MIA-PaCa-2 pancreatic cancer cell line. Therefore, directed inhibition of BCL-2 may alter diverse pathways controlling cell survival and overcome the apoptotic resistance that is the hallmark of pancreatic cancer.
Publication Type: Research Support, N.I.H., Extramural;
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21.
Blood 2008 Jan 15; 2(111):874-7.
BCL2 expression in chronic lymphocytic leukemia: lack of association with the BCL2 938A>C promoter single nucleotide polymorphism.
Majid A ,Tsoulakis O ,Walewska R ,Gesk S ,Siebert R ,Kennedy DB ,Dyer MJ ,
Medical Research Council Toxicology Unit, and Department of Haematology, University Hospitals, Leicester, UK.
High-level BCL2 expression is seen in most patients with chronic lymphocytic leukemia (CLL) in the absence of BCL2 chromosomal translocation. A single nucleotide polymorphism (SNP; -938C>A) within an inhibitory region of the BCL2 promoter has been reported to regulate BCL2 protein expression and to be associated with adverse prognostic features in CLL. We screened 276 patients with CLL for this SNP and 100 patients by quantitative Western blot for BCL2 expression. In contrast to the previous report, we found no association with BCL2 protein levels or with any clinical or laboratory parameters. BCL2 protein levels remained constant in 10 individual patients at different time points. A total of 19 patients with the lowest levels of BCL2 protein expression were biologically and clinically heterogeneous; 5 patients exhibited high-level BCL2 RNA expression and 4 were fludarabine resistant. BCL2 protein levels in CLL reflect a complex interplay of transcriptional and posttranscriptional controls, but do not appear to be associated with the -938C>A promoter SNP.
Publication Type: Research Support, Non-U.S. Gov't;
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22.
J Biol Chem 2007 Dec 14; 50(282):36496-504.
A novel calmodulin-Ca2+ target recognition activates the Bcl-2 regulator FKBP38.
Edlich F ,Maestre-Martínez M ,Jarczowski F ,Weiwad M ,Moutty MC ,Malesević M ,Jahreis G ,Fischer G ,Lücke C ,
Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, D-06120 Halle/Saale, Germany. edlich@enzyme-halle.mpg.de
The FK506-binding protein 38 (FKBP38) affects neuronal apoptosis control by suppressing the anti-apoptotic function of Bcl-2. The direct interaction between FKBP38 and Bcl-2, however, requires a prior activation of FKBP38 by the Ca2+ sensor calmodulin (CaM). Here we demonstrate for the first time that the formation of a complex between FKBP38 and CaM-Ca2+ involves two separate interaction sites, thus revealing a novel scenario of target protein regulation by CaM-Ca2+. The C-terminal FKBP38 residues Ser290-Asn313 bind to the target protein-binding cleft of the Ca2+-coordinated C-terminal CaM domain, thereby enabling the N-terminal CaM domain to interact with the catalytic domain of FKBP38 in a Ca2+-independent manner. Only the latter interaction between the catalytic FKBP38 domain and the N-terminal CaM domain activates FKBP38 and, as a consequence, also regulates Bcl-2.
Publication Type: Research Support, Non-U.S. Gov't;
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23.
Histopathology 2007 Dec ; 6(51):743-51.
Cyclooxygenase-2 expression correlates with phaeochromocytoma malignancy: evidence for a Bcl-2-dependent mechanism.
Cadden IS ,Atkinson AB ,Johnston BT ,Pogue K ,Connolly R ,McCance D ,Ardill JE ,Russell CF ,McGinty A ,
Department of Medicine, Queen's University Belfast, Belfast, UK.
AIMS: Phaeochromocytomas are rare but potentially life-threatening neuroendocrine tumours of the adrenal medulla or sympathetic nervous system ganglia. There are no histological features which reliably differentiate benign from malignant phaeochromocytomas. The aim of the study was to evaluate cyclooxygenase (COX)-2 and Bcl-2 as tissue-based biomarkers of phaeochromocytoma prognosis. METHODS AND RESULTS: COX-2 and Bcl-2 expression were examined immunohistochemically in tissue from 41 sporadic phaeochromocytoma patients followed up for a minimum of 5 years after diagnosis. There was a statistically significant association between COX-2 histoscore (intensity x proportion) and the development of tumour recurrence or metastases (P = 0.006). A significant relationship was observed between coexpression of COX-2 and Bcl-2 in the primary tumour and the presence of recurrent disease (P = 0.034). A highly significant association was observed between (i) tumour-associated expression of these two oncoproteins (P = 0.001) and (ii) COX-2 histoscore and the presence of Bcl-2 expression (P = 0.002). COX regression analysis demonstrated no significant relationship between (i) the presence or absence of either COX-2 or Bcl-2 and patient survival or (ii) COX-2 histoscore and patient survival. CONCLUSIONS: COX-2 and Bcl-2 may promote phaeochromocytoma malignancy, and these oncoproteins may be valuable surrogate markers of an aggressive tumour phenotype.
Publication Type: Research Support, Non-U.S. Gov't;
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24.
Gene 2007 Dec 1; 1-2(404):110-6.
Identification of a novel Bcl-2 promoter region that counteracts in a p53-dependent manner the inhibitory P2 region.
Bredow S ,Juri DE ,Cardon K ,Tesfaigzi Y ,
Respiratory Immunology and Asthma Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive SE, Albuquerque, NM 87108, USA.
Expression of the anti-apoptotic proto-oncogene bcl-2 is negatively affected by the pro-apoptotic p53. To understand the regulation of bcl-2 expression by p53, we studied the bcl-2 promoter regions individually and in the context of the full-length promoter. While the P1 promoter displayed the highest p53-independent activity, the P2 promoter activity was suppressed in p53-sufficient cancer cell lines. In addition, P2 activity was higher in primary airway epithelial cells from p53(-/-) mice compared to those from p53(+/+) mice. Chromatin immunoprecipitation assays confirmed that p53 interacts within a 140 bp sequence of P2 that contained the CCAAT- and TATA-elements. However, when P1 and P2 are linked in one construct, P2 suppressed P1 activity independent of p53. A potential novel promoter with a p53-dependent activity was identified located between P1 and P2, and was designated M. In the context of the full-length bcl-2 promoter, M counteracted in a p53-dependent manner the suppressive activity of P2 on P1. Collectively, these data suggest that P1 promoter is the main driving force for transcribing the bcl-2 gene and P1 activity is modulated by M and P2 in a p53-dependent and -independent manner. These findings may have implications for therapies that are geared towards inhibiting bcl-2 gene expression and inducing cell death.
Publication Type: Research Support, N.I.H., Extramural;
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25.
Cancer Biol Ther 2007 Oct ; 10(6):1553-8.
Influence of apoptosis (BCL2, FAS), cell cycle (CCND1) and growth factor (EGF, EGFR) genetic polymorphisms on survival outcome: an exploratory study in squamous cell esophageal cancer.
Jain M ,Kumar S ,Upadhyay R ,Lal P ,Tiwari A ,Ghoshal UC ,Mittal B ,
Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India.
The study aimed at investigating whether genetic polymorphisms in BCL2, FAS, CCND1, EGF and EGFR genes influence the outcome of patients of esophageal squamous cell cancer treated with radiotherapy, with or without chemotherapy. Sixty nine histologically confirmed, previously untreated, patients with a squamous cell esophageal cancer were inducted into this study. Genotyping of BCL2 (ala43thr), FAS (A-670G), CCND1 (G870A), EGF (+61A/G) and EGFR (G497A) polymorphisms were determined using the polymerase chain reaction followed by restriction fragment length polymorphism methodology. Genotyped data was analyzed using univariate and multivariate logistic regression statistical tests for predicting the survival outcome. Genotypes of BCL2, FAS, CCND1 and EGFR polymorphisms independently did not influence outcome significantly. However, patients with EGF +61AG genotype had median survival of 25.5 months (95% CI = 5.2-45.5), whereas those with EGF +61GG genotype had survival of only 3.7 months (95% CI = 0.0-9.8, p = 0.006). In univariate cox-regression analysis, interaction of genotypes EGF+61GG*radiotherapy tumor dose (< or =50 Gy) and EGF +61GG *upper third tumor location showed high hazard of death, 6.6 (95% CI = 2.0-21.5, p = 0.002) and 26.8 (95% CI = 3.7-194.2, p = 0.001) while EGF+61AG*middle third tumor location had reduced hazard 0.20 (95%CI = 0.06-0.60, p = 0.004). The pilot study suggests that EGF +61AG and +61GG genotypes may predict clinical outcome in esophageal cancer patients treated with radiotherapy with or without chemotherapy. EGF +61AG genotype was associated with improved survival, however +61GG genotype adversely affected the outcome in patients particularly with upper third location of tumor and lower dose (< or =50) of radiotherapy.
Publication Type: Research Support, Non-U.S. Gov't;
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26.
Blood 2008 Jan 15; 2(111):723-31.
Calmodulin-dependent kinase IV links Toll-like receptor 4 signaling with survival pathway of activated dendritic cells.
Illario M ,Giardino-Torchia ML ,Sankar U ,Ribar TJ ,Galgani M ,Vitiello L ,Masci AM ,Bertani FR ,Ciaglia E ,Astone D ,Maulucci G ,Cavallo A ,Vitale M ,Cimini V ,Pastore L ,Means AR ,Rossi G ,Racioppi L ,
Department of Molecular and Cellular Biology and Pathology, Federico II University of Naples, Italy.
Microbial products, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4), regulate the lifespan of dendritic cells (DCs) by largely undefined mechanisms. Here, we identify a role for calcium-calmodulin-dependent kinase IV (CaMKIV) in this survival program. The pharmacologic inhibition of CaMKs as well as ectopic expression of kinase-inactive CaMKIV decrease the viability of monocyte-derived DCs exposed to bacterial LPS. The defect in TLR4 signaling includes a failure to accumulate the phosphorylated form of the cAMP response element-binding protein (pCREB), Bcl-2, and Bcl-xL. CaMKIV null mice have a decreased number of DCs in lymphoid tissues and fail to accumulate mature DCs in spleen on in vivo exposure to LPS. Although isolated Camk4-/- DCs are able to acquire the phenotype typical of mature cells and release normal amounts of cytokines in response to LPS, they fail to accumulate pCREB, Bcl-2, and Bcl-xL and therefore do not survive. The transgenic expression of Bcl-2 in CaMKIV null mice results in full recovery of DC survival in response to LPS. These results reveal a novel link between TLR4 and a calcium-dependent signaling cascade comprising CaMKIV-CREB-Bcl-2 that is essential for DC survival.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't;
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27.
Cancer Res 2007 Oct 1; 19(67):9561-7.
Association study of 69 genes in the ret pathway identifies low-penetrance loci in sporadic medullary thyroid carcinoma.
Ruiz-Llorente S ,Montero-Conde C ,Milne RL ,Moya CM ,Cebrián A ,Letón R ,Cascón A ,Mercadillo F ,Landa I ,Borrego S ,Pérez de Nanclares G ,Alvarez-Escolá C ,Díaz-Pérez JA ,Carracedo A ,Urioste M ,González-Neira A ,Benítez J ,Santisteban P ,Dopazo J ,Ponder BA ,Robledo M , ,
Hereditary Endocrine Cancer Group, Human Genetics Group, Biomedical Research Institute, CSIC, UAM, Madrid, Spain.
To date, few association studies have been done to better understand the genetic basis for the development of sporadic medullary thyroid carcinoma (sMTC). To identify additional low-penetrance genes, we have done a two-stage case-control study in two European populations using high-throughput genotyping. We selected 417 single nucleotide polymorphisms (SNP) belonging to 69 genes either related to RET signaling pathway/functions or involved in key processes for cancer development. TagSNPs and functional variants were included where possible. These SNPs were initially studied in the largest known series of sMTC cases (n = 266) and controls (n = 422), all of Spanish origin. In stage II, an independent British series of 155 sMTC patients and 531 controls was included to validate the previous results. Associations were assessed by an exhaustive analysis of individual SNPs but also considering gene- and linkage disequilibrium-based haplotypes. This strategy allowed us to identify seven low-penetrance genes, six of them (STAT1, AURKA, BCL2, CDKN2B, CDK6, and COMT) consistently associated with sMTC risk in the two case-control series and a seventh (HRAS) with individual SNPs and haplotypes associated with sMTC in the Spanish data set. The potential role of CDKN2B was confirmed by a functional assay showing a role of a SNP (rs7044859) in the promoter region in altering the binding of the transcription factor HNF1. These results highlight the utility of association studies using homogeneous series of cases for better understanding complex diseases.
Publication Type: Research Support, Non-U.S. Gov't;
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28.
Clin Cancer Res 2007 Oct 1; 19(13):5790-7.
The AA genotype of the regulatory BCL2 promoter polymorphism ( 938C>A) is associated with a favorable outcome in lymph node negative invasive breast cancer patients.
Bachmann HS ,Otterbach F ,Callies R ,Nückel H ,Bau M ,Schmid KW ,Siffert W ,Kimmig R ,
Institute of Pharmacogenetics, University of Duisburg-Essen, Germany. hagen.bachmann@uk-essen.de
PURPOSE: Expression of the antiapoptotic and antiproliferative protein Bcl-2 has been repeatedly shown to be associated with better clinical outcome in breast cancer. We recently showed a novel regulatory (-938C>A) single-nucleotide polymorphism (SNP) in the inhibitory P2 BCL2 gene promoter generating significantly different BCL2 promoter activities. EXPERIMENTAL DESIGN: Paraffin-embedded neoplastic and nonneoplastic tissues from 274 patients (161 still alive after a follow-up period of at least 80 months) with primary unilateral invasive breast carcinoma were investigated. Bcl-2 expression of tumor cells was shown by immunohistochemistry; nonneoplastic tissues were used for genotyping. Both the Bcl-2 expression and the (-938C>A) genotypes were correlated with the patients' survival. RESULTS: Kaplan-Meier curves revealed a significant association of the AA genotype with increased survival (P = 0.030) in lymph node-negative breast cancer patients, whereas no genotype effect could be observed in lymph node-positive cases. Ten-year survival rates were 88.6% for the AA genotype, 78.4% for the AC genotype, and 65.8% for the CC genotype. Multivariable Cox regression identified the BCL2 (-938CC) genotype as an independent prognostic factor for cancer-related death in lymph node-negative breast carcinoma patients (hazard ratio, 3.59; P = 0.032). Immunohistochemical Bcl-2 expression was significantly associated with the clinical outcome of lymph node-positive but not of lymph node-negative breast cancer patients. In lymph node-negative cases, the (-938C>A) SNP was both significantly related with the immunohistochemically determined level of Bcl-2 expression (P = 0.044) and the survival of patients with Bcl-2-expressing carcinomas (P = 0.006). CONCLUSIONS: These results suggest the (-938C>A) polymorphism as a survival prognosticator as well as indicator of a high-risk group within patients with lymph node-negative breast cancer.
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29.
Int J Cancer 2008 Jan 15; 2(122):281-8.
Erythropoietin treatment of human ovarian cancer cells results in enhanced signaling and a paclitaxel-resistant phenotype.
Solar P ,Feldman L ,Jeong JY ,Busingye JR ,Sytkowski AJ ,
Laboratory for Cell and Molecular Biology, Division of Hematology and Oncology, Beth Israel Deaconess Medical Center,Department of Medicine, Harvard Medical School, Boston, MA, USA.
Erythropoietin (Epo), a glycoprotein hormone that is the principal regulator of erythropoiesis, is known to act also on nonhematopoietic cell types. Epo receptors have been reported on several normal and neoplastic human cells and tissues, including ovarian cancer cells. We found that long-term Epo treatment of A2780 cells resulted in the development of a phenotype exhibiting both enhanced Epo signaling, evidenced by increased peak levels of phospho-Erk1/2 and increased paclitaxel resistance. This phenotypic effect was specific for paclitaxel, since no change in cisplatin or carboplatin sensitivity was observed. In addition, the change in phenotype was stable, even after the removal of Epo. Measurement of mono- and oligonucleosome formation revealed that long-term Epo treated A2780 cells exhibited markedly less apoptosis than nonerythropoietin treated cells at essentially all concentrations of paclitaxel tested. Western blot analyses revealed that the long-term Epo treated cells had significantly reduced expression of apoptosis-related proteins Bcl-2 and Bcl-10. These findings may have implications for the clinical use of recombinant human Epo and other erythropoiesis stimulating agents to correct anemia in paclitaxel-treated cancer patients.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.;
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30.
J Biol Chem 2007 Nov 16; 46(282):33888-95.
Caspase-9-induced mitochondrial disruption through cleavage of anti-apoptotic BCL-2 family members.
Chen M ,Guerrero AD ,Huang L ,Shabier Z ,Pan M ,Tan TH ,Wang J ,
Department of Immunology, Baylor College of Medicine, Houston, Texas 77030, USA.
Mitochondrial disruption during apoptosis results in the release of cytochrome c that forms apoptosomes with Apaf-1 and caspase-9. Activation of caspase-9 by dimerization in apoptosomes then triggers a caspase signaling cascade. In addition, other apoptosis signaling molecules released from the mitochondrion, such as apoptosis-inducing factor and endonuclease G, may induce caspase-9-independent apoptosis. To determine the signaling events induced by caspase-9, we used chemically induced dimerization for specific activation of caspase-9. We observed that caspase-9 dimerization resulted in the loss of mitochondrial membrane potential and the cleavage of anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1. Moreover, cleavage-resistant Bcl-2, Bcl-xL, or Mcl-1 potently inhibited caspase-9-dependent loss of mitochondrial membrane potential and the release of cytochrome c. Our data suggest that a caspase-9 signaling cascade induces feedback disruption of the mitochondrion through cleavage of anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't;
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31.
Cell Cycle 2007 Sep 1; 17(6):2171-7.
By blocking apoptosis, Bcl-2 in p38-dependent manner promotes cell cycle arrest and accelerated senescence after DNA damage and serum withdrawal.
Nelyudova A ,Aksenov N ,Pospelov V ,Pospelova T ,
Laboratory of Molecular Mechanisms of Cell Differentiation, Institute of Cytology, Russian Academy of Sciences, Saint Petersburg, Russia. annanelioudova@hotmail.com
E1A+ras-transformed rodent fibroblasts are unable to be arrested in the cell cycle and die by apoptosis in response to cytostatics, ionizing radiation (IR), or serum withdrawal. Overexpression of the human antiapoptotic gene bcl-2 suppresses apoptosis and induces reversible cell cycle arrest after IR or serum withdrawal and cell senescence after adriamycin treatment. Bcl-2-sustained adriamycin-induced cell senescence requires p38 MAPK, since the knockout of p38 MAPK abrogated anti-apoptotic and senescence-inducing effects of Bcl-2 in adriamycin-treated cells. Moreover, resistance to apoptosis and cell cycle arrest were not observed in p38-/- E1A+ras+bcl-2-transformants following IR or serum deprivation. However, the pro-apoptotic effect of nocodazole in E1A+ras-transformed cells can not be prevented by Bcl-2 overexpression independently of the presence of p38 MAPK. These results allow us to conclude that p38 is necessary for Bcl-2-induced inhibition of apoptosis, induction of cell cycle arrest and accelerated senescence after DNA damage and serum starvation, but not after nocodazole treatment.
Publication Type: Research Support, Non-U.S. Gov't;
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32.
Endokrynol Diabetol Chor Przemiany Materii Wieku Rozw 2007 ; 2(13):63-70.
[Analysis of intracellular proapoptotic (Bax, Bak) and antiapoptotic (Bcl-2, Bcl-XL) proteins expression in thyrocytes from young patients with immune and non-immune thyroid disorders]
Bossowski A ,Czarnocka B ,Bardadin K ,Urban M ,Niedziela M ,Dadan J ,
ll Klinika Chorób Dzieci AM w Białymstoku.
Apoptosis one of the form of programmed cell death is a physiological occurrence, requisite to the correct function of every organism. This is an active process that proceeds with a participation of the cellular metabolism embracing the activation of genes and the synthesis of proteins. The signal to apoptosis can be started practically in every cell of our organism. Disturbances of the apoptosis regulation determine the essential link of the pathogenesis of many diseases, including autoimmune thyroid disorders. The aim of this study was to estimate the expression of proapoptotic (Bax, Bak) and antiapoptotic (Bcl-2, Bcl-XL) proteins in thyroid tissues from 12 patients with Graves' disease (GD), 10 with non-toxic nodular goitre (NTNG) and 10 with toxic nodular goitre (TNG). Criteria for qualification of Graves' patients: large goitre, ophthalmopathy, TRAb > 5 U/L, positive titre of anti-TPO and anti-TG antibodies and concentration of TSH <0.45 microIU/mL more the 2-3 months from onset of the disease. Detection of apoptotic proteins in thyroid follicular cells was performed by Western Blot. These analysis was confirmed by immunohistochemistry using monoclonal antibodies in DAB chromogene visuality and marked by Mayer's haematoxylin. Identification of antiapoptotic Bcl-2 and Bcl-XL molecules in the thyroid follicular cells revealed a higher expression of both proteins in patients with Graves' disease (+++; ++, respectively) in comparison to patients with NTNG (++/+; +) and TNG (++; +). The detection of proapoptotic molecules showed higher expression of Bak (++/+) and Bax (+) in Graves' thyroid tissues while Bax was in trace amount in NTNG (0/+) and TNG (0/+). We conclude that alteration in the expression of antiapoptotic and proapoptotic proteins on surface of thyroid follicular cells may play a role in the pathogenesis of thyroid autoimmune disorders. In addition, suppression of apoptosis in Graves' disease led to predominance for proliferation of thyroid follicular cells which is responsible for goitre formation.
Publication Type: English Abstract;
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33.
Growth Factors 2007 Apr ; 2(25):94-100.
Lack of BCL-2 confers interferon-alpha sensitivity to B-cell lymphomas.
Karauzum SB ,Yasar D ,Dirice E ,Imir N ,Luleci G ,Ozes ON ,
Department of Medical Biology and Genetics, Akdeniz University, Antalya, Turkey.
Hairy cell leukemia (HCL) is a chronic B-cell lymphoproliferative disorder with pathological manifestations usually including splenomegaly and pancytopenia. Interferons (IFNs), specifically of the alpha subtypes, have shown a significant anti-tumor effect in HCL patients, with improvement of hematological parameters within the first few months of treatment. However, the therapeutic effect of IFN-alpha is still rather limited. The mechanisms responsible for the beneficial action of IFN-alpha in HCL patients are unclear. A continuous line of cells (Eskol) from a patient diagnosed with HCL was established and shown to have several properties of HCL. Even though, Eskol cells are very resistant to anti-proliferative activity of IFN-alpha, Daudi cells, another human B-cell-derived cell line, are very sensitive to anti-proliferative activity of IFN-alpha and are commonly used as a model cell to test anti-proliferative effect of IFN-alpha. To understand the molecular reason(s) behind the observed obvious differences to IFN sensitivity of above cells, we have analyzed the expression levels of BCL2, caspase-1, Laminin and PARP in these cells. We found that Daudi cells do not express BCL2 at all, and probably because of that, these cells have constantly cleaved, and probably activated form of caspase-1. However, when we over-expressed BCL2 in these cells, they lost processed form of caspase-1 and became resistant to anti-proliferative activity of IFN-alpha. These results let us to suggest that IFN-alpha sensitivity of B-cell lymphomas, once again, depends on the presence or absence of BCL2.
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34.
J Clin Endocrinol Metab 2007 Dec ; 12(92):4845-52.
Bcl-2 overexpression in thyroid carcinoma cells increases sensitivity to Bcl-2 homology 3 domain inhibition.
Mitsiades CS ,Hayden P ,Kotoula V ,McMillin DW ,McMullan C ,Negri J ,Delmore JE ,Poulaki V ,Mitsiades N ,
Department of Medical Oncology, Dana Farber Cancer Institute, Mayer Building, Room M555, 44 Binney Street, Boston, Massachusetts 02115, USA. constantine_mitsiades@dfci.harvard.edu
CONTEXT: The Bcl-2 family of proteins regulates apoptosis in various models and may represent a promising therapeutic target in human malignancies. OBJECTIVE/METHODS: We evaluated the sensitivity of thyroid carcinoma cell lines (two papillary, one follicular, two anaplastic, three medullary) in vitro to BH3I-1 and BH3I-2', two cell-permeable inhibitors of the Bcl-2 homology (BH)-3 domain-mediated interaction between proapoptotic and antiapoptotic Bcl-2 family members. The thyroid carcinoma cell line FRO was stably transfected with cDNA for Bcl-2 or constitutively active Akt and evaluated for sensitivity to BH3-domain inhibition. RESULTS: BH3-domain inhibition disrupted the mitochondrial membrane potential in thyroid carcinoma cells, induced caspase-dependent apoptosis, and potently sensitized them to sublethal concentrations of doxorubicin and the proteasome inhibitor bortezomib (Velcade). Overexpression of constitutively active Akt suppressed BH3I-1-induced cell death. Bcl-2-overexpressing FRO cells were more resistant to conventional chemotherapeutic agents (such as doxorubicin) but significantly more sensitive to BH3I-1 than control cells and were found to overexpress caspase-9, caspase-8, Bmf, Bok, and Bik transcripts and express less A1, BRaf, and FLIP transcripts. CONCLUSIONS: Bcl-2 expression protects thyroid carcinomas against chemotherapy-induced apoptosis. Nevertheless, overexpression of Bcl-2 may result in "oncogene addiction" of the cancer cell, which can be exploited by using BH3-domain inhibitors alone or in combination with other agents, including conventional chemotherapeutics (such as doxorubicin) or novel targeted therapies (such as the proteasome inhibitor bortezomib), for the treatment of aggressive thyroid cancer, including the medullary and anaplastic types.
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35.
Bosn J Basic Med Sci 2007 Aug ; 3(7):205-11.
Non-small cell lung carcinoma: cyclin D1, bcl-2, p53, Ki-67 and HER-2 proteins expression in resected tumors.
Radović S ,Babić M ,Dorić M ,Hukić A ,Kuskunović S ,Hadzismajlović A ,Serdarević F ,
Institute of Pathology, Faculty of Medicine, University of Sarajevo, Cekalusa 90, 71000 Sarajevo, Bosnia and Herzegovina.
The aim of this study was to investigate expression of cyclin D1, bcl-2, p53, Ki-67 and HER-2 proteins in 14 cases of non-small cell lung cancer and to establish their correlation to classical clinico-pathological findings, and alleged prognostic value to estimate biological potential of tumor. Retrospective pilot study of the surgically treated non-small cell lung cancer biopsy specimen, paraffin embedded, used immunohistochemical method to demonstrate expression of cyclin D1, bcl-2, p53, Ki-67 and HER-2. Protein quantification was performed by the semi-quantitative method. Achieved results were correlated with classical clinico-pathological parameters, like tumor size, histological type, differentiation level, presence of vascular invasion and metastasis in regional lymph nodes. Out of 14 cases of non-small cell lung cancer, squamous cell carcinoma was found in 7 patients, giant cell carcinoma in 3, adenocarcinoma in 2, and 1 case of pleomorphic and mucoepidermoid carcinoma. Expression of cyclin D1 was not found, while expression of HER-2 and bcl-2 protein was established in one cases each. p53 expression was noted in 8 cases (57,1%). Statistically positive significant correlation (p<0,05) was found among: presence of lymphovascular invasion to tumor tissue and appearance of nodal metastasis; proliferation Ki-67 index and level of tumor differentiation, i.e. size of tumor. Other investigated parameters showed no significant statistically dependence. p53 expression was not correlated to any of the investigated parameters what might imply the possibility that there is an independent pathway of this protein expression. Negative expression of bcl-2 protein points out to possibility that it is not included into process of tumor apoptosis, as well as that proteins cyclin D1 and HER-2 are not included into processes of the tumor genesis. Since the proliferative activity of the tumor, measured by the expression of Ki-67, is correlated to the gradus and size of the tumor mass, Ki-67 protein can be of a prognostic value to determine biological potential of non-small cell lung cancer.
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36.
Biochem Biophys Res Commun 2007 Nov 9; 1(363):101-5.
CREB-mediated Bcl-2 expression in trichosanthin-induced Hela cell apoptosis.
Wang P ,Yan H ,Li JC ,
Institute of Cell Biology, Zhejiang University, Hangzhou 310058, PR China.
Bcl-2 plays a pivotal role in the control of cell death and is down-regulated in trichosanthin (TCS)-induced cell apoptosis. Because Bcl-2 expression is regulated by the transcription factor cyclic AMP response element-binding protein (CREB), we investigated the role of CREB activation in TCS-induced Hela cells apoptosis. Our results showed that TCS-caused Hela cell apoptosis was accompanied by the decrease of Bcl-2 and phosphorylated CREB protein levels. Interesting, this inhibitive effect can be abolished by the combined treatment of TCS/cAMP agonists. Furthermore, TCS-mediated Bcl-2 protein was abrogated by the suppression of CREB expression with antisense treatment, and blocking the interaction between CREB-binding protein and the Bcl-2 cyclic AMP-responsive element (CRE) by a CRE decoy oligonucleotide. Therefore, these data support the hypothesis that CREB plays a critical role in the regulation of Bcl-2 expression in TCS-induced Hela cell death.
Publication Type: Research Support, Non-U.S. Gov't;
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37.
J Biol Chem 2007 Nov 2; 44(282):32288-97.
A protein kinase Cepsilon-anti-apoptotic kinase signaling complex protects human vascular endothelial cells against apoptosis through induction of Bcl-2.
Steinberg R ,Harari OA ,Lidington EA ,Boyle JJ ,Nohadani M ,Samarel AM ,Ohba M ,Haskard DO ,Mason JC ,
Bywaters Center for Vascular Inflammation and Histopathology Section, Imperial College London, Hammersmith Hospital, London, W12 ONN United Kingdom.
Endothelial cell apoptosis is associated with vascular injury and predisposes to atherogenesis. Endothelial cells express anti-apoptotic genes including Bcl-2, Bcl-XL and survivin, which also contribute to angiogenesis and vascular remodeling. We report a central role for protein kinase Cepsilon (PKCepsilon) in the regulation of Bcl-2 expression and cytoprotection of human vascular endothelium against apoptosis. Using myristoylated inhibitory peptides, a predominant role for PKCepsilon in vascular endothelial growth factor-mediated endothelial resistance to apoptosis was revealed. Immunoblotting of endothelial cells infected with an adenovirus expressing a constitutively active form of PKCepsilon (Adv-PKCepsilon-CA) or control Adv-beta-galactosidase demonstrated a 3-fold, PKCepsilon-dependent increase in Bcl-2 expression, with no significant change in Bcl-XL, Bad, Bak, or Bax. The induction of Bcl-2 inhibited apoptosis induced by serum starvation or etoposide, and PKCepsilon activation attenuated etoposide-induced caspase-3 cleavage. The functional role of Bcl-2 was confirmed with Bcl-2 antagonist HA-14-1. Inhibition of phosphoinositide 3-kinase attenuated vascular endothelial growth factor-induced protection against apoptosis, and this was rescued by overexpression of constitutively active PKCepsilon, suggesting PKCepsilon acts downstream of phosphoinositide 3-kinase. Co-immunoprecipitation studies demonstrated a physical interaction between PKCepsilon and Akt, which resulted in formation of a signaling complex, leading to optimal induction of Bcl-2. This study reveals a pivotal role for PKCepsilon in endothelial cell cytoprotection against apoptosis. We demonstrate that PKCepsilon forms a signaling complex and acts co-operatively with Akt to protect human vascular endothelial cells against apoptosis through induction of the anti-apoptotic protein Bcl-2 and inhibition of caspase-3 cleavage.
Publication Type: Research Support, Non-U.S. Gov't;
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38.
J Clin Endocrinol Metab 2007 Nov ; 11(92):4459-66.
Progesterone receptor regulates Bcl-2 gene expression through direct binding to its promoter region in uterine leiomyoma cells.
Yin P ,Lin Z ,Cheng YH ,Marsh EE ,Utsunomiya H ,Ishikawa H ,Xue Q ,Reierstad S ,Innes J ,Thung S ,Kim JJ ,Xu E ,Bulun SE ,
Division of Reproductive Biology Research, Department of Obstetrics and Gynecology, Feinberg School of Medicine at Northwestern University, Chicago, Illinois 60611, USA.
CONTEXT: Uterine leiomyomas are smooth muscle cell tumors that cause irregular uterine bleeding and pregnancy loss in many reproductive-age women. Progesterone stimulates their growth, whereas treatment with progesterone receptor (PR) antagonists or selective progesterone receptor modulators shrinks these tumors. Molecular mechanisms underlying these observations are unknown. OBJECTIVE: Bcl-2 is a key protein that inhibits apoptosis. It was proposed that growth enhancement of leiomyoma cells by progesterone was mediated via bcl-2 induction. Here we test the hypothesis that PR regulates the bcl-2 gene by directly binding to its promoter. RESULTS: The pure progesterone agonist R5020 increased the total number of viable primary human leiomyoma smooth muscle (LSM) cells in culture. Progesterone or R5020 (10(-6) m) significantly increased bcl-2 mRNA levels after 2 and 4 h by 9.2- and 3.4-fold, respectively, in LSM cells. Transient transfection with deletion mutants of bcl-2 promoter showed that the -1281/-258-bp region conferred responsiveness to progesterone induction in the presence of PR-A. We identified a palindromic progesterone response element (PRE) at -553/-539 bp. EMSA showed that PR in nuclear extracts from LSM cells bound specifically to this PRE. Chromatin immunoprecipitation-PCR confirmed in situ recruitment of PR to the -629/-388-bp region bearing the PRE. In vivo, bcl-2 mRNA levels correlated significantly with total PR mRNA levels in leiomyoma tissues. CONCLUSION: Taken together, progesterone via PR interacts with the bcl-2 promoter to induce its expression in leiomyoma tissue. This may explain, in part, the progesterone-dependent enhancement of growth in uterine leiomyoma.
Publication Type: Research Support, N.I.H., Extramural;
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39.
Eur J Gynaecol Oncol 2007 ; 4(28):290-3.
The role of p53, Bcl-2 and Ki-67 in premalignant cervical lesions and cervical cancer.
Türkçüoğlu I ,Tezcan S ,Kaygusuz G ,Atabekoğlu CS ,Ortaç F ,Güngör M ,Kankaya D ,Sertçelik A ,
Department of Obstetrics and Gynecology, Ankara University School of Medicine, Ankara, Turkey.
PURPOSE: The aim of this study was to determine the role of p53, Bcl-2 and Ki-67 expression in the carcinogenesis of cervical carcinoma and aggressiveness of cervical intraepithelial neoplasia (CIN). METHODS: The pathology specimens of 63 patients with a diagnosis of normal squamous epithelium (22 cases), CIN I (14), CIN II (5), CIN III (8) and squamous cell carcinoma (14) were evaluated immunohistochemically for the expression of p53, Bcl-2 and Ki-67 in paraffin sections. RESULTS: The expression of p53 and Ki-67 increased proportionally to the grade of CIN and cervical cancer, but only the increase of p53 expression was statistically significant (p = 0.002). There was no significant correlation between Bcl-2 expression and premalignant and malignant cervical lesions. CONCLUSION: p53 expression may have a role in the carcinogenesis of squamous cell cervical carcinoma whereas Bcl-2 expression has no role. Ki-67 expression can not be used in determining the aggressiveness of CIN lesions.
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40.
Histol Histopathol 2007 Dec ; 12(22):1365-70.
Apoptosis in peripheral neuroblastic tumors. Immunohistochemical expression of bcl-2 and p53 is related to DNA fragmentation.
Mejía C ,Navarro S ,Llombart-Bosch A ,
Instituto de Neurobiologia, Universidad Nacional Autonoma de Mexico, Queretaro, Mexico. maria.c.mejia@uv.es
We examined 111 cases of neuroblastoma (NB), searching for how NB relates to apoptotic control and other prognostic factors. Immunohistochemistry using avidin-biotin-peroxidase was carried out for bcl-2 and p53 proteins. Apoptosis was analyzed by in situ detection of chromosomal breakdown. DNA ladders were detected by electrophoresis and amplification of MYCN was carried out by PCR and Southern blot. Statistical analyses were performed with Pearson's chi2 and Kruskal-Wallis tests and Cox's regression. We found expression of bcl-2 protein mainly in cases of neuroblastoma without differentiation and in stages 3 and 4. Expression of p53 protein showed a correlation with bcl-2 and the apoptotic phenomenon; apoptosis was found mainly in favorable cases. Multivariate analysis showed bcl-2 protein expression to be the most independent risk factor. The study of apoptosis could be important for the design of therapies to treat neuroblastoma.
Publication Type: Research Support, Non-U.S. Gov't;
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41.
Carcinogenesis 2007 Sep ; 9(28):2008-12.
Single-nucleotide polymorphisms at the TP53-binding or responsive promoter regions of BAX and BCL2 genes and risk of squamous cell carcinoma of the head and neck.
Chen K ,Hu Z ,Wang LE ,Sturgis EM ,El-Naggar AK ,Zhang W ,Wei Q ,
Department of Epidemiology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
Tumor protein 53 (TP53), a transcriptional factor, induces expression of the B-cell lymphoma 2-associated X protein (BAX) gene by directly binding to the TP53-binding element in the BAX promoter but inhibits B-cell lymphoma 2 (BCL2) promoter-driven transcription through a responsive region in the BCL2 promoter. Therefore, we hypothesized that single-nucleotide polymorphisms (SNPs) of BAX and BCL2 promoters and the TP53 codon 72 SNP may jointly contribute to cancer risk. We tested this hypothesis in a hospital-based case-control study of 814 patients with squamous cell carcinoma of the head and neck (SCCHN) and 934 cancer-free controls in a US non-Hispanic white population. While there was no evidence of associations between BAX (-248 G>A), BCL2 (-938 C>A) or TP53 codon 72 SNPs and SCCHN risk in single-locus analyses, further analyses showed that, among TP53 heterozygotes after adjustment for age, sex and smoking and alcohol status, the BAX AA genotype was associated with an elevated risk of SCCHN [odds ratio (OR) = 6.60, 95% confidence interval (CI) = 1.38-31.50 compared with the BAX GG genotype or OR = 6.58, 95% CI = 1.38-31.49 compared with the combined genotypes (GG + AG)], whereas BCL2 A variant genotypes were associated with a decreased risk of SCCHN (adjusted OR = 0.68, 95% CI = 0.47-0.98 for CA vs CC and OR = 0.67, 95% CI = 0.48-0.95 for AA vs CA+CC). These altered risks appeared to be consistent with the roles of the anti-apoptotic BCL2 and the pro-apoptotic BAX. Our data suggest that the risk of SCCHN may be associated with these two SNPs of BAX and BCL2 promoter regions, particularly among TP53 heterozygotes. Larger studies are needed to validate these findings.
Publication Type: Research Support, N.I.H., Extramural;
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42.
J Immunol 2007 Aug 15; 4(179):2330-8.
Up-regulation of Bcl-2 through ERK phosphorylation is associated with human macrophage survival in an estrogen microenvironment.
Subramanian M ,Shaha C ,
National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
Estrogen is a known immunomodulator with pleiotropic effects on macrophage function that partly accounts for the gender bias observed in numerous autoimmune, cardiovascular, and neurodegenerative disorders. The effect of estrogen on the survival of human macrophages is largely unknown, and in this study we demonstrate that 17beta-estradiol (E2) provokes a death response in human THP-1 macrophages by initiating Bax translocation from cytosol to the mitochondria; however, a concomitant up-regulation of Bcl-2 creates a Bax to Bcl-2 ratio favorable for Bcl-2, thus ensuring cell survival. Both Bcl-2 up-regulation and Bax translocation are estrogen receptor-dependent events; however, Bcl-2 augmentation but not Bax translocation is dependent on Ca(2+) increase, activation of protein kinase C, and ERK phosphorylation. This estrogen-induced Bcl-2 increase is crucial for the survival of THP-1 macrophages as well as that of human peripheral blood monocyte-derived macrophages, which is evident from E2-induced cell death under small interfering RNA-mediated Bcl-2 knockdown conditions. Hence, this study demonstrates that E2-induced Bcl-2 up-regulation is a homeostatic survival mechanism necessary for the manifestation of immunomodulatory effect of estrogen on human macrophages.
Publication Type: Clinical Trial; Research Support, Non-U.S. Gov't;
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43.
J Biol Chem 2007 Sep 28; 39(282):28864-73.
Breast cancer cell proliferation is inhibited by BAD: regulation of cyclin D1.
Fernando R ,Foster JS ,Bible A ,Ström A ,Pestell RG ,Rao M ,Saxton A ,Baek SJ ,Yamaguchi K ,Donnell R ,Cekanova M ,Wimalasena J ,
Graduate School of Medicine, University of Tennessee Medical Center, Knoxville, Tennessee 37920, USA.
Recent investigations suggest that functions of the proapoptotic BCL2 family members, including BAD, are not limited to regulation of apoptosis. Here we demonstrate that BAD inhibits G(1) to S phase transition in MCF7 breast cancer cells independent of apoptosis. BAD overexpression inhibited G(1) transit and cell growth as well as cyclin D1 expression. Inhibition of cyclin D1 expression was mediated through inhibition of transcription activated by AP1. Chromatin immunoprecipitation assays indicated that BAD is localized at the 12-O-tetradecanoylphorbol-13-acetate-response element (TRE) and cAMP-response element (CRE) in the cyclin D1 promoter. This was shown to reflect direct binding interactions of BAD with c-Jun, and this interaction inhibited the activity of AP1 complexes at TRE. BAD did not interact with phosphorylated forms of c-Jun. Our data suggest that inhibitory TRE/CRE-c-Jun-BAD complexes are present at the cyclin D1 promoter in quiescent cells. Estrogen stimulation displaced BAD from TRE/CRE elements in MCF7 cells, whereas BAD overexpression inhibited estrogen-induced cyclin D1 synthesis and cell proliferation. Inhibition of endogenous BAD in MCF7 cells markedly increased the proliferative fraction and DNA synthesis, activated Cdks, and increased cyclin D1 protein levels. This action of BAD required serine residues Ser(75) and Ser(99). Both phosphorylated and unphosphorylated forms of BAD localized to the nuclei of human breast epithelial cells. Thus, we demonstrate a novel role for BAD in cell cycle regulation dependent upon its phosphorylation state and independent of the BAD/BCL2 interaction and apoptosis.
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44.
Cell Mol Biol (Noisy-le-grand) 2006 ; (52 Suppl):OL915-22.
Impaired kinetics of Bax-GFP and Smac/DIABLO-GFP in caspase-8- and bid-silenced and Bcl-2 overexpressed breast cancer MCF-7 cells exposed to camptothecin.
Górka M ,Lamparska-Przybysz M ,Motyl T ,
Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Warsaw, Poland.
Smac/DIABLO, a proapoptotic protein released from mitochondrial intermembrane space during apoptosis, promotes caspases activation by IAPs neutralization. The kinetics and molecular mechanism of Smac/DIABLO release from mitochondria has remained obscure. Present study is focused on the role of Bid in the control of Bax-GFP and Smac/DIABLO-GFP kinetics in breast cancer MCF-7 cells stimulated to apoptosis with camptothecin (CPT). Minute kinetics of proteins was examined by homeostatic confocal microscopy. The release of Smac/DIABLO-GFP from mitochondria comprised two phases: initial-rapid, lasting 20-30 min and subsequent 30 min-plateau phase, followed by the decrease of Smac/DIABLO-related fluorescence due to cell destruction. The kinetics of Bax-GFP aggregation on mitochondria coincided in time with Smac/DIABLO-GFP release from these organelles. Bid knock down and Bcl-2 overexpression delayed Bax-GFP aggregation and completely inhibited Smac/DIABLO-GFP release from mitochondria. Knock down of caspase 8 (activator of Bid) delayed both Bax-GFP aggregation and Smac/DIABLO-GFP release in CPT-treated cells. In conclusion, Bid protein is crucial for the control of the release of Smac/DIABLO from mitochondria in breast cancer MCF-7 stimulated to apoptosis with CPT.
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45.
J Mol Diagn 2007 Sep ; 4(9):530-7.
''Minor'' BCL2 breakpoints in follicular lymphoma: frequency and correlation with grade and disease presentation in 236 cases.
Weinberg OK ,Ai WZ ,Mariappan MR ,Shum C ,Levy R ,Arber DA ,
Stanford University, Department of Pathology, 300 Pasteur Dr., Room L235, Stanford, CA 94305, USA. okw@stanford.edu
Follicular lymphomas are frequently associated with the t(14;18)(q32;q21). This translocation can be detected by karyotype, polymerase chain reaction (PCR), and fluorescence in situ hybridization (FISH). In addition to the breakpoints currently used for diagnosis located in the major breakpoint region (MBR) and the minor cluster region (mcr), recent studies have reported the existence of other breakpoints (3' BCL2, 5'mcr, and icr). In this study, we examined the frequency of all five breakpoints in 236 cases of follicular lymphomas by real-time PCR analysis. The distribution of breakpoint sites consisted of MBR in 118 cases (50%), mcr in 11 (5%), icr in 32 (13%), 3' BCL2 in 13 (6%), and 5' mcr in three cases (1%). These findings illustrate significantly higher frequency of the icr breakpoint as compared with the more frequently studied mcr. Correlation of breakpoints with histology showed that MBR breakpoints occur more frequently in grade 2 lymphomas (P = 0.042). A majority of the PCR-negative cases (75%) contained an IGH/BCL2 translocation with FISH methods, suggesting the presence of other BCL2 breakpoints. Correlation of breakpoints with survival did not reveal significant differences. Diagnostic laboratories should consider expanding their PCR methods to include other BCL2 breakpoints and correlating with FISH methods when appropriate.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't;
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46.
Cancer Res 2007 Jul 1; 13(67):6278-85.
Repression of BCL2 by the tumor suppressor activity of the lysyl oxidase propeptide inhibits transformed phenotype of lung and pancreatic cancer cells.
Wu M ,Min C ,Wang X ,Yu Z ,Kirsch KH ,Trackman PC ,Sonenshein GE ,
Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA. minwu@bu.edu
The gene encoding lysyl oxidase (LOX) was identified as the ras recision gene (rrg), with the ability to revert Ras-mediated transformation of NIH 3T3 fibroblasts. Mutations in RAS genes have been found in approximately 25% of lung cancers and in 85% of pancreatic cancers. In microarray analysis, these cancers were found to display reduced LOX gene expression. Thus, the ability of the LOX gene to repress the transformed phenotype of these cancer cells was tested. LOX is synthesized as a 50-kDa secreted precursor Pro-LOX that is processed to the 32-kDa active enzyme (LOX) and to an 18-kDa propeptide (LOX-PP). Recently, we mapped the rrg activity of Pro-LOX to the LOX-PP in Ras-transformed NIH 3T3 cells. Ectopic Pro-LOX and LOX-PP expression in H1299 lung cancer cells inhibited growth in soft agar and invasive colony formation in Matrigel and reduced activation of extracellular signal-regulated kinase (ERK) and Akt, with LOX-PP showing substantially higher activity. Similarly, LOX-PP expression in PANC-1 pancreatic cancer cells effectively reduced ERK and Akt activity and inhibited growth in soft agar and ability of these cells to migrate. Nuclear Factor-kappaB (NF-kappaB) and its target gene BCL2, which are overexpressed in 70% to 75% of pancreatic cancers, have recently been implicated in invasive phenotype. LOX-PP substantially reduced NF-kappaB and Bcl-2 levels. Reintroduction of Bcl-2 into PANC-1 or H1299 cells expressing LOX-PP restored the transformed phenotype, suggesting that Bcl-2 is an essential target. Thus, LOX-PP potently inhibits invasive phenotype of lung and pancreatic cancer cells, suggesting potential therapeutic applications in treatment of these cancers.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.;
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47.
Clin Cancer Res 2007 Jun 15; 12(13):3585-90.
Bcl-2 and Bax expression predict prostate cancer outcome in men treated with androgen deprivation and radiotherapy on radiation therapy oncology group protocol 92-02.
Khor LY ,Moughan J ,Al-Saleem T ,Hammond EH ,Venkatesan V ,Rosenthal SA ,Ritter MA ,Sandler HM ,Hanks GE ,Shipley WU ,Pollack A ,
Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
PURPOSE: Bcl-2 is antiapoptotic, and its overexpression has been associated with resistance to androgen deprivation and poor outcome in some patients treated with radiotherapy. Bax is proapoptotic, regulating Bcl-2 through heterodimer formation. In a prior study, Bcl-2 and Bax were not related to outcome in locally advanced patients treated with radiotherapy or short-term androgen deprivation + radiotherapy (STAD+RT) on another Radiation Therapy Oncology Group trial (86-10). A follow-up investigation was carried out here in more contemporary high-risk men treated on Radiation Therapy Oncology Group 92-02 with STAD+RT or long-term AD+RT (LTAD+RT). EXPERIMENTAL DESIGN: Adequate tissue was available to be analyzed immunohistochemically in 502 patients for Bcl-2 and 343 patients for Bax. Univariate and multivariate analyses by Cox proportional hazards models were applied to end points of failure. RESULTS: Bcl-2 was positive in 45.6% cases, and Bax expression altered in 53.9% cases. Abnormal Bcl-2 was not related to any of the failure end points tested. Altered Bax expression was significantly associated with any failure (P = 0.023) and marginally with biochemical failure (P = 0.085). The combination of negative Bcl-2/normal Bax expression seemed more robust, being significantly related to reduced biochemical failure (P = 0.036) and any failure (P = 0.046). The predictive value of negative Bcl-2/normal Bax was most pronounced in those who received STAD+RT, as opposed to LTAD+RT. CONCLUSIONS: Normal Bax expression was associated with significantly more favorable outcome. The combination of negative Bcl-2 and normal Bax was more consistently significant, particularly when STAD+RT was the treatment administered. These data suggest that LTAD+RT should be used when either Bcl-2 or Bax is abnormally expressed.
Publication Type: Clinical Trial, Phase III; Randomized Controlled Trial; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't;
Comment In: Clin Cancer Res. 2007 Jun 15;13(12):3435-8
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48.
Cancer Detect Prev 2007 ; 3(31):225-32.
Role of BCL2 (ala43thr), CCND1 (G870A) and FAS (A-670G) polymorphisms in modulating the risk of developing esophageal cancer.
Jain M ,Kumar S ,Lal P ,Tiwari A ,Ghoshal UC ,Mittal B ,
Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareilly Road, Lucknow 226014, India.
BACKGROUND: Perturbations in the cell cycle and apoptotic genes have been implicated in human malignancies. A study of BCL2 ala43thr, CCND1 G870A and FAS A-670G gene polymorphisms was undertaken to explore their role in influencing the susceptibility for development of esophageal cancer. METHODS: A total of 151 patients and age and gender matched 201 controls were investigated for BCL2 ala43thr, CCND1 G870A and FAS A-670G polymorphisms by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). RESULTS: The ala43ala genotype of BCL2 anti-apoptotic gene was significantly associated with risk of developing esophageal cancer (OR 2.1, 95%CI=1.0-4.4, P=0.03), more so in males (OR 2.6, 95%CI=P=0.03). In CCND1 G870A polymorphism, the AA genotype was marginally associated with higher risk of esophageal cancer (OR 1.5, 95%CI=0.98-2.4, P=0.05). No significant differences in genotype frequencies of FAS A-670G polymorphism were seen between esophageal cancer patients and controls (P=0.32). Interaction of BCL2 ala43ala, CCND1 870AA and FAS -670AA genotypes did not increase the risk multiplicatively. Association with clinical characteristics showed BCL2 ala43ala genotype to be at increased risk for developing tumors in the middle third location (OR 2.3, 95%CI=1.0-5.3, P=0.03), while patients with CCND1 870AA genotypes were at higher risk for the development of cancer in the upper third location (OR 3.8, 95%CI=1.6-9, P=0.002). BCL2 ala43ala genotype did not modulate the cancer risk in tobacco users. However, patients with CCND1 870AA and FAS -670AA genotypes were associated with a significantly lower number of smoking and chewing pack-years, suggesting a dose-dependent interaction in the risk for esophageal cancer (P=0.005). CONCLUSION: There appears to be an influence of BCL2 ala43ala and CCND1 870AA genotypes on esophageal cancer phenotype, particularly with regard to tumor location, which supports the theory of prevalence of site-specific genetic alterations. FAS A-670G was not associated with the risk of developing esophageal cancer. Gene-environment interaction analysis showed cancer susceptibility in CCND1 870AA and FAS -670AA genotype to be influenced by quantity of tobacco.
Publication Type: Research Support, Non-U.S. Gov't;
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49.
Tumori ; 2(93):195-7.
Mutational analysis of the BH3 domains of proapoptotic Bcl-2 family genes Bad, Bmf and Bcl-G in laryngeal squamous cell carcinomas.
Yoo NJ ,Soung YH ,Lee SH ,Jeong EG ,Lee SH ,
Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, Korea.
AIMS: There is mounting evidence that deregulation of apoptosis is involved in the mechanisms of cancer development. Somatic mutations of apoptosis-related genes have been reported in many human cancers. The aim of this study was to explore the possibility that mutation of the BH3 domains of the proapoptotic Bcl-2 genes Bad, Bmf and Bcl-G might be involved in the development of laryngeal cancer. METHODS: We analyzed the BH3 domains of Bad, Bmf and Bcl-G for the detection of somatic mutations in 33 squamous cell carcinomas of the larynx by a polymerase chain reaction-based single-strand conformation polymorphism assay. RESULTS: There were no somatic mutations of the BH3 domains of Bad, Bmfand BcI-G in the laryngeal squamous cell carcinoma samples. CONCLUSIONS: The data presented here indicate that BH3 domain mutation of the proapoptotic genes Bad, Bmf and Bcl-G is rare in laryngeal squamous cell carcinoma and may not contribute to the apoptosis-resistance mechanisms of laryngeal squamous cell carcinoma.
Publication Type: Research Support, Non-U.S. Gov't;
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50.
Appl Immunohistochem Mol Morphol 2007 Mar ; 1(15):64-9.
Immunoreactivity of p53, Mdm2, p21(WAF1/CIP1) Bcl-2, and Bax in soft tissue sarcomas: correlation with histologic grade.
Sabah M ,Cummins R ,Leader M ,Kay E ,
Department of Histopathology, Education and Research Centre, The Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9. munasabah@eircom.net
Tumor growth depends on 2 distinctive pathways: cell proliferation and apoptosis. The p53 pathway is an important regulator of the cell cycle as it triggers growth arrest or leads to apoptosis in response to cellular stress and therefore is commonly targeted during tumorigenesis. Apoptosis is also controlled by the Bcl-2 family, which includes proapoptotic and antiapoptotic proteins. The aim of this study was to investigate the expression of proteins that are involved in the p53 pathway and apoptosis in different types of soft tissue sarcomas and to correlate the expression of these proteins with the histologic grade of sarcoma cases. One hundred fifty-two cases of different types of soft tissue sarcomas were analyzed. The cases consisted of 54 low-grade, 40 intermediate-grade, and 58 high-grade sarcomas. Immunohistochemical stains for p21(WAF1/CIP1), p53, Mdm2, Bcl-2, and Bax proteins were carried out on tissue microarrays. Nuclear reactivity for p53 was detected in 49 cases (32.2%). Overexpression of Mdm2 was found in 18 cases (11.8%) and p21(WAF1/CIP1) immunostaining was seen in 28 tumors (18.4%). p53 and p21(WAF1/CIP1) expression correlated with the tumor grade (low grade, 5.6% and 3.7%; intermediate grade, 22.5% and 20%; high grade, 63.8% and 31%, respectively). Expression of Bax protein was a common finding in soft tissue sarcoma cases. It was detected in 141 cases (92.8%). Bcl-2 was identified in 59 tumors (38.8%) and was more prevalent in high-grade sarcomas (low grade, 25.9%; intermediate grade, 32.5%; high grade, 55.2%). It was concluded that alterations in the p53 pathway and genes that regulate apoptosis are common events in soft tissue sarcomas. The expression of p53, p21(WAF1/CIP1), and Bcl-2 is closely associated with the histologic grade of the tumor, and therefore these proteins may be used as prognostic markers.
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51.
Clin Orthop Relat Res 2007 Sep ; (462):32-7.
Apoptotic gene analysis in idiopathic talipes equinovarus (clubfoot).
Ester AR ,Tyerman G ,Wise CA ,Blanton SH ,Hecht JT ,
University of Texas Health Science Center, Houston Graduate School of Biomedical Sciences, Houston, TX, USA.
Idiopathic talipes equinovarus, also known as clubfoot, is a common birth defect occurring in one of 1000 live births. It is a complex disorder in which multiple genes and environmental factors may play an etiologic role. Several chromosomal deletion regions, including 2q31-33, are associated with talipes equinovarus and may harbor genes that contribute to the idiopathic talipes equinovarus phenotype. Previously, two STRs in the 2q31-33, GATA149B10 and D2S1371, showed linkage with association to idiopathic talipes equinovarus. Single nucleotide polymorphisms (SNPs) in three apoptotic genes (Casp8, Casp10, and CFLAR) near GATA149B10 were genotyped in idiopathic talipes equinovarus families. rs3731714 in Casp10 showed linkage with association, suggesting variation in the apoptotic gene pathway, which is important in limb morphogenesis, and may play a role in the development of idiopathic talipes equinovarus. We genotyped SNPs spanning seven apoptotic genes-Casp3, Casp8, Casp9, Casp10, Bid, Bcl-2 and Apaf1-in 210 simplex trios and 139 multiplex families and tested for link-age and association to idiopathic talipes equinovarus. One SNP in each of the genes provided suggestive evidence of association with idiopathic talipes equinovarus. Several haplotypes constructed from these SNPs displayed altered transmission. These data suggest genetic variation in apoptotic genes may play a role in development of idiopathic talipes equinovarus.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't;
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52.
J Rheumatol 2007 Jul ; 7(34):1580-4.
Increased Fas and Bcl-2 expression on peripheral mononuclear cells from patients with active juvenile-onset systemic lupus erythematosus.
Liphaus BL ,Kiss MH ,Carrasco S ,Goldenstein-Schainberg C ,
Children's Institute, Department of Pediatrics, and Department of Rheumatology, University of São Paulo, São Paulo, Brazil. bernadll@icr.hcnet.usp.br
OBJECTIVE: To determine expressions of Fas and Bcl-2 on peripheral blood T and B lymphocytes from patients with juvenile-onset systemic lupus erythematosus (JSLE). METHODS: Thirty-eight patients with JSLE and 21 healthy controls were studied. Eleven JSLE patients with SLEDAI score >or= 8 were categorized as active. Freshly isolated peripheral blood mononuclear cells were stained for lymphocyte markers CD3, CD4, CD8, and CD19 and for Fas and Bcl-2 molecules. Cell protein expression was measured by 3-color flow cytometry. RESULTS: Percentages of lymphocytes positively stained for Fas antigen and cytoplasmic expression of Bcl-2 measured by mean fluorescence intensity from patients were significantly increased compared to controls on CD3+, CD4+, and CD8+ T cells. Patients with active disease had higher percentages of CD19+ B cells positive for Fas antigen compared to patients with inactive lupus. A direct statistical correlation was observed between Fas and Bcl-2 expression on CD19+ B cells and SLE Disease Activity Index score. CONCLUSION: Patients with juvenile-onset SLE show upregulation of apoptosis-related proteins. Patients with active and inactive disease have a different profile of Fas and Bcl-2 expression.
Publication Type: Research Support, Non-U.S. Gov't;
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53.
Mol Cell Biochem 2007 Oct ; 1-2(304):213-8.
CDK11(p58) protein kinase activity is associated with Bcl-2 down-regulation in pro-apoptosis pathway.
Yun X ,Wu Y ,Yao L ,Zong H ,Hong Y ,Jiang J ,Yang J ,Zhang Z ,Gu J ,
Gene Research Center, Shanghai Medical College of Fudan University, Shanghai 200032, P.R. China.
CDK11(p58), a G2/M-specific protein kinase, has been shown to be associated with apoptosis in many cell lines, with largely unknown mechanisms. Our previous study proved that CDK11(p58)-enhanced cycloheximide (CHX)-induced apoptosis in SMMC-7721 hepatocarcinoma cells. Here we report for the first time that ectopic expression of CDK11(p58) down-regulates Bcl-2 expression and its Ser70, Ser87 phosphorylation in CHX-induced apoptosis in SMMC-7721 cells. Overexpression of Bcl-2 counteracts the pro-apoptotic activity of CDK11(p58). Furthermore, we confirm that the kinase activity of CDK11(p58) is essential to the down-regulation of Bcl-2 as well as apoptosis. Taken together, these results demonstrate that CDK11(p58) down-regulates Bcl-2 in pro-apoptosis pathway depending on its kinase activity, which elicits survival signal in hepatocarcinoma cells.
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54.
Mol Biol Cell 2007 Jul ; 7(18):2735-44.
Cytotoxic necrotizing factor 1 prevents apoptosis via the Akt/IkappaB kinase pathway: role of nuclear factor-kappaB and Bcl-2.
Miraglia AG ,Travaglione S ,Meschini S ,Falzano L ,Matarrese P ,Quaranta MG ,Viora M ,Fiorentini C ,Fabbri A ,
Departments of Drug Research and Evaluation and Technology, Istituto Superiore di Sanità, Rome, Italy.
Cytotoxic necrotizing factor 1 (CNF1) is a protein toxin produced by some pathogenic strains of Escherichia coli that specifically activates Rho, Rac, and Cdc42 GTPases. We previously reported that this toxin prevents the ultraviolet-B-induced apoptosis in epithelial cells, with a mechanism that remained to be defined. In this work, we show that the proteasomal degradation of the Rho GTPase is necessary to achieve cell death protection, because inhibition of Rho degradation abolishes the prosurvival activity of CNF1. We hypothesize that Rho inactivation allows the activity of Rac to become dominant. This in turn leads to stimulation of the phosphoinositide 3-kinase/Akt/IkappaB kinase/nuclear factor-kappaB prosurvival pathway and to a remarkable modification in the architecture of the mitochondrial network, mainly consisting in the appearance of elongated and interconnected mitochondria. Importantly, we found that Bcl-2 silencing reduces the ability of CNF1 to protect cells against apoptosis and that it also prevents the CNF1-induced mitochondrial changes. It is worth noting that the ability of a bacterial toxin to induce such a remodeling of the mitochondrial network is herein reported for the first time. The possible pathophysiological relevance of this finding is discussed.
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55.
Br J Cancer 2007 May 21; 10(96):1540-3.
Bcl-2 expression in rituximab refractory cutaneous B-cell lymphoma.
Wobser M ,Voigt H ,Eggert AO ,Houben R ,Kauczok CS ,Bröcker EB ,Becker JC ,
Department of Dermatology, University of Wuerzburg, Josef-Schneider-Strasse 2, 97080 Wuerzburg, Germany.
Rituximab has been established as an effective and safe therapy for cutaneous B-cell lymphoma (CBCL). Different survival pathways, that is the Raf/MEK/Erk- or the p38MAPK cascade, have been suggested as downstream mediators of rituximab and may be involved in treatment failure. Biopsies from four patients, suffering from different subtypes of CBCL, which were obtained at various time points of relapse during or after therapy with 375 mg rituximab per m2 of body surface area, were analysed for the expression of CD20, CD3, Ki-67, Raf-kinase inhibitory protein (RKIP) and bcl-2 by immunohistochemistry. No CD20-loss variants, that is the suggested main tumour escape mechanism to rituximab therapy, were observed in any specimen of relapsing CBCL. Notably, the expression of proapoptotic RKIP remained increased in these tumour samples. This was concomitated by a constant to slightly reduced proliferation status as demonstrated by Ki-67 staining. However, relapsing CBCL exhibited a strong upregulation of the antiapoptotic molecule bcl-2 in comparison to pretherapeutic levels. The immunohistochemical analyses of this case series of rituximab refractory CBCL suggest that upregulation of bcl-2 may play a major role in therapy resistance.
Publication Type: Research Support, Non-U.S. Gov't;
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56.
Scand J Infect Dis 2007 ; 5(39):441-8.
Analyses of Bcl-2, Survivin, and CD44v6 expressions and human papillomavirus infection in cervical carcinomas.
Yaqin M ,Runhua L ,Fuxi Z ,
Microbiological Laboratory, Medical College of Datong University, China.
Infection with human papillomaviruses (HPV), and suppression of apoptosis and cell adhesion are putative aetiological factors to cervical carcinogenesis. However, controversial results have been reported with respect to their relationships with cervical carcinomas. Here we analysed papillomavirus infection, apoptotic index (AI), expressions of the anti-apoptotic proteins Bcl-2 and Survivin, and expression of the cell-adhesion protein CD44 in cervical tissue samples from individuals with and without cervical carcinomas. Although both HPV16 and HPV18 are reportedly important aetiological factors, we found that cervical carcinomas were highly associated with HPV16 but not HPV18 infection. Immunohistochemistry showed that the percentages of cells expressing Bcl-2, Survivin, and CD44v6 were greatly increased in samples of cervical carcinomas. Furthermore, the expression rates of Survivin and CD44v6 increased whereas that of Bcl-2 declined as cervical cancers developed into more advanced clinical or histopathological stages. Surprisingly, there was little difference in AI between control and cervical cancer samples. These observations provide further evidence that HPV infection, apoptosis and cell adhesion abnormalities are related to cervical cancers. They also suggest that Bcl-2, Survivin and CD44v6 expressions, and HPV16 infection could be useful indices in screening of cervical carcinomas.
Publication Type: Research Support, Non-U.S. Gov't;
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57.
Clin Exp Pharmacol Physiol ; 5-6(34):450-6.
siRNA-mediated Bcl-2 and Bcl-xl gene silencing sensitizes human hepatoblastoma cells to chemotherapeutic drugs.
Lei XY ,Zhong M ,Feng LF ,Zhu BY ,Tang SS ,Liao DF ,
Institute of Pharmacy and Pharmacology, University of South China, Hengyang, Hunan, China. lei_xiaoyong@yahoo.com.cn
1. The aim of the present study was to investigate the changes in chemotherapeutic drug sensitivity of HepG2 cells transfected with Bcl-2 and Bcl-xl siRNA expression vectors. 2. Bcl-2 and Bcl-xl siRNA and negative siRNA expression vectors were constructed and stably transfected into HepG2 cells. Reverse transcriptase-polymerase chain reaction was used to detect the target gene expression, and the Bcl-2, Bcl-xl, Bax and caspase-3 protein levels were measured using western blots and immunofluorescence. The sensitivity of the cells to the chemotherapeutic drugs 5-fluorouracil (5-FU) and 10-hydroxycamptothecin (HCPT) was analysed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) and flow cytometry. 3. The Bcl-2 and Bcl-xl gene expression and corresponding protein levels in Bcl-2 siRNA, Bcl-xl siRNA and Bcl-2/Bcl-xl siRNA transfected cells were reduced compared with negative siRNA transfected or untreated cells. The Bax protein level remained unaltered but the caspase-3 level was enhanced when Bcl-2 and Bcl-xl protein levels were reduced. The MTT results demonstrated that Bcl-2 and Bcl-xl transfected cells exhibited increased sensitivity to 5-FU or HCPT. Flow cytometry demonstrated that the sub G1 cell population increased in Bcl-2/Bcl-xl siRNA co-transfected and Bcl-xl siRNA and Bcl-2 siRNA transfected cells when compared with negative siRNA or untreated cells. The latter trend was strengthened further in the presence of 5-FU or HCPT. 4. Thus, Bcl-2 and Bcl-xl siRNA-mediated gene silencing, in combination with chemotherapy, may be a potential therapeutic strategy against human hepatoblastoma.
Publication Type: Research Support, Non-U.S. Gov't;
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58.
Autophagy ; 4(3):374-6.
BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin 1 and Bcl-2/Bcl-X(L).
Maiuri MC ,Criollo A ,Tasdemir E ,Vicencio JM ,Tajeddine N ,Hickman JA ,Geneste O ,Kroemer G ,
INSERM, U848, Villejuif, France.
Beclin 1 has recently been identified as novel BH3-only protein, meaning that it carries one Bcl-2-homology-3 (BH3) domain. As other BH3-only proteins, Beclin 1 interacts with anti-apoptotic multidomain proteins of the Bcl-2 family (in particular Bcl-2 and its homologue Bcl-X(L)) by virtue of its BH3 domain, an amphipathic alpha-helix that binds to the hydrophobic cleft of Bcl-2/Bcl-X(L). The BH3 domains of other BH3-only proteins such as Bad, as well as BH3-mimetic compounds such as ABT737, competitively disrupt the inhibitory interaction between Beclin 1 and Bcl-2/Bcl-X(L). This causes autophagy of mitochondria (mitophagy) but not of the endoplasmic reticulum (reticulophagy). Only ER-targeted (not mitochondrion-targeted) Bcl-2/Bcl-X(L) can inhibit autophagy induced by Beclin 1, and only Beclin 1-Bcl-2/Bcl-X(L) complexes present in the ER (but not those present on heavy membrane fractions enriched in mitochondria) are disrupted by ABT737. These findings suggest that the Beclin 1-Bcl-2/Bcl-X(L) complexes that normally inhibit autophagy are specifically located in the ER and point to an organelle-specific regulation of autophagy. Furthermore, these data suggest a spatial organization of autophagy and apoptosis control in which BH3-only proteins exert two independent functions. On the one hand, they can induce apoptosis, by (directly or indirectly) activating the mitochondrion-permeabilizing function of pro-apoptotic multidomain proteins from the Bcl-2 family. On the other hand, they can activate autophagy by liberating Beclin 1 from its inhibition by Bcl-2/Bcl-X(L) at the level of the endoplasmic reticulum.
Publication Type: Comment; Research Support, Non-U.S. Gov't;
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59.
Carcinogenesis 2007 Oct ; 10(28):2166-71.
Bcl-2 over-expression promotes genomic instability by inhibiting apoptosis of cells exposed to hydrogen peroxide.
Cox AG ,Hampton MB ,
Free Radical Research Group, Department of Pathology, Christchurch School of Medicine & Health Sciences, University of Otago, PO Box 4345, Christchurch, New Zealand.
The anti-apoptotic oncogene bcl-2 is hypothesized to increase the antioxidant status of cells, thereby protecting them from oxidative stress. In this study, we examined hydrogen peroxide (H2O2)-mediated oxidative stress in Jurkat T lymphoma cells. Over-expression of Bcl-2 did not inhibit cytotoxicity at doses of H2O2 that caused necrosis (>200 microM), but it did block cell death at apoptotic doses (<200 microM). However, these cells exhibited the same initial level of protein and lipid oxidation following exposure to H2O2 as the parental cells, indicating that the anti-apoptotic activity is not associated with general antioxidant properties. Bcl-2 expression was able to protect against secondary protein carbonyl formation, which was linked to lysosome stabilization. Assessment of micronuclei formation in cells over-expressing Bcl-2 showed evidence of increased genomic instability, consistent with the impairment of apoptosis in damaged cells. We conclude that while Bcl-2 can block cytotoxicity associated with apoptosis-inducing levels of oxidative stress, it does not protect the cells from the stress itself. Bcl-2 may promote tumourigenesis by preventing the removal of oxidatively damaged cells.
Publication Type: Research Support, Non-U.S. Gov't;
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60.
Mol Cancer Res 2007 Apr ; 4(5):331-9.
Integrative genomic analysis of small-cell lung carcinoma reveals correlates of sensitivity to bcl-2 antagonists and uncovers novel chromosomal gains.
Olejniczak ET ,Van Sant C ,Anderson MG ,Wang G ,Tahir SK ,Sauter G ,Lesniewski R ,Semizarov D ,
Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road, Building AP-10, Department R4CD, Abbott Park, IL 60064, USA.
Cancer is a highly heterogeneous disease in terms of the genetic profile and the response to therapeutics. An early identification of a genomic marker in drug discovery may help select patients that would respond to treatment in clinical trials. Here we suggest coupling compound screening with comparative genomic hybridization analysis of the model systems for early discovery of genomic biomarkers. A Bcl-2 antagonist, ABT-737, has recently been discovered and shown to induce regression of solid tumors, but its activity is limited to a fraction of small-cell lung carcinoma (SCLC) models tested. We used comparative genomic hybridization on high-density single-nucleotide polymorphism genotyping arrays to carry out a genome-wide analysis of 23 SCLC cell lines sensitive and resistant to ABT-737. The screen revealed a number of novel recurrent gene copy number abnormalities, which were also found in an independent data set of 19 SCLC tumors and confirmed by real-time quantitative PCR. A previously unknown amplification was identified on 18q and associated with the sensitivity of SCLC cell lines to ABT-737 and another Bcl-2 antagonist. The region of gain contains Bcl-2 and NOXA, two apoptosis-related genes. Expression microarray profiling showed that the genes residing in the amplified region of 18q are also overexpressed in the sensitive lines relative to the resistant lines. Fluorescence in situ hybridization analysis of tumors revealed that Bcl-2 gain is a frequent event in SCLC. Our findings suggest that 18q21-23 copy number will be a clinically relevant predictor for sensitivity of SCLC to Bcl-2 family inhibitors. The 18q21-23 genomic marker may have a broader application in cancer because Bcl-2 is associated with apoptosis evasion and chemoresistance.
Publication Type: Evaluation Studies;
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61.
Cell 2007 Apr 6; 1(129):45-56.
Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1.
Bruey JM ,Bruey-Sedano N ,Luciano F ,Zhai D ,Balpai R ,Xu C ,Kress CL ,Bailly-Maitre B ,Li X ,Osterman A ,Matsuzawa S ,Terskikh AV ,Faustin B ,Reed JC ,
Burnham Institute for Medical Research, La Jolla, CA 92037, USA.
Caspases are intracellular proteases that cleave substrates involved in apoptosis or inflammation. In C. elegans, a paradigm for caspase regulation exists in which caspase CED-3 is activated by nucleotide-binding protein CED-4, which is suppressed by Bcl-2-family protein CED-9. We have identified a mammalian analog of this caspase-regulatory system in the NLR-family protein NALP1, a nucleotide-dependent activator of cytokine-processing protease caspase-1, which responds to bacterial ligand muramyl-dipeptide (MDP). Antiapoptotic proteins Bcl-2 and Bcl-X(L) bind and suppress NALP1, reducing caspase-1 activation and interleukin-1beta (IL-1beta) production. When exposed to MDP, Bcl-2-deficient macrophages exhibit more caspase-1 processing and IL-1beta production, whereas Bcl-2-overexpressing macrophages demonstrate less caspase-1 processing and IL-1beta production. The findings reveal an interaction of host defense and apoptosis machinery.
Publication Type: Research Support, N.I.H., Extramural;
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62.
Ann N Y Acad Sci 2007 Jan ; (1095):19-25.
Bcl-2 expression in oral squamous cell carcinoma.
Popović B ,Jekić B ,Novaković I ,Luković LJ ,Tepavcević Z ,Jurisić V ,Vukadinović M ,Milasin J ,
Institute of Biology and Human Genetics, School of Dentistry, University of Belgrade, Dr Subotica 8, 11000 Belgrade, Serbia and Montenegro. brankapo@verat.net
Apoptosis is a genetically regulated process involved in tissue size regulation, morphogenesis, and elimination of genetically damaged cells. A pallet of genes is involved in the control of apoptosis, such as bcl-2 family whose oncogenic potential has been demonstrated in oral tumorigenesis. Different members of bcl-2 family may promote or inhibit apoptosis by synthesizing anti- and proapoptotic proteins. One of antiapoptotic proteins, bcl-2, with a crucial role in apoptosis regulation was the object of our study. By means of immunohistochemistry we estimated the level of overexpression of bcl-2 proteins in a series of the 26 formalin fixed, paraffin-embedded samples of oral squamous cell carcinoma (OSCC). Analyzed tumors originated from different sites of oral cavity; 7/26 belonged to stage II, 14/26 to stage III, and 5/26 to stage IV. Immunoreactivity was scored according to the percentage and intensity of positive cytoplasmic bcl-2 staining. All tumors had low percentage of positively stained bcl-2 cells, with mean values for lower/higher intensity of 8.3 +/- 2.5/34.4 +/- 7, 7.5 +/- 1.1/31.9 +/- 4.3, and 8.4 +/- 5.8/31.5 +/- 5.8 within stages II, III, and IV, respectively. Low level of bcl-2 expression in our sample seems to be associated with higher survival rate: 77% for the 5-year follow-up period. Comparing clinicopathologic and risk factors data within each and between three groups of analyzed tumors (lip-tongue P = 0.58, tongue-floor of the mouth, P = 0.21, lip-floor of the mouth, P = 0.50) there was no significant difference. However, our results suggest that the level of bcl-2 expression could be a valuable predictor of tumor behavior and disease outcome.
Publication Type: Research Support, Non-U.S. Gov't;
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63.
Mod Pathol 2007 Apr ; 4(20):416-26.
Mcl-1, Bcl-XL and Stat3 expression are associated with progression of melanoma whereas Bcl-2, AP-2 and MITF levels decrease during progression of melanoma.
Zhuang L ,Lee CS ,Scolyer RA ,McCarthy SW ,Zhang XD ,Thompson JF ,Hersey P ,
Discipline of Pathology, Faculty of Medicine, The University of Sydney, Sydney, NSW, Australia.
Members of the Bcl-2 family of antiapoptotic proteins (Bcl-2, Bcl-XL and Mcl-1) are key regulators of apoptosis. The purpose of the present study was to examine and better define the role of Bcl-2, Bcl-XL and Mcl-1 in the progression of melanoma. Immunohistochemical staining for Bcl-2, Bcl-XL and Mcl-1 was performed on paraffin sections of 100 cases of benign nevi, primary melanoma and metastatic melanoma. Expression was correlated with histopathologic features, clinical progress and expression of transcription factors (AP-2, MITF and p-Stat3). Bcl-2 was expressed in 100% of benign nevi and thin melanoma (<or=1.0 mm) but was less in thick melanoma (>1.0 mm) (88%), subcutaneous (62%) and lymph node metastases (35%). In contrast, Bcl-XL and Mcl-1 were expressed at lower levels in nevi and thin melanoma compared to Bcl-2 but their expression was much higher in thick melanoma and in subcutaneous and lymph node metastases (P<0.0001). Bcl-2 expression was negatively associated with tumor thickness (P<0.05) but Bcl-XL expression increased with increasing tumor thickness (P<0.05) and dermal tumor mitotic rate (P<0.05). Similarly Mcl-1 expression increased with increasing tumor thickness (P<0.09) and dermal tumor mitotic rate (P<0.17). Bcl-2 expression was positively correlated with expression of the transcription factors microphthalmia transcription factor (MITF) and nuclear AP-2 whereas Bcl-XL (and Mcl-1) expression were positively correlated with p-Stat3. This study is the first to show a clear dissociation between changes in Bcl-2 expression (downregulation) and Bcl-XL, Mcl-1 expression (upregulation) during progression of melanoma. The results were also consistent with a role for AP-2 and MITF in regulation of Bcl-2 and pStat3 in regulation of Bcl-XL. These findings have important implications for the development of treatments targeting antiapoptotic proteins in patients with melanoma.
Publication Type: Research Support, Non-U.S. Gov't;
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64.
Cell Biol Int 2007 Aug ; 8(31):825-30.
Anti-apoptotic activity of Bcl-2 is enhanced by its interaction with RTN3.
Zhu L ,Xiang R ,Dong W ,Liu Y ,Qi Y ,
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Hubei, Wuhan 430072, PR China.
Bcl-2 is known as a critical inhibitor of apoptosis triggered by a broad range of stimuli, mainly acting on the mitochondria. It can interact with many members of the Bcl-2 family, influence mitochondrial membrane permeability and modulate cell apoptosis. RTN3, a member of the reticulon (RTN) family, was predominantly localized on the endoplasmic reticulum (ER). Its N- and C-termini, both facing the cytoplasm, can recruit some proteins to the ER to modulate some physiological functions. We found that RTN3, which does not belong to the Bcl-2 family, can interact with Bcl-2 on the ER. In normal HeLa cells, ectopic overexpressed Bcl-2 could reduce the cell apoptosis induced by overexpressed RTN3. When the HeLa cells stably expressing Bcl-2 were treated with tunicamycin, endogenous RTN3 increased in the cell microsomal fraction. This change increased the Bcl-2 in microsomal fractions and also in the mitochondrial fractions where the anti-apoptotic activity of Bcl-2 mainly acts. These results suggest that RTN3 could bind with Bcl-2 and mediate its accumulation in mitochondria, which modulate the anti-apoptotic activity of Bcl-2.
Publication Type: Research Support, Non-U.S. Gov't;
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65.
Ann Oncol 2007 Jun ; 6(18):1004-14.
Prognostic relevance of a novel semiquantitative classification of Bcl2 immunohistochemical expression in human infiltrating ductal carcinomas of the breast.
Treré D ,Montanaro L ,Ceccarelli C ,Barbieri S ,Cavrini G ,Santini D ,Taffurelli M ,Derenzini M ,
Department of Experimental Pathology, Unit of Clinical Pathology, University of Bologna, Italy. davide.trere@unibo.it
BACKGROUND: Bcl2 is an important prognostic parameter in human breast cancer. However, the evaluation of Bcl2 expression by immunohistochemistry is carried out using arbitrary scoring criteria. In the present study, we evaluated the clinical relevance of a novel, semiquantitative classification of the Bcl2 immunostaining based on both the distribution and the intensity of the staining reaction. PATIENTS AND METHODS: The proposed classification was first validated in 69 breast cancer specimens by comparing the Bcl2 immunostaining with the Bcl2 messenger RNA (mRNA) levels evaluated by real-time RT-PCR. Since a highly significant association was found between protein and mRNA for Bcl2, the immunohistochemical scoring system was applied to 442 patients with infiltrating ductal carcinomas of the breast with long-term follow-up (median observation time 106 months). RESULTS: In the entire series, the Bcl2 variable was an independent predictor of clinical outcome, and its prognostic independence was maintained when lymph node-negative and -positive patients were considered separately. In this regard, of particular interest was the observation of a subgroup of node-negative breast cancer patients with a negative Bcl2 immunostaining, who had a very high probability of relapse or death (respectively about five and seven times greater than patients with a positive Bcl2 immunostaining). Moreover, the Bcl2 variable retained prognostic significance also in subgroups of patients treated with either adjuvant endocrine therapy or chemotherapy. CONCLUSIONS: Our results demonstrated that in breast cancer, Bcl2 protein expression parallels its mRNA level, and it has a highly significant and independent prognostic relevance.
Publication Type: Research Support, Non-U.S. Gov't;
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66.
Surg Oncol 2006 Dec ; 4(15):211-6.
p53 and p27 as predictors of clinical outcome for rectal-cancer patients receiving neoadjuvant therapy.
Lin LC ,Lee HH ,Hwang WS ,Li CF ,Huang CT ,Que J ,Lin KL ,Lin FC ,Lu CL ,
Department of Radiation Oncology, Chi-Mei Foundation Medical Center, No. 901 Junghua Rd., Yungkang City, Tainan 710, Taiwan, ROC. 8508A6@mail.chimei.org.tw
Our aim was to examine whether certain molecular markers, specifically p53, p21, p27, and Bcl-2, could be used to predict the tumor response of rectal cancer to neoadjuvant therapy and determine the overall and disease-free survival rates of patients following neoadjuvant therapy. Seventy-seven patients with rectal cancers were used in this study. All of them received neoadjuvant therapy and 53 of them were given radical surgery. Immunohistochemical tests were performed for the four markers mentioned above using biopsy specimens obtained from 70 of the patients prior to radiation. The identical tests were performed for the same markers using excised specimens from the patients after radical surgery. For the pre-radiation specimens, the positive rate for having p27 and Bcl-2 markers was 32.7% and 16.6%, respectively. This rate increased to 73.5% and 41.6% (p=0.001 and 0.012, respectively) in the specimens obtained after the surgery. With respect to "fair response (FR)" of patients, the pre-radiation biopsy specimens showed significant difference for the p53 (-) and p27 (+) markers (p=0.006). Patients with a 3-year overall survival rate were found to have, from their surgical specimens, 92% of the p27 (+) and 75% of p27 (-) markers (p=0.0058). Our study showed: first, the rate of positive identification of molecular markers, p27 and Bcl-2, increased following neoadjuvant therapy. Second, either the p53 (-) or p27 (+) status was a good predictor for FR in the pre-radiation biopsy specimens. Third, patients with p27 (+) markers in the surgical specimens lived longer at 3 years.
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67.
Intervirology 2007 ; 3(50):224-8.
HIV Tat protein increases Bcl-2 expression in monocytes which inhibits monocyte apoptosis induced by tumor necrosis factor-alpha-related apoptosis-induced ligand.
Zheng L ,Yang Y ,Guocai L ,Pauza CD ,Salvato MS ,
Department of Infectious Diseases, The First Affiliated Hospital, Medical School, Key Laboratory of Infectious Diseases of Chinese Ministry of Public Health, Zhejiang University, Hangzhou, Zhejiang, PR China.
OBJECTIVE: To investigate the effect of HIV Tat protein on Bcl-2 expression in human monocytes, and observe apoptosis of Tat-stimulated monocytes induced by TNF-alpha-related apoptosis-induced ligand (TRAIL). METHODS: Western blot was used to detect Bcl-2 expression in monocytes stimulated by HIV Tat protein, and Annexin V and 7-AAD staining were used to detect apoptosis of monocytes induced by TRAIL. RESULTS: HIV Tat protein increased Bcl-2 expression in human monocytes in a dose-dependent manner. Annexin V staining showed that 51.54% of monocytes underwent apoptosis after being treated with 100 ng/ml recombinant TRAIL. When monocytes were prestimulated with HIV Tat, only 15.46% of monocytes underwent apoptosis. This effect can be inhibited by polyclonal anti-Tat serum. 7-AAD staining showed similar results. CONCLUSION: HIV Tat protein increases Bcl-2 expression in monocytes which inhibited apoptosis induced by TRAIL. HIV Tat protein may play an important role in the mechanisms of HIV-persistent infection in monocytes.
Publication Type: Research Support, Non-U.S. Gov't;
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68.
Med Sci Monit 2007 Mar ; 3(13):BR84-8.
Expression of Bcl-2 on oral cavity pathologies.
Niedzielska I ,Sypnilewski D ,Niedzielski Z ,
Department of Maxillofacial Surgery, Medical University of Silesia, Katowice, Poland. stomgab@wp.pl
BACKGROUND: Oral pathologies are divided by histochemical analysis. Molecular analysis is a future, and expensive, method, and in it, the role of gene BCL-2 may be of interest. MATERIAL/METHODS: Forty-five patients with tumors of the oral cavity were enrolled. Biopsies were taken from the incision line, tumor section, and healthy tissue. A three-stage molecular analysis was carried out. BCL-2 expression was investigated depending on the site of the tissue material sample and the final histopathological result: hyperplasia (group I), neoplasm in situ malignancy (group II), and carcinoma (group III). Data from four subgroups of group I were also analyzed. RESULTS: No statistically significant difference was demonstrated in BCL-2 expression among the four hyperplasia subgroups. However, statistically significant differences in BCL-2 expression were found among the three basic study groups (p=0.00002) and among the biopsy collection sites (p=0.002). The difference was the most pronounced between carcinoma and incision line, but was also noticeable between neoplasm in situ malignancies and incision line. CONCLUSIONS: Monitoring BCL-2 expression may facilitate prognosis of the progress and ultimate severity of oral cavity hyperplasias.
Publication Type: Research Support, Non-U.S. Gov't;
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69.
J Exp Clin Cancer Res 2006 Dec ; 4(25):549-55.
Correlation of expression of Ki-67, EGFR, c-erbB-2, MMP-9, p53, bcl-2, CD34 and cell cycle analysis with survival in head and neck squamous cell cancer.
Smilek P ,Dusek L ,Veselý K ,Rottenberg J ,Kostrica R ,
Clinic of Otorhinolaryngology and Head and Neck Surgery, Centre of Biostatistics and Analyses, Faculty of Medicine, Masaryk University, Faculty St. Ann's Hospital, Brno, Czech Republic. smilek@med.muni.cz
The purpose of this study was to clarify the prognostic significance of flow cytometric analysis of DNA, mitotic, apoptotic indices, Ki-67, EGF-R, c-erb-B2, matrix metalloproteinasis-9 (MMP 9), p53, bcl-2, CD 34 in Head and Neck Carcinomas (HNSCC). The analysis was carried out with a set of 217 patients suffering from HNSCC. The parameters of tumors were related to the overall survival (OS) and the event-free survival (EFS). Clinical stage, ploidy and T-N categories influence both survival measures equally and significantly. Grade score did not significantly contribute to the prediction of EFS and OS when entered to the analysis as single factor. Measure of realized proliferation significantly contributed to the risk prediction both in EFS and OS. Cytokinetic parameters generally strongly correlated with grade score and grade correlates, responsible for the increasing risk in combination with other risk factors like clinical stage and/or ploidy. Positivity in bcl-2 and MMP-9 was significantly related to OS of patients (not to EFS). Positivity of EGFR, c-erbB-2, CD34, p53 did not reached statistically significant value in association to EFS or OS.
Publication Type: Research Support, Non-U.S. Gov't;
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70.
J Biochem 2007 Mar ; 3(141):401-10.
Suppression of endoplasmic reticulum stress-induced caspase activation and cell death by the overexpression of Bcl-xL or Bcl-2.
Murakami Y ,Aizu-Yokota E ,Sonoda Y ,Ohta S ,Kasahara T ,
Department of Biochemistry, Kyoritsu University of Pharmacy, 1-5-30 Shibakoen, Tokyo 105-8512, Japan.
Continuous endoplasmic reticulum (ER) stress, such as the accumulation of unfolded proteins, results in cell death and relates to the pathogenesis of some neurodegenerative diseases. Treatment of brefeldin A, an inhibitor of transport between the ER and Golgi complex, induced cell death during 24 h, which accompanied activation of caspase-2, caspase-3 and caspase-9, starting at 12 h and increasing time-dependently up to 28 h. Caspase-2 was expressed and activated in not only mitochondria and cytosol, but also in the microsomal fraction containing ER and Golgi. Of note is that overexpression of Bcl-x(L) or Bcl-2 in PC12 cells markedly suppressed brefeldin A-induced activation of caspases and resulting cell death. Delivery of anti-Bcl-2 antibody into the Bcl-2-overexpressed cells again recovered apoptosis. While the brefeldin A-treatment induced the phosphorylation of both c-Jun N-terminal kinase (JNK) and p38 MAPK, overexpression of Bcl-x(L) or Bcl-2 reduced the prolonged phosphorylation of JNK, but not of p38 MAPK. Pretreatment with a JNK inhibitor, SP600125, suppressed the brefeldin A-induced caspase-2 activation and cell death significantly. Thus, our results suggest that protective effects of Bcl-x(L) and Bcl-2 against brefeldin A-induced cell death appear to be dependent on the regulation of JNK activation.
Publication Type: Research Support, Non-U.S. Gov't;
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71.
J Cell Biochem 2007 Aug 1; 5(101):1148-64.
Alternative production of Bcl-2 and Bax by tumor cells determines the rates of in vivo tumor progression: suggested mechanisms.
Deichman GI ,Dyakova NA ,Matveeva VA ,Kashkina LM ,
Laboratory of Antitumor Immunity, Institute of Carcinogenesis, N. N. Blokchin Cancer Research Center, Russian Academy of Medical Sciences, 115478 Moscow, Russia. antitum@space.ru
The hypothesis tested in the study suggests that mechanisms of the earlier described delayed or accelerated tumor progression may be regulated by the antiapoptotic and proapoptotic cellular programs activated in stress reactions of transformed cells to the host normal cellular environment. Therefore, spontaneously transformed hamster cell line STHE, its bcl-2-transduced line STHE-Bcl-2, and 64 of their descendant tumor cell variants naturally selected in two in vivo regimes (local tumor growth versus dissemination) were examined. The role of Bcl-2 and the possible activation of endogenous death-signaling Bax, Ras, and HSP90/HSP70 stress proteins in STHE (Bcl-2+/-) tumor cell variants were studied in dynamics of in vivo tumor progression. The data demonstrate: (1) Immediate in vivo activation of Bax and of HSP90/HSP70 stress proteins in disseminated STHE cells on the background of accelerated tumor progression; (2) No immediate activation of Bax and the gradual downregulation of Bcl-2 in STHE-Bcl-2 cells on the background of delayed tumor progression; (3) Alternative and mutually suppressive character of Bcl-2 and Bax expression in both regimes of tumor progression; (4) In the later stages of tumor progression, the regular transit of the initial Bcl-2 antiapoptotic, Bax-suppressing program, and the delayed tumor progression towards Bcl-2 loss, activation of Bax, and acceleration of tumor progression. Thus, the delay of tumor progression is apparently determined by the ability of Bcl-2-expressing tumor cells to extinguish the cell-damaging environmental stress signals and Bax activation, while its acceleration correlates with Bcl-2 loss, activation of proapoptotic Bax, and tumor cells damage.
Publication Type: Research Support, Non-U.S. Gov't;
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72.
Cell Death Differ 2007 May ; 5(14):1020-8.
Failure of stress-induced downregulation of Bcl-2 contributes to apoptosis resistance in senescent human diploid fibroblasts.
Ryu SJ ,Oh YS ,Park SC ,
Department of Biochemistry and Molecular Biology, The Aging and Apoptosis Research Center, Seoul National University College of Medicine, Seoul, Korea.
We previously reported that senescent human diploid fibroblasts (HDFs) are resistant to apoptosis induced by H(2)O(2) and staurosporine. We report here that senescent HDFs are resistant to thapsigargin-induced apoptosis as well. These agonists caused the reductions in mitochondrial membrane potential (MMP) and in the apoptosis inhibitory protein (B-cell lymphoma) only in young HDFs but not in senescent HDFs. In addition, downregulation of Bcl-2 increased the sensitivity of senescent HDFs to apoptosis induction, suggesting the significant role of Bcl-2 in apoptosis resistance of the senescent HDFs. We further found that P-cAMP response element-binding protein (CREB), a positive regulator of Bcl-2, decreased in stress-induced apoptosis of young HDFs but not in senescent HDFs, and that Bcl-2 was markedly reduced in CREB small interfering RNA (siRNA), transfected senescent HDFs. In addition, activity of protein phosphatase 2A (PP2A), which dephosphorylates p-CREB, significantly increased in young HDFs but not in senescent HDFs treated with H(2)O(2), staurosporine or thapsigargin. Taken together, these results suggest that failure of stress-induced downregulation of Bcl-2 underlies resistance of senescent HDFs to apoptosis.
Publication Type: Research Support, Non-U.S. Gov't;
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73.
Blood 2007 Jun 1; 11(109):4793-802.
Kringle 5 of human plasminogen, an angiogenesis inhibitor, induces both autophagy and apoptotic death in endothelial cells.
Nguyen TM ,Subramanian IV ,Kelekar A ,Ramakrishnan S ,
Department of Pharmacology, University of Minnesota Medical School, 321 Church Street SE, Minnesota, MN 55455, USA.
Inhibition of endothelial cell proliferation and angiogenesis is emerging as an important strategy in cancer therapeutics. Kringle 5 (K5) of human plasminogen is a potent angiogenesis inhibitor. Previous studies have shown K5 exposure promotes caspase activity and apoptosis in endothelial cells. Here we report that K5 treatment evokes an autophagic response in endothelial cells that is specific and initiated even in the absence of nutritional stress. Endothelial cells exposed to K5 up-regulated Beclin 1 levels within a few hours. Furthermore, progressively increasing amounts of antiapoptotic Bcl-2 were found to be complexed with Beclin 1, although total levels of Bcl-2 remained unchanged. Prolonged exposure to K5 ultimately led to apoptosis via mitochondrial membrane depolarization and caspase activation in endothelial cells. Knocking down Beclin 1 levels by RNA interference decreased K5 induced autophagy but accelerated K5-induced apoptosis. These studies suggest that interfering with the autophagic survival response can potentiate the antiangiogenic effects of Kringle 5 in endothelial cells.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't;
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74.
J Biol Chem 2007 Mar 23; 12(282):9279-87.
Bcl2 impedes DNA mismatch repair by directly regulating the hMSH2-hMSH6 heterodimeric complex.
Hou Y ,Gao F ,Wang Q ,Zhao J ,Flagg T ,Zhang Y ,Deng X ,
Department of Medicine, Shands Cancer Center, University of Florida, Gainesville, Florida 32610-3633, USA.
Bcl2 has been reported to suppress DNA mismatch repair (MMR) with promotion of mutagenesis, but the mechanism(s) is not fully understood. MutSalpha is the hMSH2-hMSH6 heterodimer that primarily functions to correct mutations that escape the proofreading activity of DNA polymerase. Here we have discovered that Bcl2 potently suppresses MMR in association with decreased MutSalpha activity and increased mutagenesis. Exposure of cells to nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone results in accumulation of Bcl2 in the nucleus, which interacts with hMSH6 but not hMSH2 via its BH4 domain. Deletion of the BH4 domain from Bcl2 abrogates the ability of Bcl2 to interact with hMSH6 and is associated with enhanced MMR efficiency and decreased mutation frequency. Overexpression of Bcl2 reduces formation of the hMSH2-hMSH6 complex in cells, and purified Bcl2 protein directly disrupts the hMSH2-hMSH6 complex and suppresses MMR in vitro. Importantly, depletion of endogenous Bcl2 by RNA interference enhances formation of the hMSH2-hMSH6 complex in association with increased MMR and decreased mutagenesis. Thus, Bcl2 suppression of MMR may occur in a novel mechanism by directly regulating the heterodimeric hMSH2-hMSH6 complex, which potentially contributes to genetic instability and carcinogenesis.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't;
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75.
Apoptosis 2007 Apr ; 4(12):657-69.
Cytoplasmic mutant p53 increases Bcl-2 expression in estrogen receptor-positive breast cancer cells.
Pratt MA ,White D ,Kushwaha N ,Tibbo E ,Niu MY ,
Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5. cpratt@uottawa.ca
The Bcl-2 gene is positively regulated by estrogen (E2) primarily through E2-response elements in the coding region and a putative p53 negative regulatory element (NRE) containing a short upstream open reading frame (uORF). The ability of mutant p53 to repress or induce Bcl-2 expression is controversial. In this study E2-receptor positive (ER(+))/wild-type p53 MCF-7cells were transfected with p53Delta291, which lacks a nuclear localization signal or a DNA binding domain mutant, p53(173L). Both p53 mutants but especially p53Delta291 increased Bcl-2 protein expression from a CMV-NRE-Bcl-2 cDNA construct in an NRE-position/orientation independent manner as well as from a 1.7 kb Bcl-2 promoter reporter gene. Bcl-2 protein expression prevented the p53Delta291-mediated increase in Bcl-2 promoter activity although immunoprecipitation demonstrated that only a small proportion of the wild-type p53 but not p53Delta91 protein interacts with Bcl-2. Unless levels of ectopically expressed mutant p53 were extremely high, stable expression of mutant p53 in MCF-7 cells moderately increased Bcl-2 protein levels. Expression of mutant p53 did not alter E2 regulation of Bcl-2, however, mutation of the uORF prevented regulation by both mutant p53 and E2. Adenovirus-mediated overexpression of WT p53 strongly reduced Bcl-2 expression in ER(-)/mut p53 MDA-MB-231 cells. Taken together these data support the position that mutant p53 behaves in a dominant "positive" manner relieving repression by WT p53 or another Bcl-2 transcriptional inhibitor in a manner independent of nuclear translocation.
Publication Type: Research Support, Non-U.S. Gov't;
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76.
J Neurochem 2007 Apr ; 1(101):77-86.
Simvastatin protects neurons from cytotoxicity by up-regulating Bcl-2 mRNA and protein.
Johnson-Anuna LN ,Eckert GP ,Franke C ,Igbavboa U ,Müller WE ,Wood WG ,
Department of Pharmacology, University of Minnesota School of Medicine, Geriatric Research, Education and Clinical Center, VA Medical Center, Minneapolis, Minnesota 55417, USA.
Statins are most commonly prescribed to reduce hypercholesterolemia; however, recent studies have shown that statins have additional benefits, including neuroprotection. Until now, the mechanism underlying statin-induced neuroprotection has been poorly understood. Recent in vivo studies from our lab reported the novel finding that simvastatin increased expression levels of a gene encoding for a major cell survival protein, bcl-2 [Johnson-Anuna et al., J. Pharmacol. Exp. Ther.312 (2005) 786]. The purpose of the present experiments was to determine if simvastatin could protect neurons from excitotoxicity by altering Bcl-2 levels. Neurons were pre-treated with simvastatin and challenged with a compound known to reduce Bcl-2 levels and induce cell death. Simvastatin pre-treatment resulted in a significant reduction in cytotoxicity (lactate dehydrogenase release and caspase 3 activation) following challenge compared with unchallenged neurons. In addition, chronic simvastatin treatment significantly increased Bcl-2 mRNA and protein levels while challenge resulted in a significant reduction in Bcl-2 protein abundance. G3139, an antisense oligonucleotide directed against Bcl-2, abolished the protective effects of simvastatin and eliminated simvastatin-induced up-regulation of Bcl-2 protein. These findings suggest that neuroprotection by simvastatin is dependent on the drug's previously unexplored and important effect of up-regulating Bcl-2.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't;
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77.
Respirology 2007 Jan ; 1(12):34-41.
Intronic single-nucleotide polymorphisms in Bcl-2 are associated with chronic obstructive pulmonary disease severity.
Sata M ,Takabatake N ,Inoue S ,Shibata Y ,Abe S ,Machiya J ,Wada T ,Ji G ,Kido T ,Matsuura T ,Muramatsu MA ,Kubota I ,
Department of Cardiology, Pulmonology and Nephrology, Course of Internal Medicine and Therapeutics, Yamagata University School of Medicine, Yamagata, Japan. msata@hsp.ncvc.go.jp
BACKGROUND AND OBJECTIVE: COPD is a multifactorial disease influenced by genetic and environmental factors, and gene-by-environmental interactions. There is considerable variability in the degree of airflow obstruction, moreover only 10-15% of chronic smokers develop COPD. These observations indicate that additional risk factors, possibly genetic, contribute to not only the susceptibility to COPD but also the development and severity of COPD. Recent paradigms highlight the presence and causal role of apoptosis in emphysema. There is a large amount of information on the genes involved in the regulation of apoptosis and one of the most studied is Bcl-2. The aim of this study was to investigate the genetic association of Bcl-2 gene with the level of lung function, that is, the severity, of COPD. METHODS: The genetic association of Bcl-2 polymorphisms with lung function was investigated in 261 Japanese patients with COPD using 12 single-nucleotide polymorphisms (SNPs) in Bcl-2. RESULTS: Four SNPs showed a significant association between the high and low lung function groups in a dominant trait comparison. Subsequent linkage-disequilibrium mapping and analyses of haplotype structure also showed a significant association between the level of lung function and two haplotypes comprised of the associated SNPs in Bcl-2. CONCLUSIONS: Although the linkage between Bcl-2 gene and the susceptibility to COPD remains to be clarified, the findings of the current study indicate that Bcl-2 might be influencing the level of lung function, that is, the development and severity of COPD.
Publication Type: Research Support, Non-U.S. Gov't;
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78.
Oncol Rep 2007 Feb ; 2(17):425-31.
Ethyl acetate extract of Chinese medicinal herb Sarcandra glabra induces growth inhibition on human leukemic HL-60 cells, associated with cell cycle arrest and up-regulation of pro-apoptotic Bax/Bcl-2 ratio.
Li WY ,Chiu LC ,Lam WS ,Wong WY ,Chan YT ,Ho YP ,Wong EY ,Wong YS ,Ooi VE ,
Department of Biology, The Chinese University of Hong Kong, Hong Kong, P.R. China.
Sarcandra glabra (Thunb.) Nakai, colloquially known as Caoshanhu, is a Chinese medicinal herb with reported anti-tumor, anti-inflammatory, anti-viral and non-specific immunoenhancing properties. Although the plant has been clinically used for treating a variety of diseases, its bioactive ingredients are largely unknown and its mode of action has never been investigated. In this study, the anti-tumor property of ethyl acetate (EA) extract of S. glabra was investigated by determining its in vitro growth-inhibitory effects on a panel of human cancer cell lines of different histotypes. Growth inhibition of the EA extract on the cancer cells seemed to be selective, and the leukemic HL-60 was found to be the most responsive after 48 h of treatment (IC50=58 microg/ml). Flow cytometric studies further illustrated that the extract might interfere with DNA replication and thus arrested the cell cycle at S phase in the leukemic cells, followed by DNA fragmentation and loss of phospholipid asymmetry in the plasma membrane after 72 h of treatment. Concurrently, the pro-apoptotic Bax/Bcl-2 ratio was also up-regulated by more than 178% of the control level. All these findings suggested that the extract had initiated apoptosis to kill the leukemic cells. Results from this pioneer study help to establish a scientific foundation for future research and development of the bioactive ingredients in EA extract of S. glabra as efficacious anti-cancer agents.
Publication Type: Research Support, Non-U.S. Gov't;
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79.
J Clin Invest 2007 Jan ; 1(117):112-21.
Chronic lymphocytic leukemia requires BCL2 to sequester prodeath BIM, explaining sensitivity to BCL2 antagonist ABT-737.
Del Gaizo Moore V ,Brown JR ,Certo M ,Love TM ,Novina CD ,Letai A ,
Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
Antiapoptotic B cell leukemia/lymphoma 2 (BCL2) family proteins are expressed in many cancers, but the circumstances under which these proteins are necessary for tumor maintenance are poorly understood. We exploited a novel functional assay that uses BCL2 homology domain 3 (BH3) peptides to predict dependence on antiapoptotic proteins, a strategy we call BH3 profiling. BH3 profiling accurately predicts sensitivity to BCL2 antagonist ABT-737 in primary chronic lymphocytic leukemia (CLL) cells. BH3 profiling also accurately distinguishes myeloid cell leukemia sequence 1 (MCL1) from BCL2 dependence in myeloma cell lines. We show that the special sensitivity of CLL cells to BCL2 antagonism arises from the requirement that BCL2 tonically sequester proapoptotic BIM in CLL. ABT-737 displaced BIM from BCL2's BH3-binding pocket, allowing BIM to activate BAX, induce mitochondrial permeabilization, and rapidly commit the CLL cell to death. Our experiments demonstrate that BCL2 expression alone does not dictate sensitivity to ABT-737. Instead, BCL2 complexed to BIM is the critical target for ABT-737 in CLL. An important implication is that in cancer, BCL2 may not effectively buffer chemotherapy death signals if it is already sequestering proapoptotic BH3-only proteins. Indeed, activator BH3-only occupation of BCL2 may prime cancer cells for death, offering a potential explanation for the marked chemosensitivity of certain cancers that express abundant BCL2, such as CLL and follicular lymphoma.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't;
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80.
Apoptosis 2007 Feb ; 2(12):319-28.
Reticulon 3 mediates Bcl-2 accumulation in mitochondria in response to endoplasmic reticulum stress.
Wan Q ,Kuang E ,Dong W ,Zhou S ,Xu H ,Qi Y ,Liu Y ,
The National Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, 430072, People's Republic of China.
Reticulon3 (RTN3), firstly isolated from the retina and widely expressed in human tissues with the highest expression in the brain, is presumed to play an important role in the developing axons through the transport of liquids and proteins. We have identified and characterized RTN3 as a RTN4B/ASY interaction protein. Here we demonstrated that ER-stress activated RTN3 expression. CHOP and ATF6 were sufficient to up-regulate the expression of RTN3. The down-regulation of RTN3 would induce apoptosis and attenuate the anti-apoptotic activity of Bcl-2, indicating RTN3 was required for the cellular survival and optimal anti-apoptotic activity of Bcl-2. Our present studies also indicated ER-stress induced RTN3 up-regulation could trigger Bcl-2 translocation from ER to mitochondria. Moreover, the previous studies showed that RTN4B was also a Bcl-2-interacted protein. We found that RTN3 and RTN4B could block the access of Bcl-2 to each other and thereafter determined the Bcl-2 subcellular distribution. Taken together, our findings indicate that RTN3 is directly involved in the ER-constituents trafficking events through dually acting as an essential and important ER-stress sensor, and a trigger for the Bcl-2 translocation.
Publication Type: Research Support, Non-U.S. Gov't;
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81.
Surgery 2006 Dec ; 6(140):899-905; discussion 905-6.
Patterns of expression of cell cycle/apoptosis genes along the spectrum of thyroid carcinoma progression.
Saltman B ,Singh B ,Hedvat CV ,Wreesmann VB ,Ghossein R ,
Department of Otolaryngology, Columbia University College of Physicians and Surgeons, New York, NY 10021, USA.
BACKGROUND: Genetic screening studies suggest that genetic changes underlie progression from well differentiated to anaplastic thyroid cancers. The aim of this study is to determine to what extent cell cycle/apoptosis regulators contribute to cancer progression. METHODS: Tissue microarrarys (TMAs) were constructed from well-differentiated papillary thyroid carcinoma (WDPTC; n = 41), poorly differentiated thyroid carcinoma (PDTC; n = 43), and anaplastic thyroid carcinoma (ATC; n = 22). TMAs were immunostained for 7 different cell cycle/apoptosis-related genes (p53, Ki-67, bcl-2, mdm-2, cyclin D1, p21, and p27). RESULTS: p53 (0%, 12%, 32%) and Ki-67 (5%, 49%, 82%) were expressed with increasing frequency, and bcl-2 (68%, 42%, 0%) and p21 (40%, 7%, 0%) with decreasing frequency in WDPTC to PDTC and ATC, respectively (P < .001). Interestingly, mdm-2 (54%, 5%, 0%) showed decreased expression along the progression axis (P < .001). p27 and cyclin D1 were expressed in <15% of cases, with a trend toward decreasing expression from WDPTC to PDTC to ATC. CONCLUSIONS: These data confirm the presence of increasing genetic complexity with progressive dedifferentiation in thyroid cancer, with aberrant tumor suppressor activity and increased proliferative activity being most prevalent in ATC. The data also confirm the intermediate position of PDTC in the classification scheme of thyroid carcinomas.
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82.
J Biol Chem 2007 Feb 16; 7(282):4288-300.
Activation of JNK-dependent pathway is required for HIV viral protein R-induced apoptosis in human monocytic cells: involvement of antiapoptotic BCL2 and c-IAP1 genes.
Mishra S ,Mishra JP ,Kumar A ,
Department of Pathology and Laboratory Medicine and Biochemistry, Microbiology, and Immunology, University of Ottawa K1H 8M5, Ottawa, Ontario, Canada.
Human immunodeficiency virus (HIV) accessory protein viral protein R (Vpr) plays a key role in virus replication and induces cell cycle arrest and apoptosis in various cell types including T cells and neuronal and tumor cells following infection with Vpr-expressing HIV isolates or exposure to the extracellular Vpr protein. The C-terminal Vpr peptide encompassing amino acids 52-96 (Vpr-(52-96)) is required for exerting the apoptotic effects, whereas the N-terminal Vpr-(1-45) peptide is responsible for virus transcription. We demonstrate that Vpr-(52-96) induced apoptosis in human promonocytic THP-1 cells and primary monocytes through the mitochondrial pathway in a caspase-dependent manner. To understand the regulation of Vpr-induced apoptosis, we investigated the signaling pathways, particularly the MAPKs, and the transcription factors involved. Although both Vpr-(52-96) and Vpr-(1-45) peptides induced phosphorylation of all the three members of the MAPKs, Vpr-(52-96)-activated JNK selectively induced apoptosis in monocytic cells through the mitochondrial pathway as determined by using JNK inhibitors SP60025, dexamethasone, curcumin, and JNK-specific small interfering RNAs. Furthermore Vpr-(52-96)-induced apoptosis was mediated by inhibition of downstream antiapoptotic Bcl2 and c-IAP1 genes whose expression could be restored following pretreatment with JNK-specific inhibitors. Overall the results suggest that Vpr-(52-96)-activated JNK plays a key role in inducing apoptosis through the down-regulation of antiapoptotic Bcl2 and c-IAP1 genes.
Publication Type: Clinical Trial; Research Support, Non-U.S. Gov't;
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83.
Clin Cancer Res 2006 Dec 1; 23(12):6946-51.
Detection of human papillomavirus and relevant tumor suppressors and oncoproteins in laryngeal tumors.
Manjarrez ME ,Ocadiz R ,Valle L ,Pacheco C ,Marroquin A ,De la Torre C ,Selman M ,Gariglio P ,
Instituto Nacional de Enfermedades Respiratorias, Tlalpan, México. e_manjarrez@yahoo.com
PURPOSE: The mechanism of larynx oncogenesis is complex and controlled by various factors, most of them involved in cell proliferation and apoptosis. In this study, we evaluated the levels of two suppressor proteins (pRb and p53) and two oncogenic proteins (c-Myc and Bcl-2), as well as the apoptotic levels and the presence of human papillomavirus (HPV) in both tumor types. EXPERIMENTAL DESIGN: Low- or high-risk HPV viral DNA was determined by PCR and in situ PCR; the level of cellular proteins was examined by immunohistochemistry; the presence of apoptotic cells was evaluated by in situ cell death detection. RESULTS: Most laryngeal papillomatosis samples contained low-risk HPV determined by both techniques. However, 25% of laryngeal carcinoma samples were positive for HPV employing PCR or in situ PCR. In papillomatosis, pRb and p53 levels were higher than in normal larynxes, whereas laryngeal cancer presented the lowest levels. c-Myc oncogene expression was very low in normal and cancer tissues but highly increased in papillomatosis. Bcl-2 expression was low and showed no significant difference between laryngeal papillomatosis and normal larynxes. By contrast, Bcl-2 was clearly up-regulated in cancer. Normal larynx samples and those from laryngeal papillomatosis exhibited similar relatively high numbers of apoptotic cells, whereas in malignant tumors, these cells were scarce. CONCLUSION: Our results suggest that HPV is an important risk factor in papillomatosis and in some malignant larynx tumors with a strong participation of cellular genes, specifically involved in proliferation and apoptosis. In benign papillomatosis lesions but not in larynx cancer, high p53 activity might preserve the apoptosis process. In larynx cancer, low p53 levels and high bcl-2 expression may be playing an important role to block apoptosis.
Publication Type: Research Support, Non-U.S. Gov't;
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84.
Lab Hematol 2006 ; 4(12):187-92.
Abnormal intracellular level of Bax in CD3+ cells from untreated B-cell chronic lymphocytic leukemia patients.
Scamardella F ,Maconi M ,Albertazzi L ,Gamberi B ,Gugliotta L ,Brini M ,
Department of Clinical Pathology, AO Arcispedale Santa Maria Nuova, Reggio Emilia, Italy.
B-cell chronic lymphocytic leukemia (B-CLL) is a lymphoproliferative disease caused by impaired apoptosis regulation that leads to an abnormal survival and an accumulation of B-lymphocytes. Anti-apoptotic Bcl-2 and proapoptotic Bax proteins are involved in the highly regulated mechanism of cell death. Bax and Bcl-2 intracellular levels were analyzed both in CD19+ and CD3+ cells from 28 B-CLL de novo patients and compared with cells from healthy donors. Our results were expressed as a ratio (Bax/Bcl-2) obtained by dividing Bax mean fluorescence intensity (MFI) and Bcl-2 MFI; obviously, a lower ratio is associated with an anti-apoptotic status, while a higher index correlates to apoptosis activation. In CD19+ B-CLL cells, the Bax/Bcl-2 ratio was lower than in the CD19+ normal counterpart (1.3 versus 3.51; P<.05), mainly due to a Bcl-2 over expression (17.65 versus 9.02; P<.001). In CD3+ cells from B-CLL patients, the Bax/Bcl-2 ratio was lower than in normal CD3+ cells (7.89 versus 8.96; P<.005), most importantly as a result of Bax suppression (77.22 versus 96.63; P<.001). These study data show an apoptosis inhibition not only in CD19+ cells, but also in CD3+ cells, suggesting a pivotal role of T-cells in B-CLL pathogenesis.
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85.
In Vivo ; 5(20):599-604.
Apoptotic markers p53, Bcl-2 and Bax in primary lung cancer.
Porebska I ,Wyrodek E ,Kosacka M ,Adamiak J ,Jankowska R ,Harłozińska-Szmyrka A ,
Chair and Department of Pulmonology and Lung Cancer, Wroclaw University of Medicine, ul. Grabiszyńska 105, 53-439 Wroclaw, Poland. iporebsk@poczta.onet.pl
BACKGROUND: Apoptosis is the fundamental process necessary for eliminating damaged or unwanted cells. Alterations in the apoptotic pathway appear to be key events in cancer development and progression. The aim of the study was to determine the p53, Bcl-2 and Bax expressions in lung cancer, taking into account histological heterogeneity and the adjacent bronchial resection margin. MATERIALS AND METHODS: Tissue specimens from 60 histopathologically verified lung cancer specimens and 12 bronchial stumps were evaluated. The presence of the studied markers was revealed by immunocytochemistry on paraffin-embedded tissue. RESULTS: The percentage of p53- and Bax-positive lung cancers was comparable (51.6% for both proteins), while Bcl-2 immunoreactivity was observed in fewer (31.6%) cases. There was no p53 accumulation in bronchial stumps, while Bcl-2 and Bax staining formed a repeatable specific pattern in bronchial epithelium. The differences in apoptotic marker expression between non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) were revealed, especially regarding p53 and Bax expression (60% vs. 10%, p = 0.005 and 58% vs. 20%, p = 0.04, respectively). Taking into account the histological structure of NSCLC, Bax expression appeared to be more frequent in adenocarcinoma than in squamous cell lung cancers (88% vs. 42%, p = 0.004). No interrelationship between the studied proteins in lung cancer tissue was revealed. CONCLUSION: The expression of p53, Bcl-2 and Bax was altered in lung cancer tissue compared to histologically normal bronchial epithelium. The difference between apoptotic marker expression in NSCLC and SCLC could reflect the different pathogenesis of these two lung pathologies.
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86.
J Biol Chem 2006 Dec 29; 52(281):40493-502.
Bcl-2 localized at the nuclear compartment induces apoptosis after transient overexpression.
Portier BP ,Taglialatela G ,
Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas 77555-1043, USA.
Bcl-2 is the best characterized member of a large family of proteins that regulate apoptosis. Although it is established that Bcl-2 localized at the mitochondria functions as an anti-apoptotic protein, the function of Bcl-2 at the nucleus remains unclear. Recently we showed that nuclear compartment-associated Bcl-2 inhibits transcription factor activation. Based on this observation, we hypothesized that presence of Bcl-2 at the nucleus may induce rather than protect cells from apoptosis. Here we investigated the putative apoptotic role of nuclear compartment-associated Bcl-2. Additionally, we examined the role of the Bcl-2 BH4 domain in mediating binding to FKBP38, the Bcl-2 mitochondrial chaperone. Our results demonstrate a novel, pro-apoptotic function for nuclear Bcl-2 and identify the Bcl-2 BH4 domain as a key regulator in mediating Bcl-2/FKBP38 binding. These results indicate that Bcl-2 has a dual role as both a protector and a killer and that the ability to switch roles depends on Bcl-2 subcellular localization.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't;
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87.
J Biol Chem 2007 Jan 5; 1(282):5-13.
BCL2 is a downstream effector of MIZ-1 essential for blocking c-MYC-induced apoptosis.
Patel JH ,McMahon SB ,
Biomedical Graduate Studies, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
The c-MYC oncoprotein is among the most potent transforming agents in human cells. Ironically, c-MYC is also capable of inducing massive apoptosis under certain conditions. A clear understanding of the distinct pathways activated by c-MYC during apoptosis induction and transformation is crucial to the design of therapeutic strategies aimed at selectively reactivating the apoptotic potential of c-MYC in cancer cells. We recently demonstrated that apoptosis induction in primary human cells strictly requires that c-MYC bind and inactivate the transcription factor MIZ-1. This presumably blocked the ability of MIZ-1 to activate the transcription of an unidentified pro-survival gene. Here we report that MIZ-1 activates the transcription of BCL2. More importantly, inhibition of the MIZ-1/BCL2 signal is an essential event during the apoptotic response. Furthermore, targeting BCL2 with short hairpin RNA or small molecule inhibitors restores the apoptotic potential of a c-MYC mutant that is defective for MIZ-1 inhibition. These observations suggest that repression of BCL2 transcription is the single essential consequence of targeting the MIZ-1 pathway during apoptosis induction. These data define a genetic pathway that helps to explain historical observations documenting cooperation between c-MYC and BCL2 overexpression in human cancer.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't;
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88.
Cancer Genet Cytogenet 2006 Nov ; 1(171):1-8.
Association between telomere length and BCL2 gene rearrangements in low- and high-grade non-Hodgkin lymphomas.
Haydeé Cottliar AS ,Noriega MF ,Narbaitz M ,Rodríguez A ,Slavutsky IR ,
Department of Genetics, Institute for Hematological Research, National Academy of Medicine, Instituto de Investigaciones Hematológicas Mariano R Castex, Academia Nacional de Medicina, Pacheco de Melo 3081, Buenos Aires, Argentina.
Telomere length based on terminal restriction fragment (TRF) assay was evaluated in cells of bone marrow, lymph node, or both from 53 non-Hodgkin lymphoma (NHL) patients: 44 with follicular lymphoma (FL) and 9 with secondary diffuse large B-cell lymphoma (S-DLBCL) to FL. The TRF data were correlated with BCL2 gene rearrangement evaluated by nested and long-distance polymerase chain reaction approaches. Peripheral blood cells from 12 healthy donors were studied as controls. Both groups of NHL patients revealed significant telomere shortening compared with controls (8.50 +/- 0.50 kb; P < 0.001), with significantly shorter TRFs in S-DLBCL (3.49 +/- 0.26 kb) than in FL cases (4.09 +/- 0.12 kb; P = 0.047). Patients carrying BCL2 gene rearrangements showed longer telomere length (4.19 +/- 0.14 kb) than those without (3.51 +/- 0.14 kb; P = 0.05). Among patients with FL, telomere length was shortest in patients without t(14;18), intermediate when breakpoints occurred in the minor breakpoint cluster region (3.97 +/- 0.33 kb), and greater when breakpoints occurred in the major breakpoint region (MBR) (4.24 +/- 0.15 kb), with significant differences between MBR and BCL2-negative cases (P = 0.033). Our findings support the existence of alternative genetic pathways involved in the origin of these FL subsets. Even though the number of S-DLBCL cases was small, they showed the shortest telomere length, suggesting that this parameter reflects another genetic alteration involved in the progression from FL to a higher-grade lymphoma.
Publication Type: Research Support, Non-U.S. Gov't;
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89.
Free Radic Res 2006 Nov ; 11(40):1113-23.
Induction of Bcl-2 by functional regulation of G-protein coupled receptors protects from oxidative glutamate toxicity by increasing glutathione.
Sahin M ,Saxena A ,Joost P ,Lewerenz J ,Methner A ,
Department of Neurology, Heinrich Heine Universität Düsseldorf, Moorenstreet 5, 40225, Düsseldorf, Germany.
Glutamate treatment depletes hippocampal HT22 cells of glutathione, which renders the cells incapable to reduce reactive oxygen species and ultimately cumulates in cell death by oxidative stress. HT22 cells resistant to glutamate displayed increased phosphorylation of cAMP-response-element binding (CREB) and decreased ERK1/2 suggestive of differences in signal transmission. We investigated the amount of candidate G-protein-coupled receptors involved in this resistance and found an increase in mRNA for receptors activated by the vasoactive intestinal peptide VIP (VPAC2, 12.6-fold) and glutamate like the metabotropic glutamate receptor mGlu1 (5.3-fold). Treating cells with VIP and glutamate led to the same changes in protein phosphorylation observed in resistant cells and induced the proto-oncogene Bcl-2. Bcl-2 overexpression protected by increasing the amount of intracellular glutathione and Bcl-2 knockdown by small interfering RNAs (siRNA) increased glutamate susceptibility of resistant cells. Other receptors upregulated in this paradigm might represent useful targets in the treatment of neurological diseases associated with oxidative stress.
Publication Type: Research Support, Non-U.S. Gov't;
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90.
J Immunol 2006 Oct 15; 8(177):5727-35.
Interaction of vascular endothelial growth factor 165 with neuropilin-1 protects rheumatoid synoviocytes from apoptotic death by regulating Bcl-2 expression and Bax translocation.
Kim WU ,Kang SS ,Yoo SA ,Hong KH ,Bae DG ,Lee MS ,Hong SW ,Chae CB ,Cho CS ,
Department of Internal Medicine, Division of Rheumatology, School of Medicine, Catholic University of Korea, Seoul, Korea.
Rheumatoid arthritis (RA) synoviocytes are resistant to apoptosis and exhibit a transformed phenotype, which might be caused by chronic exposure to genotoxic stimuli including reactive oxygen species and growth factors. In this study, we investigated the role of vascular endothelial growth factor165 (VEGF165), a potent angiogenic factor, and its receptor in the apoptosis of synoviocytes. We demonstrated here that neuropilin-1, rather than fms-like tyrosine kinase-1 and kinase insert domain-containing receptor, is the major VEGF165 receptor in the fibroblast-like synoviocytes. Neuropilin-1 was highly expressed in the lining layer, infiltrating leukocytes, and endothelial cells of rheumatoid synovium. The production of VEGF165, a ligand for neuropilin, was significantly higher in the RA synoviocytes than in the osteoarthritis synoviocytes. The ligation of recombinant VEGF165 to its receptor prevented the apoptosis of synoviocytes induced by serum starvation or sodium nitroprusside (SNP). VEGF165 rapidly triggered phospho-Akt and phospho-ERK activity and then induced Bcl-2 expression in the rheumatoid synoviocytes. The Akt or ERK inhibitor cancelled the protective effect of VEGF165 on SNP-induced synoviocyte apoptosis. Moreover, VEGF165 blocks SNP-induced Bcl-2 down-regulation as well as SNP-induced Bax translocation from the cytosol to the mitochondria. The down-regulation of the neuropilin-1 transcripts by short interfering RNA caused spontaneous synoviocyte apoptosis, which was associated with both the decrease in Bcl-2 expression and the increase in Bax translocation to mitochondria. Collectively, our data suggest that the interaction of VEGF165 with neuropilin-1 is crucial to the survival of rheumatoid synoviocytes and provide important implications for the abnormal growth of synoviocytes and therapeutic intervention in RA.
Publication Type: Research Support, Non-U.S. Gov't;
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91.
Nucleic Acids Res 2006 ; 18(34):5133-44.
NMR solution structure of the major G-quadruplex structure formed in the human BCL2 promoter region.
Dai J ,Chen D ,Jones RA ,Hurley LH ,Yang D ,
College of Pharmacy, The University of Arizona, 1703 E. Mabel Street, Tucson, AZ 85721, USA.
BCL2 protein functions as an inhibitor of cell apoptosis and has been found to be aberrantly expressed in a wide range of human diseases. A highly GC-rich region upstream of the P1 promoter plays an important role in the transcriptional regulation of BCL2. Here we report the NMR solution structure of the major intramolecular G-quadruplex formed on the G-rich strand of this region in K+ solution. This well-defined mixed parallel/antiparallel-stranded G-quadruplex structure contains three G-tetrads of mixed G-arrangements, which are connected with two lateral loops and one side loop, and four grooves of different widths. The three loops interact with the core G-tetrads in a specific way that defines and stabilizes the overall G-quadruplex structure. The loop conformations are in accord with the experimental mutation and footprinting data. The first 3-nt loop adopts a lateral loop conformation and appears to determine the overall folding of the BCL2 G-quadruplex. The third 1-nt double-chain-reversal loop defines another example of a stable parallel-stranded structural motif using the G3NG3 sequence. Significantly, the distinct major BCL2 promoter G-quadruplex structure suggests that it can be specifically involved in gene modulation and can be an attractive target for pathway-specific drug design.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't;
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92.
Biotechnol Lett 2006 Dec ; 23(28):1919-24.
Modeling suppression of cell death by Bcl-2 over-expression in myeloma NS0 6A1 cells.
O'Connor KC ,Muhitch JW ,Lacks DJ ,Al-Rubeai M ,
Department of Chemical and Biomolecular Engineering, Tulane University, New Orleans, LA 70118, USA.
A novel population-balance model was employed to evaluate the suppression of cell death in myeloma NS0 6A1 cells metabolically engineered to over-express the apoptotic suppressor Bcl-2. The model is robust in its ability to simulate cell population dynamics in batch suspension culture and in response to thymidine-induced growth inhibition: 89% of simulated cell concentrations are within two standard deviations of experimental data. Kinetic rate constants in model equations suggest that Bcl-2 over-expression extends culture longevity from 6 days to at least 15 days by suppressing the specific rate of early apoptotic cell formation by more than 6-fold and necrotic cell formation by at least 3-fold, despite nearly a 3-fold decrease in initial cell growth rate and no significant change in the specific rate of late apoptotic cell formation. This computational analysis supports a mechanism in which Bcl-2 is a common mediator of early apoptotic and necrotic events occurring at rates that are dependent on cellular factors accumulating over time. The model has current application to the rational design of cell cultures through metabolic engineering for the industrial production of biopharmaceuticals.
Publication Type: Research Support, U.S. Gov't, Non-P.H.S.;
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93.
Ann Oncol 2006 Nov ; 11(17):1677-86.
Assessing interactions between mdm-2, p53, and bcl-2 as prognostic variables in muscle-invasive bladder cancer treated with neo-adjuvant chemotherapy followed by locoregional surgical treatment.
Maluf FC ,Cordon-Cardo C ,Verbel DA ,Satagopan JM ,Boyle MG ,Herr H ,Bajorin DF ,
The Genitourinary Oncology Service, Division of Solid Tumor Oncology, Department of Medicine, Memorial Sloan-Kettering Cancer Center and the Department of Medicine, Joan and Sanford Weill Medical College of Cornell University, New York, USA.
BACKGROUND: Tumor proliferation and apoptosis may be influenced by the mdm-2 gene product, which can block the antiproliferative effects of p53. bcl-2, one of a family of related genes that regulates the apoptotic pathway, exhibits a negative influence. Both individual and cooperative effects of these gene products may affect the biological behavior of primary bladder cancers and long-term outcome to standard therapy. METHODS: This study retrospectively evaluated the association with survival of mdm-2, p53, and bcl-2 expression in 59 patients with muscle-invasive, node-negative transitional cell carcinoma (TCC) treated with neo-adjuvant chemotherapy followed by locoregional surgery. Each marker was defined as an altered phenotype if >or=20% malignant cells in the primary tumor exhibited staining; normal or minimal expression was defined as <20% cells exhibiting staining. RESULTS: Altered mdm-2, p53, and bcl-2 expression was observed in 37%, 54%, and 46% of patients, respectively. In single marker analysis, altered p53 expression correlated with long-term survival (P = 0.05) but mdm-2 (P = 0.42) or bcl-2 (P = 0.17) did not. In the multiple-marker analysis, a prognostic index simultaneously assessing mdm-2, p53, and bcl-2 correlated with survival (P = 0.01). The 5-year survival for patients in which all markers were normally expressed was 54% compared with 25% in those with all three markers aberrantly expressed. Patients with aberrant expression of either one or two markers had an intermediate 5-year survival (49%). There was no association of molecular markers either alone or in combination with pathologic downstaging after neo-adjuvant chemotherapy. CONCLUSION: The cooperative effects of phenotypes determined by mdm-2, p53, and bcl-2 expression may predict survival in patients with muscle-invasive TCC of the bladder.
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94.
Cancer Invest 2006 Oct ; 6(24):576-80.
Serum vascular endothelial growth factor (VEGF) and bcl-2 levels in advanced stage non-small cell lung cancer.
Tas F ,Duranyildiz D ,Oguz H ,Camlica H ,Yasasever V ,Topuz E ,
Institute of Oncology, Istanbul University, Istanbul, Turkey. faruktas2002@yahoo.com
The characteristic changes in cancer process are assumed to be genetic alterations about the imbalance of cell proliferation and apoptotic cell death. This study was conducted to determine the value of the circulating vascular endothelial growth factor (VEGF) and Bcl-2 in patients with advanced stage non-small cell lung cancer (NSCLC). These serum factors were measured of 52 NSCLC patients pathologically verified on before and after chemotherapy in comparison with 16 healthy controls by using ELISA method. Both of the serum levels of VEGF (p = 0.015) and Bcl-2 (p < 0.001) were increased significantly in NSCLC patients compared with the healthy controls. No statistically significant relationships between investigated elevated serum parameters and various characteristics of patients and disease such as stage and tumor burden were determined. Likewise, we also found no correlation between serum VEGF and Bcl-2. Cytotoxic therapy of patients was accompanied by unchanged serum levels of serum factors. The median survival of all patients was 27 weeks and one-year survival rate was 22.4 percent. With the median serum levels as the cut-off value, patients were divided into high- and low-serum parameter groups. While we found that patients' performance status (p < 0.0001), serum LDH level (p = 0.0002), response to chemotherapy (p = 0.0023), and stage of the disease (p = 0.0085) were prognostic factors for survival, serum VEGF (p = 0.48) and Bcl-2 (p = 0.91) levels were determined as ineffective on survival in patients with advanced NSCLC. In conclusion, our data suggest that these serum factors, VEGF and Bcl-2, are useful diagnostic factors, not predictive and prognostic markers for overall survival in advanced NSCLC patients.
Publication Type: Comparative Study;
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95.
Blood 2007 Jan 1; 1(109):290-7.
Association of a novel regulatory polymorphism (-938C>A) in the BCL2 gene promoter with disease progression and survival in chronic lymphocytic leukemia.
Nückel H ,Frey UH ,Bau M ,Sellmann L ,Stanelle J ,Dürig J ,Jöckel KH ,Dührsen U ,Siffert W ,
Department of Hematology, Medical Faculty, University of Duisburg-Essen, Germany. holger.nueckel@uni-essen.de
Bcl-2 plays a key role in the regulation of apoptosis. We investigated the role of a novel regulatory single-nucleotide polymorphism (-938C>A) in the inhibitory P2 BCL2 promoter in B-cell chronic lymphocytic leukemia (B-CLL). The -938C allele displayed significantly increased BCL2 promoter activity and binding of nuclear proteins compared with the A allele. Concomitantly, Bcl-2 protein expression in B cells from CLL patients carrying the -938 AA genotype was significantly increased compared with CC genotypes. Genotype distribution between 123 CLL patients (42 AA, 55 AC, 26 CC) and 120 genotyped healthy controls (36 AA, 63 AC, 21 CC) was not significantly different, suggesting that genotypes of this polymorphism do not increase the susceptibility for B-CLL. However, median time from first diagnosis to initiation of chemotherapy and median overall survival were significantly shorter in patients with -938AA genotype (38 and 199 months, respectively) compared with AC/CC genotypes (120 and 321 months, respectively; P = .008 and P = .003, respectively). Multivariable Cox regression identified the BCL2-938AA genotype as an independent prognostic factor for the time to first treatment (hazard ratio [HR] 1.9; P = .034) together with disease stage at diagnosis (HR 2.5; P = .004) and ZAP-70 status (HR 3.0; P = .001). The BCL2-938AA genotype is associated with increased Bcl-2 expression and a novel unfavorable genetic marker in patients with B-CLL.
Publication Type: Research Support, Non-U.S. Gov't;
Comment In: Blood. 2008 Jan 1;111(1):466-8
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96.
Virology ; 1-2(356):136-46.
Role of Bcl-2 expression for productive herpes simplex virus 2 replication.
Sciortino MT ,Perri D ,Medici MA ,Grelli S ,Serafino A ,Borner C ,Mastino A ,
Department of Microbiological, Genetic and Molecular Sciences, University of Messina, Salita Sperone 31, 98166 Messina, Italy.
Herpes simplex viruses infect a variety of cells in vitro. However, not all infected cells sustain a fully productive replication of these viruses. We have shown that, in U937 monocytoid cells, herpes simplex virus 2 (HSV-2) causes a low-productive infection characterized by apoptosis as cytopathic effect at a late stage of infection. This effect was associated with a down-regulation of the Bcl-2 protein. We therefore asked whether destabilization of Bcl-2 expression could act as a limiting factor for the productive HSV-2 infection. We found that overexpression of Bcl-2 in U937 cells dramatically increased the capability of these cells to sustain a fully productive infection, while protecting against apoptosis induced by HSV-2. Overall, our data indicate that Bcl-2 expression acts as a regulator of HSV-2 replication.
Publication Type: Research Support, Non-U.S. Gov't;
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97.
Hum Pathol 2007 Jan ; 1(38):103-13.
Expression of bcl2 family proteins and active caspase 3 in classical Hodgkin's lymphomas.
Bai M ,Papoudou-Bai A ,Horianopoulos N ,Grepi C ,Agnantis NJ ,Kanavaros P ,
Department of Pathology, Medical Faculty, University of Ioannina, Ioannina 45110, Greece. mbai@cc.uoi.gr
The expression of various bcl2 family proteins has been reported in Hodgkin and Reed-Sternberg cells, but the proteins bad, bid, and bim have not been analyzed in classical Hodgkin's lymphomas (HLs). This study aimed to investigate the expression of the proteins bcl2, bcl-xl, mcl1, bax, bak, bad, bid, bim, and active caspase 3, and the TUNEL (terminal deoxynucleotidyl transferase-mediated in situ labeling) index to gain further insight into the apoptosis profile of classical HLs. A high expression of the proteins bcl2, bcl-xl, mcl1, bax, bak, bad, bid, and bim in HRS cells was found in 27 of 101 (27%), 95 of 101 (94%), 27 of 97 (29%), 73 of 95 (77%), 37 of 102 (36%), 85 of 94 (90%), 19 of 109 (17%), and 43 of 91 (47%) cases, respectively. The high expression of bcl-xl, bax, and bad in HRS cells in most classical HLs indicates that these proteins may play predominant roles in the regulation of apoptosis in classical HLs. Active caspase 3-positive and TUNEL-positive Reed-Sternberg cells were detected in 47 of 70 (67%; range, 0%-12%) and 60 of 71 (85%; range, 0%-19%) cases, respectively. Significant positive correlations were found between bax/bcl2 (P = .002), bad/bcl2 (P = .020), bad/bcl-xl (P = .003), and bim/mcl1 (P = .036). Based on these findings, it could be hypothesized that the antiapoptotic proteins bcl2, bcl-xl, and mcl1 may counteract the expression of the proapoptotic proteins bax, bad, and bim, thereby contributing to the survival of Reed-Sternberg cells.
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98.
Mol Cell Biochem 2007 Jan ; 1-2(295):153-65.
Suppression of IP3-mediated calcium release and apoptosis by Bcl-2 involves the participation of protein phosphatase 1.
Xu L ,Kong D ,Zhu L ,Zhu W ,Andrews DW ,Kuo TH ,
Department of Pathology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201, USA.
The involvement and potential interdependence of inositol trisphosphate (IP3) receptors and Bcl-2 in the regulation of Ca2+ signaling is not clear. Here, we have explored the mechanism(s) of how Bcl-2 suppresses the IP3-sensitive Ca2+ release in MCF-7 cells focusing on the possible role of protein phosphatase 1 (PP1). We found that through influences on protein-protein interaction, Bcl-2 may alter the balance between the effects of phosphatase (PP1) and kinase (PKA) on the IP3 R1 signaling complex. Using various experimental approaches including phosphatase inhibition and RNAi, we show that Bcl-2 by competing with IP3R1 for the binding of PP1 can reduce the IP3-mediated calcium signal and protect cells from mitochondrial dysfunction and cell death.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't;
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99.
Ann N Y Acad Sci 2006 Jun ; (1069):364-76.
Anti-beta-2 glycoprotein I antibodies affect Bcl-2 and Bax trophoblast expression without evidence of apoptosis.
di Simone N ,Castellani R ,Raschi E ,Borghi MO ,Meroni PL ,Caruso A ,
Department of Obstetrics and Gynecology, Università Cattolica de Sacro Cuore, Rome, Italy.
Antiphospholipid antibodies (aPLs) reacting with beta-2 glycoprotein I (beta2GPI) have been associated with recurrent fetal loss and pregnancy complications. The aim of the study was to investigate whether aPLs with anti-beta2GPI specificity induce apoptosis of human trophoblasts in vitro. To this end, human anti-beta2GPI monoclonal IgM derived from a patient with antiphospholipid syndrome and a human irrelevant monoclonal IgM were incubated with human trophoblast cell cultures for 24, 48, and 72 h. In all the cultures we evaluated: (i) Bcl-2 and Bax mRNA and protein expression by Western blot and reverse transcription polymerase chain reaction (RT-PCR), respectively; (ii) DNA fragmentation by a commercial ELISA kit and by agarose gel electrophoresis; and (iii) the percentage of cells reactive with the monoclonal antibody (MAb) M30 by indirect immunofluorescence. The results were: Bcl-2/Bax ratio increased in untreated trophoblast cells during the time of culture, showing the highest values detectable after 72 h (2.68 and 2.28 at protein and mRNA levels, respectively). Cell incubation with anti-beta2GPI MAbs induced a significant Bcl-2/Bax ratio reduction in comparison with untreated cells (1.22 and 1.28 at protein and mRNA levels, respectively, after 72 h incubation). No significant difference was detected after cell exposure to irrelevant MAbs. However, neither DNA fragmentation nor increase in cells positive for the caspase-cleaved epitope of cytokeratin 18 cytoskeletal protein (M30) was found. In Conclusion, anti-beta2GPI antibodies react with trophoblast cells and reduce the Bcl-2/Bax ratio, but without any clear apoptotic effect.
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100.
Apoptosis 2006 Oct ; 10(11):1825-35.
Involvement of IL-10 and Bcl-2 in resistance against an asbestos-induced apoptosis of T cells.
Miura Y ,Nishimura Y ,Katsuyama H ,Maeda M ,Hayashi H ,Dong M ,Hyodoh F ,Tomita M ,Matsuo Y ,Uesaka A ,Kuribayashi K ,Nakano T ,Kishimoto T ,Otsuki T ,
Department of Hygiene, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 7010192, Japan.
To analyze the possibility that immunological alteration in asbestos-related diseases (ARDs) such as asbestosis (ASB) and malignant mesothelioma (MM) may affect the progression of cancers, a human adult T cell leukemia virus-immortalized T cell line (MT-2Org) was continuously exposed to 10 mug/ml of chrysotile-B (CB), an asbestos. After at least 8 months of exposure, the rate of apoptosis in the cells became very low and the resultant subline was designated MT-2Rst. The MT-2Rst cells were characterized by (i) enhanced expression of bcl-2, with regain of apoptosis-sensitivity by reduction of bcl-2 by siRNA, (ii) excess IL-10 secretion and expression, and (iii) activation of STAT3 that was inhibited by PP2, a specific inhibitor of Src family kinases. These results suggested that the contact between cells and asbestos may affect the human immune system and trigger a cascade of biological events such as activation of Src family kinases, enhancement of IL-10 expression, STAT3 activation and Bcl-2 overexpression. This speculation was partially confirmed by the detection of elevated bcl-2 expression levels in CD4 + peripheral blood T cells from patients with MM compared with those from patients with ASB or healthy donors. Further studies will be required to verify the role of T cells with enhanced bcl-2 expression in tumor progression induced by asbestos exposure.
Publication Type: Comparative Study; Research Support, Non-U.S. Gov't;
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101.
Cell Death Differ 2007 Feb ; 2(14):378-80.
BCL-2 expression is mainly regulated by JAK/STAT3 pathway in human CD34+ hematopoietic cells.
Sepúlveda P ,Encabo A ,Carbonell-Uberos F ,Miñana MD ,
Publication Type: Letter; Research Support, Non-U.S. Gov't;
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102.
Leuk Lymphoma 2006 Jun ; 6(47):1117-22.
Preferential survival of acute lymphoblastic leukemia cells at 33 degrees C is associated with up-regulation of bcl-2.
Morsi H ,Yong KL ,Jewell AP ,
School of Life Sciences, Kingston University, Kingston-Upon-Thames, Surrey, UK.
An important feature of childhood acute lymphoblastic leukemia (ALL) is the risk of testicular relapse in affected males, which may occur months or years after induction of remission. However, little is known about the factors that regulate leukemic cell survival and resistance to chemotherapy in the testis. In the present study, incubating ALL cell lines and primary cells from ALL patients at 33 degrees C resulted in increased survival, resistance to chemotherapeutic agents and upregulation of bcl-2. Acute myeloid leukemia cell lines incubated at 33 degrees C also showed increased survival and resistance to chemotherapeutic agents, but did not demonstrate upregulation of bcl-2. This may be important in determining survival of ALL cells at lower temperatures in the testis.
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103.
BMC Cancer 2006 ; (6):187.
Bcl-2 protein expression is associated with p27 and p53 protein expressions and MIB-1 counts in breast cancer.
Tsutsui S ,Yasuda K ,Suzuki K ,Takeuchi H ,Nishizaki T ,Higashi H ,Era S ,
Department of Breast Surgery, Matsuyama Red Cross Hospital, Matsuyama, Japan. tsutsui@lime.ocn.ne.jp
BACKGROUND: Recent experimental studies have shown that Bcl-2, which has been established as a key player in the control of apoptosis, plays a role in regulating the cell cycle and proliferation. The aim of this study was to investigate the relationship between Bcl-2 and p27 protein expression, p53 protein expression and the proliferation activity as defined by the MIB-1 counts. The prognostic implication of Bcl-2 protein expression in relation to p27 and p53 protein expressions and MIB-1 counts for breast cancer was also evaluated. METHODS: The immunohistochemical expression of Bcl-2 protein was evaluated in a series of 249 invasive ductal carcinomas of the breast, in which p27 and p53 protein expressions and MIB-1 counts had been determined previously. RESULTS: The Bcl-2 protein expression was found to be decreased in 105 (42%) cases. A decreased Bcl-2 protein expression was significantly correlated with a nuclear grade of III, a negative estrogen receptor, a decreased p27 protein expression, a positive p53 protein expression, positive MIB-1 counts and a positive HER2 protein expression. The incidence of a nuclear grade of III and positive MIB-1 counts increased as the number of abnormal findings of Bcl-2, p27 and p53 protein expressions increased. A univariate analysis indicated a decreased Bcl-2 protein expression to be significantly (p = 0.0089) associated with a worse disease free survival (DFS), while a multivariate analysis indicated the lymph node status and MIB-1 counts to be independently significant prognostic factors for the DFS. CONCLUSION: The Bcl-2 protein expression has a close correlation with p27 and p53 protein expressions and the proliferation activity determined by MIB-1 counts in invasive ductal carcinoma of the breast. The prognostic value of Bcl-2 as well as p27 and p53 protein expressions was dependent on the proliferation activity in breast cancer.
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104.
Int J Oncol 2006 Aug ; 2(29):349-55.
Lysosomal cathepsin initiates apoptosis, which is regulated by photodamage to Bcl-2 at mitochondria in photodynamic therapy using a novel photosensitizer, ATX-s10 (Na).
Ichinose S ,Usuda J ,Hirata T ,Inoue T ,Ohtani K ,Maehara S ,Kubota M ,Imai K ,Tsunoda Y ,Kuroiwa Y ,Yamada K ,Tsutsui H ,Furukawa K ,Okunaka T ,Oleinick NL ,Kato H ,
Department of Thoracic Surgery, Tokyo Medical University, Tokyo, Japan.
ATX-s10 is a novel and second-generation photosensitizer for photodynamic therapy (PDT). In order to conduct clinical trials of ATX-s10-PDT and/or extend its clinical applications, it is very important to elucidate the mechanisms of the action of ATX-s10-PDT. We examined the apoptic response against ATX-s10-PDT using a Bcl-2 or Bcl-2 mutant overexpressing cells. Using fluorescent microscopy, ATX-s10 localized not only to mitochondria but also to lysosomes and possibly other intracellular organelles, but not to the plasma membrane or the nucleus. These results suggest that ATX-s10-PDT can damage mitochondria and lysosomes. By Western blot analysis, ATX-s10-PDT damaged Bcl-2, which localized preferentially at mitochondrial membranes, and caused Bcl-2 to cross-link immediately after laser irradiation. However, ATX-s10-PDT was not able to rapidly induce morphologically typical apoptosis (i.e. chromatin condensation and fragmentation) as PDT using mitochondria targeted photosensitizers, such as phthalocyanine 4 (Pc 4). Pharmacological inhibitions of lysosomal cytokine protease cathepsins, such as cathepsin B and D, protected MCF-7c3 cells (human breast cancer cells expressing stably transfected procaspase-3) from apoptosis caused by ATX-s10-PDT. Overexpression of wild-type Bcl-2 or Bcl-2Delta33-54 resulted in relative resistance of cells to ATX-s10-PDT, as assessed by the degree of morphological apoptosis or loss of clonogenicity. We conclude that lysosomal damage by ATX-s10-PDT can initiate apoptotic response and this apoptotic pathway can be regulated by photodamage to Bcl-2 via mitochondrial damage.
Publication Type: Research Support, Non-U.S. Gov't;
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105.
Invest Ophthalmol Vis Sci 2006 Jul ; 7(47):2750-6.
Detection of the bcl-2 t(14;18) translocation and proto-oncogene expression in primary intraocular lymphoma.
Wallace DJ ,Shen D ,Reed GF ,Miyanaga M ,Mochizuki M ,Sen HN ,Dahr SS ,Buggage RR ,Nussenblatt RB ,Chan CC ,
Laboratory of Immunology, Division of Epidemiology and Clinical Research, National Eye Institute, National Institutes of Health, Bethesda, MD 20895-1857, USA, and Department of Ophthalmology, Tokyo Medical and Dental University, Japan.
PURPOSE: Primary intraocular lymphoma (PIOL) is a diffuse large B cell lymphoma that initially infiltrates the retina, vitreous, or optic nerve head, with or without central nervous system involvement. This study examined the expression of the bcl-2 t(14;18) translocation, the bcl-10 gene, and high expression of bcl-6 mRNA in PIOL cells. METHODS: Microdissection and PCR analysis were used to examine vitreous specimens in patients with PIOL for the presence of bcl-2 t(14;18) translocations, the bcl-10 gene, and expression of bcl-6 mRNA. A medical record review was also conducted to determine whether the bcl-2 t(14;18) translocation correlated with prognosis. RESULTS: Forty of 72 (55%) PIOL patients expressed the bcl-2 t(14;18) translocation at the major breakpoint region. Fifteen of 68 (22%) patients expressed the translocation at the minor cluster region. The bcl-10 gene was detected in 6 of 26 (23%) patients, whereas 4 of 4 (100%) PIOL patients expressed higher levels of bcl-6 mRNA compared with inflammatory lymphocytes. An analysis of clinical outcome in 23 PIOL patients revealed no significant association between bcl-2 t(14;18) translocations and survival or relapse. However, patients with the translocation were significantly younger. CONCLUSIONS: PIOL has unique molecular patterns of bcl-2, bcl-10, and bcl-6 when compared with other systemic lymphomas. This study lays the foundation for future studies aimed at exploring the genotypic classification of PIOL based on the quantitative molecular framework of gene expression profiling, with the goal of providing useful adjuncts to the pathologic diagnosis of this complex disease.
Publication Type: Research Support, N.I.H., Extramural; Research Support, N.I.H., Intramural;
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106.
Gene 2006 Sep 1; (379):127-31.
The bcl-2 major breakpoint region (mbr) possesses transcriptional regulatory function.
Zhang J ,Ma C ,Han X ,Durrin LK ,Sun Y ,
Key Laboratory of Human Functional Genomics of Jiangsu Province, PR China.
The bcl-2 major breakpoint region (mbr), located within the 3'-UTR of the bcl-2 gene, is the site of the most common chromosomal translocation, t(14;18) (q32;q21), which occurs in follicular lymphoma. The mbr forms a triplex DNA structure under physiological conditions and the transcription factor special AT-rich sequence-binding protein 1 (SATB1) binds immediately downstream of the mbr. These observations raise the possibility that the mbr may be involved in regulation of bcl-2 gene expression. We investigated the role of the bcl-2 mbr on reporter gene activity and the relevance of SATB1 to this function in a variety of cell lines. We found that the mbr up-regulated reporter gene expression. Deletion of the 37-bp AT-rich SATB1 binding site abolished the bcl-2 mbr regulation of reporter gene expression. Overexpression of SATB1 enhanced bcl-2 mbr up-regulation of the reporter gene activity. Our data strongly demonstrated that the bcl-2 mbr possessed regulatory function that was related to SATB1.
Publication Type: Research Support, Non-U.S. Gov't;
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107.
Indian J Pathol Microbiol 2005 Jul ; 3(48):325-30.
Bcl2 and ROS1 expression in human meningiomas: an analysis with respect to histological subtype.
Girish V ,Sachdeva N ,Minz RW ,Radotra B ,Mathuria SN ,Arora SK ,
Department of Immunopathology, Postgraduate Institute of Medical Education & Research, Chandigarh.
Tumors of the central nervous system account for approximately 9% of all primary neoplasm in humans, while tumors of covering elements, the meninges, account for 13-19% and constitute the second largest group of brain tumors. These are known to exhibit a variety of chromosomal abnormalities besides change in the expression level of certain oncogenes. Among oncogenes, bcl2, an anti-apoptotic factor and ROS1 that encodes a protein with a structure similar to the epidermal growth factor (EGF) and insulin receptor and has a tyrosine kinase activity, have been shown to be associated with many malignant tumors. In the present study we have analysed the expression of bcl2 using immuno-histochemistry and ROS1 expression by reverse-transcription coupled with polymerase chain reaction (RT-PCR) of the transcript using primers specific for the intra-cellular domain and then tried to correlate the findings with the subtype of the meningioma defined on the basis of histology. Out of the six bcl2 positive cases in our study, there were three transitional tumors, two fibroblastic and one recurrent meningioma subtype. bcl2 seemed to be more consistently expressed in the cytoplasm of spindle cell component of meningiomas. Thirteen meningiothelial meningiomas did not show any staining for bcl2. ROS1 gene expression could be detected in 4 tumors all of those were Grade-I meningothelial meningiomas. One of the malignant meningioma included in the study was clearly negative for bcl2 as well as ROS1. Thus bcl2 and ROS1 oncogene expression in meningiomas are not concurrent and neither can be ascribed to any histologic subtype or grade of tumor.
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108.
J Cell Sci 2006 Jun 15; Pt 12(119):2572-82.
Human Bcl-2 cannot directly inhibit the Caenorhabditis elegans Apaf-1 homologue CED-4, but can interact with EGL-1.
Jabbour AM ,Puryer MA ,Yu JY ,Lithgow T ,Riffkin CD ,Ashley DM ,Vaux DL ,Ekert PG ,Hawkins CJ ,
Children's Cancer Centre, Royal Children's Hospital, Parkville 3052, Australia.
Although the anti-apoptotic activity of Bcl-2 has been extensively studied, its mode of action is still incompletely understood. In the nematode Caenorhabditis elegans, 131 of 1090 somatic cells undergo programmed cell death during development. Transgenic expression of human Bcl-2 reduced cell death during nematode development, and partially complemented mutation of ced-9, indicating that Bcl-2 can functionally interact with the nematode cell death machinery. Identification of the nematode target(s) of Bcl-2 inhibition would help clarify the mechanism by which Bcl-2 suppresses apoptosis in mammalian cells. Exploiting yeast-based systems and biochemical assays, we analysed the ability of Bcl-2 to interact with and regulate the activity of nematode apoptosis proteins. Unlike CED-9, Bcl-2 could not directly associate with the caspase-activating adaptor protein CED-4, nor could it inhibit CED-4-dependent yeast death. By contrast, Bcl-2 could bind the C. elegans pro-apoptotic BH3-only Bcl-2 family member EGL-1. These data prompt us to hypothesise that Bcl-2 might suppress nematode cell death by preventing EGL-1 from antagonising CED-9, rather than by inhibiting CED-4.
Publication Type: Research Support, Non-U.S. Gov't;
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109.
Prostate Cancer Prostatic Dis 2006 ; 3(9):284-92.
Germline BCL-2 sequence variants and inherited predisposition to prostate cancer.
Kidd LR ,Coulibaly A ,Templeton TM ,Chen W ,Long LO ,Mason T ,Bonilla C ,Akereyeni F ,Freeman V ,Isaacs W ,Ahaghotu C ,Kittles RA ,
Cancer Prevention and Control Program, Department of Pharmacology and Toxicology, James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY, USA.
Apoptosis is an essential physiological process that regulates cellular proliferation. Here, we explored the effect of DNA sequence variation within the BCL-2 gene on prostate cancer susceptibility in three clinical populations, consisting of 428 African Americans, 214 Jamaicans and 218 European Americans. We observed a 70% reduced risk for prostate cancer among the European Americans who had possessed two copies of a promoter variant -938C/A. Additionally, common BCL-2 haplotypes appeared to influence prostate cancer risk; however, studies in larger data sets are needed to confirm our findings. Our data suggest that inherited BCL-2 variants may be associated with a decrease in prostate cancer susceptibility.
Publication Type: Comparative Study; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.;
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110.
J Biol Chem 2006 Aug 11; 32(281):23003-12.
PP2A regulates BCL-2 phosphorylation and proteasome-mediated degradation at the endoplasmic reticulum.
Lin SS ,Bassik MC ,Suh H ,Nishino M ,Arroyo JD ,Hahn WC ,Korsmeyer SJ ,Roberts TM ,
Howard Hughes Medical Institute, Department of Cancer Immunology and AIDS, Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts 02115, USA.
Anti-apoptotic activity of BCL-2 is mediated by phosphorylation at the endoplasmic reticulum (ER), but how this phosphorylation is regulated and the mechanism(s) by which it regulates apoptosis are unknown. We purified macromolecular complexes containing BCL-2 from ER membranes and found that BCL-2 co-purified with the main two subunits of the serine/threonine phosphatase, PP2A. The association of endogenous PP2A and BCL-2 at the ER was verified by co-immunoprecipitation and microcystin affinity purification. Knock down or pharmacological inhibition of PP2A caused degradation of phosphorylated BCL-2 and led to an overall reduction in BCL-2 levels. We found that this degradation was due to the action of the proteasome acting selectively at the ER. Conversely, overexpression of PP2A caused elevation in endogenous BCL-2. Most importantly, we found that PP2A knock down sensitized cells to several classes of death stimuli (including ER stress), but this effect was abolished in a genetic background featuring knock in of a non-phosphorylatable BCL-2 allele. These studies support the hypothesis that PP2A-mediated dephosphorylation of BCL-2 is required to protect BCL-2 from proteasome-dependent degradation, affecting resistance to ER stress.
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111.
Biochem Biophys Res Commun 2006 Jul 7; 3(345):1092-8.
Depression of MAD2 inhibits apoptosis of gastric cancer cells by upregulating Bcl-2 and interfering mitochondrion pathway.
Du Y ,Yin F ,Liu C ,Hu S ,Wang J ,Xie H ,Hong L ,Fan D ,
State Key Laboratory of Cancer Biology, Institute of Digestive Diseases, Xijing Hospital, The Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China.
Mitotic arrest deficient 2 (MAD2) is an essential component of the mitotic spindle checkpoint pathway. It was previously shown to be associated with drug resistance of tumor cells. To further explore the roles of MAD2 in responses of gastric cancer cells to chemotherapy drugs, we constructed the siRNA vectors of MAD2 and transfected them into gastric cancer SGC7901 cells to inhibit expression of MAD2. MTT assay showed that the downregulation of MAD2 increased the resistance of SGC7901 cells to spindle inhibitors and DNA damaging agents. The apoptosis rates of gastric cancer cells transfected with MAD2-siRNA were 10.7% and 10%, respectively, after treated by 1.0microg/ml VCR and cisplatin. In contrast, the apoptosis rates of SGC7901 and SGC7901/psilencer3.1 induced by VCR were 43.2%, 38.7%; and that induced by cispaltin were 34.1%, 31.4%. The ratio of Bcl-2 to Bax was much higher in the MAD2-siRNA transfectants compared with the SGC7901/psilencer. In SGC7901/psilencer, cytochrome c and cleaved caspase 3 protein levels increased along with the exposure time increased. However, these protein levels of SGC7901/MAD2-siRNA had no changes during the drug treatment. These results indicate that down regulation of MAD2 could promote the drug resistance of gastric cancer cells and inhibit anticancer drugs induced-apoptosis by upregulating Bcl-2 and interfering the mitochondrion apoptosis pathway.
Publication Type: Research Support, Non-U.S. Gov't;
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112.
Mol Biol Cell 2006 Aug ; 8(17):3356-68.
Association of Bcl-2 with misfolded prion protein is linked to the toxic potential of cytosolic PrP.
Rambold AS ,Miesbauer M ,Rapaport D ,Bartke T ,Baier M ,Winklhofer KF ,Tatzelt J ,
Department of Biochemistry, Neurobiochemistry, Ludwig-Maximilians-Universität München, D-80336 München, Germany.
Protein misfolding is linked to different neurodegenerative disorders like Alzheimer's disease, polyglutamine, and prion diseases. We investigated the cytotoxic effects of aberrant conformers of the prion protein (PrP) and show that toxicity is specifically linked to misfolding of PrP in the cytosolic compartment and involves binding of PrP to the anti-apoptotic protein Bcl-2. PrP targeted to different cellular compartments, including the cytosol, nucleus, and mitochondria, adopted a misfolded and partially proteinase K-resistant conformation. However, only in the cytosol did the accumulation of misfolded PrP induce apoptosis. Apoptotic cell death was also induced by two pathogenic mutants of PrP, which are partially localized in the cytosol. A mechanistic analysis revealed that the toxic potential is linked to an internal domain of PrP (amino acids 115-156) and involves coaggregation of cytosolic PrP with Bcl-2. Increased expression of the chaperones Hsp70 and Hsp40 prevented the formation of PrP/Bcl-2 coaggregates and interfered with PrP-induced apoptosis. Our study reveals a compartment-specific toxicity of PrP misfolding that involves coaggregation of Bcl-2 and indicates a protective role of molecular chaperones.
Publication Type: Research Support, Non-U.S. Gov't;
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113.
Leuk Lymphoma 2006 Apr ; 4(47):665-73.
Expression of ice, bcl-2, c-myc and p53 in different bone marrow cell populations from patients with diffuse large B-cell lymphoma.
Madrigal-Velázquez M ,Avilés A ,Neri N ,Huerta J ,Martínez-Jaramillo G ,Mayani H ,
Oncological Research Unit, Oncology Hospital, National Medical Center, IMSS, Mexico City, Mexico.
We have previously reported functional alterations in vitro in the hematopoietic compartment of patients with diffuse large B-cell lymphoma (DLBCL). In the present study, we assessed the presence of molecular alterations in hematopoietic cells derived from DLBCL marrow. Accordingly, the expression of four genes (i.e. ice, bcl-2, c-myc and p53) was assessed both, at the mRNA and protein levels, in three cell populations: (i) population I, consisting of morphologically recognizable precursor and mature cells; (ii) population II, enriched for CD34+ Lineage-negative (Lin-) cells; and (iii) population III, enriched for CD34+ CD38- Lin- cells. By using a multiplex reverse transcriptase-polymerase chain reaction system, we observed reduced expression of bcl-2 in population I, and c-myc in populations I and II from lymphoma marrow compared to their normal counterparts. On the other hand, expression of ice and p53 was not significantly different when comparing normal and DLBCL samples. At the protein level, all four molecules were expressed in a higher proportion of samples from DLBCL patients than in marrow samples from normal subjects. Interestingly, these proteins were expressed predominantly in primitive cells (population III), whereas the proportion of positive samples was reduced in population II, and even more in population I. Taken together, our results indicate that, in DLBCL, molecular alterations are present in hematopoietic cells from bone marrow, including morphologically recognizable precursor and mature cells, as well as primitive hematopoietic progenitors (CD34+ cells). To date, the physiological implications of these alterations are still unclear, and further studies should be undertaken to address this issue.
Publication Type: Research Support, Non-U.S. Gov't;
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114.
Tumour Biol 2006 ; 4(27):195-200.
Expression of the antiapoptotic proteins clusterin and bcl-2 in laryngeal squamous cell carcinomas.
Redondo M ,Esteban F ,González-Moles MA ,Delgado-Rodríguez M ,Nevado M ,Torres-Muñoz JE ,Tellez T ,Villar E ,Morell M ,Petito CK ,
Departamento de Bioquímica, Hospital Costa del Sol, Universidad de Málaga, Marbella, Spain. mredondo@hcs.es
Bcl-2 and clusterin genes have been related to the inhibition of apoptosis, an event that plays a key role in malignant transformation and in invasive disease. In this work, we determine the significance of clusterin and bcl-2 expression in a large series of laryngeal carcinomas. We used immunohistochemical methods and in situ hybridization to examine the expression of these proteins. Nontumoral epithelial laryngeal tissues did not express clusterin and bcl-2 proteins. However, 9% (14 out of 154) and 25% of these tumors (39 of 154) had positive clusterin and bcl-2 staining, respectively. Clusterin expression was significantly related to the degree of local invasion and higher bcl-2 expression was found in these clusterin-positive tumors (p < 0.05). Bcl-2 expression was significantly correlated with supraglottic localization, nodal metastases, invasion in depth, and poorly differentiated tumors. However, by multivariate analysis, bcl-2 was shown to be an independent predictor of good prognosis in these tumors (OR = 0.12, 95% CI = 0.02-0.91). These findings indicate that clusterin and bcl-2 are upregulated in laryngeal carcinomas and their expression is related to the invasiveness of these tumors.
Publication Type: Research Support, Non-U.S. Gov't;
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115.
Int J Gastrointest Cancer 2005 ; 2(36):61-8.
Relationship of Helicobacter pylori to Bcl-2 family expression, DNA content, and pathological characteristics of gastric cancer.
El-Shahat M ,El-Masry S ,Lotfy M ,El-Kenawy Ael-M ,Nasif WA ,
Molecular and Cellular Biology Department, Genetic Engineering and Biotechnology Research Institute, Minufiya University, Sadat City, Minufiya, Egypt.
BACKGROUND: Despite the fact that the association of Helicobacter pylori with an increased risk of gastric cancer has been well documented, the exact mechanisms of this association have not been fully elucidated. Scarce data on H. pylori infection and its relationship with the different pathological characteristics are available in Egypt. AIM OF THE STUDY: The rationale of the present study was to determine the prevalence of H. pylori in a group of gastric cancer patients and to analyze the relationship between H. pylori infection with the different pathological characteristics including the types of gastric cancer and tumor location within the stomach, in addition, to investigate the Bcl-2 and Bax expressions along with DNA flow cytometric analysis in the gastric cancer patients with and without H. pylori infection. METHODS: Samples were obtained from 66 consecutive patients with gastric cancer (46 males and 20 females). The youngest patient was 20 yr old, the oldest 76 yr with mean age of 52.8 yr. The samples were subjected for histopathological characterization, H. pylori detection, DNA flow cytometric analysis, and Bcl-2 and Bax expressions detection, in addition to apoptosis analysis. RESULTS: The obtained results showed that the H. pylori infection was found in 38/66 (57.6%) [Odds ratio=1.357 with 95% confidence interval (CI) 0.84-2.2]. There was a statistical significance for Bcl-2, Bax, and apoptosis with H. pylori status (p = 0.009, 0.008, 0.032, respectively). On the other hand, There was a statistical significance for H. pylori infection with the disease grade (p = 0.015) and lymph node metastasis (p = 0.05). No statistical significance was found between H. pylori status with the patients' age, gender, tumor site, tumor type, depth of invasion, and stromal reaction. CONCLUSIONS: These data may indicate that the H. pylori infection not only contributes in the disease formation through the apoptosis dysregulation but also takes a part in the disease dissemination and progression. In addition, it may reflect a biologic, pathogenic, and ethnic background affecting the relationship of H. pylori infection to gastric cancer in the Egyptian patients. A high rate of smoking in Egypt and the diet are important factors that may affect such background. Further studies are warranted.
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116.
EMBO J 2006 Jun 7; 11(25):2287-96.
Bcl-2 changes conformation to inhibit Bax oligomerization.
Dlugosz PJ ,Billen LP ,Annis MG ,Zhu W ,Zhang Z ,Lin J ,Leber B ,Andrews DW ,
Department of Biochemistry and Biomedical Sciences, McMaster University Health Sciences Centre, CDN-Hamilton, Ontario, Canada.
Bcl-2 inhibits apoptosis by regulating the release of cytochrome c and other proteins from mitochondria. Oligomerization of Bax promotes cell death by permeabilizing the outer mitochondrial membrane. In transfected cells and isolated mitochondria, Bcl-2, but not the inactive point mutants Bcl-2-G145A and Bcl-2-V159D, undergoes a conformation change in the mitochondrial membrane in response to apoptotic agonists such as tBid and Bax. A mutant Bcl-2 with two cysteines introduced at positions predicted to result in a disulfide bond that would inhibit the mobility of alpha5-alpha6 helices (Bcl-2-S105C/E152C) was only active in a reducing environment. Thus, Bcl-2 must change the conformation to inhibit tBid-induced oligomerization of integral membrane Bax monomers and small oligomers. The conformationally changed Bcl-2 sequesters the integral membrane form of Bax. If Bax is in excess, apoptosis resumes as Bcl-2 is consumed by the conformational change and in complexes with Bax. Thus, Bcl-2 functions as an inhibitor of mitochondrial permeabilization by changing conformation in the mitochondrial membrane to bind membrane-inserted Bax monomers and prevent productive oligomerization of Bax.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't;
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117.
Clin Cancer Res 2006 Apr 15; 8(12):2545-54.
Association of DNA repair and steroid metabolism gene polymorphisms with clinical late toxicity in patients treated with conformal radiotherapy for prostate cancer.
Damaraju S ,Murray D ,Dufour J ,Carandang D ,Myrehaug S ,Fallone G ,Field C ,Greiner R ,Hanson J ,Cass CE ,Parliament M ,
Cross Cancer Institute, Department of Oncology, University of Alberta, Edmonton, Alberta, Canada.
OBJECTIVE: To explore the possible relationship between single nucleotide polymorphisms (SNP) in candidate genes encoding DNA damage recognition/repair/response and steroid metabolism proteins with respect to clinical radiation toxicity in a retrospective cohort of patients previously treated with three-dimensional conformal radiotherapy (3-DCRT) for prostate cancer. EXPERIMENTAL DESIGN: One hundred twenty-four patients with prostate cancer underwent 3-DCRT at our institution between September 1996 and December 2000. Of these, 83 consented for follow-up of blood sampling and SNP analysis. Twenty-eight patients were documented as having experienced grade >/=2 late bladder or rectal toxicity (scoring system of Radiation Therapy Oncology Group) on at least one follow-up visit. We analyzed 49 SNPs in BRCA1, BRCA2, ESR1, XRCC1, XRCC2, XRCC3, NBN, RAD51, RAD52, LIG4, ATM, BCL2, TGFB1, MSH6, ERCC2, XPF, NR3C1, CYP1A1, CYP2C9, CYP2C19, CYP3A5, CYP2D6, CYP11B2, and CYP17A1 genes using the Pyrosequencing technique. RESULTS: Significant univariate associations with late rectal or bladder toxicity (grade >/=2) were found for XRCC3 (A>G 5' untranslated region NT 4541), LIG4 (T>C Asp(568)Asp), MLH1 (C>T, Val(219)Ile), CYP2D6*4 (G>A splicing defect), mean rectal and bladder dose, dose to 30% of rectum or bladder, and age <60 years. On Cox multivariate analysis, significant associations with toxicity were found for LIG4 (T>C, Asp(568)Asp), ERCC2 (G>A, Asp(711)Asp), CYP2D6*4 (G>A, splicing defect), mean bladder dose >60 Gy, and dose to 30% of rectal volume >75 Gy. CONCLUSIONS: In this study, we identified SNPs in LIG4, ERCC2, and CYP2D6 genes as putative markers to predict individuals at risk for complications arising from radiation therapy in prostate cancer.
Publication Type: Research Support, Non-U.S. Gov't;
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118.
Clin Cancer Res 2006 Apr 15; 8(12):2468-75.
Bcl-2 is a prognostic marker in breast cancer independently of the Nottingham Prognostic Index.
Callagy GM ,Pharoah PD ,Pinder SE ,Hsu FD ,Nielsen TO ,Ragaz J ,Ellis IO ,Huntsman D ,Caldas C ,
Cancer Genomics Program, Department of Oncology, Hutchison-Medical Research Council Research Centre, University of Cambridge, United Kingdom.
PURPOSE: Prognostication of breast cancer using clinicopathologic variables, although useful, remains imperfect. Many reports suggest that gene expression profiling can refine the current approach. Alternatively, it has been shown that panels of proteins assessed by immunohistochemistry might also be useful in this regard. We evaluate the prognostic potential of a panel of markers by immunohistochemistry in a large case series to establish if either a single marker or a panel could improve the prognostic power of the Nottingham Prognostic Index (NPI). We validated the results in an independent series. Experimental Design and RESULTS: The expression of 13 biomarkers was evaluated in 930 breast cancers on a tissue microarray. Eight markers [estrogen receptor (ER), progesterone receptor (PR), Bcl-2, cyclin E, p53, MIB-1, cytokeratin 5/6, and HER2] showed a significant association with survival at 10 years on univariate analysis. On multivariate analysis that included these eight markers and the NPI, only the NPI [hazard ratio (HR), 1.35; 95% confidence interval (95% CI), 1.16-1.56; P = 0.0005], ER (HR, 0.59; 95% CI, 0.39-0.88; P = 0.011), and Bcl-2 (HR, 0.68; 95% CI, 0.46-0.99; P = 0.055) were significant. In a subsequent multivariate analysis that included the NPI, ER, and Bcl-2, only Bcl-2 (HR, 0.62; 95% CI, 0.44-0.87; P = 0.006) remained independent of NPI (HR, 1.50; 95% CI, 1.16-1.56; P = 0.004). In addition, Bcl-2, used as a single marker, was more powerful than the use of a panel of markers. Based on these results, an independent series was used to validate the prognostic significance of Bcl-2. ER and PR were also evaluated in this validation series. Bcl-2 (HR, 0.83; 95% CI, 0.71-0.96; P = 0.018) retained prognostic significance independent of the NPI (HR, 2.04; 95% CI, 1.67-2.51; P < 0.001) with an effect that was maximal in the first 5 years. CONCLUSION: Bcl-2 is an independent predictor of breast cancer outcome and seems to be useful as a prognostic adjunct to the NPI, particularly in the first 5 years after diagnosis.
Publication Type: Research Support, Non-U.S. Gov't;
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119.
Oncogene 2006 Sep 14; 41(25):5601-11.
Identification of an ataxia telangiectasia-mutated protein mediated surveillance system to regulate Bcl-2 overexpression.
Zhang J ,Lahti JM ,Bruce A ,He L ,Parihar K ,Fan C ,Grenet J ,Liu L ,Kidd VJ ,Cormier S ,Tang D ,
Division of Nephrology, Department of Medicine, McMaster University, Hamilton, Ontario, Canada.
Bcl-2 can both promote and attenuate tumorigenesis. Although the former function is relatively well characterized, the mechanism of the latter remains elusive. We report here that enforced Bcl-2 expression in MCF7 cells stabilizes p53, induces phosphorylation of p53 serine 15 (p53pSer15) and inhibits MCF7 cell growth. Consistent with p53 Ser15 being a target of ataxia telangiectasia mutated protein(ATM)/ATR (ATM- and rad3-related) in the DNA damage response, Bcl-2 activates ATM by inducing ATM Ser1981 phosphorylation, which is accompanied with the phosphorylaton of two additional ATM substrates, Chk2 Thr68 and H2AX Ser139. Downregulation of ATM using a specific small interference RNA fragment (ATMRNAi) abolished Bcl-2-induced p53pSer15 and Bcl-2-mediated growth inhibition of MCF7 cells. Ectopic expression of a dominant-negative p53 mutant, p53175H, partially rescued this growth inhibition. Taken together, these observations demonstrate the contribution of ATM-p53 function to Bcl-2-mediated inhibition of MCF7 cell growth, indicating an ATM-mediated surveillance system for regulating Bcl-2 overexpression. Consistent with this concept, we found that MCF7 cells express Bcl-2 heterogeneously with 34.5% of cells being Bcl-2 negative. In general, Bcl-2-positive MCF7 cells proliferate slower than those of Bcl-2 negative. Thus, we provide evidence suggesting that activation of ATM suppresses Bcl-2-induced tumorigenesis, and that attenuation of ATM function may be an important event in breast cancer progression.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't;
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120.
J Assist Reprod Genet 2006 Mar ; 3(23):149-56.
Changes in the ratio of Bax and Bcl-2 mRNA expression and their cellular localization throughout the ovulatory cycle in the human oviduct.
Briton-Jones C ,Lok IH ,Po AL ,Cheung CK ,Chiu TT ,Haines C ,
The Department of Obstetrics and Gynaecology, The Prince of Wales Hospital, Shatin, New Territories, Hong Kong, People's Republic of China.
PURPOSE: To examine changes in the ratio of Bax and Bcl-2 mRNA expression throughout the ovulatory cycle in the ampullary region of the human oviduct. METHODS: The mucosal layer was isolated from the human oviduct tissue and semiquantitative reverse-transcriptase polymerase chain-reaction (RT-PCR) analysis of mRNA of Bax and Bcl-2 was performed. Immunohistochemistry provided the cellular localization of the Bax and Bcl-2 proteins. The ratio of expression of Bax and Bcl-2 mRNA was examined in the ampullary region of the oviduct in samples obtained in the follicular, periovulatory, and luteal phases of the ovulatory cycle. RESULTS: Bax expression was constant in the follicular and periovulatory phase but showed a significant increase in the luteal phase. The Bax protein was present in all oviduct mucosal epithelial cells and the intensity of staining increased in luteal phase samples. Bcl-2 was expressed at a relatively constant level throughout the ovulatory cycle. The Bcl-2 protein was present in some but not all mucosal epithelial cells and the proportion of positive cells remained constant throughout the ovulatory cycle. CONCLUSION: The proapoptotic gene Bax shows a significant increase in mRNA expression in the luteal phase of the ovulatory cycle while the expression level of the antiapoptotic gene Bcl-2 remains constant throughout the ovulatory cycle. The ratio of Bax:Bcl-2 increases significantly in the luteal phase consistent with cells undergoing apoptosis.
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121.
Oncogene 2006 Aug 17; 36(25):5046-55.
Bcl-2 is a key regulator for the retinoic acid-induced apoptotic cell death in neuroblastoma.
Niizuma H ,Nakamura Y ,Ozaki T ,Nakanishi H ,Ohira M ,Isogai E ,Kageyama H ,Imaizumi M ,Nakagawara A ,
Division of Biochemistry, Chiba Cancer Center Research Institute, Chuoh-ku, Chiba, Japan.
Retinoic acid (RA) has been shown to induce neuronal differentiation and/or apoptosis, and is widely used as a chemotherapeutic agent for treating the patients with neuroblastoma. However, the therapeutic effect of RA is still limited. To unveil the molecular mechanism(s) inducing differentiation and apoptosis in neuroblastoma cells, we compared CHP134 and NB-39-nu cell lines, in which all-trans-RA (ATRA) induces apoptosis, with LA-N-5 and RTBM1 cell lines, in which it induces neuronal differentiation. Here, we found that Bcl-2 was strongly downregulated in CHP134 and NB-39-nu cells, whereas it was abundantly expressed in LA-N-5 and RTBM1 cells. ATRA-mediated apoptosis in CHP134 and NB-39-nu cells was associated with a significant activation of caspase-9 and caspase-3 as well as cytoplasmic release of cytochrome c from mitochondria in a p53-independent manner. Enforced expression of Bcl-2 significantly inhibited ATRA-mediated apoptosis in CHP134 cells. In addition, treatment of RTBM1 cells with a Bcl-2 inhibitor, HA14-1, enhanced apoptotic response induced by ATRA. Of note, two out of 10 sporadic neuroblastomas expressed bcl-2 at undetectable levels and underwent cell death in response to ATRA in primary cultures. Thus, our present results suggest that overexpression of Bcl-2 is one of the key mechanisms to give neuroblastoma cells the resistance against ATRA-mediated apoptosis. This may provide a new therapeutic strategy against the ATRA-resistant and aggressive neuroblastomas by combining treatment with ATRA and a Bcl-2 inhibitor.
Publication Type: Research Support, Non-U.S. Gov't;
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122.
Exp Cell Res 2006 Jun 10; 10(312):1865-75.
Regulation of bcl-2 expression by Ubc9.
Lu Z ,Wu H ,Mo YY ,
Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, 801 N. Rutledge, PO Box 19626, Springfield, IL 62794, USA.
Posttranslational modifications mediated by ubiquitin-like proteins have been implicated in regulating a variety of cellular pathways. Although small ubiquitin-like modifier (SUMO) is a new member of this family, it has caught a great deal of attention recently because of its novel and distinguished functions. Sumoylation is a multiple-step process, involving maturation, activation, conjugation and ligation. Ubc9 is an E2 conjugating enzyme essential for sumoylation. We have previously shown that suppression of sumoylation by a dominant negative Ubc9 mutant (Ubc9-DN) in the estrogen receptor (ER) positive MCF-7 cells is associated with alterations of tumor cell's response to anticancer drugs as well as tumor growth in a xenograft mouse carcinoma model. To dissect the underlying mechanism of Ubc9-associated alterations of drug responsiveness and tumor growth, we profiled gene expression for the cells expressing wild type Ubc9 (Ubc9-WT) and Ubc9-DN. We found that several tumorigenesis-related genes were downregulated in the Ubc9-DN cells. Within this group, we found that over 10 genes are known to be regulated by ER. Experiments using the estrogen response element fused to the luciferase reporter showed that the basal level of luciferase activity was significantly reduced in the Ubc9-DN cells when compared to the vector alone or the Ubc9-WT cells. Furthermore, we found that both the stability and the subcellular localization of steroid hormone receptor coactivator-1 (SRC-1) were altered in the Ubc9-DN cells. Together, these results suggest that Ubc9 might regulate bcl-2 expression through the ER signaling pathway, which ultimately contributes to the alterations of drug responsiveness and tumor growth.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't;
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123.
J Biol Chem 2006 May 19; 20(281):14446-56.
Bcl2 suppresses DNA repair by enhancing c-Myc transcriptional activity.
Jin Z ,May WS ,Gao F ,Flagg T ,Deng X ,
University of Florida Shands Cancer Center, Department of Medicine, Gainesville, Florida 32610-0232, USA.
Bcl2 and c-Myc are two major oncogenic proteins that can functionally promote DNA damage, genetic instability, and tumorigenesis. However, the mechanism(s) remains unclear. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most potent carcinogen contained in cigarette smoke that induces cellular DNA damage. Here we found that Bcl2 potently suppresses the repair of NNK-induced abasic sites of DNA lesions in association with increased c-Myc transcriptional activity. The Bcl2 BH4 domain (amino acids 6-31) was found to bind directly to c-Myc MBII domain (amino acids 106-143), and this interaction is required for Bcl2 to enhance c-Myc transcriptional activity and inhibit DNA repair. In addition to mitochondria, Bcl2 is also expressed in the nucleus, where it co-localizes with c-Myc. Expression of nuclear-targeted Bcl2 enhances c-Myc transcriptional activity with suppression of DNA repair but fails to prolong cell survival. Depletion of c-Myc expression from cells overexpressing Bcl2 significantly accelerates the repair of NNK-induced DNA damage, indicating that c-Myc may be essential for the Bcl2 effect on DNA repair. It is known that apurinic/apyrimidinic endonuclease (APE1) plays a crucial role in the repair of abasic sites of DNA lesions. That overexpression of Bcl2 results in up-regulation of c-Myc and down-regulation of APE1 suggests APE1 may function as the downstream target of Bcl2/c-Myc in the DNA repair machinery. Thus, Bcl2, in addition to its survival function, may also suppress DNA repair in a novel mechanism involving c-Myc and APE1, which may lead to an accumulation of DNA damage in living cells, genetic instability, and tumorigenesis.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't;
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124.
Int J Cancer 2006 Aug 15; 4(119):765-70.
Increasing cyclooxygenase-2 (cox-2) gene expression in the progression of Barrett's esophagus to adenocarcinoma correlates with that of Bcl-2.
Shimizu D ,Vallböhmer D ,Kuramochi H ,Uchida K ,Schneider S ,Chandrasoma PT ,Shimada H ,DeMeester TR ,Danenberg KD ,Peters JH ,DeMeester SR ,Danenberg PV ,
Department of Biochemistry and Molecular Biology, University of Southern California/Norris Comprehensive Cancer Center, Los Angeles, CA 90033, USA.
Previous studies from our laboratory and others have suggested that increased expression of cox-2 is important in the genesis of esophageal adenocarcinoma. In vitro studies suggest that cox-2 regulates expression of the anti-apoptotic protein bcl-2, thus possibly accounting for reduced apoptosis in carcinogenesis. The aim of this study was to investigate the relationship of these 2 genes in the development of Barrett's-associated adenocarcinoma. Histologic sections from endoscopic biopsies or esophagectomy specimens were classified as non-dysplastic Barrett's (n = 30), intraepithelial neoplasia (n = 12) and adenocarcinoma (n = 48). The desired tissue was isolated by laser capture microdissection and expression levels of cox-2 and bcl-2 were measured by quantitative real-time PCR (Taqman). Gene expression levels were compared to samples of the distal esophageal squamous epithelium (n = 55) and reflux-esophagitis (n = 25), without Barrett's or cancer. Expression of both bcl-2 and cox-2 were increased in non-dysplastic Barrett's (p = 0.0077, p = 0.0037), intraepithelial neoplasia (p = 0.0053, p = 0.0220) and adenocarcinoma (p < 0.0001, p < 0.0001) compared to squamous epithelium or reflux-esophagitis. Furthermore, there is a significant correlation between these two genes, especially in carcinoma (p < 0.0001).
Publication Type: Research Support, N.I.H., Extramural;
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125.
Free Radic Biol Med 2006 Apr 1; 7(40):1161-9.
Bcl-2 protects against oxidative stress while inducing premature senescence.
López-Diazguerrero NE ,López-Araiza H ,Conde-Perezprina JC ,Bucio L ,Cárdenas-Aguayo MC ,Ventura JL ,Covarrubias L ,Gutiérrez-Ruíz MC ,Zentella A ,Königsberg M ,
Departamento Ciencias de la Salud, División de Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana-Iztapalapa. A.P. 55-535, C.P 09340, México D.F., México.
Replicative senescence is a cellular response to stress that has been postulated to serve as a tumor suppression mechanism and a contributor to aging. Numerous experimental studies have demonstrated that an apparently identical senescent state can also be prematurely induced in vitro in different cell types following sublethal oxidative stress stimuli. The former suggests a molecular link between cell cycle regulation and cell survival that could involve regulatory proteins such as Bcl-2. There is strong evidence that, in addition to its well-known effects on apoptosis, Bcl-2 is involved in antioxidant protection and regulation of cell cycle progression. The aim of this work was to determine if the protection against oxidative stress mediated by Bcl-2 could prevent or delay oxidative stress-induced senescence. Using a retroviral infection system, Bcl-2 was overexpressed in primary, nonembryonic mice fibroblasts obtained from lungs derived from 2-month-old CD1 mice. Fibroblasts overexpressing Bcl-2 were exposed to 75 microM H2O2 for 2 h to induce SIPS. The rate of proliferation and the increment of senescent cells were then determined. Our results indicate that overexpression of Bcl-2 protected primary fibroblasts against oxidative stress-mediated reduction in cell proliferation, but did not prevent premature senescence.
Publication Type: Research Support, Non-U.S. Gov't;
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126.
Breast Cancer Res Treat 2006 Sep ; 1(99):77-83.
Bcl-2 expression correlates with lymphovascular invasion and long-term prognosis in breast cancer.
Neri A ,Marrelli D ,Roviello F ,DeMarco G ,Mariani F ,DeStefano A ,Megha T ,Caruso S ,Corso G ,Cioppa T ,Pinto E ,
Department of General Surgery and Surgical Oncology, Surgical Oncology Unit, University of Siena, Siena, Italy. neria@unisi.it
Alterations in the mechanisms of apoptosis are responsible not only for the progression of breast cancer, but for different responses to treatment as well. Among the genes regulators of apoptosis, the tumor suppressor gene p53 and the bcl-2 gene have raised interest for their possible role as predictors of response to therapy and markers of prognosis. The purpose of our study was to prospectively analyze the prognostic value of the expression of p53 and bcl-2 genes in a series of 235 consecutive patients operated on for breast cancer at the Department of General Surgery and Surgical Oncology of the University of Siena, Italy.p53 and bcl-2 expression were evaluated by immunohistochemistry, their association with conventional clinicopathological factors was analyzed by univariate analysis and their prognostic impact was evaluated by multivariate analysis.p53 and bcl-2 were detected respectively in 15.7 and 75.7% of cases, and resulted significantly related to presence of estrogen receptors for p53 over-expression and presence of peritumor lymphovascular invasion (LVI) for bcl-2 expression.With a median follow-up of 79 months, an independent negative prognostic impact on disease free and overall survival was observed for presence of LVI, absence of bcl-2 expression and number of involved axillary lymphnodes. The expression of bcl-2 improved the prognosis of LVI positive tumors up to values similar to LVI negative cases, while its absence associated to presence of LVI resulted in a poor outcome with only 28% of patients alive at 8 years.These data may indicate that expression of bcl-2 is a marker of breast cancers with reduced capability of distant colonization, even in presence of LVI, and may be particularly useful in the clinical setting, allowing to identify a subset of patients with an high risk of relapse.
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127.
Apoptosis 2006 Mar ; 3(11):413-25.
p38 Mitogen-activated protein kinases is required for counteraction of 2-methoxyestradiol to estradiol-stimulated cell proliferation and induction of apoptosis in ovarian carcinoma cells via phosphorylation Bcl-2.
Bu SZ ,Huang Q ,Jiang YM ,Min HB ,Hou Y ,Guo ZY ,Wei JF ,Wang JW ,Ni X ,Zheng SS ,
Department of Physiology, Basic Medicine College Second Military Medical University, Shanghai, PR China.
INTRODUCTION: 2-Methoxyestradiol (2ME2), a natural endogenous product of estradiol (E2) metabolism, has been shown to be a selective apoptotic agent for cancer cells but not for normal cells. In this study, we determined that 2ME2 counteracts E2-stimulated cell growth and induces apoptosis in ovarian carcinoma cells. In addition, we demonstrate that 2ME2 induces apoptosis via p38 and phospho-Bcl2 pathway. METHODS: 2ME2 and/or E2 were administered to the OVCAR-3 (human ovarian cancer) cell line. Cell growth inhibition was analyzed by [3H] Thymidine incorporation assay and DNA fluorometric assay. Cell apoptosis was tested by DNA fragmentation analysis and FACS. The signaling pathway was determined by a series of biochemical assays. RESULTS: 2ME2 inhibited estradiol-stimulated cell growth and induced apoptosis in an ovarian carcinoma cell line. MAPK and p38, but not JNK, were found to be critical mediators in this process. Expression of a dominant negative mutant of p38 kinase or p38 specific inhibitor, SB 203580, almost completely blocked the process. Furthermore, Bcl-2 phosphorylation was required for 2ME2-induced effects. CONCLUSION: Our data suggest that 2ME2 inhibits E2-stimulated proliferation and induces apoptosis in ovarian carcinoma cells. Furthermore, activation of p38 and phosphorylation of Bcl-2 plays a critical role in the mechanism. 2ME2 therefore, may have a clinical application for the treatment of ovarian cancer.
Publication Type: Research Support, Non-U.S. Gov't;
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128.
J Biol Chem 2006 Jun 16; 24(281):16207-19.
Apoptosis induction by activator protein 2alpha involves transcriptional repression of Bcl-2.
Wajapeyee N ,Britto R ,Ravishankar HM ,Somasundaram K ,
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India.
Activator protein 2alpha (AP-2alpha) induces cytotoxicity by inducing cell cycle arrest and apoptosis. In this study we investigated the mechanism of apoptosis induction by AP-2alpha. We found that AP-2alpha induced apoptosis efficiently in cells treated with benzyloxycar-bonyl-IETD-fluoromethyl ketone or FADD-silenced cells but failed to do so in benzyloxycarbonyl-LEHD-fluoromethyl ketone-treated or apoptosis protease activation factor-1 (Apaf1)-silenced cells, suggesting the central role of mitochondria in AP-2alpha-induced apoptosis. In good correlation, cells overexpressing AP-2alpha showed a reduction in mitochondrial membrane potential (Deltapsi(m)), cytochrome c and Smac/DIABLO release into cytosol, and Bax translocation into mitochondria. We found that the pro-apoptotic protein Bax is important for AP-2alpha-induced apoptosis as adenovirus AP2 failed to induce apoptosis in HCT116 Bax(-/-) cells. However, we found the IAP (inhibitor of apoptosis) inhibitor Smac/DIABLO may have a limited role in AP-2alpha-induced apoptosis as we found the IAP member Survivin down-regulated by AP-2alpha. Although the total Bax level remains unaltered, we found a time-dependent increase in the activated form of Bax in adenovirus AP2-infected cells. In addition, we show that AP-2alpha transcriptionally represses Bcl-2 by binding to its promoter both in vitro and in vivo and that this is essential for AP-2alpha-induced apoptosis as ectopic expression of Bcl-2 efficiently inhibited apoptosis induced by AP-2alpha. Furthermore, we show that chemotherapy-induced endogenous AP-2alpha down-regulates Bcl-2 and induces apoptosis in an AP-2alpha-dependent manner. Moreover, we demonstrate that inhibition of okadaic acid or staurosporine-sensitive pathways in AP-2alpha overexpressing breast cancer cells resulted in AP-2alpha-dependent apoptosis induction. These results suggest that AP-2alpha induces apoptosis by down-regulating Bcl-2 and utilizing a bax/cytochrome c/Apaf1/caspase 9-dependent mitochondrial pathway.
Publication Type: Research Support, Non-U.S. Gov't;
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129.
Bosn J Basic Med Sci 2006 Feb ; 1(6):39-45.
Expression of p53, bcl-2, and Ki-67 proteins in the inflammatory regenerative and dysplastic epithelial lesions of flat colonic mucosa.
Radović S ,Vukobrat-Bijedić Z ,Selak I ,Babić M ,
Institute of Pathology, Faculty of Medicine, University of Sarajevo, Cekalusa 90, 71000 Sarajevo, Bosnia and Herzegovina.
The aim of the study was to define the distribution of p53, bcl-2 and Ki-67 proteins in the inflammatory-regenerative and dysplastic lesions of the colon mucosa. The relationship between the presentation of p53, bcl-2 and Ki-67 proteins and the intensity of the inflammatory-regenerative and dysplastic lesions in the colon flat mucosa was investigated as well. Biopsy specimens from 270 patients were examined: 74 were classified as inflammatory-regenerative and 196 as dysplastic lesions (108 mild, 58 moderate, and 30 severe dysplasia). The expression of all three proteins was assessed on the basis of location, quantity, and intensity of immunostaining, by counting antigen positive cells, in comparison with normal mucosa and adenocarcinoma. p53 protein appears only in sporadic cases (6.6%) of severe dysplasia. Bcl-2 expression appears significantly (p<0.005) more often in cases of mild dysplasia (61.1%) compared to inflammatory-regenerative mucosa (14.8%). In cases of mild dysplasia, bcl-2 positive cells were spreading from the lower third to the middle third of the crypts. Bcl-2 expression was maintained through the stadiums of moderate and severe dysplasia (75.8%), where antigen positive cells were found all along the crypts. A significant increase (p<0.005) in the expression of nuclear protein Ki-67 was noticed in the stadiums of moderate (labelling index =26.3) compared to mild dysplasia (labelling index=16.7), and severe (labelling index=36.7) compared to moderate dysplasia, where the zone of cellular proliferation was widen along the whole crypt length. In the process of the development of epithelial dysplasia in the flat mucosa of colon a degree of the gene p53 alteration is low and appears only in sporadic cases of severe dysplasia. Mutation of the bcl-2 gene is involved in the genesis of the lesion but not in its progression to carcinoma. Increased expression of Ki-67 protein speaks in favour of an increased cellular proliferation which, together with the above mentioned mechanisms, is involved in the process of occurrence and progression of epithelial dysplasia in the flat mucosa of colon.
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130.
Apoptosis 2006 Mar ; 3(11):389-99.
Increasing ornithine decarboxylase activity is another way of prolactin preventing methotrexate-induced apoptosis: crosstalk between ODC and BCL-2.
Hsu PC ,Hour TC ,Liao YF ,Hung YC ,Liu CC ,Chang WH ,Kao MC ,Tsay GJ ,Hung HC ,Liu GY ,
Department of Medicine, Da-Chien General Hospital, Miao-Li, Taiwan, ROC.
Prolactin has more than 300 separate functions including affecting mammary growth, differentiation, secretion and anti-apoptosis. In the previous studies, prolactin induced Bcl-2 expression to prevent apoptosis and also provoked the activity of ornithine decarboxylase (ODC). Our previous data showed that ODC overexpression upregulates Bcl-2 and prevents tumor necrosis factor alpha (TNF-alpha)- and methotrexate (MTX)-induced apoptosis. Here, we further investigate whether prolactin prevents MTX-induced apoptosis through inducing ODC activity and the relationship between ODC and Bcl-2 upon prolactin stimulation. Prolactin prevented MTX-induced apoptosis in a dose-dependent manner in HL-60 cells. Following prolactin stimulation, ODC enzyme activity also shows an increase in a dose-dependent manner, expressing its maximum level at 3 h, and rapidly declining thereafter. Prolactin-induced ODC activity is completely blocked by a protein kinase C delta (PKCdelta) inhibitor, rottlerin. However, there are no changes in the expressions of ODC mRNA and protein level after prolactin stimulus. It indicates that prolactin may induce ODC activity through the PCKdelta pathway. Besides, Bcl-2 expresses within 1 h of prolactin treatment and this initiating effect of prolactin is not inhibited by alpha-difluoromethylornithine (DFMO). However, Bcl-2 is further enhanced following prolactin stimulation for 4 h and this enhancement is blocked by DFMO. Bcl-2 has no effect on ODC activity and protein levels, but ODC upregulates Bcl-2, which is inhibited by DFMO. Overall, there are two different forms of prolactin effect, it induces Bcl-2 primarily, and following this it stimulates ODC activity. Consequently induced ODC activity further enhances the expression of Bcl-2. The anti-apoptotic effect of prolactin is diminished by DFMO and recovered by putrescine. Obviously, ODC activity is one basis for the anti-apoptotic mechanisms of prolactin. A Bcl-2 inhibitor, HA14-1, together with DFMO, completely blocks the anti-apoptotic effects of prolactin. These results suggest that increasing ODC activity is another way of prolactin preventing MTX-induced apoptosis and that this induction of ODC activity enhances the expression of Bcl-2 strongly enough to bring about the anti-apoptotic function.
Publication Type: Research Support, Non-U.S. Gov't;
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131.
J Biol Chem 2006 Apr 14; 15(281):10066-72.
The mitochondrial effects of small organic ligands of BCL-2: sensitization of BCL-2-overexpressing cells to apoptosis by a pyrimidine-2,4,6-trione derivative.
Milanesi E ,Costantini P ,Gambalunga A ,Colonna R ,Petronilli V ,Cabrelle A ,Semenzato G ,Cesura AM ,Pinard E ,Bernardi P ,
Department of Biomedical Sciences and Consiglio Nazionale delle Ricerche Institute of Neurosciences, University of Padova, Viale Giuseppe Colombo 3, I-35121 Padua, Italy.
We have investigated the mitochondrial effects of BH3I-2', Chelerythrine, and HA14-1, small organic molecules that share the ability to bind the BH3 domain of BCL-2. All compounds displayed a biphasic effect on mitochondrial respiration with uncoupling at low concentrations and respiratory inhibition at higher concentrations, the relative uncoupling potency being BH3I-2' (half-maximal uncoupling at about 80 nm) > Chelerythrine (half-maximal uncoupling at about 2 microm) > HA14-1 (half-maximal uncoupling at about 20 microm). At concentrations lower than required for uncoupling all compounds sensitized the permeability transition pore (PTP) to opening both in isolated mitochondria and intact cells. To assess whether the effects on BCL-2 binding, PTP induction and respiration could be due to different structural determinants we have tested a set of HA14-1 analogs from the Hoffmann-La Roche chemical library. We have identified 5-(6-chloro-2,4-dioxo-1,3,4,10-tetrahydro-2H-9-oxa-1,3-diaza-anthracen-10-yl)-pyrimidine-2,4,6-trione (EM20-25) as a molecule devoid of effects on respiration that is able to induce PTP opening, to disrupt the BCL-2/BAX interactions in situ and to activate caspase-9 in BCL-2-overexpressing cells. EM20-25 neutralized the antiapoptotic activity of overexpressed BCL-2 toward staurosporine and sensitized BCL-2-expressing cells from leukemic patients to the killing effects of staurosporine, chlorambucil, and fludarabine. These results provide a proof of principle that the potentially toxic effects of BCL-2 ligands on mitochondrial respiration are not essential for their antiapoptotic activity and represent an important step forward in the development of tumor-selective drugs acting on BCL-2.
Publication Type: Research Support, Non-U.S. Gov't;
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132.
J Korean Med Sci 2006 Feb ; 1(21):35-9.
Prognostic value of immunohistochemical staining of p53, bcl-2, and Ki-67 in small cell lung cancer.
Paik KH ,Park YH ,Ryoo BY ,Yang SH ,Lee JC ,Kim CH ,Ki SS ,Kim JM ,Park MJ ,Ahn HJ ,Choi W ,Chung JH ,
Department of Internal Medicine, Korea Institute of Radiological and Medical Sciences, Seoul, Korea.
Small cell lung cancer (SCLC) is one of the most fatal cancers in humans and many factors are known to be related to its poor prognosis. Immunohistochemical (IHC) stainings were done on SCLC specimens in order to investigate the prognostic value of the apoptosis-related gene expression and the tumor proliferative maker, and the relationships among these IHC results and patients clinical characteristics, chemoresponsiveness, and survival were analyzed. The medical records of 107 patients were reviewed retrospectively. IHC stainings for p53, bcl-2 and Ki-67 expressions were performed in the 66 paraffin-embedded biopsy samples. Sixty-six out of the 107 patients were evaluable for response rate and survival. The overall response rate was 75% (95% Confidence Interval=74-76%) and the median survival time was 14 months. The median survival time of limited stage was 16 months and that of extensive stage was 10 months. The prevalence of p53, bcl-2 and Ki-67 expression was 62%, 70%, and 49%, respectively. There were no correlations among the immunoreactivities of p53, bcl-2 and Ki-67 with clinical stage, chemoresponsiveness or overall survival. The clinical stage was the only prognostic factor influencing survival. The expression rates of p53, bcl-2, and Ki-67 were relatively high in SCLC without any prognostic significance. The exact clinical role of these markers should be defined through further investigations.
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133.
Biochem Biophys Res Commun 2006 Mar 31; 1(342):206-13.
Alzheimer's amyloid beta-peptide (1-42) induces cell death in human neuroblastoma via bax/bcl-2 ratio increase: an intriguing role for methionine 35.
Clementi ME ,Pezzotti M ,Orsini F ,Sampaolese B ,Mezzogori D ,Grassi C ,Giardina B ,Misiti F ,
CNR-ICRM, Institute of Chimica del Riconoscimento Molecolare, c/o Institute of Biochemistry and Clinical Biochemistry, Catholic University School of Medicine, Largo F. Vito 1, 00168 Rome, Italy.
The beta amyloid (Abeta), the major protein component of brain senile plaques in Alzheimer's disease, is known to be directly responsible for the production of free radicals toxic to brain tissue and the redox state of Met-35 residue seems to play a particular and critical role in peptide's neurotoxic actions. In this study, we investigated, in human neuroblastoma cells (IMR-32), the relationship between the oxidative state of methionine, and both neurotoxic and pro-apoptotic actions induced by Abeta-peptide, comparing the effects of native peptide, in which the Met-35 is present in the reduced state, with those of a modified peptide with oxidized Met-35 (Abeta(1-42)(35Met-ox)), as well as an Abeta-derivative with Met-35 substituted with norleucine (Abeta(1-42)(35Nle)). The obtained results show that Abeta induces a time-dependent decrease in cell viability; Abeta(1-42)(35Met-ox) was significantly less potent, though inducing a remarkable decrease in cell viability compared to control. On the contrary, no toxic effects were observed after treatment with Abeta(1-42)(35Nle). Abeta-peptide as well as the amyloid modified peptide with oxidized Met-35 induced the pro-apoptotic gene bax over-expression after 24 h, whereas Abeta(1-42)(35Nle) had no effect. Conversely, bcl-2, an anti-apoptotic gene, became highly down-regulated by Abeta peptide treatment, in contrast to that evidenced by the Abeta(1-42)(35Met-ox) peptide. Finally, Abeta caused an increase in caspase-3 activity to be higher with respect to that shown by Abeta(1-42)(35Met-ox) while Abeta(1-42)(35Nle) had no effect. These results support the hypothesis that Abeta-induced neurotoxicity occurs via bax over-expression, bcl-2 down-regulation, and caspase-3 activation, first indicating that methionine 35 redox state may alter this cell death pathway.
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134.
FASEB J 2006 Apr ; 6(20):800-2.
Transcription factor gata4 regulates cardiac BCL2 gene expression in vitro and in vivo.
Kobayashi S ,Lackey T ,Huang Y ,Bisping E ,Pu WT ,Boxer LM ,Liang Q ,
Cardiovascular Research Institute, University of South Dakota School of Medicine, South Dakota Health Research Foundation, Sioux Falls, South Dakota 57105, USA.
The transcription factor GATA-4 protects cardiomyocytes against doxorubicin-induced cardiotoxicity. Here, we report the identification of Bcl2 as a direct target gene of GATA4 that may mediate the prosurvival function of GATA4 in cardiomyocytes. Bcl2 transcript and protein levels were reduced by doxorubicin in neonatal rat ventricular cardiomyocytes (NRVC) and in mouse heart as determined by RT-PCR and Western blot analysis. The reduction in Bcl2 was prevented by overexpression of GATA4 in NRVC and in transgenic mouse heart. Also, expression of GATA4 increased baseline Bcl2 levels by 30% in NRVC and 2.7-fold in transgenic heart, indicating the sufficiency of GATA4 to up-regulate Bcl2 gene expression. GATA4 knockdown by siRNA reduced Bcl2 levels by 48% in NRVC, suggesting that GATA4 is required for Bcl2 constitutive gene expression. Transfection of HEK cells with GATA4 plasmids activated Bcl2 promoter and elevated Bcl2 protein levels. Deletion and mutagenesis analysis revealed that a consensus GATA motif at base -266 on the promoter conserved across multiple species is partially responsible for the promoter activity. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrate that GATA4 directly bound to this GATA site. Together, these results indicate that GATA4 positively regulates cardiac Bcl2 gene expression in vitro and in vivo.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't;
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135.
J Biol Chem 2006 Mar 31; 13(281):8600-6.
WT p53, but not tumor-derived mutants, bind to Bcl2 via the DNA binding domain and induce mitochondrial permeabilization.
Tomita Y ,Marchenko N ,Erster S ,Nemajerova A ,Dehner A ,Klein C ,Pan H ,Kessler H ,Pancoska P ,Moll UM ,
Department of Pathology, Stony Brook University, Stony Brook, New York 11794-8691, USA.
The induction of apoptosis by p53 in response to cellular stress is its most conserved function and crucial for p53 tumor suppression. We recently reported that p53 directly induces oligomerization of the BH1,2,3 effector protein Bak, leading to outer mitochondrial membrane permeabilization (OMMP) with release of apoptotic activator proteins. One important mechanism by which p53 achieves OMMP is by forming an inhibitory complex with the anti-apoptotic BclXL protein. In contrast, the p53 complex with the Bcl2 homolog has not been interrogated. Here we have undertaken a detailed characterization of the p53-Bcl2 interaction using structural, biophysical, and mutational analyses. We have identified the p53 DNA binding domain as the binding interface for Bcl2 using solution NMR. The affinity of the p53-Bcl2 complex was determined by surface plasmon resonance analysis (BIAcore) to have a dominant component KD 535 +/- 24 nm. Moreover, in contrast to wild type p53, endogenous missense mutants of p53 are unable to form complexes with endogenous Bcl2 in human cancer cells. Functionally, these mutants are all completely or strongly compromised in mediating OMMP, as measured by cytochrome c release from isolated mitochondria. These data implicate p53-Bcl2 complexes in contributing to the direct mitochondrial p53 pathway of apoptosis and further support the notion that the DNA binding domain of p53 is a dual function domain, mediating both its transactivation function and its direct mitochondrial apoptotic function.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't;
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136.
Acta Chir Belg ; 6(105):644-8.
Expression of bcl-2 in papillary thyroid cancers and its prognostic value.
Aksoy M ,Giles Y ,Kapran Y ,Terzioglu T ,Tezelman S ,
Department of General Surgery, Medical Faculty of Istanbul University. maksoy@turk.net
PURPOSE: Papillary thyroid cancer has a good prognosis. This favourable prognosis may be attributed to the apoptotic tendency of the cancer cells. This study aims to evaluate the expression of bcl-2, which is an antidote of apoptosis, and aims to evaluate the value of bcl-2 as a prognostic marker in papillary thyroid cancer. MATERIAL-METHODS: Bcl-2 expression in the archival materials of 31 patients with papillary thyroid cancer was examined with immunohistochemical methods using bcl-2 and p-53 stains. The results were compared with 31 normal thyroid tissue specimens, which consisted of the contralateral lobes of these patients. The results were then analyzed in accordance with the clinical features of the patients. RESULTS: Thirty (96.7%) patients of the control group were positive for bcl-2 whilst one (3.3%) was negative. The staining for bcl-2 was positive in 12 (%75) patients with microcarcinomas (p < 0.05) and 13 (86.6%) with papillary cancers (p > 0.05). Two cases of the papillary cancer group were admitted to the hospital with local recurrence (6.4%) and both were positive for bcl-2 (p > 0.05). All cases (4/31), whose MACIS scores were higher than 7 were positive for bcl-2. Twenty-one of 27 cases whose MACIS scores were lower than 7 (77.7%) were positive for bcl-2 (p > 0.05). All tumours of this series were negative for p-53 immunstaining. CONCLUSION: The rate of bcl-2 expression in microcarcinomas of papillary thyroid cancer decreases when compared to normal thyroid tissue. This may be an early sign of oncogenesis, and a reason for the favourable prognosis in microcarcinomas. However, bcl-2 cannot be used as a prognostic marker.
Publication Type: Research Support, Non-U.S. Gov't;
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137.
J Zhejiang Univ Sci B 2005 Dec ; 12(6):1163-9.
Prognostic significance of bcl-2 and p53 expression in colorectal carcinoma.
Zhao DP ,Ding XW ,Peng JP ,Zheng YX ,Zhang SZ ,
Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China.
OBJECTIVE: This study was designed to detect the expression of bcl-2 and p53 proteins in colorectal carcinomas and to determine their association with the patient survival and stage of the diseases. METHODS: Immunohistochemistry method was used to detect the expression of bcl-2 and p53 proteins in 93 cases of colorectal carcinoma. The stain results were obtained by analyzing the clinic-pathological characteristics of patients. RESULTS: Fifty-seven percent (53/93) of the colorectal carcinomas were bcl-2 protein positive. The positive rate of bcl-2 protein in lymph node involvement cases was lower (15/37) than the cases without node involvement (38/58, P<0.01). The positive rate of p53 protein was 43% (40/93) in colon-rectum carcinomas. No significant correlation was observed between p53 protein expression and clinic-pathological manifestations (P>0.05) but the survival was significantly worse (P=0.0001) in the p53 protein positive cases. Neither bcl-2 nor p53 alone was correlated with stage of the disease. When combined bcl-2/p53 status was analyzed, a group with bcl-2(+) and p53(-) had the best prognosis. This group was significantly associated with earlier Dukes' stages (P=0.1763). In multivariate Cox regression analysis, lymph node involvement and p53 protein expression were two independent factors correlated with survival time. CONCLUSION: The expression of bcl-2 and p53 represent biological characteristics of colorectal carcinomas. Assessment of both bcl-2 and p53 status may be valuable in predicting the prognosis of patients.
Publication Type: Clinical Trial;
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138.
Cell Oncol 2005 ; 4(27):245-53.
p53, Bcl-2 and C-Myc expressions in colorectal carcinoma associated with schistosomiasis in Egypt.
Zalata KR ,Nasif WA ,Ming SC ,Lotfy M ,Nada NA ,El-Hak NG ,Leech SH ,
Department of Pathology, Faculty of Medicine, Mansoura University, Mansoura, Egypt. kzalata@yahoo.com
BACKGROUND AND AIMS: Oncogenes and tumor suppressor genes expression are well described in bladder cancer associated with schistosomiasis especially in Egypt. Scarce studies were directed to colorectal cancer (CRC) associated with Schistosoma mansoni (S. mansoni). Apoptosis (programmed cell death) and the genes regulating this process (e.g., Bcl-2) have recently become a focus of interest in the study of cancer development and progression. In the present study, we aimed to investigate the expression pattern of p53, Bcl-2 and C-Myc in CRC tissues obtained from Egyptian colorectal cancer patients divided in two different groups, one associated with Schistosoma mansoni (CRC-Sm) and the other without Schistosoma mansoni (CRC-NSm). METHODS: Seventy-five CRC tumors containing 36 draining lymph node metastatic tumors were immunohistochemically stained using specific monoclonal antibodies for p53, Bcl-2 and C-Myc, in addition the apoptotic activity of these tumors were analyzed. RESULTS AND CONCLUSIONS: Regardless of the S. mansoni infection, the obtained results showed that the apoptotic activity was more evident in p53 diffuse positive tumors (P = 0.021). There was a significant correlation between p53 diffuse positive staining and Bcl-2 positive immunostaining (P = 0.011). Signet ring cell carcinoma and mucinous adenocarcinoma exhibited both intense C-Myc expression than non-mucinous carcinoma (P = 0.001). When adjusting for S. mansoni infection, 58.3% of CRC-Sm cases were Bcl-2 positive compared to only (33.3%) of CRC-NSm (P = 0.046). Apoptotic activity was more evident in the latter group than of CRC-Sm tumors (P = 0.009). p53 and C-Myc expressions were found insignificantly different in CRC-Sm compared with CRC-NSm (P > 0.05). These observations suggest that the genotoxic agents produced endogenously through the course of schistosomiasis mansoni may play a role in CRC-Sm pathogenesis through the dysregulation of apoptosis by alteration the expression pattern of Bcl-2 protein differently from CRC-NSm suggesting a different biological behavior.
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139.
Food Chem Toxicol 2006 Apr ; 4(44):567-73.
T-2 toxin induces apoptosis, and selenium partly blocks, T-2 toxin induced apoptosis in chondrocytes through modulation of the Bax/Bcl-2 ratio.
Chen J ,Chu Y ,Cao J ,Yang Z ,Guo X ,Wang Z ,
Institute of Endemic Diseases, Medical School of Xi'an Jiaotong University, Key Laboratory of Environment and Genes related to Diseases, Ministry of Education, Xi'an 710061 Shaanxi, China.
T-2 toxin is one of the mycotoxins, a group of type A trichothecenes produced by several fungal genera including Fusarium species. In the present study, we have investigated the apoptotic effects of T-2 toxin on chondrocytes and the relationship between T-2 toxin induced chondrocyte apoptosis and its influence on Bcl-2/Bax protein and mRNA expression. We have also examined the inhibitory effects of selenium on chondrocyte apoptosis induced by T-2 toxin. We have combined morphological and biological techniques to establish the relevance of apoptosis in human chondrocyte death induced by T-2 toxin. Treatment with T-2 toxin caused accelerated apoptosis in a concentration dependent manner. The apoptosis induced by T-2 toxin involved an increased Bax/Bcl-2 ratio. Bcl-2 mRNA expression remained unchanged in chondrocyte apoptosis induced by T-2 toxin treatment, while Bax mRNA expression increased following treatment with T-2 toxin. Selenium could partly block the apoptosis of chondrocytes induced by T-2 toxin through decreasing the Bax/Bcl-2 ratio. These results suggest that, under our experimental conditions, apoptosis of chondrocytes can be induced by T-2 toxin (1-20ng/mL) via the Bcl-2 and Bax proteins, and the Bax/Bcl-2 ratio may play a critical role in governing the susceptibility to apoptosis induced by T-2 toxin in human chondrocytes.
Publication Type: Research Support, Non-U.S. Gov't;
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140.
Oncol Rep 2005 Dec ; 6(14):1527-31.
Cell-cycle-associated markers and clinical outcome in human epithelial cancers: a tissue microarray study.
Abdulkader I ,Sánchez L ,Cameselle-Teijeiro J ,Gude F ,Chávez JE ,López-López R ,Forteza J ,Fraga M ,
Department of Pathology, School of Medicine, Clinical University Hospital, 15706 Santiago de Compostela, Spain. ihab.abdulkader.nallib@sergas.es
The development and progression of epithelial cancers are the result of an imbalance in signals promoting and inhibiting cellular proliferation and apoptosis. The aim of this study is to evaluate the expression of cell-cycle and apoptosis regulators and correlate them with clinical outcome in the most frequent carcinomas, in order to establish common prognostic biomarkers independent of cancer origin. Using tissue microarrays (TMAs), we have analysed the immuno-expression of Ki-67, Bcl-2, Bax, cyclin D1, cyclin D3, CDK1, CDK2, CDK6, p16, p21, and p27 in a series of 205 carcinomas of the large bowel, breast, lung and prostate (80, 73, 37 and 15 cases, respectively). By univariate analysis, positivity for p27, p16 and Bcl-2 was associated with better overall survival (P<0.0135, P<0.0442 and P<0.0001, respectively). The risk of mortality was 2.3-fold greater in patients without Bcl-2 expression. TMA immunohistochemical analysis identified a subset of epithelial cancers with overlapping alterations in cell-cycle checkpoints, apoptosis regulators and tumour suppressor pathways. We found that in most common epithelial cancers, regardless of origin, Bcl-2 appears to be the key biological factor influencing clinical behaviour.
Publication Type: Comparative Study; Research Support, Non-U.S. Gov't;
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141.
J Gastroenterol Hepatol 2005 Nov ; 11(20):1674-8.
Bax and Bcl-2 protein expression in gastric precancerous lesions: immunohistochemical study.
Anagnostopoulos GK ,Stefanou D ,Arkoumani E ,Sakorafas G ,Pavlakis G ,Arvanitidis D ,Tsianos E ,Agnantis NJ ,
Gastroenterology Clinic, 251 Hellenic Air Force and Veterans General Hospital, Athens, Greece. gkanagnostopoulos@yahoo.gr
BACKGROUND AND AIMS: Bcl-2 protein prolongs cell survival in the face of classical apoptotic stimuli, and is considered to be a suppressor of apoptosis. Bax plays a key role in apoptosis by accelerating cell death after an apoptotic stimulus. The aim of our study was to determine the roles of the Bax proapoptotic gene and the Bcl-2 antiapoptotic gene in the carcinogenesis of gastric cancer. METHODS: One hundred and forty-five gastric biopsy specimens of chronic gastritis, atrophic gastritis, intestinal metaplasia and gastric dysplasia were studied. Using immunohistochemical methods, Bax and Bcl-2 protein expression was observed. RESULTS: Bax was expressed in epithelial cells in all cases of chronic gastritis. Bax was not detected in 26% of specimens of atrophic gastritis. As intestinal metaplasia develops, Bax is further suppressed. In biopsy samples with dysplasia, Bax expression was demonstrated only in 12% of biopsy samples. Although Bcl-2 protein was not detected in chronic gastritis, aberrant expression was found in gastric epithelial intestinal metaplasia and dysplasia. CONCLUSIONS: The suppression of Bax and overexpression of Bcl-2 protein is an early event in gastric tumorigenesis, before gastric dysplastic changes occur.
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142.
Clin Cancer Res 2005 Oct 15; 20(11):7255-63.
Expression of phosphorylated Ser70 of Bcl-2 correlates with malignancy in human colorectal neoplasms.
Kondo E ,Miyake T ,Shibata M ,Kimura T ,Iwagaki H ,Nakamura S ,Tanaka T ,Ohara N ,Ichimura K ,Oka T ,Yanai H ,Shibasaki F ,Yoshino T ,
Department of Pathology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan. ekondo@md.okayama-u.ac.jp
PURPOSE: Bcl-2 is a model apoptosis suppressor postulated to promote tumorigenesis. Recently, it has been reported that Bcl-2 undergoes phosphoregulation of its Ser70 to substantially alter its molecular function. Previous studies further suggest that such phospho-Bcl-2 regulation may influence tumor progression in colorectal and other cancers; however, phosphorylation status of the Ser70 of Bcl-2 (pSer70) in vivo in tumors remains obscure. To elucidate this question that may suggest the biological role, we molecularly screened a panel of human colorectal adenomas and adenocarcinomas for endogenous expression of pSer70 Bcl-2. EXPERIMENTAL DESIGN: An antibody specific against pSer70 Bcl-2 was generated for thorough immunohistochemical examination of paraffin-embedded tumor specimens, allowing detection of the endogenously expressed antigen among a range of Bcl-2-positive colorectal neoplasms, including 75 tubular adenomas, 114 adenocarcinomas, and 15 cases of cancer in adenomas. RESULTS: Loss of pSer70 Bcl-2 expression was observed in adenocarcinomas in a differentiation-dependent manner (positivities: well differentiated 63%, moderately differentiated 52%, and poorly differentiated 12%), whereas tubular adenomas maintained their expression (positivity 88%). Interestingly, an inverse correlation was found between expression of pSer70 Bcl-2 and Ki-67 antigen in those cases of cancer in adenoma (P < 0.01). It was further observed that loss of pSer70 Bcl-2 expression was associated with significantly shorter survival (P < 0.05) and correlated with clinical stages and lymph node metastasis (P < 0.05 and P < 0.05, respectively). CONCLUSIONS: Loss of pSer70 Bcl-2 expression is closely linked to biological aggressiveness in colorectal tumors and represents a statistically significant molecular index for prognosis of patients with these tumors.
Publication Type: Comparative Study;
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143.
Apoptosis 2005 Dec ; 6(10):1419-31.
p73 modulates HIV-1 Tat transcriptional and apoptotic activities in human astrocytes.
Saunders M ,Eldeen MB ,Del Valle L ,Reiss K ,Peruzzi F ,Mameli G ,Gelman BB ,Khalili K ,Amini S ,Sawaya BE ,
Department of Neuroscience & Center for Neurovirology, Temple University, 1900 North 12th Street, 015-96, Philadelphia, PA 19122, USA.
HIV-1 Tat is a potent transcriptional activator of the viral promoter with the ability to modulate a number of cellular regulatory circuits including apoptosis. Tat exerts its effects through interaction with viral as well as cellular proteins. Here, we studied the influence of p73, a protein that is implicated in apoptosis and cell cycle control, on Tat apoptotic function in the central nervous system. We recently demonstrated the ability of Tat to associate with p73, and that this association modulates Tat transcriptional activity (Amini et al., Mol Cell Biol 2005; 18: 8126-8138). We demonstrated that p73 interferes with Tat-mediated apoptosis by preventing the up-regulation of Bax and down-regulation of Bcl-2 proteins in astrocytes. Thus, the interplay between Tat and p73 may affect Tat contribution to apoptotic events in the brain, limiting its involvement in the neuropathology often observed in the brains of HIV-1 patients.
Publication Type: Research Support, N.I.H., Extramural;
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144.
J Biomed Sci 2005 Dec ; 6(12):999-1011.
BCL-2 antisense and cisplatin combination treatment of MCF-7 breast cancer cells with or without functional p53.
Basma H ,El-Refaey H ,Sgagias MK ,Cowan KH ,Luo X ,Cheng PW ,
Department of Biochemistry and Molecular Biology, College of Medicine, Omaha, NE 68198-5870, USA.
Chemotherapy has been used for treatment of breast cancer but with limited success. We characterized the effects of bcl-2 antisense and cisplatin combination therapy in two human isogenic breast carcinoma cells p53(+)MCF-7 and p53(-)MCF-7/E6. The transferrin-facilitated lipofection strategy we have developed yielded same transfection efficiency in both cells. Bcl-2 antisense delivered with this strategy significantly induced more cell death, apoptosis, and cytochrome c release in MCF-7/E6 than in MCF-7, but did not affect Fas level in both cells and activated caspase-8 equally. Cisplatin exerted same effects on cell viability and apoptosis in both cells, but released smaller amounts of cytochrome c while activated more caspase-8 in MCF-7/E6. The combination treatment yielded greater effects on cell viability, apoptosis, cytochrome c release, and caspase-8 activation than individual treatments in both cells although p53(-) cells were more sensitive. The potentiated activation of caspase-8 in the combination treatment suggested that caspase-8-mediated (but cytochrome c-independent) apoptotic pathway is the major contributor of the enhanced cell killing. Thus, bcl-2 antisense delivered with transferrin-facilitated lipofection can achieve the efficacy of killing breast cancer cells and sensitizing them to chemotherapy. Bcl-2 antisense and cisplatin combination treatment is a potentially useful therapeutic strategy for breast cancer irrespective of p53 status.
Publication Type: Research Support, N.I.H., Extramural;
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145.
Mol Cancer Ther 2005 Oct ; 10(4):1475-83.
Sulindac sulfide-induced apoptosis is enhanced by a small-molecule Bcl-2 inhibitor and by TRAIL in human colon cancer cells overexpressing Bcl-2.
Sinicrope FA ,Penington RC ,
Division of Gastroenterology, Mayo Clinic, 200 First Street Southwest, Rochester, MN 55905, USA. sinicrope.frank@mayo.edu
Sulindac is a nonsteroidal anti-inflammatory drug (NSAID) that induces apoptosis in cultured colon cancer cells and in intestinal epithelia in association with its chemopreventive efficacy. Resistance to sulindac is well documented in patients with familial adenomatous polyposis; however, the molecular mechanisms underlying such resistance remain unknown. We determined the effect of ectopic Bcl-2 expression upon sulindac-induced apoptotic signaling in SW480 human colon cancer cells. Sulindac sulfide activated both the caspase-8-dependent and mitochondrial apoptotic pathways. Ectopic Bcl-2 attenuated cytochrome c release and apoptosis induction compared with SW480/neo cells. Coadministration of sulindac sulfide and the small-molecule Bcl-2 inhibitor HA14-1 increased apoptosis induction and enhanced caspase-8 and caspase-9 cleavage, Bax redistribution, and cytochrome c and second mitochondria-derived activator of caspase release. Given that sulindac sulfide activated caspase-8 and increased membrane death receptor (DR4 and DR5) protein levels, we evaluated its combination with the endogenous death receptor ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Coadministration of sulindac sulfide and TRAIL cooperatively enhanced apoptotic signaling as effectively as did HA14-1. Together, these data indicate that HA14-1 or TRAIL can enhance sulindac sulfide-induced apoptosis and represent novel strategies for circumventing Bcl-2-mediated apoptosis resistance in human colon cancer cells.
Publication Type: Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, P.H.S.;
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146.
Apoptosis 2005 Dec ; 6(10):1333-43.
Bcl-2 attenuates anticancer agents-induced apoptosis by sustained activation of Akt/protein kinase B in U937 cells.
Woo KJ ,Yoo YH ,Park JW ,Kwon TK ,
Department of Immunology, School of Medicine, Keimyung University, 194 DongSan-Dong Jung-Gu, Taegu 700-712, Korea.
Aberrant overexpression of antiapoptotic members of the Bcl-2 protein family contributes to resistance to anticancer therapeutic drugs. Thus, this protein represent attractive target for novel anticancer agents. In the present study, we determined the effect of the anti-apoptosis protein Bcl-2 on caspase-3 activation, PLC-gamma1 degradation and Akt activation during the various anticancer agents-induced apoptosis. Treatment with chrysin for 12 h produced morphological features of apoptosis in U937 cells, which was associated with caspase-3 activation and PLC-gamma1 degradation. Induction of apoptosis was also accompanied by down-regulation of XIAP and inactivation of Akt. Chrysin-induced caspase-3 activation, PLC-gamma1 degradation and apoptosis were significantly attenuated in Bcl-2 overexpressing U937/Bcl-2 cells. Ectopic expression of Bcl-2 appeared to inhibit ceramide-, and Akt specific inhibitor (SH-6)-induced apoptosis by sustained Akt activation. Thus, our findings imply that some of the biological functions of Bcl-2 may be attributed to their ability to inhibit anticancer agents-induced apoptosis through the sustained Akt activation.
Publication Type: Research Support, Non-U.S. Gov't;
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147.
J Biol Chem 2005 Dec 16; 50(280):41137-45.
Catalase plays a critical role in the CSF-independent survival of human macrophages via regulation of the expression of BCL-2 family.
Komuro I ,Yasuda T ,Iwamoto A ,Akagawa KS ,
Department of Immunology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan.
M-colony-stimulating factor (M-CSF)-induced monocyte-derived macrophages (M-Mphi) required continuous presence of M-CSF for their survival, and depletion of M-CSF from the culture induced apoptosis, whereas human alveolar macrophages (A-Mphi) and granulocyte-macrophage (GM)-CSF-induced monocyte-derived macrophages (GM-Mphi) survived even in the absence of CSF. The expression of BCL-2 was higher in M-Mphi, and M-CSF withdrawal down-regulated the expression. The expression of BCL-X(L) was higher in A-Mphi and GM-Mphi, and the expression was CSF-independent. The expression of MCL-1 and BAX were not different between M-Mphi and GM-Mphi and were CSF-independent. Down-regulation of the expression of BCL-2 and BCL-X(L) by RNA interference showed the important role of BCL-2 and BCL-X(L) in the survival of M-Mphi and GM-Mphi, respectively. Human erythrocyte catalase (HEC) and conditioned medium obtained from GM-Mphi or A-Mphi cultured in the absence of GM-CSF prevented the M-Mphi from apoptosis and restored the expression of BCL-2. The activity of the conditioned medium was abrogated by pretreatment with anti-HEC antibody. Anti-HEC antibody also induced the apoptosis of M-Mphi cultured in the presence of M-CSF and GM-Mphi and A-Mphi cultured in the presence or absence of GM-CSF and down-regulated the expression of BCL-2 and BCL-X(L) in these Mphis. GM-Mphi and A-Mphi, but not M-Mphi, can produce both extracellular catalase and cell-associated catalase in a CSF-independent manner. Intracellular glutathione levels were kept equivalent in these Mphis, both in the presence or absence of CSF. These results indicate a critical role of extracellular catalase in the survival of human macrophages via regulation of the expression of BCL-2 family genes.
Publication Type: Research Support, Non-U.S. Gov't;
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148.
J Biol Chem 2005 Dec 9; 49(280):41047-56.
Pretreatment of acetylsalicylic acid promotes tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by down-regulating BCL-2 gene expression.
Kim KM ,Song JJ ,An JY ,Kwon YT ,Lee YJ ,
Department of Surgery and Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to be selective in the induction of apoptosis in cancer cells with minimal toxicity to normal tissues. However, not all cancers are sensitive to TRAIL-mediated apoptosis. Thus, TRAIL-resistant cancer cells must be sensitized first to become responsive to TRAIL. In this study, we observed that pretreatment by acetylsalicylic acid (ASA) augmented TRAIL-induced apoptotic death in human prostate adenocarcinoma LNCaP and human colorectal carcinoma CX-1 cells. Western blot analysis showed that pretreatment of ASA followed by TRAIL treatment activated caspases (8, 9, and 3) and cleaved poly(ADP-ribose) polymerase, the hallmark feature of apoptosis. Most interestingly, at least 12 h of pretreatment with ASA was prerequisite for promoting TRAIL-induced apoptosis and was related to down-regulation of BCL-2. Biochemical analysis revealed that ASA inhibited NF-kappaB activity, which is known to regulate BCL-2 gene expression, by dephosphorylating IkappaB-alpha and inhibiting IKKbeta activity but not by affecting the HER-2/neu phosphatidylinositol 3-kinase-Akt signal pathway. Overexpression of BCL-2 suppressed the promotive effect of ASA on TRAIL-induced apoptosis and changes in mitochondrial membrane potential. Taken together, our studies suggested that ASA-promoted TRAIL cytotoxicity is mediated through down-regulating BCL-2 and by decreasing mitochondrial membrane potential.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.;
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149.
FASEB J 2005 Oct ; 12(19):1617-26.
Ischemic preconditioning modulates the expression of several genes, leading to the overproduction of IL-1Ra, iNOS, and Bcl-2 in a human model of liver ischemia-reperfusion.
Barrier A ,Olaya N ,Chiappini F ,Roser F ,Scatton O ,Artus C ,Franc B ,Dudoit S ,Flahault A ,Debuire B ,Azoulay D ,Lemoine A ,
Inserm 602; Service de Biochimie et Biologie Moléculaire; Hôpital Universitaire Paul Brousse; Université Paris-Sud/XI, Villejuif Cedex; Assistance Publique-Hôpitaux de Paris, France.
Ischemia triggers an inflammatory response that precipitates cell death during reperfusion. Several studies have shown that tissues are protected by ischemic preconditioning (IP) consisting of 10 min of ischemia followed by 10 min of reperfusion just before ischemia. The molecular basis of this protective effect is poorly understood. We used cDNA arrays (20K) to compare global gene expression in liver biopsies from living human liver donors who underwent IP (n=7) or not (n=7) just before liver devascularization. Microarray data were analyzed using pairedt test with a type I error rate fixed at alpha = 2.5 10(6) (Bonferroni correction). We found that 60 genes were differentially expressed (36 over- and 24 underexpressed in preconditioning group). After IP, the most significantly overexpressed gene was IL-1Ra. This was confirmed by immunoblotting. Differentially expressed were genes involved in apoptosis (NOD2, ephrin-A1, and calpain) and in the carbohydrate metabolism. A significant increase in the amount of the anti-apoptotic protein Bcl-2 in preconditioned livers but no change in the cleavage of procaspase-3, -8, and -9 was observed. We also observed an increase in the amount in the inducible nitric oxide synthase. Therefore, the benefits of IP may be associated with the overproduction of IL-1Ra, Bcl-2, and NO countering the proinflammatory and proapoptotic effects generated during ischemia-reperfusion.
Publication Type: Research Support, Non-U.S. Gov't;
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150.
Leuk Lymphoma 2005 Oct ; 10(46):1513-6.
Translocation of BCL2 and BCL6 to the same immunoglobulin heavy chain locus in a case of follicular lymphoma.
Rack K ,Delannoy A ,Ravoet C ,Vannuffel P ,Hamels J ,Gillerot Y ,
Centre de Génétique Humaine, Institut de Pathologie et de Génétique, Allée des Templiers, Loverval 6280, Belgium. katrina.rack@ipg.be
Follicular Lymphoma is a low grade malignancy of mature B-cells. The hallmark chromosome abnormality is the translocation t(14;18) which is observed in 70 - 80% of cases with a translocation t(3;14) present in a further 10%. Rarely both of these translocations, or one of their variants, may be present. These co-incident translocations usually involve different Ig loci or different Ig alleles. We present here a case of Follicular Lymphoma with leukemic presentation and a complex translocation involving the IgH, BCL2 and BCL6 loci. Double oncogene translocations to a single immunoglobulin locus are extremely rare in lymphomas with few cases described to date. To our knowledge this is the first reported case with a complex translocation involving these loci.
Publication Type: Case Reports;
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151.
Oncogene 2006 Feb 9; 6(25):888-98.
Oct transcription factors mediate t(14;18) lymphoma cell survival by directly regulating bcl-2 expression.
Heckman CA ,Duan H ,Garcia PB ,Boxer LM ,
Center for Molecular Biology in Medicine, Palo Alto VAHCS, Palo Alto, CA, USA.
Oct-1 and Oct-2 are members of the POU homeodomain family of transcriptional regulators and are critical for normal embryonic development. Gene-targeting studies showed that Oct-1 and Oct-2 are largely dispensable for B-cell development and immunoglobulin production, although both Oct-2 and Bob-1 are required for a proper immune response and germinal center formation. In these studies, we investigated the role of Oct factors in B-cell lymphomas. Recent investigations have shown increased expression of Oct-2 and Bob-1 in lymphomas, and we observed greatly increased levels of Oct-2 in lymphoma cells with the t(14;18) translocation. Decreased expression of Oct-1, Oct-2, or Bob-1 by RNA interference resulted in apoptosis and down-regulation of bcl-2 expression. Furthermore, Oct-2 induced bcl-2 promoter activity and mediated this effect through three regions in the bcl-2 P2 promoter. Although these regions did not contain canonical octamer motifs, we observed the direct interaction of Oct-2 with all three sites both in vitro by EMSA and in vivo by chromatin immunoprecipitation assay. Moreover, by mutation analysis we found that the ability of Oct-2 to activate bcl-2 required C/EBP, Cdx, and TATA-binding sites. Oct-2, therefore, acts as a cell survival factor in t(14;18) lymphoma cells by directly activating the antiapoptotic gene bcl-2.
Publication Type: Research Support, N.I.H., Extramural;
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152.
J Mol Neurosci 2005 ; 2(27):167-74.
bcl2, bax, and nestin in the brains of patients with neurodegeneration and those of normal aging.
Lu G ,Kwong WH ,Li Q ,Wang X ,Feng Z ,Yew DT ,
Department of Anatomy, Chinese University of Hong Kong.
This study was conducted by employing specimens from the frontal cortices of Alzheimer, multiple-infarct dementia patients, and those of normal aging (age matched to patients). The objective was to evaluate and compare the bcl2, bax, and nestin patterns in these three groups. Using immunocytochemistry, it was observed that bcl2 and bax active sites were colocalized in 45% of cells in Alzheimer, 52% of cells in multiple infarct, and 30% of cells in normal aging. bcl2 and bax could also be separately located in cells of all three groups. bax cells were most prominent in number in Alzheimer patients and least prominent in normal aging. nestin was found in all three groups but was most prominent in the multiple-infarct patients. Both astrocytes and neurons demonstrated positive nestin sites. The difference in pattern between groups will lead to further understanding of cellular changes in neurodegenerative patients and those of normal aging.
Publication Type: Research Support, Non-U.S. Gov't;
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153.
J Neurochem 2005 Oct ; 1(95):230-43.
TAT-mediated endocytotic delivery of the loop deletion Bcl-2 protein protects neurons against cell death.
Soane L ,Fiskum G ,
Department of Anesthesiology, University of Maryland, School of Medicine, Baltimore, Maryland 21201, USA.
Protein delivery mediated by protein transduction domains (PTD) such as the HIV-1 TAT-PTD has emerged as a promising approach for neuroprotection. The objective of this study was to generate and evaluate the neuroprotective potential of TAT fusion proteins using constructs based on Bcl-2 anti-death family proteins. A TAT-Bcl-2 construct with the loop domain deleted (TAT-Bcl-2Deltaloop) was tested for its ability to transduce neuronal cells and to promote survival. The potential mechanism of TAT-mediated protein internalization in neural cells was also investigated. The purified TAT-Bcl-2Deltaloop binds to neural cell and rat brain mitochondria, and transduces cultured neural cell lines and primary cortical neurons when used at nm concentrations. Effective internalization of TAT-Bcl-2Deltaloop occurs at 37 degrees C but not at 4 degrees C, consistent with an endocytotic process. Both cell association and internalization require interaction of TAT-Bcl-2Deltaloop with cell surface heparan sulfate proteoglycans. TAT-mediated protein delivery in neuronal cells occurs through a lipid raft-dependent endocytotic process, inhibited by the cholesterol-sequestering agent nystatin. Transducible loop deleted Bcl-2 increases the survival of cortical neurons following trophic factor withdrawal and also rescues neural cell lines from staurosporine-induced death. These results support the concept of using protein transduction of Bcl-2 constructs for neuroprotection.
Publication Type: Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, P.H.S.;
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154.
J Cell Biochem 2006 Jan 1; 1(97):98-108.
Apoptotic index or a combination of Bax/Bcl-2 expression correlate with survival after resection of pancreatic adenocarcinoma.
Magistrelli P ,Coppola R ,Tonini G ,Vincenzi B ,Santini D ,Borzomati D ,Vecchio F ,Valeri S ,Castri F ,Antinori A ,Nuzzo G ,Caraglia M ,Picciocchi A ,
Department of Surgery, Catholic University School of Medicine, Rome, Italy.
In the present study, the prognostic impact of factors involved in the apoptosis pathway were tested on 67 consecutive patients treated with surgical resection. Included in the study were all patients resected for pancreatic adenocarcinoma from 1988 to 2003. Expression analysis for p53, Bax, and Bcl-2 were performed by immunohistochemical staining. Apoptotic cells were identified by the TUNEL method. These data were correlated with survival. Sixty-seven tumor specimens were included in the study. A strong positive correlation was recorded between p53 overexpression and Bax expression levels (P < 0.001). By univariate analysis, overall survival seemed to be improved with Bcl-2 and Bax expression (respectively, P = 0.0379 and 0.0311). The median survival time in patients with low apoptotic index was better versus those with a high index (P = 0.0127). Lymph node involvement was the only clinico-pathologic parameter that significantly correlated with overall survival (P = 0.0202). By a multivariate Cox regression analysis, the only immunohistochemical parameter that influenced overall survival was the apoptotic index (P = 0.040). Tumor's overexpression of both Bax and Bcl-2 resulted the strongest independent prognostic factor (P = 0.013). This is the first study to report a statistically significant association of apoptosis to overall survival for pancreatic cancer patients treated with surgical resection. The contemporary overexpression of Bax and Bcl-2 represents the strongest prognostic factor.
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155.
J Bioenerg Biomembr 2005 Jun ; 3(37):179-90.
Inhibition of mitochondrial neural cell death pathways by protein transduction of Bcl-2 family proteins.
Soane L ,Fiskum G ,
Department of Anesthesiology, School of Medicine, University of Maryland, 685 W. Baltimore Street, Baltimore, MD 21201, USA.
Bcl-2 and other closely related members of the Bcl-2 family of proteins inhibit the death of neurons and many other cells in response to a wide variety of pathogenic stimuli. Bcl-2 inhibition of apoptosis is mediated by its binding to pro-apoptotic proteins, e.g., Bax and tBid, inhibition of their oligomerization, and thus inhibition of mitochondrial outer membrane pore formation, through which other pro-apoptotic proteins, e.g., cytochrome c, are released to the cytosol. Bcl-2 also exhibits an indirect antioxidant activity caused by a sub-toxic elevation of mitochondrial production of reactive oxygen species and a compensatory increase in expression of antioxidant gene products. While classic approaches to cytoprotection based on Bcl-2 family gene delivery have significant limitations, cellular protein transduction represents a new and exciting approach utilizing peptides and proteins as drugs with intracellular targets. The mechanism by which proteins with transduction domains are taken up by cells and delivered to their targets is controversial but usually involves endocytosis. The effectiveness of transduced proteins may therefore be limited by their release from endosomes into the cytosol.
Publication Type: Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.; Review;
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156.
Proc Natl Acad Sci U S A 2005 Sep 27; 39(102):13944-9.
miR-15 and miR-16 induce apoptosis by targeting BCL2.
Cimmino A ,Calin GA ,Fabbri M ,Iorio MV ,Ferracin M ,Shimizu M ,Wojcik SE ,Aqeilan RI ,Zupo S ,Dono M ,Rassenti L ,Alder H ,Volinia S ,Liu CG ,Kipps TJ ,Negrini M ,Croce CM ,
Department of Molecular Virology, Immunology, and Medical Genetics and Comprehensive Cancer Center, Ohio State University, Columbus OH 43210, USA.
Chronic lymphocytic leukemia (CLL) is the most common human leukemia and is characterized by predominantly nondividing malignant B cells overexpressing the antiapoptotic B cell lymphoma 2 (Bcl2) protein. miR-15a and miR-16-1 are deleted or down-regulated in the majority of CLLs. Here, we demonstrate that miR-15a and miR-16-1 expression is inversely correlated to Bcl2 expression in CLL and that both microRNAs negatively regulate Bcl2 at a posttranscriptional level. BCL2 repression by these microRNAs induces apoptopsis in a leukemic cell line model. Therefore, miR-15 and miR-16 are natural antisense Bcl2 interactors that could be used for therapy of Bcl2-overexpressing tumors.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.;
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157.
FASEB J 2005 Nov ; 13(19):1899-901.
Regulation of Bcl-2 family proteins, neurotrophic factors, and APP processing in the neurorescue activity of propargylamine.
Bar-Am O ,Weinreb O ,Amit T ,Youdim MB ,
Eve Topf and USA National Parkinson Foundation Centers of Excellence for Neurodegenerative Diseases Research, and Department of Pharmacology, Rappaport Family Research Institute, Technion-Faculty of Medicine, Haifa, Israel.
The anti-Parkinson drug, rasagiline (N-propargyl-(1R)-aminoindan) promotes neuronal survival, via neuroprotective activity related to its propargyl moiety (propargylamine). We have investigated the neurorescue effects of propargylamine, in a progressive neuronal death model, induced by long-term serum deprivation in human SH-SY5Y neuroblastoma cells. Propargylamine (0.1-10 microM) dose-dependently reduced the levels of the early apoptosis-associated phosphorylated protein, H2A-X (ser 139), as well as decreased the cleavage of caspase-3 and its substrate poly-ADP ribose polymerase (PARP). In addition, the compound markedly reversed the apoptotic effects induced by long-term serum withdrawal, including down-regulation of the antiapoptotic protein, Bcl-2, as well as up-regulation of the proapoptotic proteins, Bax, Bad, and Bim. Real-time RT-PCR demonstrated that propargylamine elevated gene expression levels of Bcl-2, and the neurotrophic factors glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) and reduced Bax gene expression. Serum deprivation increased mRNA and protein levels of holo-amyloid precursor protein (APP), which was markedly decreased by propargylamine. This was accompanied by inducing the release of the nonamyloidogenic alpha-secretase form of soluble APP (sAPPalpha) into the medium. Similar effects on cell survival and APP regulation/processing were demonstrated for rasagiline. These results indicate that both rasagiline and propargylamine possess neurorescue activity, associated with regulation of Bcl-2 family proteins, neurotrophic factors, and APP metabolism.
Publication Type: Research Support, Non-U.S. Gov't;
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158.
Acta Histochem 2005 ; 5(107):345-55.
Changed Bcl:Bax ratio in endometrium of patients with unexplained infertility.
Vatansever HS ,Lacin S ,Ozbilgin MK ,
Department of Histology and Embryology, School of Medicine, Celal Bayar University, Manisa, Turkey. hsedav@yahoo.com
Apoptosis has been shown to be an important regulator of endometrial function during the menstrual cycle and implantation. Recently, some possible implantation defects were identified in patients with unexplained infertility. In this study, we investigated the role of spontaneous apoptosis, which is regulated by death regulatory genes, such as Bcl-2, Bax, p53, and isoenzymes of nitric oxide synthases; eNOS and iNOS during the implantation window in women with unexplained infertility. Endometrial samples were evaluated from fertile (n=15) and unexplained-infertile women (n=15) during post-ovulatory 7th or 8th day of their menstrual cycles. Apoptotic cells were detected using the dUTP nick-end labelling assay and Bcl-2, Bax, p53, iNOS and eNOS were assessed immunohistochemically. Reduced apoptotic cells, weak immunoreactivity of p53 and strong immunoreactivity of Bcl-2 were observed in the unexplained-infertile group compared with the fertile group (p<0.001). Bax intensity was similar in both groups. While weak iNOS immunoreactivity was detected in both groups, moderately increased eNOS immunoreactivity was observed in infertile cases. Spontaneous apoptosis is reduced in the endometrium of unexplained-infertile women, and is associated with the changed Bcl-2:Bax ratio. This finding may be a contributing factor to defective implantation causing infertility in this group of patients.
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159.
J Clin Immunol 2005 Jul ; 4(25):353-64.
Mechanisms of apoptosis of T-cells in human tuberculosis.
Hirsch CS ,Johnson JL ,Okwera A ,Kanost RA ,Wu M ,Peters P ,Muhumuza M ,Mayanja-Kizza H ,Mugerwa RD ,Mugyenyi P ,Ellner JJ ,Toossi Z ,
Case Western Reserve University, University Hospitals of Cleveland, Cleveland, Ohio 44106-4984, USA. cxh40@po.cwru.edu
The role of TGF-beta TNF-alpha FasL and Bcl-2 in apoptosis of CD4 T-cells during active TB was studied. Coculture of PBMC from TB patients with neutralizing antibodies to TGF-beta or TNF-alpha decreased spontaneous (P < or = 0.05) and MTB-induced (P < or = 0.02) T-cell apoptosis by 50-90%, but effects were not additive. Interestingly, only levels of TGF-beta in supernatants correlated with rates of spontaneous and MTB-induced apoptosis. FasL surface and mRNA expression were higher in unstimulated and MTB-stimulated PBMC from patients than controls, and neutralization of FasL abrogated apoptosis of T-cells from patients only. Intracellular Bcl-2 protein was lower among unstimulated CD4 T-cells from patients than those from controls (P < or = 0.02), and MTB stimulation reduced intracellular Bcl-2 content in CD4 T-cells from patients only (P < or = 0.001). These findings may indicate that, during TB, predisposition of CD4 T-cells to apoptosis may involve both low expression of Bcl-2, and excessive expression of TGF-beta TNF-alpha and FasL.
Publication Type: Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, P.H.S.;
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160.
Mol Cell Biochem 2005 Sep ; 1-2(277):81-7.
Overexpression of human GPX1 modifies Bax to Bcl-2 apoptotic ratio in human endothelial cells.
Faucher K ,Rabinovitch-Chable H ,Cook-Moreau J ,Barrière G ,Sturtz F ,Rigaud M ,
School of Medicine, University of Limoges, EA3839, Molecular Medicine Laboratory, 2, Rue du Dr Marcland, 87025, Limoges Cedex, France. karine.faucher@unilim.fr
As they scavenge reactive oxygen species, antioxidants were studied for their ability to interfere with apoptotic processes. However, their mechanisms of action remain unclear. In this study, we measured the expression of two Bcl-2 family members, Bax and Bcl-2, in a human endothelial like cell-line overexpressing the organic hydroperoxide-scavenging enzyme glutathione peroxidase (GPX1), in the absence of any apoptotic/oxidant stimulus. ECV304 were stably transfected with the GPX1 cDNA and used for quantification of Bax (pro-apoptotic) and Bcl-2 (antiapoptotic) mRNA and protein levels, by quantitative RT-PCR and Western-blot. We found that, compared to control cells, cells from a clone showing a 13.2 fold increase in GPX1 activity had unchanged mRNA or protein Bcl-2 levels but expressed 42.6% and 46.1% less Bax mRNA and Bax protein respectively. Subsequently to Bax decrease, the Bax/Bcl-2 ratio, reflecting the apoptotic state of the cells, was also lower in cells overexpressing GPX1. Noticeably, the mRNA and the protein level of the cell-cycle protein p53, known to activate Bax expression, was unchanged. Our study showed that overexpressing an antioxidant gene such as GPX1 in endothelial cells is able to change the basal mRNA and protein Bax levels without affecting those of p53 and Bcl-2. This phenomenon could be useful to antiatherogenic therapies which use antioxidants with the aim of protecting the vascular wall against oxidative stress injury.
Publication Type: Research Support, Non-U.S. Gov't;
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161.
Mitochondrion 2004 Jul ; 2-3(4):223-33.
Mitochondrial membrane permeabilization by HIV-1 Vpr.
Deniaud A ,Brenner C ,Kroemer G ,
CNRS FRE 2445, Université de Versailles/St Quentin, 45, avenue des Etats-Unis, 78035 Versailles, France.
The mitochondrion is a privileged target for apoptosis-modulatory proteins of viral origin. Thus, viral protein R (Vpr) can target mitochondria and induce apoptosis via a specific interaction with the permeability transition pore complex (PTPC). Vpr cooperates with the adenine nucleotide translocator (ANT) to form large conductance channels and to trigger all the hallmarks of mitochondrial membrane permeabilization (MMP). The Vpr/ANT interaction is direct, since it is abolished by the addition of a peptide corresponding to the Vpr binding site of ANT, ADP, ATP, or by Bcl-2. Accordingly, Vpr modulates MMP through direct structural and functional interactions with PTPC proteins.
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162.
Mol Cell Biochem 2005 Apr ; 1-2(272):187-99.
Regulation of Ca2+-induced permeability transition by Bcl-2 is antagonized by Drpl and hFis1.
Kong D ,Xu L ,Yu Y ,Zhu W ,Andrews DW ,Yoon Y ,Kuo TH ,
Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
The regulation of mitochondrial permeability transition (MPT) is essential for cell survival. Un-controlled opening of the MPT pore is often associated with cell death. Anti-death protein Bcl-2 can block MPT as assessed by the enhanced capacity of mitochondria to accumulate and retain Ca2+. We report here that two proteins of the mitochondrial fission machinery, dynamin-related protein (Drp1) and human mitochondrial fission protein (hFis1), have an antagonistic effect on Bcl-2. Drp1, with the assistance of hFis1, sensitizes cells to MPT by reducing the mitochondrial Ca2+ retention capacity (CRC). While the reduction of CRC by Drp1/hFis1 is linked to mitochondrial fission, the antagonism between Bcl-2 and Drp1 appears to be mediated by mutually exclusive interactions of the two proteins with hFis1 . The complexity of protein-protein interactions demonstrated in the present study suggests that in addition to the previously described role of Bcl-2 in the control of apoptosis, Bcl-2 may also participate directly or indirectly in the regulation of mitochondrial fission.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.;
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163.
J Bone Miner Res 2005 Aug ; 8(20):1414-29.
Effect of osteoblast-targeted expression of bcl-2 in bone: differential response in male and female mice.
Pantschenko AG ,Zhang W ,Nahounou M ,McCarthy MB ,Stover ML ,Lichtler AC ,Clark SH ,Gronowicz GA ,
Department of Orthopaedic Surgery, University of Connecticut Health Center, Farmington, Connecticut 06030-3105, USA.
Transgenic mice (Col2.3Bcl-2) with osteoblast-targeted human Bcl-2 expression were established. Phenotypically, these mice were smaller than their wildtype littermates and showed differential effects of the transgene on bone parameters and osteoblast activity dependent on sex. The net effect was an abrogation of sex differences normally observed in wildtype mice and an inhibition of bone loss with age. Ex vivo osteoblast cultures showed that the transgene had no effect on osteoblast proliferation, but decreased bone formation. Estrogen was shown to stimulate endogenous Bcl-2 message levels. These studies suggest a link between Bcl-2 and sex regulation of bone development and age-related bone loss. INTRODUCTION: Whereas Bcl-2 has been shown to be an important regulator of apoptosis in development, differentiation, and disease, its role in bone homeostasis and development is not well understood. We have previously showed that the induction of glucocorticoid-induced apoptosis occurred through a dose-dependent decrease in Bcl-2. Estrogen prevented glucocorticoid-induced osteoblast apoptosis in vivo and in vitro by preventing the decrease in Bcl-2 in osteoblasts. Therefore, Bcl-2 may be an important regulator of bone growth through mechanisms that control osteoblast longevity and function. MATERIALS AND METHODS: Col2.3Bcl-2 mice were developed carrying a 2.3-kb region of the type I collagen promoter driving 1.8 kb of human Bcl-2 (hBcl-2). Tissue specific expression of hBcl-2 in immunoassays validated the transgenic animal model. Histomorphometry and DXA were performed. Proliferation, mineralization, and glucocorticoid-induced apoptosis were examined in ex vivo cultures of osteoblasts. The effect of estrogen on mouse Bcl-2 in ex vivo osteoblast cultures was assayed by RT-PCR and Q-PCR. RESULTS AND CONCLUSIONS: Two Col2.3Bcl-2 (tg/+) founder lines were established and appeared normal except that they were smaller than their nontransgenic wildtype (+/+) littermates at 1, 2, and 6 months of age, with the greatest differences at 2 months. Immunohistochemistry showed hBcl-2 in osteoblasts at the growth plate and cortical surfaces. Nontransgenic littermates were negative. Western blots revealed hBcl-2 only in type I collagen-expressing tissues. Histomorphometry of 2-month-old mice showed a significant decrease in tg/+ calvaria width with no significant differences in femoral trabecular area or cortical width compared with +/+. However, tg/+ males had significantly more trabecular bone than tg/+ females. Female +/+ mice showed increased bone turnover with elevated osteoblast and osteoclast parameters compared with +/+ males. Col2.3Bcl-2 mice did not show such significant differences between sexes. Male tg/+ mice had a 76.5 +/- 1.5% increase in ObS/BS with no significant differences in bone formation rate (BFR) or mineral apposition rate (MAR) compared with male +/+ mice. Transgenic females had a significant 48.4 +/- 0.1% and 20.1 +/- 5.8% decrease in BFR and MAR, respectively, compared with +/+ females. Osteoclast and osteocyte parameters were unchanged. By 6 months, femurs from female and male +/+ mice had lost a significant amount of their percent of trabecular bone compared with 2-month-old mice. There was little to no change in femoral bone in the tg/+ mice with age. Ex vivo cultures of osteoblasts from +/+ and Col2.3Bcl-2 mice showed a decrease in mineralization, no effect on proliferation, and an inhibition of glucocorticoid-induced apoptosis in Col2.3Bcl-2 cultures. Estrogen was shown to increase mouse Bcl-2 transcript levels in osteoblast cultures of wildtype mice, supporting a role for Bcl-2 in the sex-related differences in bone phenotype regulated by estrogen. Therefore, Bcl-2 differentially affected bone phenotype in male and female transgenic mice, altered bone cell activity associated with sex-related differences, and decreased bone formation, suggesting that apoptosis is necessary for mineralization. In addition, Bcl-2 targeted to mature osteoblasts seemed to delay bone development, producing a smaller transgenic mouse compared with wildtype littermates. These studies suggest that expression of Bcl-2 in osteoblasts is important in regulating bone mass in development and in the normal aging process of bone.
Publication Type: Comparative Study; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, P.H.S.;
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164.
J Clin Immunol 2005 May ; 3(25):224-9.
CD4+ T cells downregulate Bcl-2 in germinal centers.
Schenka AA ,Müller S ,Fournié JJ ,Capila F ,Vassallo J ,Delsol G ,Valitutti S ,Brousset P ,
Department of Pathology and Inserm U563, Centre de Physiopathologie de Toulouse, Purpan, France. schenka@hotmail.com
Germinal centers (GCs) are the main site of T cell-dependent antibody responses. Upon antigen challenge, GCs comprise mostly B cells undergoing proliferation, somatic hypermutation and antigen-affinity selection. GC B cells down-modulate the expression of Bcl-2 protein and are highly sensitive to apoptosis to eliminate autoreactive or low-affinity cells. Bcl-2 is still expressed in a few GC cells, whose identity remains unclear. To address this issue, we examined by confocal microscopy the expression of Bcl-2 by different GC lymphocyte subsets in hyperplastic tonsils. We found that the vast majority of Bcl-2(+) GC cells are T lymphocytes. Conversely, while in the mantle zone and in the interfollicular areas T cells are almost exclusively Bcl-2(+), in the GC, most T lymphocytes are Bcl-2(-). In addition, most of the CD4(+) GC T cells are Bcl-2(-), while nearly 100% of the CD8(+) GC T cells are Bcl-2(+). The Bcl-2 downregulation by both B and CD4(+) T GC cells supports the concept that these two subsets may undergo a selection process in this microenvironment.
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165.
Med Oncol 2005 ; 2(22):139-43.
The value of serum Bcl-2 levels in advanced lung cancer patients.
Tas F ,Duranyildiz D ,Oguz H ,Camlica H ,Oral EN ,Yasasever V ,Topuz E ,
Institute of Oncology, University of Istanbul, Turkey. faruktas2002@yahoo.com
Overexpression of the Bcl-2 protein was associated with a favorable prognostic factor for survival in lung cancer patients, especially nonsmall cell lung carcinoma. The present study was conducted to investigate the value of serum Bcl-2 levels in advanced lung cancer patients. Fifty patients with advanced lung carcinoma pathologically verified and 18 healthy controls were investigated. Serum samples were obtained on the first admission before the chemotherapeutic treatment were given. Serum Bcl-2 levels were determined by using anti-Bcl-2 monoclonal coating antibody. The baseline serum Bcl-2 levels were significantly higher in patients with lung cancer than in the control group (p<0.001). Serum Bcl-2 levels were elevated in 48 (96%) advanced lung cancer patients. None of the prognostic parameters analyzed, such as age of patient, gender, histology, stage of disease, erythrocyte sedimentation rate, serum albumin, hemoglobin, CEA, NSE, LDH, performance of patient, weight loss, and response to chemotherapy, was significantly correlated with Bcl-2 serum concentrations. The serum Bcl-2 concentrations were not changed with cisplatin-based cytotoxic chemotherapy regardless of response (p=0.76). No prognostic value of serum Bcl-2 was determined. In conclusion, the results of the present study, which is the first study to determine serum Bcl-2 levels in lung cancer, suggest that decreased apoptosis occurred due to the effect of serum Bcl-2 elevation in lung cancer patients. Serum Bcl-2 level was of diagnostic but not prognostic value in lung cancer patients. However, more studies are needed to define the role of Bcl-2 in the diagnosis and prognosis of lung cancer.
Publication Type: Comparative Study;
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166.
World J Gastroenterol 2005 Jun 7; 21(11):3293-6.
Expression of bcl-2 oncogene in gastric precancerous lesions and its correlation with syndromes in traditional Chinese medicine.
Hu L ,Lao SX ,Tang CZ ,
Institute of the Spleen and Stomach, Guangzhou University of Traditional Chinese Medicine, 12 Jichang Road, Guangzhou 510405, Guangdong Province, China. pqhl@yahoo.com.cn
AIM: To observe the protein and mRNA expression of bcl-2 oncogene in gastric precancerous lesions (GPL) and to analyze its correlation with syndromes in traditional Chinese medicine (TCM). METHODS: Sixty-seven patients with GPL confirmed by gastroscopy and pathology were studied, including 39 cases of moderate gastric mucosal dysplasia, 19 cases of severe gastric mucosa dysplasia, 9 cases of incomplete colon metaplasia. In syndrome differentiation of TCM, 17 cases belonged to the syndrome of qi and yin deficiency of the spleen and stomach complicated by qi stagnation, 21 cases belonged to the syndrome of qi and yin deficiency of the spleen and stomach complicated by stomach heat, 29 cases belonged to the syndrome of qi and yin deficiency of the spleen and stomach complicated by blood stasis. Protein and mRNA expression of bcl-2 oncogene were detected by labeled streptavidin biotin (LSAB) immunohistochemistry and in situ hybridization respectively. RESULTS: Abnormal expression of protein and mRNA on bcl-2 oncogene was found in GPL, which increased gradually with the course of lesions. In moderate and severe gastric mucosal dysplasia and incomplete colon metaplasia, there was no difference in the expression of bcl-2 oncogene (P>0.05). In different accompanying syndromes, the expression of protein and mRNA on bcl-2 oncogene increased gradually in the following order: deficiency of both qi and yin of the spleen and stomach accompanying qi stagnation-->stomach heat-->blood stasis. In GPL, compared with accompanying blood stasis, there was an obvious difference in the expression of bcl-2 oncogene between the syndrome of qi and yin deficiency of the spleen and stomach and accompanying stomach heat, so did accompanying qi stagnation (the level of protein: chi(2) = 8.45, P<0.05; the level of mRNA: chi(2) = 7.35, P<0.05). CONCLUSION: Apoptosis-associated bcl-2 oncogene is abnormally expressed in GPL, which correlates with different accompanying syndromes in TCM.
Publication Type: Research Support, Non-U.S. Gov't;
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167.
Mol Cell Neurosci 2005 Jun ; 2(29):202-21.
Enhanced Purkinje cell survival but compromised cerebellar function in targeted anti-apoptotic protein transgenic mice.
Goswami J ,Martin LA ,Goldowitz D ,Beitz AJ ,Feddersen RM ,
Department of Veterinary Biomedical Sciences, University of Minnesota, 295 AnSci/VetMed Building, 1988 Fitch Avenue, St. Paul, MN 55108, USA.
Regulation of Purkinje cell (PC) number is critical for proper assembly and function of the cerebellum. Murine cerebellar neurogenesis yields supernumerary populations of cells that are subject to programmed cell death during development and aging. This study focuses on the control of mouse PC number during development and the consequences of interrupting normal cell death. Purkinje cell-specific regulatory elements from the pcp2 gene were employed to target expression of two anti-apoptotic proteins, human BCL-2 and adenovirus E1B 19k to the PCs of transgenic mice. Comparative morphometric analyses indicated no significant difference in PC numbers in the strongest BCL-2 expressing line, while a 14.2% increase was noted in the pcp2/E1B 19k transgenic line. The temporal transgene expression patterns of several mouse lines indicated that PC numbers are normally adjusted during the first postnatal week. Crossbreeding studies demonstrated that both Bcl-2 and E1B 19k transgenes provided Purkinje cell protection from SV40 Tag-induced cell death. Interestingly, RotaRod behavioral analysis demonstrated that 'rescued' Purkinje cells degrade cerebellar function. Furthermore, aged E1B 19k and Bcl-2 mice exhibited decreased RotaRod performance despite increased PC numbers. These findings have implications regarding neuronal death during development and aging as well as cellular and genetic strategies to circumvent neuronal degeneration.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.;
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168.
BMC Cancer 2005 ; 1(5):50.
Maspin overexpression modulates tumor cell apoptosis through the regulation of Bcl-2 family proteins.
Zhang W ,Shi HY ,Zhang M ,
Department of Molecular & Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA. wgzhang8@yahoo.com
BACKGROUND: Maspin is a member of serpin family with tumor suppressing activity. Recent studies of maspin in animal models strongly support maspin's role as an inhibitor against the growth of primary tumor sand the process of metastasis. However, the molecular mechanism underlying this inhibition has not been fully elucidated. In this report, we analyze the effect of maspin on tumor cell apoptosis under several stress conditions. METHODS: Stable clones overexpressing maspin are established in the mouse mammary tumor TM40D cells. They are treated with staurosporine, TNF-alpha, and serum starvation. The rates of cell apoptosis are analyzed by TUNEL assay. Inhibitors against caspase 8 and 9 are used in the apoptosis assay. Western blot analysis and ribonuclease protection assay (RPA) are performed to examine the expression of Bcl2 family genes. RESULTS: We report that maspin expressing tumor cells have increased rate of apoptosis when they are treated with staurosporine and serum starvation. The effect is not through extracellular maspin. Maspin-mediated apoptosis is partially blocked by caspase 8 and 9 inhibitors, and is accompanied by changes in the Bcl-2 family proteins. Maspin-expressing tumor cells have a reduced level of anti-apoptotic protein Bcl-2, and an increased level of pro-apoptotic protein Bax. The regulation is not controlled at the transcriptional level but is through selective control of Bcl-2 and Bax protein stability. CONCLUSION: Maspin overexpression modulates tumor cell apoptosis through the regulation of Bcl2 family proteins. Such change results in an increased release of cytochrome c from mitochondria, thus the increased apoptosis in maspin-expressing cells. This evidence strongly suggests that the induction of apoptosis in maspin-overexpressing cells represents a major mechanism by which maspin inhibits breast tumor progression.
Publication Type: Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.;
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169.
Genes Dev 2005 Jun 1; 11(19):1294-305.
Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins.
Willis SN ,Chen L ,Dewson G ,Wei A ,Naik E ,Fletcher JI ,Adams JM ,Huang DC ,
The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.
Commitment of cells to apoptosis is governed largely by the interaction between members of the Bcl-2 protein family. Its three subfamilies have distinct roles: The BH3-only proteins trigger apoptosis by binding via their BH3 domain to prosurvival relatives, while the proapoptotic Bax and Bak have an essential downstream role involving permeabilization of organellar membranes and induction of caspase activation. We have investigated the regulation of Bak and find that, in healthy cells, Bak associates with Mcl-1 and Bcl-x(L) but surprisingly not Bcl-2, Bcl-w, or A1. These interactions require the Bak BH3 domain, which is also necessary for Bak dimerization and killing activity. When cytotoxic signals activate BH3-only proteins that can engage both Mcl-1 and Bcl-x(L) (such as Noxa plus Bad), Bak is displaced and induces cell death. Accordingly, the BH3-only protein Noxa could bind to Mcl-1, displace Bak, and promote Mcl-1 degradation, but Bak-mediated cell death also required neutralization of Bcl-x(L) by other BH3-only proteins. The results indicate that Bak is held in check solely by Mcl-1 and Bcl-x(L) and induces apoptosis only if freed from both. The finding that different prosurvival proteins have selective roles has notable implications for the design of anti-cancer drugs that target the Bcl-2 family.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.;
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170.
Biomed Res 2005 Apr ; 2(26):87-90.
Influence of prostaglandin A2 and 2-methoxyestradiol on Bax and Bcl-2 expression levels in cervical carcinoma cells.
Joubert A ,Maritz C ,Joubert F ,
Department of Physiology, University of Pretoria, Pretoria, South Africa. annie.joubert@up.ac.za
Proteins of the Bcl-2 family are key regulators of apoptosis. Bax can be regarded as pro-apoptotic, whereas Bcl-2 is perceived as anti-apoptotic. It has been proposed that an increased ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2 can be associated with apoptosis. Since prostaglandin A2 (PGA2) and 2-methoxyestradiol (2-ME) play an active role in the induction of apoptosis, the influence of 20 microg/ml PGA2 and 1 microM 2-ME was investigated on Bax and Bcl-2 expression levels in cervical carcinoma cells. Both PGA2 and 2-ME exposure led to statistically significant increases in Bax expression levels. Cells were shown to be more susceptible to the effects of 2-ME than to the effects caused by PGA2. In contrast, no statistically significant effects were observed on Bcl-2 expression levels after exposure to PGA2 and 2-ME. The Bax/Bcl-2 ratios for PGA2- and 2-ME-exposed cells were 2.06 and 1.87 respectively, normalised against Bcl-2 levels. Further investigation of the function and regulation of the Bcl-2 family will allow researchers to consider potential pathways of apoptosis signaling mechanisms for diseases where apoptosis can potentially be controlled.
Publication Type: Research Support, Non-U.S. Gov't;
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171.
Acta Otolaryngol 2005 Feb ; 2(125):191-5.
Cyclooxygenase-2 regulates the degree of apoptosis by modulating bcl-2 protein in pleomorphic adenoma and mucoepidermoid carcinoma of the parotid gland.
Sakamoto T ,Uozaki H ,Kondo K ,Imauchi Y ,Yamasoba T ,Sugasawa M ,Kaga K ,
Department of Otorhinolaryngology, Mutual Aid Association for Tokyo Metropolitan Teachers and Officials, Sanraku Hospital, Tokyo, Japan. ta-saka@nifty.com
CONCLUSION: These results suggest that COX-2 and bcl-2 protein were overexpressed and that apoptosis was reduced in MEC compared to PMA, and that COX-2 may regulate the degree of apoptosis by modulating bcl-2 protein in PMA and MEC. OBJECTIVE: Cyclooxygenase (COX)-2 plays a crucial role in tumorigenesis and overexpression of COX-2 in vitro accompanied by overexpression of bcl-2 protein has been shown to reduce apoptosis. The purpose of this study was to verify that COX-2 regulates the degree of apoptosis by modulating bcl-2 protein in benign and malignant parotid gland tumors. : We examined archival formalin-fixed, paraffin-embedded tissue sections of 10 pleomorphic adenomas (PMAs) and 10 mucoepidermoid carcinomas (MECs) by immunostaining with anti-COX-2, anti-bcl-2 and anti-single-stranded DNA (ssDNA) antibodies. Labeling indices of the three antibodies were calculated using computer-assisted image analysis. RESULTS: Labeling indices (mean+/-SD) of anti-COX-2 antibody in PMA and MEC were 2.05+/-1.30 and 11.2+/-2.95, respectively (p < 0.001), those of anti-bcl-2 antibody were 2.00+/-1.28 and 9.68+/-4.05, respectively (p < 0.001) and those of anti-ssDNA antibody were 8.06+/-2.54 and 2.08+/-1.47; respectively (p <0.001). Correlation coefficients between the labeling indices of anti-COX-2 antibody and anti-bcl-2 antibody, anti-bcl-2 antibody and anti-ssDNA antibody and anti-COX-2 antibody and anti-ssDNA antibody were 0.88, -0.75 and -0.76, respectively (p <0.001).
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172.
Cell Cycle 2005 Apr ; 4(4):590-6.
Localization of phosphorylated forms of Bcl-2 in mitosis: co-localization with Ki-67 and nucleolin in nuclear structures and on mitotic chromosomes.
Barboule N ,Truchet I ,Valette A ,
Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération, Université Paul Sabatier, Toulouse cédex, France. barboule@cict.fr
Bcl-2 phosphorylation is a normal physiological process occurring at mitosis or during mitotic arrest induced by microtubule damaging agents. The consequences of Bcl-2 phosphorylation on its function are still controversial. To better understand the role of Bcl-2 phosphorylation in mitosis, we studied the subcellular localization of phosphorylated forms of Bcl-2. Immunofluorescence experiments performed in synchronized HeLa cells indicate for the first time that mitotic phosphorylated forms of Bcl-2 can be detected in nuclear structures in prophase cells together with nucleolin and Ki-67. In later mitotic stages, as previously described, phosphorylated forms of Bcl-2 are localized on mitotic chromosomes. In addition, we demonstrate that Bcl-2 in these structures is at least in part phosphorylated on the T56 residue. Then, coimmunoprecipitation experiments reveal that, in cells synchronized at the onset of mitosis, Bcl-2 is present in a complex with nucleolin, cdc2 kinase and PP1 phosphatase. Taken together, these data further support the idea that Bcl-2 could have a new function at mitosis.
Publication Type: Research Support, Non-U.S. Gov't;
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173.
J Biol Chem 2005 Jul 1; 26(280):24698-705.
p21-activated Kinase 1 (Pak1)-dependent phosphorylation of Raf-1 regulates its mitochondrial localization, phosphorylation of BAD, and Bcl-2 association.
Jin S ,Zhuo Y ,Guo W ,Field J ,
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Raf-1 protects cells from apoptosis, independently of its signals to MEK and ERK, by translocating to the mitochondria where it binds Bcl-2 and displaces BAD. However, the answer to the question of how Raf-1 is normally lured to the mitochondria and becomes activated remains elusive. p21-activated protein kinases (Paks) are serine/threonine protein kinases that phosphorylate Raf-1 at Ser-338 and Ser-339. Here we elucidate the molecular mechanism through which Pak1 signals to BAD through a Raf-1-activated pathway. Upon phosphorylation by Pak1, Raf-1 translocates to mitochondria and phosphorylates BAD at Ser-112. Moreover, the mitochondrial translocation of Raf-1 and the interaction between Raf-1 and Bcl-2 are regulated by Raf-1 phosphorylation at Ser-338/Ser-339. Notably, we show that formation of a Raf-1-Bcl-2 complex coincides with loss of an interaction between Bcl-2 and BAD. These signals are specific for Pak1, because Src-activated Raf-1 only stimulates the MAP kinase cascade. Thus, our data identify the molecular connections of a Pak1-Raf-1-BAD pathway that is involved in cell survival signaling.
Publication Type: Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.;
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174.
J Biol Chem 2005 Jun 17; 24(280):22749-60.
Evidence for a triplex DNA conformation at the bcl-2 major breakpoint region of the t(14;18) translocation.
Raghavan SC ,Chastain P ,Lee JS ,Hegde BG ,Houston S ,Langen R ,Hsieh CL ,Haworth IS ,Lieber MR ,
Norris Comprehensive Cancer Center, Zilka Neurogenetics Institute, University of Southern California Keck School of Medicine, Los Angeles, 90033, USA.
The most common chromosomal translocation in cancer, t(14;18), occurs at the bcl-2 major breakpoint region (Mbr) in follicular lymphomas. The 150-bp bcl-2 Mbr, which contains three breakage hotspots (peaks), has a single-stranded character and, hence, a non-B DNA conformation both in vivo and in vitro. Here, we use gel assays and electron microscopy to show that a triplex-specific antibody binds to the bcl-2 Mbr in vitro. Bisulfite reactivity shows that the non-B DNA structure is favored by, but not dependent upon, supercoiling and suggests a possible triplex conformation at one portion of the Mbr (peak I). We have used circular dichroism to test whether the predicted third strand of that suggested structure can indeed form a triplex with the duplex at peak I, and it does so with 1:1 stoichiometry. Using an intracellular minichromosomal assay, we show that the non-B DNA structure formation is critical for the breakage at the bcl-2 Mbr, because a 3-bp mutation that disrupts the putative peak I triplex also markedly reduces the recombination of the Mbr. A three-dimensional model of such a triplex is consistent with bond length, bond angle, and energetic restrictions (stacking and hydrogen bonding). We infer that an imperfect purine/purine/pyrimidine (R.R.Y) triplex likely forms at the bcl-2 Mbr in vitro, and in vivo recombination data favor this as the major DNA conformation in vivo as well.
Publication Type: Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, P.H.S.;
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175.
Neurobiol Dis ; 1-2(19):183-93.
Transplantation of embryonic stem cells overexpressing Bcl-2 promotes functional recovery after transient cerebral ischemia.
Wei L ,Cui L ,Snider BJ ,Rivkin M ,Yu SS ,Lee CS ,Adams LD ,Gottlieb DI ,Johnson EM Jr,Yu SP ,Choi DW ,
Center for the Study of Nervous System Injury and Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110, USA. weil@musc.edu
The study tested the hypothesis that transplantation of embryonic stem (ES) cells into rat cortex after a severe focal ischemia would promote structural repair and functional recovery. Overexpression of the human anti-apoptotic gene bcl-2 in ES cells was tested for increasing survival and differentiation of transplanted cells and promoting functional benefits. Mouse ES cells, pretreated with retinoic acid to induce differentiation down neural lineages, were transplanted into the post-infarct brain cavity of adult rats 7 days after 2-h occlusion of the middle cerebral artery (MCA). Over 1-8 weeks after transplantation, the lesion cavity filled with ES cell-derived cells that expressed markers for neurons, astrocytes, oligodendrocytes, and endothelial cells. ES cell-derived neurons exhibited dendrite outgrowth and formed a neuropil. ES cell-transplanted animals exhibited enhanced functional recovery on neurological and behavioral tests, compared to control animals injected with adult mouse cortical cells or vehicle. Furthermore, transplantation with ES cells overexpressing Bcl-2 further increased the survival of transplanted ES cells, neuronal differentiation, and functional outcome. This study supports that ES cell transplantation and gene modification may have values for enhancing recovery after stroke.
Publication Type: Comparative Study; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.;
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176.
Int J Biol Markers ; 1(20):18-27.
Transcriptional inactivation of p53, Bax, Bcl-2 and Mdm2 correlates with malignant transformation of the uterine cervix.
Soufla G ,Baritaki S ,Sifakis S ,Zafiropoulos A ,Spandidos DA ,
Department of Virology, Medical School, University of Crete, Heraklion, Crete, Greece.
Deregulation of the apoptotic machinery plays a major role in cell death, cellular transformation and cancer. p53, Bcl-2, Bcl-XL, Bax and Mdm2 mRNA expression patterns were evaluated in tissue samples with cervical intraepithelial neoplasia (CIN) and cervical cancer compared to those of normal cervical tissues, and correlated with the underlying cervical lesions. Transcript levels of the above genes were assessed by RT-PCR analysis in a total of 44 cervical specimens. p53, Bcl-2, Bax and Mdm2 transcript levels were significantly different in the normal, CIN and cancer specimen groups (p=0.003, p=0.009, p=0.040 and p=0.001, respectively). Specifically, p53, Bax and Bcl-2 exhibited substantially lower transcript levels in CIN lesions compared to controls, whereas Bax mRNA levels showed a significant decrease in cancer compared to normal specimens. Mdm2 mRNA expression was considerably lower in cancer than in CIN lesions or normal cervix. High-grade squamous intraepithelial lesions exhibited lower p53 and Bcl-2 mRNA levels than controls (p=0.002, p=0.016). Coexpression analysis revealed more correlations between the above apoptosis-related molecules in normal tissues compared to CIN or cancer specimens. p53 showed significant coexpression with Bax, Bcl-2 and Mdm2 (p=0.040, p=0.013 and p=0.015, respectively) in normal cervical specimens. Bax and Bcl-XL mRNA expression was negatively correlated. Mdm2 transcriptional levels correlated significantly with those of Bax, Bcl-XL and Bcl-2. Our findings show that p53, Bax, Bcl-2 and Mdm2 mRNA expression levels correlate with the malignant transformation of the uterine cervix. mRNA coexpression patterns of the members of the pro- and anti-apoptotic family examined in cervical carcinogenesis were found to be disrupted in CIN and cancer, as already demonstrated at the protein level.
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177.
Cell Death Differ 2005 Aug ; (12 Suppl 1):962-70.
Human immunodeficiency virus type 1 (HIV-1) Vpr-regulated cell death: insights into mechanism.
Muthumani K ,Choo AY ,Premkumar A ,Hwang DS ,Thieu KP ,Desai BM ,Weiner DB ,
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
The destruction of CD4(+) T cells and eventual induction of immunodeficiency is a hallmark of the human immunodeficiency virus type 1 infection (HIV-1). However, the mechanism of this destruction remains unresolved. Several auxiliary proteins have been proposed to play a role in this aspect of HIV pathogenesis including a 14 kDa protein named viral protein R (Vpr). Vpr has been implicated in the regulation of various cellular functions including apoptosis, cell cycle arrest, differentiation, and immune suppression. However, the mechanism(s) involved in Vpr-mediated apoptosis remains unresolved, and several proposed mechanisms for these effects are under investigation. In this review, we discuss the possibility that some of these proposed pathways might converge to modulate Vpr's behavior. Further, we also discuss caveats and future directions for investigation of the interesting biology of this HIV accessory gene.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.; Review;
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178.
Ann Clin Lab Sci 2005 ; 1(35):91-6.
Immunohistochemical expression of Bax and Bcl-2 in penile carcinoma.
Saeed S ,Keehn CA ,Khalil FK ,Morgan MB ,
Department of Pathology, University of South Florida College of Medicine, Tampa, FL 33612, USA. mbkmmorgan@aol.com
There is a complex interplay between the pro-apoptotic Bax and anti-apoptotic Bcl-2 family of proteins and the tumor suppressor gene p53. The pathogenic role of Bax and Bcl-2 protein expression in penile carcinomas has not previously been investigated. We examined Bax and Bcl-2 expression in verrucous (VC) and squamous cell carcinoma (SCC) of the penis. Herein we also present a concise review of p53, Bcl-2/Bax ratios, and their relationship to apoptosis. Fourteen cases of penile carcinoma, including 7 VC and 7 well-differentiated SCC, were analyzed for Bax and Bcl-2 expression by immunohistochemical analysis of paraffin embedded archived tissues. The number of positively staining tumor cells was enumerated per 100 tumor cells within non-overlapping high power fields. The Bax immunoreactivity was similar in VC (19+/-3%) and well-differentiated SCC (15+/-4%) (p = 0.69). The expression of Bcl-2 protein was significantly higher in well-differentiated SCC (69+/-12%) compared to VC (36+/-14%) (p = 0.04). The mean Bcl-2/Bax ratio was significantly lower in VC (1.89) compared to well-differentiated SCC (4.6) (p = 0.05). These findings indicate that penile VC and SCC are immunophenotypically distinct. Bax expression is comparable in verrucous and low-grade squamous cell carcinomas, but Bcl-2 expression of Bcl-2 is significantly higher in the squamous cell carcinomas.
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179.
Eur Urol 2005 May ; 5(47):703-9.
Inhibition of bcl-2 enhances the efficacy of chemotherapy in renal cell carcinoma.
Kausch I ,Jiang H ,Thode B ,Doehn C ,Krüger S ,Jocham D ,
Department of Urology, University of Lubeck, Medical School, Ratzeburger Allee 160, 23538 Lubeck, Germany. ikausch@aol.com
OBJECTIVES: Renal cell cancer (RCC) is highly resistant to chemotherapy. Increased expression of the antiapoptotic gene bcl-2 in tumors is known to be associated with poor responses to systemic treatment of cancer. Down-regulation of bcl-2 expression using antisense oligonucleotides (asON) has been shown to increase chemosensitivity in clinical phase I-III studies with various cancers. However, no studies on the efficacy of this approach in RCC have been reported so far. This study aimed to evaluate whether bcl-2 asON could enhance efficacy of chemotherapy in human RCC. MATERIAL AND METHODS: Expression of bcl-2 mRNA and protein was analyzed in different RCC cell lines by RT-PCR and Western blot. Cells with high or low bcl-2 mRNA and protein expression were treated with different concentrations of bcl-2 asON in combination with cisplatin. AsON-induced down-regulation of bcl-2 mRNA and protein was documented by RT-PCR and Western blot. Treatment effects on cell viability were analyzed by colorimetric tetrazolium (MTT) assay. Immunohistochemical staining of M30-positive cells was performed for quantification of apoptotic cells. RESULTS: Transfection of high bcl-2 expressing cells with bcl-2 asON alone induced no reduction of cell viability at a concentration range from 100-1000 nM. In combination therapy, pretreatment with asON significantly enhanced MTT reduction after cisplatin treatment. IC50 concentrations of cisplatin were 1 microg/ml with and 2.7 microg/ml without prior incubation. The marked reduction of cell viability correlated with an 8-fold increase of apoptotic cells after combination treatment. Only a minor increase of cisplatin effectivity was noted after asON preincubation of cells with lower bcl-2 expression. CONCLUSIONS: The combination of cisplatin and bcl-2 antisense ON exerts significantly greater effects on cell viability and apoptosis than either agent used alone on human RCC cells. These data indicate that inhibition of bcl-2 expression may be an attractive therapeutic strategy in RCC tumors with high bcl-2 expression.
Publication Type: Comparative Study; In Vitro; Research Support, Non-U.S. Gov't;
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180.
J Biol Chem 2005 Jun 24; 25(280):23758-65.
Bcl-2 rescues ceramide- and etoposide-induced mitochondrial apoptosis through blockage of caspase-2 activation.
Lin CF ,Chen CL ,Chang WT ,Jan MS ,Hsu LJ ,Wu RH ,Fang YT ,Tang MJ ,Chang WC ,Lin YS ,
Department of Microbiology and Immunology, National Cheng Kung University Medical College, Tainan, Taiwan.
Recent studies indicate that caspase-2 is involved in the early stage of apoptosis before mitochondrial damage. Although the activation of caspase-2 has been shown to occur in a large protein complex, the mechanisms of caspase-2 activation remain unclear. Here we report a regulatory role of Bcl-2 on caspase-2 upstream of mitochondria. Stress stimuli, including ceramide and etoposide, caused caspase-2 activation, mitochondrial damage followed by downstream caspase-9 and -3 activation, and cell apoptosis in human lung epithelial cell line A549. When A549 cells were pretreated with the caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(-OMe)-Val-Ala-Asp(-OMe)-fluoromethyl ketone or transfected with caspase-2 short interfering RNA, both ceramide- and etoposide-induced mitochondrial damage and apoptosis were blocked. Overexpression of Bcl-2 prevented ceramide- and etoposide-induced caspase-2 activation and mitochondrial apoptosis. Furthermore, caspase-2 was activated when A549 cells were introduced with Bcl-2 short interfering RNA or were treated with Bcl-2 inhibitor, which provided direct evidence of a negative regulatory effect of Bcl-2 on caspase-2. Cell survival was observed when caspase-2 was inhibited in Bcl-2-silencing cells. Blockage of the mitochondrial permeability transition pore and caspase-9 demonstrated that Bcl-2-modulated caspase-2 activity occurred upstream of mitochondria. Further studies showed that Bcl-2 was dephosphorylated at serine 70 after ceramide and etoposide treatment. A protein phosphatase inhibitor, okadaic acid, rescued Bcl-2 dephosphorylation and blocked caspase-2 activation, mitochondrial damage, and cell death. Taken together, ceramide and etoposide induced mitochondria-mediated apoptosis by initiating caspase-2 activation, which was, at least in part, regulated by Bcl-2.
Publication Type: Research Support, Non-U.S. Gov't;
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181.
J Cutan Pathol 2005 May ; 5(32):356-9.
Comparison of benign keratoses using p53, bcl-1, and bcl-2.
Ko CJ ,Shintaku P ,Binder SW ,
Department of Pathology and Laboratory Medicine, University of California, Los Angeles, CA 90095, USA.
While cell-cycle markers have been used to differentiate benign vs. malignant lesions and to classify malignant lesions, benign keratoses have not been well studied using such markers. We hypothesized that inflammation or irritation of benign keratoses may be related to a shift in the cell cycle. We compared the immunohistochemical staining patterns of 10 seborrheic keratoses (SKs), 10 inflamed seborrheic keratoses (iSKs), and 10 inverted follicular keratoses (IFKs) using antibodies to p53, bcl-1, and bcl-2. Staining with antibodies to p53 was slightly increased in IFKs compared with iSKs or non-inflamed seborrheic keratoses. Bcl-1 staining was similar in all lesions. A population of bcl-2-positive dendritic cells was seen within the epidermal portion of IFKs. Keratinocyte bcl-2 staining was significantly higher in SKs compared with the other two keratoses. Bcl-2 may be increased in SKs as an anti-apoptotic mechanism.
Publication Type: Comparative Study;
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182.
Nat Cell Biol 2005 Apr ; 4(7):326-7.
Dual role for Bcl-2 in antibody affinity maturation.
Dunn-Walters D ,Spencer J ,
Publication Type: Comment; Letter;
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183.
Pathol Oncol Res 2005 ; 1(11):45-9.
Expression of p53, Ki-67 and Bcl-2 in parathyroid adenoma and residual normal tissue.
Hadar T ,Shvero J ,Yaniv E ,Ram E ,Shvili I ,Koren R ,
Department of Otolaryngology--Head and Neck Surgery, Rabin Medical Center, Petah Tikva and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
The aim of this study was to investigate the expression of Ki-67, bcl-2 and p53 in parathyroid adenomas and their residual rim of normal parathyroid tissue. Specimens from 26 parathyroid adenomas were studied by immunohistochemical analysis for Ki-67, bcl-2 and p53 expression. Positive findings were noted for p53 in 4 (15%) adenomas and none of the residual rims of normal parathyroid tissue (p = 0.055); for Ki-67 in 15 (56%) adenomas and none of the residual rims of normal parathyroid tissue (p = 0.00002); and for bcl-2 in 19 (73%) adenomas and 8 (31%) residual rims of normal parathyroid tissue (p < 0.01). The high rate of Ki-67 expression may indicate susceptibility of parathyroid adenomas to clonal proliferation. The weak immunoreactive expression of p53, combined with a relatively strong expression of bcl-2, may contribute to the characteristic slow progression of these tumors.
Publication Type: Review;
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184.
Radiat Res 2005 Apr ; 4(163):384-90.
Medium from irradiated cells induces dose-dependent mitochondrial changes and BCL2 responses in unirradiated human keratinocytes.
Maguire P ,Mothersill C ,Seymour C ,Lyng FM ,
Radiation and Environmental Science Centre, Dublin Institute of Technology, Dublin, Ireland. pelcg_30@yahoo.com
Exposure of unirradiated human keratinocytes to irradiated cell conditioned medium (ICCM) is known to cause a cascade of events that leads to reproductive death and apoptosis. This study investigates the effect of ICCM on clonogenic survival, mitochondrial mass and BCL2 expression in unirradiated keratinocytes. Exposure to 5 mGy, 0.5 Gy and 5 Gy ICCM resulted in a significant decrease in clonogenic survival. Human keratinocytes incubated with ICCM containing an antioxidant, N-acetylcysteine, showed no significant decrease in clonogenic survival. HPV-G cells incubated with ICCM containing a caspase 9 inhibitor showed no significant decrease in clonogenic survival when the ICCM dose was < or =0.5 Gy. A significant increase in mitochondrial mass per cell was observed after exposure to 5 mGy and 0.5 Gy ICCM. A change in the distribution of the mitochondria from a diffuse cytoplasmic distribution to a more densely concentrated perinuclear distribution was also observed at these doses. No significant increase in mitochondrial mass or change in distribution of the mitochondria was found for 5 Gy ICCM. Low BCL2 expression was observed in HPV-G cells exposed to 5 mGy or 0.5 Gy ICCM, whereas a large significant increase in BCL2 expression was observed in cells exposed to 5 Gy ICCM. This study has shown that low-dose irradiation can cause cells to produce medium-borne signals that can cause mitochondrial changes and the induction of BCL2 expression in unirradiated HPV-G cells. The dose dependence of the mitochondrial changes and BCL2 expression suggests that the mechanisms may be aimed at control of response to radiation at the population level through signaling pathways.
Publication Type: Research Support, Non-U.S. Gov't;
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185.
Steroids 2005 Mar ; 3(70):153-60.
Estrogen receptor-alpha, bcl-2 and c-myc gene expression in fibroadenomas and adjacent normal breast: association with nodule size, hormonal and reproductive features.
Cericatto R ,Pozzobon A ,Morsch DM ,Menke CH ,Brum IS ,Spritzer PM ,
Laboratory of Molecular Endocrinology, Department of Physiology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rua Sarmento Leite, 500, Porto Alegre 90050-170, RS, Brazil.
Fibroadenomas are the most common benign lump in females. The study of gene alterations and/or deregulation in reproductive years may help explain hormonal physiological processes involved in nodule development and evolution. The objective was to compare ER-alpha, c-myc, and bcl-2 gene expression in breast fibroadenomas and in normal tissue and evaluate menstrual cycle, parity, and oral contraceptive influences. Fifty-seven premenopausal women (14-49 years) undergoing surgical removal of fibroadenomas were selected. Samples from fibroadenomas and circumjacent normal tissue were obtained for RT-PCR paired analysis. Patients were divided in groups according to menstrual cycle, use of contraceptives and parity. Tissue from 32 patients was adequate for RT-PCR. Paired analysis showed higher expression of ER-alpha (P=0.012) and bcl-2 (P=0.001) in fibroadenomas than in normal breast, while c-myc presented a similar expression (P=0.655). ER-alpha was higher in fibroadenomas of patients in follicular phase versus contraceptive users and normal tissue (P=0.003); bcl-2 was higher in fibroadenomas of patients in luteal phase than in the normal samples from all groups (P=0.007). c-myc did not differ according to menstrual cycle, but was higher in fibroadenomas>3 cm versus<3 cm (P=0.015) and in nulliparous women (P=0.04). A positive correlation between c-myc levels and fibroadenoma diameter was demonstrated (r=0.536; P=0.007). Nulliparous mean nodule diameter was superior than parous women (P=0.008). In conclusion, the expression of ER-alpha, bcl-2 and c-myc depends on hormonal and reproductive factors, with a possible contribution to lump formation and evolution.
Publication Type: Research Support, Non-U.S. Gov't;
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186.
Hum Mol Genet 2005 Apr 15; 8(14):1029-40.
Muscle-specific BCL2 expression ameliorates muscle disease in laminin {alpha}2-deficient, but not in dystrophin-deficient, mice.
Dominov JA ,Kravetz AJ ,Ardelt M ,Kostek CA ,Beermann ML ,Miller JB ,
Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472, USA. dominov@bbri.org
To examine the role of apoptosis in neuromuscular disease progression, we have determined whether pathogenesis in dystrophin-deficient (mdx) and laminin alpha2-deficient (Lama2-null) mice is ameliorated by overexpression of the anti-apoptosis protein BCL2 in diseased muscles. The mdx mice are a model for the human disease, Duchenne muscular dystrophy (DMD), and the Lama2-null mice are a model for human congenital muscular dystrophy type 1A (MDC1A). For these studies, we generated transgenic mice that overexpressed human BCL2 under control of muscle-specific MyoD or MRF4 promoter fragments. We then used cross-breeding to introduce the transgenes into diseased mdx or Lama2-null mice. In mdx mice, we found that overexpression of BCL2 failed to produce any significant differences in muscle pathology. In contrast, in the Lama2-null mice, we found that muscle-specific expression of BCL2 led to a several-fold increase in lifespan and an increased growth rate. Thus, BCL2-mediated apoptosis appears to play a significant role in pathogenesis of laminin alpha2 deficiency, but not of dystrophin deficiency, suggesting that therapies designed to ameliorate disease by inhibition of apoptosis are more likely to succeed in MDC1A than in DMD.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.;
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187.
World J Gastroenterol 2005 Feb 28; 8(11):1228-31.
Relationship between expression and distribution of cyclooxygenase-2 and bcl-2 in human gastric adenocarcinoma.
Chen XL ,Su BS ,Sun RQ ,Zhang J ,Wang YL ,
Department of Pathology, Second Hospital of Xi'an Jiaotong University, Xi'an 710004, Shaanxi Province, China. chenxiaoli64.student@sina.com
AIM: To explore expression and distribution features of COX-2 and bcl-2 in human gastric adenocarcinoma tissues and to study its biological significance. METHODS: Totally 36 human gastric carcinoma samples were enrolled in this study (cardiac adenocarcinoma 16 cases, distal gastric adenocarcinoma 20 cases). The expressions of COX-2 and bcl-2 in cancerous tissues and corresponding para-cancerous tissues were investigated by immunohistochemistry using COX-2 polyclonal antibody and bcl-2 monoclonal antibody. The normal gastric mucosa tissues were used as control. RESULTS: The expressions of COX-2 and bcl-2 in gastric carcinoma were significantly higher than that in the para-cancerous tissues (77.8% vs 47.2%, P<0.01, 80.56% vs 58.33%, P<0.05). The expression of COX-2 in cardiac adenocarcinoma was remarkably higher than that in the distal gastric carcinoma (93.8% vs 65.0%, P<0.01). The expression of COX-2 was mainly localized in the cytoplasm of tumor cells and partly in the nucleus. There is a transition of the COX-2 cytoplasmic positivity to nucleic in tumor cells with the increase of gastric carcinoma pathological grade. Interstitial macrophages, fibroblasts and vascular endothelial cells also expressed COX-2. The tissues with higher expression of COX-2 also expressed high level of bcl-2 protein. CONCLUSION: Abnormal expression pattern of COX-2 within the tissues of human gastric cancer is correlated with tumor location and lymph node metastasis. COX-2 may regulate expression of apoptosis suppressor gene (bcl-2) through interaction of tumor cells and stromal cells and play an important role in the generation and development of tumors, which will be of great help in developing new methods for antitumor therapy.
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188.
Oncogene 2005 May 5; 20(24):3339-47.
Arsenic trioxide (As(2)O(3)) induces apoptosis through activation of Bax in hematopoietic cells.
Zheng Y ,Yamaguchi H ,Tian C ,Lee MW ,Tang H ,Wang HG ,Chen Q ,
The Laboratory of Apoptosis and Cancer Biology, The National Key Laboratory of Biomembrane and Membrane Biotechnology, The Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China.
This study explores the roles of Bax and other Bcl-2 family members play in arsenic trioxide (As(2)O(3))-induced apoptosis. We showed that As(2)O(3) treatment triggered Bax conformational change and subsequent translocation from cytosol to mitochondria to form various multimeric homo-oligomers in IM-9 cells. On the other hand, human leukemic Jurkat cells deficient in Bax showed dramatically reduced apoptosis in response to As(2)O(3). Stable overexpression of Bcl-2 in IM-9 cells (IM-9/Bcl-2) inhibited As(2)O(3)-mediated Bax activation and apoptosis, and this inhibition could be partially averted by cell-permeable Bid-Bcl-2 homology (BH)3 peptide. Meanwhile, Bax conformational change and oligomerization induced by As(2)O(3) were not inhibited by the pancaspase inhibitor z-VAD-fmk, although Bid cleavage could be completely abolished. Bax activation by As(2)O(3) seemed to require stress-induced intracellular reactive oxygen species (ROS), since the ROS scavengers (N-acetyl-L-cysteine and lipoic acid) could completely block the conformational change and translocation of Bax from cytosol to mitochondria. These data suggest that As(2)O(3) might exert the cell killing in part by inducing Bax activation through a Bcl-2-suppressible pathway in hematopoietic cells that is caspase independent and intracellular ROS regulated.
Publication Type: Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.;
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189.
FEBS Lett 2005 Feb 28; 6(579):1469-76.
The flexible loop of Bcl-2 is required for molecular interaction with immunosuppressant FK-506 binding protein 38 (FKBP38).
Kang CB ,Tai J ,Chia J ,Yoon HS ,
Division of Structural and Computational Biology, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637511, Singapore.
Bcl-2 contains an unusually long loop between the first and the second helices. This loop has been shown to be highly flexible based on NMR and X-ray crystallographic analyses of this region. Bcl-2 is regulated at the posttranslational level through phosphorylation of specific residues within the flexible loop. The biological role and posttranslational modifications of the loop of Bcl-2 is currently unclear. FK-506 binding protein 38 (FKBP38) has been reported to interact with Bcl-2, suggesting that FKBP38 could act as a docking molecule to localize Bcl-2 at the mitochondrial membrane [Shirane, M. and Nakayama, K.I. (2003) Inherent calcineurin inhibitor FKBP38 targets Bcl-2 to mitochondria and inhibits apoptosis. Nat. Cell Biol. 5, 28-37]. Here, we investigated the molecular interaction between FKBP38 and Bcl-2, and demonstrated that Bcl-2 interacts with FKBP38 through the unstructured loop, and the interaction appears to regulate phosphorylation in the loop of Bcl-2.
Publication Type: Research Support, Non-U.S. Gov't;
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190.
Histopathology 2005 Mar ; 3(46):328-33.
CD10 and Bcl-2 expression combined with the International Prognostic Index can identify subgroups of patients with diffuse large-cell lymphoma with very good or very poor prognoses.
Biasoli I ,Morais JC ,Scheliga A ,Milito CB ,Romano S ,Land M ,Pulcheri W ,Spector N ,
Haematology and Pathology Services, University Hospital, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
AIMS: Diffuse large B-cell lymphoma (DLBCL) is characterized by marked biological heterogeneity. The identification of reproducible parameters that can be combined with the International Prognostic Index (IPI) to better predict outcome could lead to the development of effective risk-adaptive strategies. METHODS AND RESULTS: Bcl-2 and CD10 expression was determined by immunohistochemistry. The impact of the positivity on survival was evaluated in combination with the IPI in 86 patients with a confirmed diagnosis of DLBCL. Patients were divided according to the IPI into low-risk (no to two factors) or high-risk (three to five factors) groups. Positivity rates were 25% for CD10 and 42% for Bcl-2. In a Cox analysis, the high-risk IPI group [hazard ratio (HR) 5.98, P < 0.0001) and Bcl-2 expression (HR 2.43, P = 0.02) were independent poor prognostic factors, and expression of CD10 (HR 0.41, P = 0.052) predicted a favourable outcome. Among patients in the low-risk IPI group, CD10 positivity was associated with an excellent 8-year overall survival (92% versus 45%, P = 0.06). In the high-risk IPI group, Bcl-2 positivity identified a subgroup with invariably fatal disease. CONCLUSIONS: The expression of CD10 in the low-risk IPI group, and the expression of Bcl-2 in the high-risk IPI group can identify two subgroups of patients who might benefit from new risk-adaptive treatment approaches.
Publication Type: Research Support, Non-U.S. Gov't;
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191.
Histopathology 2005 Mar ; 3(46):270-9.
The expression of p53 and bcl-2 in gastrointestinal stromal tumours is associated with anatomical site, and p53 expression is associated with grade and clinical outcome.
Feakins RM ,
Department of Histopathology, Barts and the London NHS Trust and Queen Mary, University of London, London, UK. r.m.feakins@qmul.ac.uk
AIMS: To compare the expression of p53 and bcl-2 in gastrointestinal stromal tumours (GISTs) with anatomical site, National Institutes of Health (NIH) risk category (grade), pathological features, and clinical outcome. METHODS AND RESULTS: The immunohistochemical expression of p53 and bcl-2 in 105 GISTs (71 gastric, 20 small intestinal, four colonic, 10 rectal) was recorded. When all GISTs were assessed, there was p53 positivity in 28% and bcl-2 positivity in 77%. Gastric tumours had a lower prevalence of p53 positivity (20%) than intestinal (40-50%). Rectal GISTs had the lowest prevalence of bcl-2 positivity (20%) and gastric and small intestinal the highest (80% and 90%, respectively). In GISTs from all sites, p53 positivity was associated with size > 50 mm, epithelioid cell shape, nuclear atypia, mucosal invasion, and mitotic count > 5/50 high-power fields. In gastric GISTs the associations were the same, apart from size and mitotic count. In GISTs from all sites and in gastric GISTs, p53 expression correlated with NIH risk category. When GISTs from all sites were subjected to univariate survival analysis, an adverse outcome was associated with p53 positivity, NIH risk category, and several established prognostic factors. When gastric GISTs were assessed, the associations were similar although size was not prognostic. In multivariate survival analysis, p53 expression was independently prognostic for gastric GISTs in some models, while it was never independently prognostic for GISTs from all sites. Whether all GISTs or gastric GISTs were assessed, bcl-2 showed no association with clinical outcome or risk category. CONCLUSIONS: Anatomical site influences p53 and bcl-2 expression in GISTs. p53 expression is associated with NIH risk category, various pathological features, and clinical outcome, and may be independently prognostic for gastric GISTs. Bcl-2 expression has no prognostic value.
Publication Type: Comparative Study; Research Support, Non-U.S. Gov't;
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192.
Leukemia 2005 Apr ; 4(19):659-63.
Concurrent translocation of BCL2 and MYC with a single immunoglobulin locus in high-grade B-cell lymphomas.
Knezevich S ,Ludkovski O ,Salski C ,Lestou V ,Chhanabhai M ,Lam W ,Klasa R ,Connors JM ,Dyer MJ ,Gascoyne RD ,Horsman DE ,
Department of Pathology and Laboratory Medicine, British Columbia Cancer Agency, University of British Columbia, Vancouver, BC, Canada.
B-cell leukaemia or lymphoma with a combination of t(8;14)(q24;q32) of Burkitt leukaemia/lymphoma and t(14;18)(q32;q21) of follicular lymphoma may present clinically as de novo acute lymphoblastic leukaemia or transformation of follicular lymphoma to aggressive histology diffuse lymphoma. A number of cell lines have been reported with a complex t(8;14;18) with fusion of MYC, IGH and BCL2 on the same derivative 8 chromosome. The objective of this study was to determine the frequency and chromosomal features of this der(8)t(8;14;18) in a series of acute leukaemias and malignant lymphomas. A database of 1350 leukaemia and lymphoma karyotypes was searched for cases with structural alterations affecting both 8q24 and 18q21. A total of 55 cases were identified, of which eight revealed a complex der(8)t(8;14;18) with an MYC-IGH-BCL2 rearrangement resulting from translocation of BCL2 and MYC with a single disrupted IGH allele. Molecular cytogenetic investigation is essential to identify cases of high-grade leukaemia/lymphoma with concurrent translocations affecting the BCL2 and MYC loci.
Publication Type: Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.;
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193.
Mol Cell 2005 Feb 4; 3(17):393-403.
Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function.
Chen L ,Willis SN ,Wei A ,Smith BJ ,Fletcher JI ,Hinds MG ,Colman PM ,Day CL ,Adams JM ,Huang DC ,
The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville Victoria 3050, Australia.
Apoptosis is initiated when Bcl-2 and its prosurvival relatives are engaged by proapoptotic BH3-only proteins via interaction of its BH3 domain with a groove on the Bcl-2-like proteins. These interactions have been considered promiscuous, but our analysis of the affinity of eight BH3 peptides for five Bcl-2-like proteins has revealed that the interactions vary over 10,000-fold in affinity, and accordingly, only certain protein pairs associate inside cells. Bim and Puma potently engaged all the prosurvival proteins comparably. Bad, however, bound tightly to Bcl-2, Bcl-xL, and Bcl-w but only weakly to A1 and not to Mcl-1. Strikingly, Noxa bound only Mcl-1 and A1. In accord with their complementary binding, Bad and Noxa cooperated to induce potent killing. The results suggest that apoptosis relies on selective interactions between particular subsets of these proteins and that it should be feasible to discover BH3-mimetic drugs that inactivate specific prosurvival targets.
Publication Type: In Vitro; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.;
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194.
Mol Cancer Ther 2005 Jan ; 1(4):23-31.
(-)-Gossypol acts directly on the mitochondria to overcome Bcl-2- and Bcl-X(L)-mediated apoptosis resistance.
Oliver CL ,Miranda MB ,Shangary S ,Land S ,Wang S ,Johnson DE ,
Department of Otolaryngology, University of Pittsburgh Eye and Ear, 200 Lothrop Street, Suite 500 Pittsburgh, PA 15213-2546, USA. olivercl@msx.upmc.edu
Aberrant overexpression of antiapoptotic members of the Bcl-2 protein family, including Bcl-2 and Bcl-X(L), contributes to malignant transformation and subsequent resistance to traditional chemotherapeutics. Thus, these proteins represent attractive targets for novel anticancer agents. The small molecule, gossypol, was initially investigated as a contraceptive agent, but subsequently has been shown to possess anticancer properties in vitro and in vivo. Recently gossypol has been found to bind to Bcl-X(L) and, with less affinity, to Bcl-2. Here we investigate the ability of the (-) enantiomer of gossypol, (-)-gossypol, to overcome the apoptosis resistance conferred by Bcl-2 or Bcl-X(L) overexpression in Jurkat T leukemia cells. (-)-Gossypol potently induced cell death in Jurkat cells overexpressing Bcl-2 (IC50, 18.1+/-2.6 micromol/L) or Bcl-X(L) (IC50, 22.9+/-3.7 micromol/L). Vector-transfected control cells were also potently killed by (-)-gossypol (IC50, 7.0+/-2.7 micromol/L). By contrast, the chemotherapy drug etoposide only induced efficient killing of vector-transfected cells (IC50, 9.6+/-2.3 micromol/L). Additionally, (-)-gossypol was more efficient than etoposide at inducing caspase-3 activation and phosphatidylserine externalization in the setting of Bcl-2 or Bcl-X(L) overexpression. (-)-Gossypol-induced apoptosis was associated with Bak activation and release of cytochrome c from mitochondria, suggesting a mitochondrial-mediated apoptotic mechanism. Moreover, (-)-gossypol treatment of isolated mitochondria purified from Bcl-2-overexpressing cells also resulted in cytochrome c release, indicating a possible direct action on Bcl-2 present in the mitochondrial outer membrane. Taken together, these results suggest that (-)-gossypol is a potent and novel therapeutic able to overcome apoptosis resistance by specifically targeting the activity of antiapoptotic Bcl-2 family members. (-)-Gossypol may be a promising new agent to treat malignancies that are resistant to conventional therapies.
Publication Type: Comparative Study; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.;
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195.
Mol Cancer Ther 2005 Jan ; 1(4):13-21.
Preclinical studies of a nonpeptidic small-molecule inhibitor of Bcl-2 and Bcl-X(L) [(-)-gossypol] against diffuse large cell lymphoma.
Mohammad RM ,Wang S ,Aboukameel A ,Chen B ,Wu X ,Chen J ,Al-Katib A ,
Department of Internal Medicine, Division of Hematology and Oncology, Karmanos Cancer Institute, Wayne State University School of Medicine, 724 HWCRC, 4100 John R. Street, Detroit, MI 48201, USA. Mohammad@karmanos.org
Overexpression of Bcl-2/Bcl-X(L) protein has been observed in more than 80% of B-cell lymphomas. Diffuse large cell lymphoma (DLCL) is the most common subtype of non-Hodgkin's lymphoma. (-)-Gossypol, a natural product isolated from cottonseeds, was discovered as a potent small-molecule inhibitor of Bcl-2 and Bcl-X(L) proteins, with a Ki value in the nanomole per liter range for both. In vitro, (-)-gossypol showed significant growth inhibition effect against WSU-DLCL2 lymphoma cell line and fresh cells obtained from a lymphoma patient with no effect on normal peripheral blood lymphocytes. As expected (-)-gossypol induced complete cytochrome c release from mitochondria, increased caspases-3 and -9 activity, and caused apoptotic death without affecting protein levels of Bcl-2, Bcl-X(L), Bax, and Bak. The addition of cyclophosphamide-Adriamycin-vincristine-prednisolone (CHOP) regimen to lymphoma cells preexposed to (-)-gossypol enhanced killing significantly. The maximum tolerated dose of (-)-gossypol in severe combined immunodeficient (SCID) mice was 40 mg/kg for three i.v. injections when given alone and 20 mg/kg x 3 when given in combination with CHOP. Using WSU-DLCL2-SCID mouse xenograft model, the tumor growth inhibition, the tumor growth delay, and the log10 kill of mice treated with (-)-gossypol + CHOP were better than CHOP or (-)-gossypol alone. We conclude that adding Bcl-2/Bcl-X(L) small-molecule inhibitor to standard chemotherapy may prove an effective strategy in lymphoma therapy.
Publication Type: Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.;
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196.
Rocz Akad Med Bialymst 2004 ; (49 Suppl 1):22-4.
Bcl-2 and Bax protein expression in human optic nerve axons in the eyeballs after severe trauma and in the eyes with absolute glaucoma.
Zalewska R ,Reszeć J ,Mariak Z ,Sulkowski S ,Proniewska-Skretek E ,
Department of Ophthalmology, Medical University of Bialystok, Poland. renata-zalewska@wp.pl
The aim of our study was to evaluate the immunohistochemical expression of Bcl-2 and Bax protein in optic nerve axons after severe eyeball injury and in the eyes with absolute glaucoma. A series of 19 eyeballs, enucleated because of absolute glaucoma and 41 eyeballs, enucleated, following extensive injury, at the Department of Ophthalmology of the Medical University of Bialystok, were taken into our study. The immunohistochemical reaction was performed with Bcl-2 and Bax protein antibodies and by the LSAB technique. DAB was used in order to visualise the reaction. The optic nerve axons in glaucomatous eyeballs showed statistically significant higher Bax protein expressions than those of Bcl-2 proteins. In the optic nerve axons after severe eyeball injury, a non-significantly higher Bcl-2 protein expression was observed.
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197.
J Immunol 2005 Jan 15; 2(174):671-9.
Phagocytosis-induced apoptosis in macrophages is mediated by up-regulation and activation of the Bcl-2 homology domain 3-only protein Bim.
Kirschnek S ,Ying S ,Fischer SF ,Häcker H ,Villunger A ,Hochrein H ,Häcker G ,
Institute for Medical Microbiology, Immunology and Hygiene, Technical University Munich, Munich, Germany.
Cell death by apoptosis is important in immune cell homeostasis and in the defense against infectious microorganisms. The physiological event of uptake and intracellular destruction of bacteria is a powerful apoptotic stimulus to macrophages and neutrophil granulocytes. In this study, we provide a molecular analysis of phagocytosis-induced apoptosis. Apoptosis was blocked by Bcl-2 in a mouse macrophage cell line and in primary mouse macrophages. Analysis of the upstream mechanisms revealed that apoptosis was triggered by the Bcl-2 homology domain 3-only protein Bim/Bod. Contact with bacteria or bacterial components induced a strong increase in Bim-expression through TLR and MyD88. Inhibition of the MAPK p38 and JNK reduced both up-regulation of Bim and apoptosis. Phosphorylation of Bim was further observed in mouse macrophages, which appeared to be the result of TLR-dependent phosphatase inhibition. Although TLR-induced Bim was, unlike Bim in resting cells, not bound to the microtubuli cytoskeleton, the up-regulation of Bim was not sufficient to cause apoptosis. A second signal was required that was generated in the process of phagocytosis. Phagocytosis-induced apoptosis was strongly reduced in Bim(-/-) macrophages. These data provide the molecular context of a form of apoptosis that may serve to dispose of terminally differentiated phagocytes.
Publication Type: Research Support, Non-U.S. Gov't;
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198.
World J Gastroenterol 2005 Jan 14; 2(11):204-7.
Specific COX-2 inhibitor NS398 induces apoptosis in human liver cancer cell line HepG2 through BCL-2.
Huang DS ,Shen KZ ,Wei JF ,Liang TB ,Zheng SS ,Xie HY ,
Department of Hepatobiliary Pancreatic Surgery, The First Affiliated Hospital, Zhejiang University, College of Medicine, Hangzhou 310003, Zhejiang Province, China.
AIM: To evaluate the effects of NS-398, a cyclooxygenase-2 (COX-2) inhibitor, on the proliferation and apoptosis of HepG2 cells. METHODS: The effects of NS-398 on the proliferation of HepG2 cells were evaluated by MTT. DNA fragmentation gel analysis was used to analyze the apoptotic cells. DNA ploidy and apoptotic cell percentage were calculated by flow cytometry. The expression of COX-2 and Bcl-2 mRNA was identified by competitive RT-PCR. Furthermore, expression level of Bcl-2 was detected using Western blot in HepG2 after treated with NS-398. RESULTS: NS-398 inhibited cell proliferation and induced apoptosis of HepG2 cells in a concentration-dependent manner. DNA ploidy analysis showed that S phase cells were significantly decreased with increase of NS-398 concentration. The quiescent G0/G1 phase was accumulated with decrease of Bcl-2 mRNA. Whereas NS-398 had no effect on the expression of COX-2 mRNA, and no correlations were found between COX-2 mRNA and HepG2 cell proliferation and apoptosis induced by NS-398 (r = 0.056 and r = 0.119, respectively). Bcl-2 protein level was inhibited after treated with NS-398. CONCLUSION: NS-398 significantly inhibits the proliferation and induces apoptosis of HepG2 cells. Mechanisms involved may be accumulation of quiescent G0/G1 phase and decrease of Bcl-2 expression.
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199.
Nat Cell Biol 2005 Feb ; 2(7):137-47.
Bcl-2 expression suppresses mismatch repair activity through inhibition of E2F transcriptional activity.
Youn CK ,Cho HJ ,Kim SH ,Kim HB ,Kim MH ,Chang IY ,Lee JS ,Chung MH ,Hahm KS ,You HJ ,
Department of Pharmacology, School of medicine, Chosun University, 375 Seusuk-dong, Gwangju 501-759, South Korea.
Bcl-2 stimulates mutagenesis after the exposure of cells to DNA-damaging agents. However, the biological mechanisms of Bcl-2-mediated mutagenesis have remained largely obscure. Here we demonstrate that the Bcl-2-mediated suppression of hMSH2 expression results in a reduced cellular capacity to repair mismatches. The pathway linking Bcl-2 expression to the suppression of mismatch repair (MMR) activity involves the hypophosphorylation of pRb, and then the enhancement of the E2F-pRb complex. This is followed by a decrease in hMSH2 expression. MMR has a key role in protection against deleterious mutation accumulation and in maintaining genomic stability. Therefore, the decreased MMR activity by Bcl-2 may be an underlying mechanism for Bcl-2-promoted oncogenesis.
Publication Type: Research Support, Non-U.S. Gov't;
Comment In: Nat Cell Biol. 2005 Apr;7(4):326-7
Nat Cell Biol. 2005 Apr;7(4):326-7
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200.
Proc Natl Acad Sci U S A 2005 Jan 4; 1(102):105-10.
Proapoptotic BAX and BAK regulate the type 1 inositol trisphosphate receptor and calcium leak from the endoplasmic reticulum.
Oakes SA ,Scorrano L ,Opferman JT ,Bassik MC ,Nishino M ,Pozzan T ,Korsmeyer SJ ,
Howard Hughes Medical Institute, Dana-Farber Cancer Institute, Brigham and Women's Hospital, Departments of Pathology and Medicine, Harvard Medical School, Boston, MA 02115, USA.
Proapoptotic BCL-2 family members BAX and BAK are required for the initiation of mitochondrial dysfunction during apoptosis and for maintaining the endoplasmic reticulum (ER) Ca(2+) stores necessary for Ca(2+)-dependent cell death. Conversely, antiapoptotic BCL-2 has been shown to decrease Ca(2+) concentration in the ER. We found that Bax(-/-)Bak(-/-) double-knockout (DKO) cells have reduced resting ER Ca(2+) levels because of increased Ca(2+) leak and an increase in the Ca(2+)-permeable, hyperphosphorylated state of the inositol trisphosphate receptor type 1 (IP3R-1). The ER Ca(2+) defect of DKO cells is rescued by RNA interference reduction of IP3R-1, supporting the argument that this channel regulates the increased Ca(2+) leak in these cells. BCL-2 and IP3R-1 physically interact at the ER, and their binding is increased in the absence of BAX and BAK. Moreover, knocking down BCL-2 decreases IP3R-1 phosphorylation and ER Ca(2+) leak rate in the DKO cells. These findings support a model in which BCL-2 family members regulate IP3R-1 phosphorylation to control the rate of ER Ca(2+) leak from intracellular stores.
Publication Type: Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.;
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201.
J Mol Med 2005 Apr ; 4(83):296-307.
Downregulation of Skp2 and p27/Kip1 synergistically induces apoptosis in T98G glioblastoma cells.
Lee SH ,McCormick F ,
Cancer Research Institute and Comprehensive Cancer Center, University of California, San Francisco, CA 94143, USA.
S-phase kinase associated protein (Skp) 2 is an F-box protein required for substrate recognition of the SCF(Skp2) ubiquitin ligase complex. Skp2 is often overexpressed in transformed cells and in various types of tumors. Downregulation or inhibition of Skp2 inhibits growth of breast cancer cells and small-cell lung carcinoma cells. We downregulated Skp2 in T98G glioblastoma cells using small interfering RNA (siRNA). Downregulation induced p27 and caused growth arrest and apoptosis. Downregulation of both Skp2 and p27 increased apoptosis synergistically. Cyclin E levels and cyclin E-CDK2 kinase activity increased dramatically when both Skp2 and p27 were downregulated. Coincidently, Bcl-2 but not Bcl-xL expression decreased, and caspase-3 was activated. Inhibition of cyclin E-CDK2 kinase activity by forced expression of p21 reversed these effects. Moreover, stable expression of Bcl-2 also abrogated apoptosis induced by downregulation of Skp2 and p27. We suggest that Skp2 in tumor cells suppresses apoptosis through Bcl-2 expression, potentially through regulation of cyclin E-CDK2 activity.
Publication Type: Research Support, Non-U.S. Gov't;
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202.
Arch Virol 2004 Sep ; 9(149):1745-59.
Human papillomavirus type 16 E5 protein colocalizes with the antiapoptotic Bcl-2 protein.
Auvinen E ,Alonso A ,Auvinen P ,
Department of Virology, Helsinki University Central Hospital, University of Helsinki, Helsinki, Finland. eeva.auvinen@helsinki.fi
Human papillomavirus type 16 E5 protein contributes to cellular transformation by increasing the mitogenic stimulus from growth factor receptors to the nucleus. In order to study the biological mechanisms of the E5 protein we performed site-directed mutagenesis of the E5 gene. Wild-type as well as mutant E5 proteins were transiently expressed in human cervical epithelial cells, and cell morphology, expression of proteins involved in cell adhesion, and localization of the different proteins were studied. Little differences in cell morphology or expression kinetics were observed between the different E5 proteins, except for relocalization of a mutant E5 protein where a hydrophobic leucine membrane anchor was mutated to positively charged amino acids. This mutant E5 protein localized to lamellipodia, which are motility-associated structures at the leading edge of motile cells. In our experimental conditions, 100% of E5-expressing epithelial cells died by four days of expression, possibly due to toxicity or disturbance of the membrane compartment by the E5 protein. Most interestingly, a remarkable colocalization of the E5 protein with the Bcl-2 antiapoptotic protein on intracellular membranes was established.
Publication Type: Research Support, Non-U.S. Gov't;
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203.
J Neurochem 2004 Dec ; 6(91):1275-83.
BCL-2-induced glioma cell invasiveness depends on furin-like proteases.
Wick W ,Wild-Bode C ,Frank B ,Weller M ,
Laboratory of Molecular Neuro-Oncology, Department of General Neurology, Hertie Institute for Clinical Brain Research, University of Tübingen, Medical School, Tübingen, Germany. wolfgang.wick@uni-tuebingen.de
Migration and invasion are prerequisites for the neoplastic phenotype of malignant glioma. Ectopic expression of BCL-2 enhances migration and invasion of glioma cells and promotes their synthesis of transforming growth factor-beta2 (TGF-beta2). We here report that BCL-2-expressing cells show enhanced expression and activity of the proprotein convertase, furin, which processes metalloproteinases (MMP) and TGF-beta. Consistent with a biological role for a BCL-2-dependent increase in furin-like protease (FLP) activity, BCL-2-expressing cells exhibit enhanced MMP activity. Both a pseudosubstrate furin inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk), or alpha 1-anti-trypsin Portland (PDX), a recombinant furin-inhibitory protein, suppress constitutive and BCL-2-mediated MMP activity and invasion. This inhibition is not overcome by TGF-beta or hepatocyte growth factor (HGF). A neutralizing TGF-beta antibody attenuates, but not abrogates, the invasive properties conferred by exogenous expression of BCL-2, whereas the MMP inhibitor o-phenantroline (o-PA) abolishes the pro-invasive action of BCL-2. Exogenous HGF results in enhanced, and expression of dominant-negative ezrin in reduced, FLP activity, and dec-RVKR-cmk blunts the HGF-induced expression of mature TGF-beta2. Consequently, HGF and BCL-2 family proteins use a furin-dependent pathway to promote invasion via TGF-beta and MMP in human malignant glioma cells and the pro-invasive properties of TGF-beta require furin- dependent MMP activity.
Publication Type: Research Support, Non-U.S. Gov't;
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204.
Melanoma Res 2004 Dec ; 6(14):543-6.
Serum bcl-2 and survivin levels in melanoma.
Tas F ,Duranyildiz D ,Argon A ,Oguz H ,Camlica H ,Yasasever V ,Topuz E ,
Institute of Oncology, University of Istanbul, Capa, Istanbul, Turkey. faruktas2002@yahoo.com
This study was conducted to investigate the serum levels of bcl-2 and survivin in patients with melanoma and the relationship with tumour progression and known prognostic parameters. Forty-four patients with cutaneous melanoma were investigated. Serum samples were obtained on first admission before adjuvant and metastatic treatment were given and at follow-up. Serum bcl-2 and survivin levels were determined using enzyme immunometric assay (EIA) and enzyme-linked immunosorbent assay (ELISA). The baseline serum bcl-2 levels were significantly higher in patients with melanoma than in the control group (P=0.01). For the serum survivin levels, no difference was found (P=0.6). No significant correlations were found between the prognostic parameters analysed and the serum survivin concentrations. The same was true of the serum bcl-2 values, except for the age of the patient (P=0.025) and nodal involvement (P=0.003). No significant relationship was found between the serum levels of bcl-2 and survivin (r=-0.13, P=0.4). In node-positive patients (n=8) both of these anti-apoptotic substances were unchanged after interferon-alpha-2b therapy. However, serum survivin concentrations were significantly increased in 10 patients with metastatic melanoma who underwent dacarbazine (DTIC)-based cytotoxic chemotherapy (P=0.047). A similar finding was not determined for the serum bcl-2 levels. In conclusion, the results of this study suggest that decreased apoptosis is associated partly with an increase in serum bcl-2. However, much research continues in this field, and exciting new knowledge will ultimately emerge.
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205.
Mol Cell 2004 Dec 3; 5(16):807-18.
The first alpha helix of Bax plays a necessary role in its ligand-induced activation by the BH3-only proteins Bid and PUMA.
Cartron PF ,Gallenne T ,Bougras G ,Gautier F ,Manero F ,Vusio P ,Meflah K ,Vallette FM ,Juin P ,
INSERM U601, 9 Quai Moncousu, 44035 Nantes Cedex 035, France.
The mechanism by which some BH3-only proteins of the Bcl-2 family directly activate the "multidomain" proapoptotic member Bax is poorly characterized. We report that the first alpha helix (Halpha1) of Bax specifically interacts with the BH3 domains of Bid and PUMA but not with that of Bad. Inhibition of this interaction, by a peptide comprising Halpha1 or by a mutation in this helix, prevents ligand-induced activation of Bax by Bid, PUMA, or their BH3 peptides. Halpha1-mutated Bax, which can mediate death induced by Bad or its BH3 peptide, does not mediate that induced by Bid, PUMA, or their BH3 peptides. The response of Halpha1-mutated Bax to Bid can be restored by a compensating mutation in Bid BH3. Thus, a specific interaction between Bax Halpha1 and their BH3 domains allows Bid and PUMA to function as "death agonists" of Bax, whereas Bad recruits Bax activity through a distinct pathway.
Publication Type: Research Support, Non-U.S. Gov't;
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206.
J Clin Endocrinol Metab 2005 Feb ; 2(90):953-61.
Progesterone receptor modulator CDB-2914 down-regulates proliferative cell nuclear antigen and Bcl-2 protein expression and up-regulates caspase-3 and poly(adenosine 5'-diphosphate-ribose) polymerase expression in cultured human uterine leiomyoma cells.
Xu Q ,Takekida S ,Ohara N ,Chen W ,Sitruk-Ware R ,Johansson ED ,Maruo T ,
Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, Chuo-Ku, Kobe 650-0017, Japan.
The present study was conducted to evaluate the effects of the progesterone receptor modulator CDB-2914 on proliferative activity and apoptosis in cultured human uterine leiomyoma cells. Isolated leiomyoma cells were subcultured in phenol red-free DMEM supplemented with 10% fetal bovine serum for 120 h and then stepped down to serum-free conditions for 12, 24, 48, and 96 h in the absence or presence of graded concentrations of CDB-2914 (10(-9), 10(-8), 10(-7), and 10(-6) M). The number of viable cultured leiomyoma cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazodium bromide assay. Proliferating cell nuclear antigen (PCNA) expression was evaluated by immunocytochemistry and Western blot analysis. Apoptosis was examined by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) assay. Caspase-3, cleaved poly(ADP-ribose) polymerase (PARP), and Bcl-2 expression were assessed by Western blot analysis. Compared with untreated control cultures, treatment with CDB-2914 decreased the number of viable cultured leiomyoma cells and the PCNA-positive rate in those cells and increased the TUNEL-positive rate in cultured leiomyoma cells in a dose-dependent manner. Western blot analysis revealed that treatment with CDB-2914 significantly decreased the expression of PCNA and Bcl-2 protein and increased the expression of cleaved caspase-3 and cleaved PARP in a dose-dependent manner compared with untreated control cultures. These results suggest that CDB-2914 inhibits the proliferation of cultured leiomyoma cells by down-regulating PCNA expression and induces apoptosis by up-regulating cleaved caspase-3 and PARP expression and down-regulating Bcl-2 protein expression in those cells.
Publication Type: Research Support, Non-U.S. Gov't;
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207.
Mol Cancer Res 2004 Nov ; 11(2):620-31.
Bcl-2 overexpression leads to increases in suppressor of cytokine signaling-3 expression in B cells and de novo follicular lymphoma.
Vanasse GJ ,Winn RK ,Rodov S ,Zieske AW ,Li JT ,Tupper JC ,Tang J ,Raines EW ,Peters MA ,Yeung KY ,Harlan JM ,
Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street, WWW-403, Box 208021, New Haven, CT 06520, USA. gary.vanasse@yale.edu
The t(14;18)(q32;q21), resulting in deregulated expression of B-cell-leukemia/lymphoma-2 (Bcl-2), represents the genetic hallmark in human follicular lymphomas. Substantial evidence supports the hypothesis that the t(14;18) and Bcl-2 overexpression are necessary but not solely responsible for neoplastic transformation and require cooperating genetic derangements for neoplastic transformation to occur. To investigate genes that cooperate with Bcl-2 to influence cellular signaling pathways important for neoplastic transformation, we used oligonucleotide microarrays to determine differential gene expression patterns in CD19+ B cells isolated from Emu-Bcl-2 transgenic mice and wild-type littermate control mice. Fifty-seven genes were induced and 94 genes were repressed by > or =2-fold in Emu-Bcl-2 transgenic mice (P < 0.05). The suppressor of cytokine signaling-3 (SOCS3) gene was found to be overexpressed 5-fold in B cells from Emu-Bcl-2 transgenic mice. Overexpression of Bcl-2 in both mouse embryo fibroblast-1 and hematopoietic cell lines resulted in induction of SOCS3 protein, suggesting a Bcl-2-associated mechanism underlying SOCS3 induction. Immunohistochemistry with SOCS3 antisera on tissue from a cohort of patients with de novo follicular lymphoma revealed marked overexpression of SOCS3 protein that, within the follicular center cell region, was limited to neoplastic follicular lymphoma cells and colocalized with Bcl-2 expression in 9 of 12 de novo follicular lymphoma cases examined. In contrast, SOCS3 protein expression was not detected in the follicular center cell region of benign hyperplastic tonsil tissue. These data suggest that Bcl-2 overexpression leads to the induction of activated signal transducer and activator of transcription 3 (STAT3) and to the induction of SOCS3, which may contribute to the pathogenesis of follicular lymphoma.
Publication Type: Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.;
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208.
J Cell Biochem 2005 Feb 15; 3(94):611-26.
BMRP is a Bcl-2 binding protein that induces apoptosis.
Chintharlapalli SR ,Jasti M ,Malladi S ,Parsa KV ,Ballestero RP ,González-García M ,
Departments of Biology and Chemistry, Texas A&M University-Kingsville, 700 University Blvd., Kingsville, Texas 78363, USA.
Members of the Bcl-2 family of proteins play important roles in the regulation of cell death by apoptosis. The yeast Two-Hybrid system was utilized to identify a protein that interacts with the anti-apoptotic protein Bcl-2, designated BMRP. This protein corresponds to a previously known mitochondrial ribosomal protein (MRPL41). Binding experiments confirmed the interaction of BMRP to Bcl-2 in mammalian cells. Subcellular fractionation by differential centrifugation studies showed that both Bcl-2 and BMRP are localized to the same fractions (fractions that are rich in mitochondria). Northern blot analysis revealed a major bmrp mRNA band of approximately 0.8 kb in several human tissues. Additionally, a larger 2.2 kb mRNA species was also observed in some tissues. Western blot analysis showed that endogenous BMRP runs as a band of 16-17 kDa in SDS-PAGE. Overexpression of BMRP induced cell death in primary embryonic fibroblasts and NIH/3T3 cells. Transfection of BMRP showed similar effects to those observed by overexpression of the pro-apoptotic proteins Bax or Bad. BMRP-stimulated cell death was counteracted by co-expression of Bcl-2. The baculoviral caspase inhibitor p35 also protected cells from BMRP-induced cell death. These findings suggest that BMRP is a mitochondrial ribosomal protein involved in the regulation of cell death by apoptosis, probably affecting pathways mediated by Bcl-2 and caspases.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.;
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209.
IUBMB Life 2004 Aug ; 8(56):483-90.
Emerging role of p53, bcl-2 and telomerase activity in Egyptian breast cancer patients.
Swellam M ,Ismail M ,Eissa S ,Hamdy M ,Mokhtar N ,
Biochemistry Department, National Research Center, Dokki, Giza, Egypt. menha_m_swellam@hotmail.com
Apoptotic cell death represents an important mechanism for the precise regulation of cell numbers, and a defense mechanism against tumor cells. Both bcl-2 and mutant p53 gene products have been involved in apoptotic pathways. On the other hand, cell proliferation capacity and tumorgenesis have been controlled by telomerase. The purpose of our study is to assess the prognostic significance of additional markers implicated in apoptosis and tumorgenesis. Fifty-one fresh tissue samples of primary breast carcinoma and 26 tissue samples of benign breast lesions were included in this study. Expression of bcl-2 in cell lysates and mutant p53 protein in nuclear fraction were measured by Oncogene Science EIA procedures. Telomerase activity was analyzed using the Telomerase-PCR-ELISA based on the TRAP (telomerase repeat amplification protocol) method. On the same specimens, steroid hormone receptors (ER and PgR) were measured in cytosol fraction using Abbott EIA assays. In addition, information regarding surgical-pathological features of the tumor was obtained. Univariate and Multivariate analysis was done to identify variables predictive of poor prognosis. Significant expression of bcl-2, mutant p53 proteins and relative telomerase activity were observed in malignant cases when compared to benign ones. Univariate analysis revealed significant association in the level of both mutant p53 and relative telomerase activity with tumor size and disease recurrence. Moreover, telomerase activity was significantly expressed in late stages than early ones. Multivariate analysis revealed that bcl-2, mutant p53, telomerase activity, PgR and age were independent prognostic factors. Among a panel of molecular genetic factors investigated, mutant p53 and relative telomerase activity were strongly associated with disease recurrence; hence they exert a significant prognostic role in breast cancer.
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210.
Regul Pept 2005 Jan 15; 1-3(124):45-51.
Overexpression of Bcl-2 inhibits nuclear localization of annexin I during tumor necrosis factor-alpha-mediated apoptosis in porcine renal LLC-PK1 cells.
Ishido M ,
Endocrine Disruptors Research Projects, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba 305-8506, Japan. ishidou@nies.go.jp
The addition of tumor necrosis factor (TNF)-alpha into the cultured porcine kidney LLC-PK1 cells caused apoptosis concomitantly with caspase-3 activation and the inductions of an endogenous Bcl-2 protein. An SDS-polyacrylamide electrophoretic analysis revealed that a 37-kDa protein in a nuclear fraction was increased during TNF-alpha-induced apoptosis. Partial amino acid sequence of the protein was A-L-T-G-H-L-E-E-V, perfectly matching that of annexin I. Immunocytochemistry revealed that annexin I migrated to the nucleus and/or peri-nucleus region upon exposure to TNF-alpha. Overexpression of Bcl-2 proteins inhibited the nuclear localization of annexin I during TNF-alpha-induced apoptosis. Antisense oligodeoxynucleotides complementary to annexin I-inhibited TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labelling) staining in TNF-alpha-treated cells, suggesting that annexin I expression is a possible prerequisite for the induction of apoptosis by the cytokine. Thus, it is first time to show that annexin I is regulated by an anti-apoptotic Bcl-2 protein in TNF-alpha-induced renal apoptotic events.
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211.
J Dermatol Sci 2004 Dec ; 3(36):183-5.
Dominant Bcl-2 expression during telogen-anagen transition phase in human hair.
Soma T ,Hibino T ,
Publication Type: Letter;
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212.
J Immunol 2004 Nov 15; 10(173):6179-88.
B cells expressing Bcl-2 and a signaling-impaired BAFF-specific receptor fail to mature and are deficient in the formation of lymphoid follicles and germinal centers.
Rahman ZS ,Manser T ,
Department of Microbiology and Immunology and The Kimmel Cancer Institute, Thomas Jefferson Medical College, Philadelphia, PA 19017, USA.
The TNF family cytokine B cell-activating factor belonging to the TNF family (BAFF) (BLyS) plays a fundamental role in regulating peripheral B cell survival and homeostasis. A BAFF-specific receptor (BAFF-R; BR3) appears to mediate these functions via activation of the NF-kappaB2 pathway. Signaling by the BAFF-R is also required to sustain the germinal center (GC) reaction. Engagement of this receptor results in the induction of Bcl-2, suggesting that this antiapoptotic factor acts downstream of the BAFF-R and NF-kappaB2 pathway to promote peripheral B cell survival during primary and Ag-driven development. To test this idea, we created lines of mice coexpressing a Bcl-2 transgene and a signaling-deficient form of the BAFF-R derived from the B lymphopenic A/WySnJ strain. Surprisingly, although dramatically elevated numbers of B cells accumulate in the periphery of these mice, these B cells exhibit extremely perturbed primary development, formation of lymphoid microenvironments, and GC and IgG responses. Moreover, mice expressing the bcl-2 transgene alone display a loss of marginal zone B cells, an expansion of follicular B cells that appear immature, and alterations of the GC reaction. These results suggest that the BAFF-R and Bcl-2 regulate key and nonoverlapping aspects of peripheral B cell survival and development.
Publication Type: Research Support, U.S. Gov't, P.H.S.;
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213.
Cancer Res 2004 Nov 1; 21(64):7947-53.
Breast cancer cells can evade apoptosis-mediated selective killing by a novel small molecule inhibitor of Bcl-2.
Real PJ ,Cao Y ,Wang R ,Nikolovska-Coleska Z ,Sanz-Ortiz J ,Wang S ,Fernandez-Luna JL ,
Unidad de Genetica Molecular and Servicio de Oncologia Medica, Hospital Universitario Marques de Valdecilla, Servicio Cantabro de Salud, Santander, Spain.
Proteins of the Bcl-2 family are key regulators of caspase activation and apoptosis. Some members of this family, notably Bcl-2 and Bcl-x(L), are overexpressed in cancer cells, which have been associated with chemoresistance. We have designed and synthesized a small molecule inhibitor of Bcl-2, named YC137, and studied its role in cancer cells. In vitro studies showed that YC137 inhibits the binding of the Bid BH3 peptide to Bcl-2, thus disrupting an interaction essential for the antiapoptotic activity of Bcl-2. This inhibitor induces apoptosis of hematopoietic progenitors overexpressing Bcl-2 but not Bcl-x(L) and breast cancer cells that express high levels of Bcl-2. On the contrary, a variety of normal primary cells, including CD34(+) progenitors, myoblasts, and peripheral blood mononuclear cells, do not respond to the inhibitor. A breast cancer cell line resistant to YC137 was generated. Analysis of resistant cells revealed a reduced expression of Bcl-2, which correlated with low activation of signal transducer and activator of transcription-3 (Stat3) and reduced expression of the human epidermal growth factor receptor-2 (HER2). Of note, YC137-resistant cells were more sensitive to apoptosis induced by chemotherapy. Because HER2 has not been linked previously to the Stat3-Bcl-2 transcriptional pathway, we additionally confirmed that specific blockade of HER2 in breast cancer cells resulted in down-regulation of Stat3 activity and reduced levels of Bcl-2. Consistently, HER2 blockade led to YC137 resistance. These data provide evidence for the selective killing of tumor cells by YC137 and represent the first example of in vitro selection of cancer cells refractory to a Bcl-2 inhibitor.
Publication Type: Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.;
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214.
Mol Cancer Res 2004 Oct ; 10(2):551-6.
Bcl-2-mediated cell survival promotes metastasis of EpH4 betaMEKDD mammary epithelial cells.
Pinkas J ,Martin SS ,Leder P ,
Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, USA. jan.pinkas@genzyme.com.
The majority of patients who succumb to cancer die from metastatic disease progression rather than from the primary tumor. Elucidation of the mechanisms underlying tissue-specific metastasis is essential to the development of effective therapies. The mitogen-activated protein kinase kinase (MEK) pathway is frequently activated in human tumors and has been shown to regulate genes involved in proliferation, migration, and invasion. Studies with MEK-transformed EpH4 mouse mammary epithelial cells showed that these cells are highly tumorigenic but have a limited metastatic ability. Detachment of epithelial cells from the extracellular matrix causes disruption of the actin cytoskeleton and induces apoptosis. Several metastatic breast carcinoma cell lines have been shown to be resistant to cell death following actin disruption. This death-resistant phenotype can be modeled by overexpressing the antiapoptotic Bcl-2 protein in cells. This suggests that mechanisms that regulate survival of extravasated tumor cells may enhance metastatic efficiency. Therefore, we examined whether expression of Bcl-2 in MEK-transformed EpH4 mammary epithelial cells could provide a survival advantage and promote metastasis. Expression of Bcl-2 in parental EpH4 mammary epithelial cells or MEK-transformed cells was insufficient to induce increased migration, invasion, or tumor development. However, Bcl-2 expression markedly enhanced spontaneous lung metastasis from orthotopically implanted primary tumors. These results clearly show that mechanisms that regulate primary tumor development are distinct from those that promote metastasis and that assays designed to isolate genes involved in transformation may fail to identify genes that are critical regulators of metastasis.
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215.
Mol Pharmacol 2005 Jan ; 1(67):319-26.
Retinoid-induced apoptosis in HL-60 cells is associated with nucleolin down-regulation and destabilization of Bcl-2 mRNA.
Otake Y ,Sengupta TK ,Bandyopadhyay S ,Spicer EK ,Fernandes DJ ,
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, P.O. Box 250509, Charleston, SC 29425, USA.
All-trans retinoic acid (ATRA) induces differentiation of promyelocytic leukemia cells, but the mechanisms by which cellular differentiation leads to apoptosis are not well understood. Studies were done to address the question whether ATRA-induced apoptosis is a consequence of destabilization of bcl-2 mRNA and decreased cellular levels of the anti-apoptotic protein, bcl-2. ATRA induced differentiation of HL-60 cells along the granulocytic pathway within 48 h. The half-lives of bcl-2 mRNA in HL-60 cells incubated with ATRA for 48 or 72 h were reduced to 39 and 7% of the corresponding untreated control values, respectively. Cellular differentiation was accompanied by down-regulation of the cytoplasmic levels of nucleolin, a bcl-2 mRNA-stabilizing protein. Binding of a bcl-2 mRNA instability element (AU-rich element-1) to nucleolin in S100 extracts from ATRA-treated cells was decreased to 15% of control within 72 h. The decay of 5' capped, polyadenylated bcl-2 mRNA transcripts containing ARE-1 was more rapid in S100 extracts from ATRA-treated cells compared with untreated cells. However, when recombinant nucleolin was added to extracts of ATRA-treated cells, the rate of bcl-2 mRNA decay was similar to the rate in extracts of untreated cells. These results provide evidence that ATRA-induced apoptosis is a consequence of cellular differentiation, which leads to nucleolin down-regulation and bcl-2 mRNA instability.
Publication Type: Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.;
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216.
Oncogene 2004 Dec 2; 56(23):9102-10.
The apoptotic pathway triggered by the Fhit protein in lung cancer cell lines is not affected by Bcl-2 or Bcl-x(L) overexpression.
Roz L ,Andriani F ,Ferreira CG ,Giaccone G ,Sozzi G ,
Department of Experimental Oncology, Istituto Nazionale Tumori, Via Venezian 1, Milan 20133, Italy. luca.roz@istitutotumori.mi.it
The expression of the tumour suppressor protein fragile histidine triad (Fhit) is often impaired in many human cancers and its restoration in Fhit-negative cancer cell lines suppresses tumorigenicity and induces apoptosis. Although the proapoptotic function of Fhit is well documented, little is known about its precise mechanism of action and further studies are needed in order to elucidate the putative therapeutic properties of this protein. To this end, we have engineered the lung cancer cell line NCI-H460 in order to express different molecules involved in the control of apoptotic pathways. Infection of these cells with an adenoviral vector transducing the Fhit gene (Ad-Fhit) revealed that complete protection from apoptosis was conferred by the inhibitor of caspases Cytokine response modifier A (CrmA) and by a dominant-negative form of the adapter protein Fas-associated death domain (FADD) and partial protection by a dominant-negative form of caspase-8, while cells over expressing mitochondrial mediators of the apoptotic response such as Bcl-2 or Bcl-x(L) that are resistant to treatment with cisplatin, remained highly susceptible to cell death triggered by Fhit gene transfer. In line to what was observed in H460 cells, Ad-Fhit efficacy was not affected by Bcl-2 overexpression also in two other lung cancer cell lines (A549 and Calu-1). Analysis of cytochrome c release also confirmed that in Bcl-2- or Bcl-x(L)-expressing cells apoptosis could be detected by terminal deoxynucleotidyl-transferase mediated dUTP nick-end labelling (TUNEL) assay before any evidence of mitochondrial membrane perturbation. In conclusion, our analysis indicates that the Fhit protein exerts its oncosuppressor activity through induction of an apoptotic mechanism that seems to be FADD dependent, caspase-8 mediated and independent from mitochondrial amplification.
Publication Type: Research Support, Non-U.S. Gov't;
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217.
Genome Res 2004 Oct ; 10B(14):2121-7.
The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).
Gerhard DS ,Wagner L ,Feingold EA ,Shenmen CM ,Grouse LH ,Schuler G ,Klein SL ,Old S ,Rasooly R ,Good P ,Guyer M ,Peck AM ,Derge JG ,Lipman D ,Collins FS ,Jang W ,Sherry S ,Feolo M ,Misquitta L ,Lee E ,Rotmistrovsky K ,Greenhut SF ,Schaefer CF ,Buetow K ,Bonner TI ,Haussler D ,Kent J ,Kiekhaus M ,Furey T ,Brent M ,Prange C ,Schreiber K ,Shapiro N ,Bhat NK ,Hopkins RF ,Hsie F ,Driscoll T ,Soares MB ,Casavant TL ,Scheetz TE ,Brown-stein MJ ,Usdin TB ,Toshiyuki S ,Carninci P ,Piao Y ,Dudekula DB ,Ko MS ,Kawakami K ,Suzuki Y ,Sugano S ,Gruber CE ,Smith MR ,Simmons B ,Moore T ,Waterman R ,Johnson SL ,Ruan Y ,Wei CL ,Mathavan S ,Gunaratne PH ,Wu J ,Garcia AM ,Hulyk SW ,Fuh E ,Yuan Y ,Sneed A ,Kowis C ,Hodgson A ,Muzny DM ,McPherson J ,Gibbs RA ,Fahey J ,Helton E ,Ketteman M ,Madan A ,Rodrigues S ,Sanchez A ,Whiting M ,Madari A ,Young AC ,Wetherby KD ,Granite SJ ,Kwong PN ,Brinkley CP ,Pearson RL ,Bouffard GG ,Blakesly RW ,Green ED ,Dickson MC ,Rodriguez AC ,Grimwood J ,Schmutz J ,Myers RM ,Butterfield YS ,Griffith M ,Griffith OL ,Krzywinski MI ,Liao N ,Morin R ,Morrin R ,Palmquist D ,Petrescu AS ,Skalska U ,Smailus DE ,Stott JM ,Schnerch A ,Schein JE ,Jones SJ ,Holt RA ,Baross A ,Marra MA ,Clifton S ,Makowski KA ,Bosak S ,Malek J , ,
The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.
Publication Type: Comparative Study; Research Support, U.S. Gov't, P.H.S.;
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218.
World J Gastroenterol 2004 Nov 15; 22(10):3251-4.
Detection of bcl-2 and bax expression and bcl-2/JH fusion gene in intrahepatic cholangiocarcinoma.
Guo LL ,Xiao S ,Guo Y ,
Department of Pathology, Zhujiang Hospital, Guangzhou 510282, Guangdong Province, China. linlangg@yahoo.com
AIM: To investigate the relationship between bcl-2 gene and its related protein bax and intrahepatic cholangiocellular carcinoma (CCC). METHODS: Semi-nested in situ PCR (SNISPCR) and immunohistochemistry were performed to detect bcl-2/JH fusion gene and bcl-2, bax protein expression in 29 cases of CCC. RESULTS: No bcl-2/JH fusion gene was found in all cases of CCC, 72.4% of 29 cases expressed bcl-2 protein. Bcl-2 protein expression was related to histopathological grades (P<0.05). There was no corresponding relationship between bcl-2/JH fusion gene formation and bcl-2 protein expression in CCC (P<0.05). Bax was expressed in 10.3% of 29 cases. The ratio of bcl-2 to bax in normal liver tissues (3.5 to 1) was different from that in tumor tissues (7.0 to 1). CONCLUSION: It is suggested that bcl-2/JH fusion gene formation is not a frequent event and may not play an important role in the pathogenesis of CCC. However, aberrant ratio of bcl-2 to bax protein expression may be involved in the course of tumorigenesis of CCC. Abnormal bcl-2 protein expression may not be solely resulted from bcl-2/JH fusion gene.
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219.
Folia Morphol (Warsz) 2004 Aug ; 3(63):337-41.
Expression of the apoptotic markers in normal breast epithelium, benign mammary dysplasia and in breast cancer.
Koda M ,Kanczuga-Koda L ,Reszec J ,Sulkowska M ,Famulski W ,Baltaziak M ,Kisielewski W ,Sulkowski S ,
Department of General Pathology, Medical University, Białystok, Poland. kodamar@zeus.amb.edu.pl
Apoptosis and proliferation are processes associated with the development and progression of breast cancer. The sensitivity of tumour cells to the induction of apoptosis depends on the balance between pro- and anti-apoptotic proteins. The expression of Bak and Bcl-2 was examined using an immunohistochemical method in 71 primary breast cancers. Furthermore, Bcl-2 and Bak were assessed in the normal mammary gland as well as in benign mammary dysplasia adjacent to breast cancer. Positive immunostaining for Bcl-2 was observed in 77.8% of cases of normal breast epithelium (NBE), 93% of benign dysplasia without intraductal proliferation (BBD) as well as in 94% of intraductal proliferative lesions of the breast (BIPL). Expression of Bak was detected in 39% of cases of NBE, 45% of BBD and in 67% of BIPL. In breast cancer Bcl-2 and Bak expression was found in 83% and 70% of the cases studied, respectively. Increased Bcl-2 expression in primary tumours significantly correlated with favourable prognostic factors, namely pT1, G2 and lack of metastases to the regional lymph nodes (p < 0.01, p < 0.03, p < 0.02, respectively). There were no relationships between Bak and the clinicopathological features studied, but our results indicate changes in the expression of Bak during breast cancer development and progression. It would appear to be important to assess and compare pro- and anti-apoptotic proteins between normal mammary gland, benign mammary dysplasia and the primary tumours of breast cancer. This knowledge should be helpful in understanding breast cancer development and progression.
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220.
Cell Cycle 2004 Oct ; 10(3):1312-9.
JNK is associated with Bcl-2 and PP1 in mitochondria: paclitaxel induces its activation and its association with the phosphorylated form of Bcl-2.
Brichese L ,Cazettes G ,Valette A ,
Université Paul Sabatier, Toulouse, France.
It has been shown that the activation of JNK after paclitaxel-induced microtubule damage is parallel to Bcl-2 phosphorylation, cell cycle arrest in mitosis and apoptosis. Using subcellular fractionation and immunocytochemistry, we found here that a pool of activated JNK is located in mitochondria of HeLa cells treated with paclitaxel. Furthermore, whereas the JNK protein is present in a tripartite complex with the anti-apoptotic Bcl-2 protein and the PP1 phosphatase in mitochondria isolated from control cells, the activated form of JNK was associated with the phosphorylated form of Bcl-2, but devoid of PP1, in mitochondria isolated from paclitaxel-treated cells. Moreover, using an original cell-free system, we evidenced a direct involvement of JNK as the kinase responsible for the phosphorylation of mitochondrial Bcl-2 in mitotic arrested cells. Indeed, cytosols prepared from mitotic arrested cells led to a dose-dependent phosphorylation of mitochondrial Bcl-2. Bcl-2 phosphorylation was inhibited by CEP 11004, a JNK pathway inhibitor and by immunodepletion of JNK. Taken together, these data show that JNK activation provides a molecular linkage from microtubule damages to the mitochondrial apoptotic machinery and also point to a pivotal role for the JNK/Bcl-2/PP1 complex in the control of apoptosis following paclitaxel treatment.
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221.
Cancer Biol Ther 2004 Oct ; 10(3):983-8.
Alterations of p53, Bcl-2, and hMSH2 protein expression in the normal breast, benign proliferative breast disease, in situ and infiltrating ductal breast carcinomas in the upper Egypt.
Hussein MR ,Ismael HH ,
Pathology Department, Faculty of Medicine, Assuit University, Assuit, Egypt. mrh17@swissinfo.org
BACKGROUND: Tumorigenesis involves alterations in the tumor suppressor genes (p53), protooncogenes (Bcl-2) and housekeeping genes [human MutS homologue-2 (hMSH2)]. We hypothesized that mammary carcinogenesis involves interactions among p53, Bcl-2 and hMSh2 proteins. In the Upper Egypt, the clinicopathologic features and genetic changes during mammary carcinogenesis are unknown. METHODS: To test our hypothesis and to examine these issues, 53 mastectomy specimens, each entailing normal breast, benign proliferative breast disease (BPBD), duct carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC) were immunostained for p53, Bcl-2 and hMSH2 protein expression. RESULTS: The average age incidence of ductal carcinomas was 43.2 +/- 7.06 years. The tumors were common in the left than right side (1.2:1, respectively, p > 0.05). Infiltrating ductal carcinoma of non-specific type was the most common histologic type. Examination of the average weighted scores in the normal breast, BPBD, DCIS and IDC, respectively, showed: (1) significant upregulation of p53 proteins (0.00 +/- 0.00, 0.00 +/- 0.00, 6.25 +/- 2.42, 6.62 +/- 2.15, p = 0.001), (2) insignificant downregulation of Bcl-2 (6.67 +/- 1.33, 5.17 +/- 2.20, 4.79 +/- 2.27 and 4.42 +/- 2.83, p = 0.37), and (3) significant downregulation of hMSH2 (11.3 +/- 0.75, 10.70 +/- 1.27, 7.11 +/- 1.50 and 7.0 +/- 1.33, p = 0.0006). There were insignificant negative correlations between p53 and both Bcl-2 (r = -0.20, p > 0.05) and hMSH2 (r = -0.15, p > 0.05) protein expression. CONCLUSIONS: In the Upper Egypt: (1) breast cancer had similar clinicopathologic features to those in the high risk regions, and (2) alterations of the p53, Bcl-2 and hMSH2 proteins occur during mammary carcinogenesis.
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222.
Int J Mol Med 2004 Oct ; 4(14):633-40.
Hypoxia-induced renal epithelial cell death through caspase-dependent pathway: role of Bcl-2, Bcl-xL and Bax in tubular injury.
Yamamoto K ,Tomita N ,Yoshimura S ,Nakagami H ,Taniyama Y ,Yamasaki K ,Ogihara T ,Morishita R ,
Division of Clinical Gene Therapy, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita 565-0871, Japan.
Although injury of epithelial cells has been reported to be responsible for renal disease such as acute renal failure, its molecular mechanisms are largely unknown. As hypoxia has been postulated as the initial trigger of epithelial injury, we studied the molecular mechanisms of apoptosis induced by hypoxia in human renal epithelial cells. Severe hypoxia caused epithelial cell death, accompanied by a significant increase in LDH release (p<0.01). In addition, hypoxic treatment of epithelial cells resulted in a significant increase in apoptotic cells as assessed by cell morphology (p<0.01). The apoptotic change in epithelial cells under hypoxic condition was also confirmed by a significant increase in caspase-3-like activity and release of cytochrome c (p<0.01). The decrease in epithelial cell number was completely abolished by addition of a wide-spectrum caspase inhibitor, Z-VAD, rather than Z-DEVD, a specific caspase-3 inhibitor (p<0.01). Thus, we further studied the molecular mechanisms of apoptosis induced by hypoxia. Anti-apoptotic factors, Bcl-2 and Bcl-xL, were significantly decreased in epithelial cells under a hypoxic condition as assessed by Western blotting (p<0.01). In contrast, hypoxia did not alter their location. Of particular importance, translocation of a proapoptotic factor, Bax, from the cytoplasm to the mitochondrial membrane was observed in response to hypoxia, whereas total Bax protein was not changed by hypoxia. Overall, this study demonstrated that hypoxia caused epithelial cell death induced by caspase-3-like activity-dependent apoptosis. The pro-apoptotic mechanisms of hypoxia in epithelial cells largely depend on a significant decrease in Bcl-2 and Bcl-xL. In addition, the present results demonstrate that translocation of Bax from the cytosol to the mitochondrial membrane occurred under hypoxia, thereby leading to pathological tissue destruction.
Publication Type: Research Support, Non-U.S. Gov't;
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223.
Blood 2005 Jan 15; 2(105):728-34.
Immunogenicity of Bcl-2 in patients with cancer.
Andersen MH ,Svane IM ,Kvistborg P ,Nielsen OJ ,Balslev E ,Reker S ,Becker JC ,Straten PT ,
Tumor Immunology Group, Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Demmark. mha@cancer.dk
B-cell lymphoma 2 (Bcl-2) is a pivotal regulator of apoptotic cell death and it is overexpressed in many cancers. Consequently, the Bcl-2 protein is an attractive target for drug design, and Bcl-2-specific antisense oligonucleotides or small-molecule Bcl-2 inhibitors have shown broad anticancer activities in preclinical models and are currently in several clinical trials. The clinical application of immunotherapy against cancer is rapidly moving forward in multiple areas, including the adoptive transfer of anti-tumor-reactive T cells and the use of "therapeutic" vaccines. The overexpression of Bcl-2 in cancer and the fact that immune escape by down-regulation or loss of expression of this protein would impair sustained tumor growth makes Bcl-2 a very attractive target for anticancer immunotherapy. Herein, we describe spontaneous T-cell reactivity against Bcl-2 in peripheral blood from patients suffering from unrelated tumor types (ie, pancreatic cancer, breast cancer, acute myeloid leukemia [AML], and chronic lymphocytic leukemia [CLL]). Additionally, we show that these Bcl-2-reactive T cells are indeed peptide-specific, cytotoxic effector cells. Thus, Bcl-2 may serve as an important and widely applicable target for anticancer immunotherapeutic strategies (eg, in the combination with conventional radiotherapy and chemotherapy).
Publication Type: Research Support, Non-U.S. Gov't;
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224.
Front Biosci 2004 Sep 1; (9):2388-413.
Role of viral regulatory and accessory proteins in HIV-1 replication.
Seelamgari A ,Maddukuri A ,Berro R ,de la Fuente C ,Kehn K ,Deng L ,Dadgar S ,Bottazzi ME ,Ghedin E ,Pumfery A ,Kashanchi F ,
Department of Biochemistry and Molecular Biology, School of Medicine and Health Sciences, The George Washington University, Washington, DC 20037, USA.
Human immunodeficiency virus-1 (HIV-1) is the causative agent of acquired immune deficiency syndrome (AIDS), a disease characterized by CD4+ T lymphocyte depletion. HIV-1 replicates actively in a variety of cells by encoding several regulatory (Tat and Rev) and accessory (Vpr, Vif, Vpu, and Nef) proteins. Accessory proteins, thought initially to be dispensable for infection, have now been shown to be important for efficient infection in vivo. Recent evidence suggests that certain viral proteins, like Vif, have evolved to overcome the antiviral mechanisms of the host, while proteins like Nef, which are markers for disease pathogenesis in vivo, help to increase pathogenesis by targeting bystander cells. Thus, these proteins control many aspects of the virus life cycle as well as host cell function, namely gene regulation and apoptosis. Understanding the mechanisms by which the virus is able to successfully replicate in host cells and subsequently cause gradual destruction of the immune system may yield new approaches for therapeutic strategies. In this review, we attempt to integrate information on the role of these regulatory and accessory proteins, emphasizing their interactions with other viral and cellular components, and the subsequent effect on viral replication.
Publication Type: Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.; Review;
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225.
Endocr J 2004 Aug ; 4(51):399-405.
Immunohistochemical analysis of bcl-2, Bax and Bak expression in thyroid glands from patients with Graves' disease.
Hiromatsu Y ,Kaku H ,Mukai T ,Miyake I ,Fukutani T ,Koga M ,Shoji S ,Toda S ,Koike N ,
Division of Endocrinology and Metabolism, Department of Medicine, Kurume University School of Medicine, Kurume, Japan.
In order to clarify the role of apoptosis and the expression of Bcl-2 family proteins in the pathology of Graves' disease (GD), we evaluated the apoptosis by in situ end-labeling of fragmented DNA and the expression of Bcl-2, Bax and Bak by immunohistochemistry in thyroid tissues from 20 patients with GD and in normal thyroid tissues from 6 patients with follicular adenoma (N). Apoptotic nuclei were found in thyrocytes and in germinal center of lymphoid follicles. Bcl-2 was strongly expressed in both GD and N thyrocytes. Bax was not expressed in either GD or N thyrocytes. Bak was expressed in thyrocytes from 5 of 20 patients with GD, while it was detected in all N thyrocytes. In lymphoid follicles Bcl-2 was expressed in the mantle zone, while Bax and Bak were both expressed in the germinal center. The percentage of apoptotic nuclei in GD thyrocytes was low (0~3.6%), and negatively correlated with the weight of the thyroid glands resected (rs = -0.43, P<0.05). It was greater in Bak-positive GD thyrocytes than in Bak-negative ones (mean +/- SD; 1.7 +/- 0.7% vs. 0.7 +/- 0.9%, P<0.05). These findings suggest that the differential expression of Bcl-2 family proteins in both thyrocytes and lymphoid follicles may be involved in the pathology of GD.
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226.
Circ Res 2004 Oct 1; 7(95):734-41.
Transgenic expression of Bcl-2 modulates energy metabolism, prevents cytosolic acidification during ischemia, and reduces ischemia/reperfusion injury.
Imahashi K ,Schneider MD ,Steenbergen C ,Murphy E ,
Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
The antiapoptotic protein Bcl-2 is targeted to the mitochondria, but it is uncertain whether Bcl-2 affects only myocyte survival after ischemia, or whether it also affects metabolic functions of mitochondria during ischemia. Hearts from mice overexpressing human Bcl-2 and from their wild-type littermates (WT) were subjected to 24 minutes of global ischemia followed by reperfusion. During ischemia, the decrease in pH(i) and the initial rate of decline in ATP were significantly reduced in Bcl-2 hearts compared with WT hearts (P<0.05). The reduced acidification during ischemia was dependent on the activity of mitochondrial F1F0-ATPase. In the presence of oligomycin (Oligo), an F1F0-ATPase inhibitor, the decrease in pH(i) was attenuated in WT hearts, but in Bcl-2 hearts, Oligo had no additional effect on pH(i) during ischemia. Likewise, addition of Oligo to WT hearts slowed the rate of decline in ATP during ischemia to a level similar to that observed in Bcl-2 hearts, but addition of Oligo had no significant effect on the rate of decline in ATP in Bcl-2 hearts during ischemia. These data are consistent with Bcl-2-mediated inhibition of consumption of glycolytic ATP. Furthermore, mitochondria from Bcl-2 hearts have a reduced rate of consumption of ATP on uncoupler addition. This could be accomplished by limiting ATP entry into the mitochondria through the voltage-dependent anion channel, and/or the adenine nucleotide transporter, or by direct inhibition of the F1F0-ATPase. Immunoprecipitation showed greater interaction between Bcl-2 and voltage-dependent anion channel during ischemia. These data indicate that Bcl-2 modulation of metabolism contributes to cardioprotection.
Publication Type: Research Support, U.S. Gov't, P.H.S.;
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227.
Biol Pharm Bull 2004 Sep ; 9(27):1340-7.
Over-expressed Bcl-2 cannot suppress apoptosis via the mitochondria in buprenorphine hydrochloride-treated NG108-15 cells.
Kugawa F ,Nakamura M ,Ueno A ,Aoki M ,
Department of Biological Pharmaceutical Sciences, College of Pharmacy, Nihon University, 7-7-1 Narashino-dai, Funabashi, Chiba 274-8555, Japan. jfkugawa@pha.nihon-u.ac.jp
We previously reported that the morphine alkaloid derivative buprenorphine hydrochloride (Bph) induces rapid apoptosis in NG108-15 nerve cells accompanied by the activation of caspase-3. Here, we found this kind of apoptosis was also accompanied by rapid loss of the mitochondrial membrane potential, followed by the efflux of cytochrome c from the mitochondria to the cytosol and the activation of caspases-9 and -3. Together, these results strongly suggested the Bph death signal was routed through the mitochondrial pathway in NG108-15 cells. In these cells, serum-starvation induces a different apoptosis, which we exploited to investigate Bcl-2's role as an apoptosis inhibitor. We made an NG108-15 transfectant, Bcl-2(P2), that stably expressed human Bcl-2, and used it to test Bcl-2's effect on the serum-starvation-induced apoptosis in NG108-15 cells. Cell viability, DNA-ladder formation, and efflux of cytochrome c from the mitochondria were all detected, showing that the human Bcl-2 functioned normally in the Bcl-2(P2) cells. Although the apoptotic events tested were identical in the parental cells and transformants, Bcl-2 expression completely failed to inhibit Bph-induced apoptosis in the Bcl-2(P2) cells.
Publication Type: Research Support, Non-U.S. Gov't;
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228.
Haematologica 2004 Aug ; 8(89):934-9.
Combined analysis of bcl-2 and MDR1 proteins in 256 cases of acute myeloid leukemia.
Venditti A ,Del Poeta G ,Maurillo L ,Buccisano F ,Del Principe MI ,Mazzone C ,Tamburini A ,Cox C ,Panetta P ,Neri B ,Ottaviani L ,Amadori S ,
Cattedra di Ematologia, Università di Roma Tor Vergata, Divisione di Ematologia, Osp.S.Eugenio, p.le dell'Umanesimo 10, 00144 Rome, Italy. adriano.venditti@uniroma2.it
BACKGROUND AND OBJECTIVES: The objective of this study was to investigate the expression of MDR1 and bcl-2 proteins in de novo acute myeloid leukemia (AML). DESIGN AND METHODS: The expression of MDR1 and bcl-2 was analyzed by flow cytometry in a large series of 256 consecutive cases of AML. The results were recorded as percentage of positivity and relative mean fluorescence intensity (rMFI). To determine individual protein levels, an index which equals the product of the percentage of positive cells and rMFI was generated. RESULTS: Using cut-offs of >or=800 and 300 of the index value for bcl-2 and MDR1 expression, respectively, we identified 4 different classes of AML: 1) double negative; 2) single positive bcl-2+/MDR1-; 3) single positive bcl-2-/MDR1+; 4) double positive. The highest incidence of double negative cases was observed in the M2 class whereas double positive cases occurred more frequently in the M4, M5 and M6 subgroups. Seventy-eight percent and 71% of M0 and M1, respectively, showed single positive bcl-2+/MDR1- expression (p = 0.00001). Twenty-eight percent of patients belonging to the double positive category achieved complete remission, whereas for double negative, single positive bcl-2+MDR1- and single positive bcl-2-/MDR1+ category, the complete remission rate was 69%, 52% and 56%, respectively (p = 0.00038). In multivariate analysis, the double positive status independently affected frequency of complete remission (p = 0.008). INTERPRETATION AND CONCLUSIONS: Bcl-2 is over-expressed in CD34+ AML; conversely, MDR1 is over-expressed in CD34- AML. However, the combined expression of the two proteins defines a subset of AML with a very poor prognosis.
Publication Type: Research Support, Non-U.S. Gov't;
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229.
Clin Biochem 2004 Sep ; 9(37):798-802.
Incidence of Bcl-2 expression in bladder cancer: relation to schistosomiasis.
Swellam M ,Abd-Elmaksoud N ,Halim MH ,Khatab H ,Khiry H ,
Biochemistry Department, National Research Center [corrected] Cairo, Egypt. menha_m_swellam@hotmail.com
OBJECTIVES: Apoptosis (programmed cell death) and the genes regulating this process (e.g., Bcl-2) have recently become a focus of interest in the study of cancer development and progression. The bcl-2 gene product plays a role as an inhibitor of apoptosis; it contributes to oncogenesis by suppressing signals that induce apoptotic cell death. The aim of this study was to determine the expression of bcl-2 in schistosomal bladder cancer and to compare it with the established clinicopathological factors. METHODS: Tumor tissues from 118 patients with bladder cancer were examined [57 with squamous cell carcinoma (SCC) and the remaining 61 with transitional cell carcinoma (TCC)]. Of 118 patients, 60 had schistosomiasis associated with bladder cancer. Bcl-2 expression was determined by enzyme immunoassay and the results were confirmed by Western blot and immunodot blot techniques. RESULTS: Bcl-2 was significantly expressed in SCC compared to those with TCC type in the presence of schistosomiasis. Moreover, bcl-2 was associated with clinical stages and lymph node involvement but not with histological grades. CONCLUSIONS: These observations detect a potential role for bcl-2 expression in schistosomal carcinogenesis, and hence selecting patients for future anti-bcl-2 therapy.
Publication Type: Comparative Study;
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230.
Prostate Cancer Prostatic Dis 2004 ; 4(7):321-6.
Abnormalities of apoptotic and cell cycle regulatory proteins in distinct histopathologic components of benign prostatic hyperplasia.
Gandour-Edwards R ,Mack PC ,Devere-White RW ,Gumerlock PH ,
Department of Pathology, University of California Davis Health System, 4400V Street, Sacramento, CA 95817, USA. regina.gandour-edwards@ucdmc.ucdavis.edu
INTRODUCTION: Benign prostatic hyperplasia (BPH) is a slowly progressive abnormal glandular enlargement with heterogeneous morphology. Disruption of apoptotic pathways has been suggested as an important regulatory mechanism in this common and significantly morbid disease. METHODS: Prostatic tissue from 20 patients with BPH and no prior or subsequent prostatic carcinoma was obtained by transurethral prostatectomy (TURP) at the University of California Davis. Apoptotic regulatory proteins: BCL2, BAX and p27 were analyzed by immunohistochemistry and evaluated for expression in four distinct histologic patterns: hyperplastic epithelium, nodules, dilated glands and atrophic/inflammatory glands. RESULTS: Particularly striking was the decreased expression of BAX and an abnormal BCL2 : BAX ratio within all nodules relative to expression in other epithelial patterns. p27 expression was decreased in 35% of the hyperplastic epithelial areas and 10% of the nodules. DISCUSSION: Overall, abnormal expression of BCL2, BAX and/or p27 was identified in the hyperplastic epithelium of 19 (90%) of specimens and all 12 (100%) of the hyperplastic nodules. The high frequency of abnormalities in apoptosis regulatory genes, suggests that alteration of apoptotic pathways is important for the development of this condition.
Publication Type: Comparative Study; Research Support, U.S. Gov't, P.H.S.;
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231.
Apoptosis 2004 Sep ; 5(9):619-27.
Over-expression of Bcl-2 against Pteris semipinnata L-induced apoptosis of human colon cancer cells via a NF-kappa B-related pathway.
Chen GG ,Liang NC ,Lee JF ,Chan UP ,Wang SH ,Leung BC ,Leung KL ,
Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, NT, Hong Kong. gchen@cuhk.edu.hk
Ent-11 alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F), an antitumor component, is a chemical compound isolated from Pteris semipinnata L (PsL), a Chinese traditional herb. We examined whether 5F could affect apoptosis in human colon cancer HT-29 cells, and test whether and how the over-expression of Bcl-2 and Bcl-xL could offset the effect of 5F on cell growth. The result demonstrated that 5F significantly induced apoptosis of HT-29, as shown by MTT assay and DNA fragmentation measurement. Treatment of HT-29 with 5F increased both p38 and iNOS levels, suggesting these two molecules may contribute to the apoptotic effect of 5F. Over-expression of Bcl-2 or Bcl-xL attenuated the increase of p38 and iNOS induced by 5F. The cells with Bcl-2 or Bcl-xL over-expression showed an elevation of nuclear factor kappa B (NF-kappa B) activity, accompanying a significant reduction of 5F-induced apoptosis. Furthermore, inhibition of NF-kappa B by I k B alpha SR, which is a powerful inhibitor of NF-kappa B, restored the ability of 5F to induce apoptosis in the cells transfected with Bcl-2. These data strongly indicated that the apoptotic effect of 5F on HT-29 was closely associated with the activity of NF-kappa B, which was up-regulated by Bcl-2 and Bcl-xL. In conclusion, 5F induced apoptosis in HT-29 cells and this apoptotic effect was associated with the high level of p38 and iNOS expression. The apoptotic effect of 5F could be significantly offset by over-expression of either Bcl-2 or Bcl-xL. Bcl-2, and to the less extent, Bcl-xL, were able to increase the activity of NF-kappa B, which was a known anti-apoptotic molecule in human colon cancer cells.
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232.
J Int Med Res ; 4(32):367-74.
Low frequency of bcl-2 expression in large non-polypoid colonic neoplasms.
Yamada H ,Hasegawa H ,Iino H ,Eguchi H ,Fujii H ,
First Department of Surgery, Yamanashi University, School of Medicine, Yamanashi, Japan.
Large non-polypoid colorectal adenomas that spread over the mucosa and morphologically flat lesions are included in a group called granule-aggregating tumours. These are uncommon in the West. We aimed to clarify the biological differences between granule-aggregating tumours and colorectal polypoid adenomas. We evaluated the extent of apoptotic cell loss and expression of bcl-2 and p53 oncoproteins in 26 granule-aggregating tumours and 19 polypoid adenomas. The mean apoptosis index value of granule-aggregating tumours was significantly higher than that of polypoid adenomas. Only two (7.7%) granule-aggregating tumours and 13 (68.4%) polypoid colorectal adenomas expressed bcl-2, while 12 (46.2%) granule-aggregating tumours and six (31.6%) polypoid colorectal adenomas were p53-positive. Our results show that the higher apoptosis index and frequent expression of bcl-2 oncoprotein differentiates granule-aggregating tumours from polypoid colorectal adenomas. We propose that large non-polypoid granule-aggregating tumours of the colorectum are a biologically distinct entity.
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233.
J Biol Chem 2004 Oct 15; 42(279):43920-8.
Bcl-2 homodimerization involves two distinct binding surfaces, a topographic arrangement that provides an effective mechanism for Bcl-2 to capture activated Bax.
Zhang Z ,Lapolla SM ,Annis MG ,Truscott M ,Roberts GJ ,Miao Y ,Shao Y ,Tan C ,Peng J ,Johnson AE ,Zhang XC ,Andrews DW ,Lin J ,
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190, USA.
The homo- and heterodimerization of Bcl-2 family proteins is important for transduction and integration of apoptotic signals and control of the permeability of mitochondria and endoplasmic reticulum membranes. Here we mapped the interface of the Bcl-2 homodimer in a cell-free system using site-specific photocross-linking. Bcl-2 homodimer-specific photoadducts were detected from 11 of 17 sites studied. When modeled into the structure of Bcl-2 core, the interface is composed of two distinct surfaces: an acceptor surface that includes the hydrophobic groove made by helices 2 and 8 and the loop connecting helices 4 and 5 and a donor surface that is made by helices 1-4 and the loop connecting helices 2 and 3. The two binding surfaces are on separate faces of the three-dimensional structure, explaining the formation of Bcl-2 homodimers, homo-oligomers, and Bcl-2/Bax hetero-oligomers. We show that in vitro the Bcl-2 dimer can still interact with activated Bax as a larger oligomer. However, formation of a Bax/Bcl-2 heterodimer is favored, since this interaction inhibits Bcl-2 homodimerization. Our data support a simple model mechanism by which Bcl-2 interacts with activated Bax during apoptosis in an effective manner to neutralize the proapoptotic activity of Bax.
Publication Type: Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.;
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