Bcl-2 inhibits apoptosis induced by mitochondrial uncoupling but does not prevent mitochondrial transmembrane depolarization

Bcl-2 overexpression protects cells from apoptosis induced by many cytotoxic agents. In this study, we investigated the effects of uncoupling mitochondrial electron transport in both HL60 wild-type and Bcl-2-overexpressing cells using the protonophore carbonyl cyanide m-chlorophenylhydrazone. We found that uncoupling mitochondrial electron transport induced apoptosis in wild-type, but not in Bcl-2-overexpressing cells. To investigate the mechanism of action of Bcl-2-mediated inhibition of cyanide m-chlorophenylhydrazone-induced apoptosis, we measured the mitochondrial transmembrane potential (DeltaPsi(m)) after uncoupling mitochondrial electron transport and found that both HL-60 wild-type and Bcl-2-overexpressing cells similarly depolarize following cyanide m-chlorophenylhydrazone exposure. Western blot analysis demonstrated that Bcl-2 overexpression did not completely block cytochrome c release from mitochondria after uncoupling mitochondrial electron transport. Since Bcl-2 may act as an antioxidant, we studied the effect of altering the cellular redox state prior to uncoupling mitochondrial electron transport in Bcl-2-overexpressing cells. Depletion of mitochondrial (but not cytosolic) glutathione induced apoptosis in Bcl-2-overexpressing cells and negated the protective effect of Bcl-2. Furthermore, following glutathione depletion, Bcl-2-overexpressing cells were sensitized to undergo cyanide m-chlorophenylhydrazone-induced apoptosis. These data suggest that the action of Bcl-2 is dependent, in part, on the cellular and mitochondrial redox state.     

A novel apoptotic cascade mediated by CDK4 in rat pheochromocytoma PC12 cells.

Apoptosis induced by serum withdrawal in pheochromocytoma PC12 cells is promoted by overexpression of cyclin-dependent kinase 4 (CDK4). We compared CDK4-promoted apoptosis with that induced by serum withdrawal alone in PC12 cells. Protein synthesis inhibitors did not prevent apoptosis in parental cells, but prevented the promotion of apoptosis by CDK4 overexpression. Nerve growth factor, basic-fibroblast growth factor, and Bcl-2 proteins protected both parental and CDK4-overexpressing cells from apoptosis. However, insulin-like growth factor-I and Bcl-X(L) protein only partially inhibited apoptosis in the CDK4-overexpressing cells. Bcl-2 or Bcl-X(L) had no significant effect on CDK4 kinase activity in both cell lines. These results suggest a novel CDK4-mediated apoptotic cascade which is normally restrained, but which is activated by CDK4 overexpression. This apoptotic cascade should eventually converge with the cascade induced by serum withdrawal in normal PC12 cells. We discuss the interactions among these apoptotic cascades and the points where anti-apoptotic agents act.     

Blocking granule-mediated death by primary human NK cells requires both protection of mitochondria and inhibition of caspase activity

Human GraB (hGraB) preferentially induces apoptosis via Bcl-2-regulated mitochondrial damage but can also directly cleave caspases and caspase substrates in cell-free systems. How hGraB kills cells when it is delivered by cytotoxic lymphocytes (CL) and the contribution of hGraB to CL-induced death is still not clear. We show that primary human natural killer (hNK) cells, which specifically used hGraB to induce target cell death, were able to induce apoptosis of cells whose mitochondria were protected by Bcl-2. Purified hGraB also induced apoptosis of Bcl-2-overexpressing targets but only when delivered at 5- to 10-fold the concentration required to kill cells expressing endogenous Bcl-2. Caspases were critical in this process as inhibition of caspase activity permitted clonogenic survival of Bcl-2-overexpressing cells treated with hGraB or hNK cells but did not protect cells that only expressed endogenous Bcl-2. Our data therefore show that hGraB triggers caspase activation via mitochondria-dependent and mitochondria-independent mechanisms that are activated in a hierarchical manner, and that the combined effects of Bcl-2 and direct caspase inhibition can block cell death induced by hGraB and primary hNK cells.     

Transforming growth factor-beta1 protects against pulmonary artery endothelial cell apoptosis via ALK5

Transforming growth factor (TGF)-beta1 has been reported to cause endothelial cell apoptosis. However, conflicting data have also demonstrated that TGF-beta1 promotes endothelial cell survival. In this study, the effect of TGF-beta1 on apoptosis of cultured bovine pulmonary artery endothelial cells (PAEC) induced by multiple stimuli was investigated. TGF-beta1 protected against apoptosis of bovine PAEC induced by serum deprivation or the VEGF receptor inhibitor SU-5416, but not by UV light exposure or TNFalpha. Neither caspase-8 nor caspase-12 was activated by serum deprivation or the VEGF receptor blocker. However, blockade of VEGF receptors activated caspase-9, an effect that was abolished by TGF-beta1. Furthermore, serum deprivation and inhibition of VEGF receptors significantly decreased the protein level of Bcl-2, an effect that was also abrogated by TGF-beta1. In addition, the baseline level of Bcl-2 was enhanced by TGF-beta1 and reduced by inhibition of activin receptor-like kinase 5 (ALK5), a TGF-beta1 type I receptor. Furthermore, inhibition of ALK5 caused apoptosis of bovine PAEC. These results suggest that TGF-beta1 signaling is critical for maintenance of bovine PAEC survival. Finally, the protective effects of TGF-beta1 on bovine PAEC apoptosis and Bcl-2 reduction were abolished by ALK5 inhibition, but not by inhibition of non-SMAD signaling pathways. Also, TGF-beta1 activated SMAD2 and SMAD1/5, an effect that was abolished by ALK5 inhibition. The results of this study suggest that TGF-beta1 protects against bovine PAEC apoptosis, possibly through ALK5-mediated Bcl-2 induction and subsequent inhibition of the mitochondria-mediated intrinsic pathway of apoptosis. Understanding the mechanism by which TGF-beta1 promotes endothelial cell survival may provide a better treatment for apoptosis-dependent vascular diseases, such as emphysema.     

The cell death inhibitor Bcl-2 and its homologues influence control of cell cycle entry.

The effect of the cell death inhibitor Bcl-2 and its homologues on cell cycle regulation was explored in lymphocytes and cell lines. Expression of a bcl-2 transgene reduced proliferation of thymocytes and delayed cell cycle entry of mitogen-stimulated B and T lymphocytes. Overexpression of Bcl-2, Bcl-xL or adenovirus E1B19kD substantially delayed serum stimulation-induced S phase entry of quiescent NIH 3T3 fibroblasts. Bcl-2-mediated cell survival and growth inhibition are both antagonized by Bax. Bcl-2, Bcl-xL and E1B19kD, but not Bcl-2 mutants that are defective in blocking apoptosis, suppress growth of colon carcinoma cells. This evidence that regulation of cell survival is coupled to control of cell growth has implications for normal cell turnover and tumorigenesis.     

Evidence of a direct role for Bcl-2 in the regulation of articular chondrocyte apoptosis under the conditions of serum withdrawal and retinoic acid treatment

The regulation of chondrocyte apoptosis in articular cartilage may underlay age-associated changes in cartilage and the development of osteoarthritis. Here we demonstrate the importance of Bcl-2 in regulating articular chondrocyte apoptosis in response to both serum withdrawal and retinoic acid treatment. Both stimuli induced apoptosis of primary human articular chondrocytes and a rat chondrocyte cell line as evidenced by the formation of DNA ladders. Apoptosis was accompanied by decreased expression of aggrecan, a chondrocyte specific matrix protein. The expression of Bcl-2 was downregulated by both agents based on Northern and Western analysis, while the level of Bax expression remained unchanged compared to control cells. The importance of Bcl-2 in regulating chondrocyte apoptosis was confirmed by creating cell lines overexpressing sense and antisense Bcl-2 mRNA. Multiple cell lines expressing antisense Bcl-2 displayed increased apoptosis even in the presence of 10% serum as compared to wild-type cells. In contrast, chondrocytes overexpressing Bcl-2 were resistant to apoptosis induced by both serum withdrawal and retinoic acid treatment. Finally, the expression of Bcl-2 did not block the decreased aggrecan expression in IRC cells treated with retinoic acid. We conclude that Bcl-2 plays an important role in the maintenance of articular chondrocyte survival and that retinoic acid inhibits aggrecan expression independent of the apoptotic process. J. Cell. Biochem. 71:302–309, 1998. © 1998 Wiley-Liss, Inc.     

紫草素对人脑胶质瘤U251 细胞增殖和凋亡的影响

目的:观察紫草素对体外培养的人脑胶质瘤U251细胞的增殖和凋亡的影响。方法:应用MTT法和Annexin V-FITC/PI双染流式细胞术分别检测2.5、5、10μM/L的紫草素对U251细胞的体外抑杀作用以及凋亡诱导作用,进一步应用Western blot方法检测紫草素对凋亡相关蛋白Bcl-2及Bax表达水平的影响。结果:紫草素对人U251胶质瘤细胞具有明显的增殖抑制作用,且呈一定的剂量依赖性和时间依赖性。紫草素可明显上调U251细胞Bax的表达,下调Bcl-2的表达,与对照组相比存在显著性差异(P0.05)。结论:紫草素对人胶质瘤U251细胞具有明显的抑制增殖和促进凋亡作用。     

Transforming growth factor-beta 1 induces apoptosis independently of p53 and selectively reduces expression of Bcl-2 in multipotent hematopoietic cells

Transforming growth factor-beta1 (TGF-beta1) can inhibit cell proliferation or induce apoptosis in multipotent hematopoietic cells. To study the mechanisms of TGF-beta1 action on primitive hematopoietic cells, we used the interleukin-3 (IL-3)-dependent, multipotent FDCP-Mix cell line. TGF-beta1-mediated growth inhibition was observed in high concentrations of IL-3, while at lower IL-3 concentrations TGF-beta1 induced apoptosis. The proapoptotic effects of TGF-beta1 occur via a p53-independent pathway, since p53(null) FDCP-Mix demonstrated the same responses to TGF-beta1. IL-3 has been suggested to enhance survival via an increase in (antiapoptotic) Bcl-x(L) expression. In FDCP-Mix cells, neither IL-3 nor TGF-beta1 induced any change in Bcl-x(L) protein levels or the proapoptotic proteins Bad or Bax. However, TGF-beta1 had a major effect on Bcl-2 levels, reducing them in the presence of high and low concentrations of IL-3. Overexpression of Bcl-2 in FDCP-Mix cells rescued them from TGF-beta1-induced apoptosis but was incapable of inhibiting TGF-beta1-mediated growth arrest. We conclude that TGF-beta1-induced cell death is independent of p53 and inhibited by Bcl-2, with no effect on Bcl-x(L). The significance of these results for stem cell survival in bone marrow are discussed.     

Dual anticancer activity of 8-Cl-cAMP: inhibition of cell proliferation and induction of apoptotic cell death

8-Cl-cAMP induces apoptotic cell death in human cancer cells. To look at this more closely, we examined the changes in the levels of Bcl-2 family proteins during 8-Cl-cAMP-induced apoptosis of SH-SY5Y human neuroblastoma cells. Following the treatment with 8-Cl-cAMP, Bcl-2 was transiently down-regulated and Bad was increased continuously up to day 5. In addition, overexpression of Bcl-2 efficiently blocked the 8-Cl-cAMP-induced apoptosis, suggesting Bcl-2 family proteins may be involved in the 8-Cl-cAMP-induced apoptosis. The contribution of the apoptotic cell death and the inhibition of cell proliferation in the 8-Cl-cAMP-induced growth inhibition was closely monitored in the Bcl-2-overexpressing cells. Though the apoptosis was reduced significantly, no significant difference was observed in the inhibition of cell proliferation up to day 2 of 8-Cl-cAMP treatment. These results suggest that 8-Cl-cAMP exerts anticancer activity by two distinct mechanisms, i.e. , through the inhibition of cell proliferation as well as the induction of apoptosis. Supporting this notion was the observations that (1) suppression of apoptosis by zVAD did not abrogate 8-Cl-cAMP-induced inhibition of cell proliferation, and (2) 8-Cl-cAMP did not show additive inhibition of cell proliferation in RIIbeta-overexpressing cells.     

Death-signaling pathways in human myeloid cells by oxLDL and its cytotoxic components 7beta-hydroxycholesterol and cholesterol-5beta,6beta-epoxide

Oxidized low-density lipoprotein contains many potentially proatherogenic molecules, including oxysterols, which have been shown to induce apoptosis in various cell lines. The aim of this study was to investigate the pathway of apoptosis induced by oxidized low-density lipoprotein and the oxysterols, 7beta-hydroxycholesterol and cholesterol-5beta,6beta-epoxide, in two human monocytic cell lines. The HL-60 cells appeared to be more sensitive to oxidized low-density lipoprotein than U937 cells, whereas the isolated oxysterols were more potent inducers of apoptosis in the U937 cells. Caspase-2 inhibition decreased the number of viable cells in oxidized low-density lipoprotein-treated samples; however, it protected against cholesterol-5beta,6beta-epoxide-induced cell death. Western blot analysis was utilized to examine the effect of caspase-2 inhibition on the expression of the antiapoptotic protein Bcl-2. Pretreatment with the inhibitor protected against the decrease in Bcl-2 expression in oxidized low-density lipoprotein- and 7beta-hydroxycholesterol-treated U937 cells. In HL-60 cells, Bcl-2 was overexpressed in oxidized low-density lipoprotein-treated cells, but in the presence of the inhibitor Bcl-2 expression was returned to control levels. Depleted ATP concentrations in the cells suggest that both apoptosis and necrosis may have occurred simultaneously. Our results highlight differences in the signaling pathways induced by oxidized low-density lipoprotein, 7beta-hydroxycholesterol, and cholesterol-5beta,6beta-epoxide in U937 and HL-60 cells.     

Indomethacin inhibits cell growth of medullary thyroid carcinoma by reducing cell cycle progression into S phase

Indomethacin, a non-steroidal anti-inflammatory drug (NSAID), has been reported to inhibit the growth of medullary thyroid carcinoma (MTC) cells in vitro. However, the mechanism of inhibition of MTC cell growth by indomethacin and its potency have yet to be revealed. We examined the effect of indomethacin on three different MTC cell lines (TT cells, DRO 81-1 cells and HRO 85-1 cells) and two non-MTC cells. The mechanism of indomethacin action in MTC cells was investigated by analyzing intracellular prostaglandin level, apoptosis, and cell cycle in TT cells. Indomethacin inhibited cell growth of all three MTC cell lines but not normal thyroid cells or anaplastic thyroid carcinoma cells. Indomethacin at 10 microM or greater showed a dose response inhibition of cell growth. Indomethacin at 25 muM, a putative therapeutic serum indomethacin level, showed potency similar to 100 to 200 nM sunitinib, a receptor tyrosine kinase inhibitor. To examine whether prostaglandin depletion might determine the inhibition of MTC cell growth, we created different prostaglandin E2 (PGE2) levels in TT cells using three different NSAIDs. A profound PGE2 depletion by indomethacin-ester, a potent cyclooxygenase (COX) II inhibitor, showed the least inhibition of cell growth. Indomethacin did not increase apoptosis of TT cells. Indomethacin, but not naproxen or indomethacin-ester, reduced cell cycle progression into S phase; this was unrelated to the degree of PGE2 depletion. The expression of phosphorylated retinoblastoma (pRb) protein that shifts cells from G(1) to S phase was reduced after exposure to indomethacin. In conclusion, indomethacin has specific anti-tumor effect on MTC cells, probably by reducing cell cycle progression into S phase rather than by prostaglandin depletion. Since no drug therapy is currently available for MTC, indomethacin may be one of the therapeutic candidates.     

扁蒴藤素对人鼻咽癌HNE2 细胞增殖的抑制作用研究

目的：探讨扁蒴藤素对人鼻咽癌HNE2 细胞增殖的抑制作用,明确HSP70 在肿瘤发展过程中的抑制作用。方法：噻唑蓝法 （MTT）检测扁蒴藤素对HNE2 细胞生长抑制作用,流式细胞术检测扁蒴藤素诱导HNE2 细胞凋亡,免疫印迹法检测Caspase、 PARP、酪氨酸激酶、AKT 及Bcl-2 家族蛋白表达。结果：扁蒴藤素抑制HNE2 细胞的生长,促使Caspase 9,Caspase 3和PARP 蛋 白被切割,上调Bim 等促凋亡蛋白的表达,减少Bcl-xL等抗凋亡蛋白的表达,下调EGFR 等受体酪氨酸激酶, 抑制AKT 磷酸化, 上调热休克蛋白70（HSP70）的表达。用热休克反应抑制剂KNK437 抑制HSP70 的表达可以增强扁蒴藤素促进细胞凋亡的能力。 结论：扁蒴藤素通过下调受体酪氨酸激酶,激活caspase 介导的凋亡通路抑制鼻咽癌HNE2 细胞的增殖,抑制HSP70 的表达可增 强其抗肿瘤作用。     

Flavonoids inhibit cell growth and induce apoptosis in B16 melanoma 4A5 cells

We investigated the growth inhibitory activity of several flavonoids, including apigenin, luteolin, kaempherol, quercetin, butein, isoliquiritigenin, naringenin, genistein, and daizein against B16 mouse melanoma 4A5 cells. Isoliquiritigenin and butein, belonging to the chalcone group, markedly suppressed the growth of B16 melanoma cells and induced cell death. The other flavonoids tested showed little growth inhibitory activity and scarcely caused cell death. In cells treated with isoliquiritigenin or butein, condensation of nuclei and fragmentation of nuclear DNA, which are typical phenomena of apoptosis, were observed by Hoechst 33258 staining and by agarose gel electrophoresis of DNA. Flowcytometric analysis showed that isoliquiritigenin and butein increased the proportion of hypodiploid cells in the population of B16 melanoma cells. These results demonstrate that isoliquiritigenin and butein inhibit cell proliferation and induce apoptosis in B16 melanoma cells. Extracellular glucose decreased the proportion of hypodiploid cells that appeared as a result of isoliquiritigenin treatment. p53 was not detected in cells treated with either of these chalcones, however, protein of the Bcl-2 family were detected. The level of expression of Bax in cells treated with either of these chalcones was markedly elevated and the level of Bcl-XL decreased slightly. Isoliquiritigenin did not affect Bcl-2 expression, but butein down-regulated Bcl-2 expression. From these results, it seems that the pathway by which the chalcones induce apoptosis may be independent of p53 and dependent on proteins of the Bcl-2 family. It was supposed that isoliquiritigenin induces apoptosis in B16 cells by a mechanism involving inhibition of glucose transmembrane transport and promotion of Bax expression. On the other hand, it was suggested that butein induces apoptosis via down-regulation of Bcl-2 expression and promotion of Bax expression. This mechanism differs from the isoliquiritigenin induction pathway.     

Mechanisms of arsenic trioxide induced apoptosis of human cervical cancer HeLa cells and protection by Bcl-2

It was recently reported that arsenic trioxide (As_2O_3) can induce complete remission in patients with acute promyelocytic leukemia (APL). In this present article, the biological effect of As_2O_3 on human cervical cancer HeLa cells and HeLa cells overexpressing Bcl-2 is studied. By MTT and colony forming ability assays, morphology alteration, flow cytometric analysis, DNA gel electrephoresis and in situ cell death detection (TUNEL), it was found that As_2O_3 inhibited the growth of HeLa cells and induced G2/M arrest and apoptosis of the cells. RT-PCR, Northern blot, Western blot analysis revealed that As_2O_3 induced HeLa cell apoptosis possibly via decreasing the expression of c-myc and viral genes. HeLa cells overexpressing Bcl-2 partly resist As_2O_3 induced apoptosis, which might be relative to preventing the cells from As_2O_3 caused G2/M block, downregulation of c-myc gene expression and inhibition of viral gene expression was also noted, However, it was found that As_2O_3 at a high concentratio     

罗汉果醇抗肿瘤活性及其作用机制研究

罗汉果醇是罗汉果皂苷的苷元,有研究报道罗汉果皂苷V具有防癌抑癌作用。该研究采用噻唑蓝实验( MTT法)检测罗汉果醇对不同肿瘤细胞增殖的抑制情况,以及不同浓度的罗汉果醇对CNE1细胞的增殖抑制率;应用细胞克隆形成实验进一步验证罗汉果醇对CNE1细胞增殖的抑制作用;采用Annexin V/PI 双染法检测罗汉果醇对CNE1细胞凋亡的影响;以实时定量PCR技术检测罗汉果醇对CNE1细胞中Caspase-3、Sur-vivin、Bax和Bcl-2基因的mRNA 表达水平的影响。结果表明：罗汉果醇能显著抑制DU145、HepG2、A549、CNE1、CNE2细胞的增殖,其中对CNE1细胞增殖的抑制作用最为显著,并呈剂量依赖性,其半数抑制浓度IC50为(81.48±4.73)μmol·L-1;通过对CNE1细胞进一步的克隆形成实验,也验证了这一点;Annexin V/PI 双染法可见随着浓度的增加,凋亡比例增加;实时定量PCR技术检测显示罗汉果醇处理后,促凋亡基因Caspase-3、Bax的表达增加,抗凋亡基因Survivin、Bcl-2的表达减少。因此,罗汉果醇可能是通过促进Caspase-3、Bax等促凋亡基因和抑制Survivin、Bcl-2等抗凋亡基因的表达,来诱导肿瘤细胞凋亡,进而发挥抗肿瘤活性。     

PK11195, a Ligand of the Mitochondrial Benzodiazepine Receptor, Facilitates the Induction of Apoptosis and Reverses Bcl-2-Mediated Cytoprotection

One critical step of the apoptotic process is the opening of the mitochondrial permeability transition (PT) pore leading to the disruption of mitochondrial membrane integrity and to the dissipation of the inner transmembrane proton gradient (ΔΨ_m). The mitochondrial PT pore is a polyprotein structure which is inhibited by the apoptosis-inhibitory oncoprotein Bcl-2 and which is closely associated with the mitochondrial benzodiazepine receptor (mBzR). Here we show that PK11195, a prototypic ligand of the 18-kDa mBzR, facilitates the induction of ΔΨ_mdisruption and subsequent apoptosis by a number of different agents,including agonists of the glucocorticoid receptor,chemotherapeutic agents (etoposide, doxorubicin),gamma irradiation, and the proapoptotic second messenger ceramide. Whereas PK11195 itself has no cytotoxic effect, it enhances apoptosis induction by these agents. This effect is not observed for benzodiazepine diazepam, whose binding site in the mBzR differs from PK11195. PK11195 partially reverses Bcl-2 mediated inhibition of apoptosis in two different cell lines. Thus, transfection-enforced Bcl-2 overexpression confers protection against glucocorticoids and chemotherapeutic agents, and this protection is largely reversed by the addition of PK11195. This effect is observed at the level of ΔΨ_mdissipation as well as at the level of nuclear apoptosis. To gain insights into the site of action of PK11195, we performed experiments on isolated organelles. PK11195 reverses the Bcl-2-mediated mitochondrial retention of apoptogenic factors which cause isolated nuclei to undergo apoptosis in a cell-free system. Mitochondria from control cells, but not mitochondria from Bcl-2-overexpressing cells, readily release such apoptogenic factors in response to atractyloside, a ligand of the adenine nucleotide translocator. However, control and Bcl-2-overexpressing mitochondria respond equally well to a combination of atractyloside and PK11195. Altogether, these findings indicate that PK11195 abolishes apoptosis inhibition by Bcl-2 via a direct effect on mitochondria. Moreover, they suggest a novel strategy for enhancing the susceptibility of cells to apoptosis induction and, concomitantly, for reversing Bcl-2-mediated cytoprotection.     

瘦素对卵巢癌细胞 SKOV3 增殖及凋亡的影响

目的:探讨瘦素对人卵巢癌SKOV3细胞增殖及凋亡的影响及其作用机制。方法:用不同浓度的瘦素(0、50、100、200 ng/m L)处理人卵巢癌SKOV3细胞48 h后,采用MTT法检细胞的生长;以血清饥饿诱导细胞凋亡,同时给予瘦素刺激,Annexin V/PI双染法检测细胞凋亡的变化;western blotting分析p21、cyclin D1、Bcl-2、Bax蛋白的表达水平和ERK1/2通路的活化情况。结果:瘦素以剂量依赖性的方式促进人卵巢癌SKOV3细胞的增殖,同时抑制血清饥饿诱导的细胞凋亡。瘦素处理可下调p21和上调cyclin D1的表达,抑制促凋亡分子Bax的表达和上调抗凋亡分子Bcl-2的表达。瘦素可诱导细胞中ERK1/2通路的活化,其抑制剂PD98059可明显抑制瘦素诱导的促细胞增殖和抗凋亡作用,同时伴随有cyclin D1、Bcl-2蛋白表达的下调和Bax的上调。结论:瘦素可能通过活化ERK1/2通路调节细胞有丝分裂进程,进而促进卵巢癌细胞的增殖;同时通过调节凋亡相关蛋白Bcl-2和Bax的表达抑制卵巢癌细胞的凋亡。     

Restoration of G1/S Arrest in E1A+c-Ha-ras-Transformed Cells by Bcl-2 Overexpression

Here we show that introduction of human bcl-2 gene into E1A+c-Ha-ras-transformed rat embryo fibroblasts, which are highly susceptible to proapoptotic stimuli and fail to be arrested at the G1/S boundary following genotoxic stresses, results not only in inhibition of apoptosis, but also in restoration of the G1/S arrest. Overexpression of Bcl-2 did not affect proliferation rate and saturation density of E1A+c-Ha-ras transformants. Genotoxic stresses caused prolong G1/S arrest in Bcl-2-overexpressing transformants. Remarkably, levels and activities of Cdk2, cyclins E/A, cyclin E-Cdk2 and cyclin A-Cdk2 were unchanged during G1/S arrest. Introduction of Bcl-2 into E1A+c-Ha-ras-transformants resulted in accumulation of p21/Waf-1 without inhibiting cyclin-Cdk complexes. In both parental and Bcl-2-overexpressing cells, p21/Waf-1 was co-immunoprecipitated with ERK 1,2 and JNK 1,2, whereas p38 was found in complexes with p21/Waf-1 only in Bcl-2-overexpressing transformants. JNK 1,2 and p38 but not ERK 1,2 were detected in complexes with the exogenous Bcl-2. However, Bcl-2 did not affect phosphorylation of ERK 1,2, JNK 1,2 and p38. G1/S arrest induced by adriamycin and serum withdrawal (but not by IR) was accompanied by release of active forms of p38 from complexes with Bcl-2. We suggest that Bcl-2 restores stress-induced G1/S arrest without inhibiting cyclin-Cdk2 complexes and MAPK pathways.