Expression of Bcl-2 family proteins and spontaneous apoptosis in normal human testis.
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We investigated the frequency of spontaneous apoptosis and expression of the Bcl-2 family of proteins during normal spermatogenesis in man. Testicular tissue with both normal morphology and DNA content was obtained from necro-donors and fixed in Bouin's solution. A TdT-mediated dUTP end-labelling method (TUNEL) was used for the detection of apoptotic cells. Expression of apoptosis regulatory Bcl-2 family proteins and of p53 and p21(Waf1) was assessed by immunohistochemistry. Germ cell apoptosis was detected in all testes and was mainly seen in primary spermatocytes and spermatids and in a few spermatogonia. Bcl-2 and Bak were preferentially expressed in the compartments of spermatocytes and differentiating spermatids, while Bcl-x was preferentially expressed in spermatogonia. Bax showed a preferential expression in nuclei of round spermatids, whereas Bad was only seen in the acrosome region of various stages of spermatids. Mcl-1 staining was weak without a particular pattern, whereas expression of Bcl-w, p53 and p21(Waf1) proteins was not detected by immunohistochemistry. The results show that spontaneous apoptosis occurs in all male germ cell compartments in humans. Bcl-2 family proteins are distributed preferentially within distinct germ cell compartments suggesting a specific role for these proteins in the processes of differentiation and maturation during human spermatogenesis. (+info)
Cisplatin induces the proapoptotic conformation of Bak in a deltaMEKK1-dependent manner.
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In a panel of four human melanoma cell lines, equitoxic doses of cisplatin induced the proapoptotic conformation of the Bcl-2 family protein Bak prior to the execution phase of apoptosis. Because cisplatin-induced modulation of the related Bax protein was seen in only one cell line, a degree of specificity in the signal to Bak is indicated. Little is known about upstream regulation of Bak activity. In this study, we examined whether the apoptosis-specific pathway mediated by a kinase fragment of MEKK1 (DeltaMEKK1) is involved in the observed Bak modulation. We report that expression of a kinase-inactive fragment of MEKK1 (dominant negative MEKK [dnMEKK]) efficiently blocked cisplatin-induced modulation of Bak and cytochrome c release and consequently also reduced DEVDase activation and nuclear fragmentation. Accordingly, expression of a kinase-active MEKK1 fragment (dominant positive MEKK) was sufficient to induce modulation of Bak in three cell lines and to induce apoptosis in two of these. dnMEKK did not block cisplatin-induced c-Jun N-terminal kinase (JNK) activation, in agreement with a specifically proapoptotic role for the DeltaMEKK1 pathway. Finally, we show that reduction of Bak expression by antisense Bak reduced cisplatin-induced loss of mitochondrial integrity and caspase cleavage activity in breast cancer cell lines. In summary, we have identified Bak as a cisplatin-regulated component downstream in a proapoptotic, JNK-independent DeltaMEKK1 pathway. (+info)
Progressive resistance to apoptosis in a cell lineage model of human proliferative breast disease.
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BACKGROUND: Proliferative breast disease (PBD) may increase a woman's risk of developing breast cancer, perhaps by decreasing cellular sensitivity to apoptosis. To determine whether resistance to apoptosis develops during PBD, we investigated apoptosis initiated through the Fas pathway in a series of cell lines that recapitulates the morphologic changes of PBD in nude/beige mice. METHODS: The series of cell lines used was MCF-10A cells (parental preneoplastic human breast epithelial cells), MCF-10AT cells (transformed with T(24) Ha-ras), and MCF-10ATG3B cells (derivative cells that progress to carcinoma). Fas-mediated apoptosis, induced when a Fas monoclonal antibody bound to and activated the Fas receptor on these cells, was assessed morphologically and by flow cytometry. Levels of proteins involved in Fas-mediated apoptosis and cleavage of poly(adenosine diphosphate-ribose) polymerase (PARP), an end product of caspase activation, were determined by immunoblotting. Bcl-2 and Bax heterodimerization was examined by coimmunoprecipitation. All statistical tests were two-sided. RESULTS: Sensitivity to Fas-mediated apoptosis decreased with the tumorigenic potential of cells: MCF-10A cells were extremely susceptible, MCF-10AT cells were less susceptible, and MCF-10ATG3B cells were resistant. The percentage of apoptotic cells declined, from 24% to 8% to 6%, respectively. All lines produced Fas ligand (FasL) and had comparable levels of Fas receptor, FasL, Fas-associated death-domain protein, and caspases 3 and 6. Levels of caspase 8 were similar in MCF-10A and MCF-10AT cells but about 30% lower in MCF-10ATG3B cells (P>.01 but <.05). Levels of caspase 10 were about 20% lower in MCF-10AT cells (P>.005 but <.01) and about 59% lower in MCF-10ATG3B cells than in MCF-10A cells (P>.01 but <.05). PARP cleavage was detected in MCF-10A and MCF-10AT cells but not in MCF-10ATG3B cells. Levels of Bax, Bid, and Bak proteins were similar in all lines, but levels of Bcl-2 were lower in MCF-10AT and MCF-10ATG3B cells than in MCF-A cells, and Bcl-2-Bax heterodimerization progressively declined in the series. CONCLUSION: Resistance to Fas-mediated apoptosis appears to develop progressively in the MCF-10AT cell series. (+info)
Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis.
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Bax is a member of the Bcl-2 family of proteins known to regulate mitochondria-dependent programmed cell death. Early in apoptosis, Bax translocates from the cytosol to the mitochondrial membrane. We have identified by confocal and electron microscopy a novel step in the Bax proapoptotic mechanism immediately subsequent to mitochondrial translocation. Bax leaves the mitochondrial membranes and coalesces into large clusters containing thousands of Bax molecules that remain adjacent to mitochondria. Bak, a close homologue of Bax, colocalizes in these apoptotic clusters in contrast to other family members, Bid and Bad, which circumscribe the outer mitochondrial membrane throughout cell death progression. We found the formation of Bax and Bak apoptotic clusters to be caspase independent and inhibited completely and specifically by Bcl-X(L), correlating cluster formation with cytotoxic activity. Our results reveal the importance of a novel structure formed by certain Bcl-2 family members during the process of cell death. (+info)
BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak.
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The BH3-only proteins Bim and Bad bind to the antiapoptotic Bcl-2 proteins and induce apoptosis in wild-type cells and cells from either bax(-/-) or bak(-/-) animals. In contrast, constitutively active forms of Bim and Bad failed to induce apoptosis in bax(-/-)bak(-/-) cells. Expression of Bax restored susceptibility of the cells to Bim and Bad. In addition, Bax but not Bim or Bad sensitized the bax(-/-)bak(-/-) cells to a wide variety of cell death stimuli including UV irradiation, chemotherapeutic agents, and ER stress. These results suggest that neither activation of BH3-only proteins nor suppression of pro-survival Bcl-2 proteins is sufficient to kill cells in the absence of both Bax and Bak. Furthermore, whereas mouse embryo fibroblasts (MEF) expressing only Bax or Bak displayed resistance to transformation, bax(-/-)bak(-/-) MEF were nearly as prone to oncogenic transformation as p53(-/-) MEF. Thus, the function of either Bax or Bak appears required to initiate most forms of apoptosis and to suppress oncogenic transformation. (+info)
Induction of apoptosis in prostate carcinoma cells by BH3 peptides which inhibit Bak/Bcl-2 interactions.
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Interactions between proteins of the Bcl-2 family play an important role in the regulation of apoptosis. Anti-apoptotic family members can heterodimerize with pro-apoptotic family members and antagonize their function, thus protecting against death. In cells protected from death by overexpression of Bcl-2 much of the Bax is present in Bax/Bcl-2 hetero-multimers and its death signal is blocked as it cannot homodimerize. This led us to use the Bcl-2/Bax heterodimer as a target for new compounds which may provide a therapy particularly suited to tumour cells for which resistance to conventional therapy is associated with elevated expression of Bcl-2. We assessed whether apoptosis could be induced in prostate tumour cells by blocking this heterodimerization with synthetic peptide sequences derived from the BH3 domain of pro-apoptotic Bcl-2 family members. Prostate cells were found to undergo up to 40% apoptosis 48 h following the introduction of synthetic peptides from the BH3 domains of Bax and Bak. The caspase inhibitor z-VAD.fmk provided protection against apoptosis mediated by these peptides. Immunoprecipitation studies revealed that introduction of peptides derived from the BH3 regions of Bak and Bax into cells blocked Bak/Bcl-2 heterodimerization. These data suggest that by blocking the dimerization through which Bcl-2 would normally inhibit apoptosis the apoptotic pathway driven by Bak was re-opened. (+info)
A role for mitochondrial Bak in apoptotic response to anticancer drugs.
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In the present study a clonal Jurkat cell line deficient in expression of Bak was used to analyze the role of Bak in cytochrome c release from mitochondria. The Bak-deficient T leukemic cells were resistant to apoptosis induced by UV, staurosporin, VP-16, bleomycin, or cisplatin. In contrast to wild type Jurkat cells, these Bak-deficient cells did not respond to UV or treatment with these anticancer drugs by membranous phosphatidylserine exposure, DNA breaks, activation of caspases, or release of mitochondrial cytochrome c. The block in the apoptotic cascade was in the mitochondrial mechanism for cytochrome c release because purified mitochondria from Bak-deficient cells failed to release cytochrome c or apoptosis-inducing factor in response to recombinant Bax or truncated Bid. The resistance of Bak-deficient cells to VP-16 was reversed by transduction of the Bak gene into these cells. Also, the cytochrome c releasing capability of the Bak-deficient mitochondria was restored by insertion of recombinant Bak protein into purified mitochondria. Following mitochondrial localization, low dose recombinant Bak restored the mitochondrial release of cytochrome c in response to Bax; at increased doses it induced cytochrome c release itself. The function of Bak is independent of Bid and Bax because recombinant Bak induced cytochrome c release from mitochondria purified from Bax(-/-), Bid(-/-), or Bid(-/-) Bax(-/-) mice. Together, our findings suggest that Bak plays a key role in the apoptotic machinery of cytochrome c release and thus in the chemoresistance of human T leukemic cells. (+info)
Inducible overexpression of Bak sensitizes HCC-9204 cells to apoptosis induced by doxorubicin.
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AIM: To investigate the role of overexpression of Bak in apoptotic pathways and drug susceptibility using doxorubicin and vinorelbine in human HCC-9204 cells. METHODS: An inducible system, MT-II regulatory system which allowed controlled expression of protein upon addition of ZnSO4(100 mumol/L) as an external inducer was used. Stable transfection of pMD-Bak gene was performed on HCC-9204 cells. Apoptotic cells were measured by morphological criteria, as well as by TUNEL assay and flow cytometry. The ability of Bak to decrease clonogenic cell survival was studied by colony-forming assays, while decrease in cell viability was assessed by MTT assay. RESULTS: Cells overexpressing Bak showed extensive cell death with nucleus fragmentation detected by TUNEL assay. FACS analyses showed that Bak could induce significant G1 accumulation and apoptosis in 19.29% cells 24 h after induction. Bak significantly decreased the clonogenic survival following exposure to adriamycin, but not vinorelbine. Furthermore, the time-course of cell viability rates following exposure of HCC-9204/Bak cells to adriamycin and vinorelbine was in agreement with the above findings. Bak selectively sensitized HCC-9204 cells to death induced by adriamycin while resisted to vinorelbine. CONCLUSION: Bak may prolong cell cycle in G1 phase, leading to apoptosis and decrease clonogenic survival of HCC-9204 cells in a drug-specific manner. (+info)