Casp8p41 generated by HIV protease kills CD4 T cells through direct Bak activation

Previous studies have shown that human immunodeficiency virus (HIV) protease cleaves procaspase 8 to a fragment, termed Casp8p41, that lacks caspase activity but nonetheless contributes to T cell apoptosis. Herein, we show that Casp8p41 contains a domain that interacts with the BH3-binding groove of pro-apoptotic Bak to cause Bak oligomerization, Bak-mediated membrane permeabilization, and cell death. Levels of active Bak are higher in HIV-infected T cells that express Casp8p41. Conversely, targeted mutations in the Bak-interacting domain diminish Bak binding and Casp8p41-mediated cell death. Similar mutations in procaspase 8 impair the ability of HIV to kill infected T cells. These observations support a novel paradigm in which HIV converts a normal cellular constituent into a direct activator that functions like a BH3-only protein.     

Infected cell killing by HIV-1 protease promotes NF-kappaB dependent HIV-1 replication

Acute HIV-1 infection of CD4 T cells often results in apoptotic death of infected cells, yet it is unclear what evolutionary advantage this offers to HIV-1. Given the independent observations that acute T cell HIV-1 infection results in (1) NF-kappaB activation, (2) caspase 8 dependent apoptosis, and that (3) caspase 8 directly activates NF-kappaB, we questioned whether these three events might be interrelated. We first show that HIV-1 infected T cell apoptosis, NF-kappaB activation, and caspase 8 cleavage by HIV-1 protease are coincident. Next we show that HIV-1 protease not only cleaves procaspase 8, producing Casp8p41, but also independently stimulates NF-kappaB activity. Finally, we demonstrate that the HIV protease cleavage of caspase 8 is necessary for optimal NF-kappaB activation and that the HIV-1 protease specific cleavage fragment Casp8p41 is sufficient to stimulate HIV-1 replication through NF-kappaB dependent HIV-LTR activation both in vitro as well as in cells from HIV infected donors. Consequently, the molecular events which promote death of HIV-1 infected T cells function dually to promote HIV-1 replication, thereby favoring the propagation and survival of HIV-1.     

Pseudomonas aeruginosa homoserine lactone triggers apoptosis and Bak/Bax‐independent release of mitochondrial cytochrome C in fibroblasts

Pseudomonas aeruginosa use N‐(3‐oxododecanoyl)‐homoserine lactone (C12) as a quorum‐sensing molecule to regulate gene expression in the bacteria. It is expected that in patients with chronic infections with P. aeruginosa, especially as biofilms, local [C12] will be high and, since C12 is lipid soluble, diffuse from the airways into the epithelium and underlying fibroblasts, capillary endothelia and white blood cells. Previous work showed that C12 has multiple effects in human host cells, including activation of apoptosis. The present work tested the involvement of Bak and Bax in C12‐triggered apoptosis in mouse embryo fibroblasts (MEF) by comparing MEF isolated from embryos of wild‐type (WT) and Bax^?/?/Bak^?/? (DKO) mice. In WT MEF C12 rapidly triggered (minutes to 2 h): activation of caspases 3/7 and 8, depolarization of mitochondrial membrane potential (Δψ_mito), release of cytochrome C from mitochondria into the cytosol, blebbing of plasma membranes, shrinkage/condensation of cells and nuclei and, subsequently, cell killing. A DKO MEF line that was relatively unaffected by the Bak/Bax‐dependent proapoptotic stimulants staurosporine and etoposide responded to C12 similarly to WT MEF: activation of caspase 3/7, depolarization of Δψ_mito and release of cytochrome C and cell death. Re‐expression of Bax or Bak in DKO MEF did not alter the WT‐like responses to C12 in DKO MEF. These data showed that C12 triggers novel, rapid proapoptotic Bak/Bax‐independent responses that include events commonly associated with activation of both the intrinsic pathway (depolarization of Δψ_mito and release of cytochrome C from mitochondria into the cytosol) and the extrinsic pathway (activation of caspase 8). Unlike the proapoptotic agonists staurosporine and etoposide that release cytochrome C from mitochondria, C12's effects do not require participation of either Bak or Bax.     

Infected Cell Killing by HIV-1 Protease Promotes NF-κB Dependent HIV-1 Replication

Acute HIV-1 infection of CD4 T cells often results in apoptotic death of infected cells, yet it is unclear what evolutionary advantage this offers to HIV-1. Given the independent observations that acute T cell HIV-1 infection results in (1) NF-κB activation, (2) caspase 8 dependent apoptosis, and that (3) caspase 8 directly activates NF-κB, we questioned whether these three events might be interrelated. We first show that HIV-1 infected T cell apoptosis, NF-κB activation, and caspase 8 cleavage by HIV-1 protease are coincident. Next we show that HIV-1 protease not only cleaves procaspase 8, producing Casp8p41, but also independently stimulates NF-κB activity. Finally, we demonstrate that the HIV protease cleavage of caspase 8 is necessary for optimal NF-κB activation and that the HIV-1 protease specific cleavage fragment Casp8p41 is sufficient to stimulate HIV-1 replication through NF-κB dependent HIV-LTR activation both in vitro as well as in cells from HIV infected donors. Consequently, the molecular events which promote death of HIV-1 infected T cells function dually to promote HIV-1 replication, thereby favoring the propagation and survival of HIV-1.     

Loss of caspase-9 provides genetic evidence for the type I/II concept of CD95-mediated apoptosis

The death receptor CD95 triggers apoptosis upon formation of a death-inducing signaling complex and the activation of caspase-8. Two types of CD95-mediated apoptosis have been distinguished that differ in their efficiency of death-inducing signaling complex formation and the requirement of mitochondria for caspase activation. The validity of the type I/II model, however, has been challenged, as Bcl-2 expression or the use of various CD95 agonists resulted in different apoptosis effects. By identifying a caspase-9-deficient T cell line, we now provide genetic evidence for the two-pathway model of CD95-mediated apoptosis and demonstrate that type II cells strongly depend on caspase-9. Caspase-9-deficient cells revealed strongly impaired apoptosis, caspase activation, and mitochondrial membrane depolarization upon CD95 triggering, whereas, surprisingly, activation of Bak and cytochrome c release were not inhibited. Furthermore, caspase-9-deficient cells did not switch to necrosis, and reconstitution of caspase-9 expression restored CD95 sensitivity. Finally, we also show that different death receptors have a distinct requirement for caspase-9.     

Curcumin induces Apaf-1-dependent,p21-mediated caspase activation and apoptosis

Previous studies have demonstrated that curcumin induces mitochondria-mediated apoptosis. However, understanding of the molecular mechanisms underlying curcumin-induced cell death remains limited. In this study, we demonstrate that curcumin treatment of cancer cells caused dose- and time-dependent caspase-3 activation, which is required for apoptosis as confirmed using the pan caspase inhibitor, z-VAD. Knockdown experiments and knockout cells excluded a role of caspase-8 in curcumin-induced caspase-3 activation. In contrast, Apaf-1 deficiency or silencing inhibited the activity of caspase-3, pointing to a requisite role of Apaf-1 in curcumin-induced apoptotic cell death. Curcumin treatment led to Apaf-1 upregulation both at the protein and mRNA levels. Cytochrome c release from mitochondria to the cytosol in curcumin-treated cells was associated with upregulation of proapoptotic proteins such as Bax, Bak, Bid, and Bim. Crosslinking experiments demonstrated Bax oligomerization during curcumin-induced apoptosis, suggesting that induced expression of Bax, Bid, and Bim causes Bax-channel formation on the mitochondrial membrane. The release of cytochrome c was unaltered in p53-deficient cells, whereas absence of p21 blocked cytochrome c release, caspase activation, and apoptosis. Importantly, p21-deficiency resulted in reduced expression of Apaf-1 during curcumin treatment, indicating a requirement of p21 in Apaf-1 dependent caspase activation and apoptosis. Together, our findings demonstrate that Apaf-1, Bax, and p21 as novel potential targets for curcumin or curcumin-based anticancer agents.     

Tumor necrosis factor-alpha induces Bax-Bak interaction and apoptosis, which is inhibited by adenovirus E1B 19K

Tumor necrosis factor (TNF)-alpha-mediated death signaling induces oligomerization of proapoptotic Bcl-2 family member Bax into a high molecular mass protein complex in mitochondrial membranes. Bax complex formation is associated with the release of cytochrome c, which propagates death signaling by acting as a cofactor for caspase-9 activation. The adenovirus Bcl-2 homologue E1B 19K blocks TNF-alpha-mediated apoptosis by preventing cytochrome c release, caspase-9 activation, and apoptosis of virus-infected cells. TNF-alpha induces E1B 19K-Bax interaction and inhibits Bax oligomerization. Oligomerized Bax may form a pore to release mitochondrial proteins, analogous to the homologous pore-forming domains of bacterial toxins. E1B 19K can also bind to proapoptotic Bak, but the functional significance is not known. TNF-alpha signaling induced Bak-Bax interaction and both Bak and Bax oligomerization. E1B 19K was constitutively in a complex with Bak, and blocked the Bak-Bax interaction and oligomerization of both. The TNF-alpha-mediated cytochrome c and Smac/DIABLO release from mitochondria was inhibited by E1B 19K expression in adenovirus-infected cells. Since either Bax or Bak is essential for death signaling by TNF-alpha, the interaction between E1B 19K and both Bak and Bax may be required to inhibit their cooperative or independent oligomerization to release proteins from mitochondria which promote caspase activation and cell death.     

Curcumin induces Apaf-1-dependent,p21-mediated caspase activation and apoptosis

Previous studies have demonstrated that curcumin induces mitochondria-mediated apoptosis. However, understanding of the molecular mechanisms underlying curcumin-induced cell death remains limited. In this study, we demonstrate that curcumin treatment of cancer cells caused dose- and time-dependent caspase 3 activation, which is required for apoptosis as confirmed using the pan-caspase inhibitor, z-VAD. Knockdown experiments and knockout cells excluded a role for caspase 8 in curcumin-induced caspase 3 activation. In contrast, Apaf-1 deficiency or silencing inhibited the activity of caspase 3, pointing to a requisite role of Apaf-1 in curcumin-induced apoptotic cell death. Curcumin treatment led to Apaf-1 upregulation, both at the protein and mRNA levels. Cytochrome c release from mitochondria to the cytosol in curcumin-treated cells was associated with upregulation of pro-apoptotic proteins, such as Bax, Bak, Bid and Bim. Cross-linking experiments demonstrated Bax oligomerization during curcumin-induced apoptosis, suggesting that induced expression of Bax, Bid and Bim causes Bax channel formation on the mitochondrial membrane. The release of cytochrome c was unaltered in p53-deficient cells, whereas absence of p21 blocked cytochrome c release, caspase activation and apoptosis. Importantly, p21 deficiency resulted in reduced expression of Apaf-1 during curcumin treatment, indicating a requirement for p21 in Apaf-1-dependent caspase activation and apoptosis. Together, our findings identify Apaf-1, Bax and p21 as novel potential targets for curcumin or curcumin-based anticancer agents.Key words: curcumin, mitochondria, cytochrome c, Apaf-1, caspase, p21     

Activation of Bak and Bax through c-abl-protein kinase Cdelta-p38 MAPK signaling in response to ionizing radiation in human non-small cell lung cancer cells

Intracellular signaling molecules and apoptotic factors seem to play an important role in determining the radiation response of tumor cells. However, the basis for the link between signaling pathway and apoptotic cell death machinery after ionizing irradiation remains still largely unclear. In this study, we showed that c-Abl-PKCdelta-Rac1-p38 MAPK signaling is required for the conformational changes of Bak and Bax during ionizing radiation-induced apoptotic cell death in human non-small cell lung cancer cells. Ionizing radiation induced conformational changes and subsequent oligomerizations of Bak and Bax, dissipation of mitochondrial membrane potential, and cytochrome c release from mitochondria. Small interference (siRNA) targeting of Bak and Bax effectively protected cells from radiation-induced mitochondrial membrane potential loss and apoptotic cell death. p38 MAPK was found to be selectively activated in response to radiation treatment. Inhibition of p38 MAPK completely suppressed radiation-induced Bak and Bax activations, dissipation of mitochondrial membrane potential, and cell death. Moreover, expression of a dominant negative form of protein kinase Cdelta (PKCdelta) or siRNA targeting of PKCdelta attenuated p38 MAPK activation and conformational changes of Bak and Bax. In addition, ectopic expression of RacN17, a dominant negative form of Rac1, markedly inhibited p38 MAPK activation but did not affect PKCdelta activation. Upon stimulation of cells with radiation, PKCdelta was phosphorylated dramatically on tyrosine. c-Abl-PKCdelta complex formation was also increased in response to radiation. Moreover, siRNA targeting of c-Abl attenuated radiation-induced PKCdelta and p38 MAPK activations, and Bak and Bax modulations. These data support a notion that activation of the c-Abl-PKCdelta-Rac1-p38 MAPK pathway in response to ionizing radiation signals conformational changes of Bak and Bax, resulting in mitochondrial activation-mediated apoptotic cell death in human non-small cell lung cancer cells.     

Plasminogen K5 activates mitochondrial apoptosis pathway in endothelial cells by regulating Bak and Bcl-x<Subscript>L</Subscript> subcellular distribution

Plasminogen Kringle 5(K5) is a proteolytic fragment of plasminogen, which displays potent anti-angiogenic activities. K5 has been shown to induce apoptosis in proliferating endothelial cells; however the exact mechanism has not been well explored. The present study was designed to elucidate the possible molecular mechanism of K5-induced endothelial cell apoptosis. Our results showed that K5 inhibited basic fibroblast growth factors activated in human umbilical vein endothelial cells (HUVECs), indicating proliferation in a dose-dependent manner and induced endothelial cell death via apoptosis. K5 exposure activated caspase 7, 8 and 9. These results suggested that both the intrinsic mitochondrial apoptosis pathway and extrinsic pathway might be involved in K5-induced apoptosis. K5 reduced mitochondrial membrane potential (MMP) of HUVECs, demonstrating mitochondrial depolarization in HUVECs. K5 increased the ratio of Bak to Bcl-x_L on mitochondria, decreased the ratio in cytosol, and had no effect on the total amounts of these proteins. K5 also did not effect on Bax/Bcl-2 distribution. K5 increased the ratio of Bak to Bcl-x_L on mitochondrial that resulted in mitochondrial depolarization, cytochrome c release and consequently the cleavage of caspase 9. These results suggested that K5 induces endothelial cell apoptosis at least in part via activating mitochondrial apoptosis pathway. The regulation of K5 on Bak and Bcl-x_L distribution may play an important role in endothelial cell apoptosis. These results provide further insight into the anti-angiogenesis roles of K5 in angiogenesis-related ocular diseases and solid tumors.     

Apigeninidin induces apoptosis through activation of Bak and Bax and subsequent mediation of mitochondrial damage in human promyelocytic leukemia HL-60 cells

Treatment of human promyelocytic leukemia HL-60 cells with apigeninidin could induce cytotoxicity (IC₅₀ = ～80 μM), along with apoptotic sub-G₁ cells, TUNEL-positive apoptotic DNA fragmentation, activation of the multidomain pro-apoptotic Bcl-2 proteins (Bak and Bax), mitochondrial membrane potential (Δψ_m) loss, release of mitochondrial cytochrome c and AIF into the cytoplasm, activation of caspase-9, -3, -8, and -7, and cleavage of PARP and lamin B. These induced apoptotic events were accompanied by decrease of Bcl-2 level and increase of Bak and Bax levels. Apigeninidin-induced sub-G₁ cells and activation of Bak and Bax were also detected in human acute leukemia Jurkat T cells, but not in Jurkat T cells overexpressing Bcl-2. Pretreatment of HL-60 cells with the pan-caspase inhibitor z-VAD-fmk reduced significantly apigeninidin-induced sub-G₁ cells and caspase cascade activation, whereas it failed to suppress Bak and Bax activations, Δψ_m loss, and release of mitochondrial cytochrome c and AIF. None of FADD and caspase-8 deficiencies affected the sensitivity of Jurkat T cells to apigeninidin-induced cytotoxicity. These results demonstrated that apigeninidin-induced apoptosis was mediated by activation of Bak and Bax, mitochondrial damage and resultant release of not only cytochrome c, causing caspase cascade activation, but also caspase-independent death effector AIF in HL-60 cells.     

Necrosis-like death can engage multiple pro-apoptotic Bcl-2 protein family members

Necroptosis is a physiologically relevant mode of cell death with some well-described initiating events, but largely unknown executioners. Here we investigated necrostatin-1 (Nec-1) sensitive death elicited by different necroptosis stimuli in L929 mouse fibrosarcoma cells, mouse embryonic fibroblasts (MEF) and bone marrow-derived macrophages. We found that TNFα- or zVAD-induced necroptosis occurs independently of the recently implicated executioners Bmf or PARP-2, but can involve the Bcl-2 family proteins Bid and Bak. Furthermore, this type of necroptosis is associated with mitochondrial cytochrome c release and partly sensitive to cyclosporine A inhibition, suggesting a cross talk with the mitochondrial permeability transition pore. Necroptosis triggered by cadmium (Cd) exposure caused fully Nec-1-sensitive and caspase-independent death in L929 cells that was associated with autocrine TNFα-mediated feed-forward signalling. In MEF Cd-exposure elicited a mixed mode of cell death that was to some extent Nec-1-sensitive but also displayed features of apoptosis. It was partly dependent on Bmf and Bax/Bak, proteins typically considered to act pro-apoptotic, but ultimately insensitive to caspase inhibition. Overall, our study indicates that inducers of “extrinsic” and “intrinsic” necroptosis can both trigger TNF-receptor signalling. Further, necroptosis may depend on mitochondrial changes engaging proteins considered critical for MOMP during apoptosis that ultimately contribute to caspase-independent necrotic cell death.     

Caspase-2 triggers Bax-Bak-dependent and -independent cell death in colon cancer cells treated with resveratrol

Polyphenol phytoalexin (resveratrol), found in grapes and red wine is a strong chemopreventive agent with promising safety records with human consumption and unique forms of cell death induction in a variety of tumor cells. However, the mechanism of resveratrol-induced apoptosis upstream of mitochondria is still not defined. The results from this study suggest that caspase-2 activation occurs upstream of mitochondria in resveratrol-treated cells. The upstream activation of caspase-2 is not dependent on its antioxidant property or NF-kappaB inhibition. The activated caspase-2 triggers mitochondrial apoptotic events by inducing conformational changes in Bax/Bak with subsequent release of cytochrome c, apoptosis-inducing factor, and endonuclease G. Caspase-8 activation seems to be independent of these events and does not appear to be mediated by classical death receptor processing or downstream caspases. Both caspase-2 and caspase-8 contribute toward the mitochondrial translocation of Bid, since neither caspase-8 inhibition nor caspase-2 inhibition could prevent translocation of Bid DsRed into mitochondria. Caspase-2 inhibitors or antisense silencing of caspase-2 prevented cell death induced by resveratrol and partially prevented processing of downstream caspases, including caspase-9, caspase-3, and caspase-8. Studies using mouse embryonic fibroblasts deficient for both Bax and Bak indicate the contribution of both Bax and Bak in mediating cell death induced by resveratrol and the existence of Bax/Bak-independent cell death possibly through caspase-8- or caspase-2-mediated mitochondria-independent downstream caspase processing.     

Execution of apoptosis signal-regulating kinase 1 (ASK1)-induced apoptosis by the mitochondria-dependent caspase activation

ASK1 activates JNK and p38 mitogen-activated protein kinases and constitutes a pivotal signaling pathway in cytokine- and stress-induced apoptosis. However, little is known about the mechanism of how ASK1 executes apoptosis. Here we investigated the roles of caspases and mitochondria in ASK1-induced apoptosis. We found that benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), a broad-spectrum caspase inhibitor, mostly inhibited ASK1-induced cell death, suggesting that caspases are required for ASK1-induced apoptosis. Overexpression of ASK1DeltaN, a constitutively active mutant of ASK1, induced cytochrome c release from mitochondria and activation of caspase-9 and caspase-3 but not of caspase-8-like proteases. Consistently, caspase-8-deficient (Casp8 (-/-)) cells were sensitive to ASK1-induced caspase-3 activation and apoptosis, suggesting that caspase-8 is dispensable for ASK1-induced apoptosis, whereas ASK1 failed to activate caspase-3 in caspase-9-dificient (Casp9 (-/-)) cells. Moreover, mitochondrial cytochrome c release, which was not inhibited by zVAD-fmk, preceded the onset of caspase-3 activation and cell death induced by ASK1. ASK1 thus appears to execute apoptosis mainly by the mitochondria-dependent caspase activation.     

Mouse Cytotoxic T Cell-derived Granzyme B Activates the Mitochondrial Cell Death Pathway in a Bim-dependent Fashion

Cytotoxic T cells (Tc) use perforin and granzyme B (gzmB) to kill virus-infected cells and cancer cells. Recent evidence suggests that human gzmB primarily induces apoptosis via the intrinsic mitochondrial pathway by either cleaving Bid or activating Bim leading to the activation of Bak/Bax and subsequent generation of active caspase-3. In contrast, mouse gzmB is thought to predominantly induce apoptosis by directly processing pro-caspase-3. However, in certain mouse cell types gzmB-mediated apoptosis mainly occurs via the mitochondrial pathway. To investigate whether Bim is involved under the latter conditions, we have now employed ex vivo virus-immune mouse Tc that selectively kill by using perforin and gzmB (gzmB⁺Tc) as effector cells and wild type as well as Bim- or Bak/Bax-deficient spontaneously (3T9) or virus-(SV40) transformed mouse embryonic fibroblast cells as targets. We show that gzmB⁺Tc-mediated apoptosis (phosphatidylserine translocation, mitochondrial depolarization, cytochrome c release, and caspase-3 activation) was severely reduced in 3T9 cells lacking either Bim or both Bak and Bax. This outcome was related to the ability of Tc cells to induce the degradation of Mcl-1 and Bcl-X_L, the anti-apoptotic counterparts of Bim. In contrast, gzmB⁺Tc-mediated apoptosis was not affected in SV40-transformed mouse embryonic fibroblast cells lacking Bak/Bax. The data provide evidence that Bim participates in mouse gzmB⁺Tc-mediated apoptosis of certain targets by activating the mitochondrial pathway and suggest that the mode of cell death depends on the target cell. Our results suggest that the various molecular events leading to transformation and/or immortalization of cells have an impact on their relative resistance to the multiple gzmB⁺Tc-induced death pathways.     

Bax and Bak independently promote cytochrome C release from mitochondria

Pro-apoptotic Bax and Bak have been implicated in the regulation of p53-dependent apoptosis. We assessed the ability of primary baby mouse kidney (BMK) epithelial cells from bax(-/-), bak(-/-), and bax(-/-) bak(-/-) mice to be transformed by E1A alone or in conjunction with dominant-negative p53 (p53DD). Although E1A alone transformed BMK cells from p53-deficient mice, E1A alone did not transform BMK cells from bax(-/-), bak(-/-), or bax(-/-) bak(-/-) mice. Thus, the loss of both Bax and Bak was not sufficient to relieve p53-dependent suppression of transformation in epithelial cells. To test the requirement for Bax and Bak in other death signaling pathways, stable E1A plus p53DD-transformed BMK cell lines were derived from the bax(-/-), bak(-/-), and bax(-/-) bak(-/-) mice and characterized for their response to tumor necrosis factor-alpha (TNF-alpha)-mediated apoptosis. The loss of both Bax and Bak severely impaired TNF-alpha-mediated apoptosis, but the presence of either Bax or Bak alone was sufficient for cell death. Cytochrome c was released from mitochondria, and caspase-9 was activated in Bax- or Bak-deficient cells in response to TNF-alpha but not in cells deficient in both. Thus, either Bax or Bak is required for death signaling through mitochondria in response to TNF-alpha, but both are dispensable for p53-dependent transformation inhibition.     

Human immunodeficiency virus type 1 protease cleaves procaspase 8 in vivo

Human immunodeficiency virus type 1 (HIV-1) infection causes apoptosis of infected CD4 T cells as well as uninfected (bystander) CD4 and CD8 T cells. It remains unknown what signals cause infected cells to die. We demonstrate that HIV-1 protease specifically cleaves procaspase 8 to create a novel fragment termed casp8p41, which independently induces apoptosis. casp8p41 is specific to HIV-1 protease-induced death but not other caspase 8-dependent death stimuli. In HIV-1-infected patients, casp8p41 is detected only in CD4(+) T cells, predominantly in the CD27(+) memory subset, its presence increases with increasing viral load, and it colocalizes with both infected and apoptotic cells. These data indicate that casp8p41 independently induces apoptosis and is a specific product of HIV-1 protease which may contribute to death of HIV-1-infected cells.     

Benzo[a]pyrene-7,8-diol-9,10-epoxide causes caspase-mediated apoptosis in H460 human lung cancer cell line

We have shown previously that wild-type p53 renders H460 human lung cancer cells more sensitive to apoptosis induction by environmental carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), but the mechanism of cell death is not fully understood. The present study provides insights into the mechanism by which BPDE causes apoptosis in H460 cells. Exposure of H460 cells to BPDE resulted in a concentration-dependent apoptotic cell death characterized by cleavage of poly(ADP-ribose)polymerase, DNA condensation, and apoptotic histone-associated DNA fragments released into the cytosol. The BPDE-mediated release of apoptotic histone-associated DNA fragments into the cytosol was also observed in a normal bronchial epithelial cell line BEAS-2B. The BPDE-induced apoptosis in H460 cells correlated with up-regulation of pro-apoptotic protein Bak, down-regulation of anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xL, release of cytochrome c from mitochondria to the cytosol without a change in mitochondrial membrane potential or mitochondrial morphology (electron microscopy), and cleavage of caspase-8, -9, and -3. Ectopic expression of Bcl-2 failed to confer significant protection against BPDE-induced apoptosis in H460 cells. The SV40 immortalized mouse embryonic fibroblasts (MEFs) derived from Bak and Bax double knockout mice, but not Bid knockout mice, were significantly more resistant to BPDE-induced apoptosis compared with the MEFs derived from wild-type mice. The BPDE-induced apoptosis was partially but statistically significantly attenuated in the presence of specific inhibitors of caspase-9 (z-LEHDfmk) and caspase-8 (z-IETDfmk). In conclusion, the present study reveals that BPDE-induced apoptosis in H460 cells is associated with Bak induction and caspase activation but independent of Bcl-2.     

c-Myc functionally cooperates with Bax to induce apoptosis

c-Myc promotes apoptosis by destabilizing mitochondrial integrity, leading to the release of proapoptotic effectors including holocytochrome c. Candidate mediators of c-Myc in this process are the proapoptotic members of the Bcl-2 family. We show here that fibroblasts lacking Bak remain susceptible to c-Myc-induced apoptosis whereas bax-deficient fibroblasts are resistant. However, despite this requirement for Bax, c-Myc activation exerts no detectable effects on Bax expression, localization, or conformation. Moreover, susceptibility to c-Myc-induced apoptosis can be restored in bax-deficient cells by ectopic expression of Bax or by microinjection of a peptide comprising a minimal BH3 domain. Microinjection of BH3 peptide also restores sensitivity to c-Myc-induced apoptosis in p53-deficient primary fibroblasts that are otherwise resistant. By contrast, there is no synergy between BH3 peptide and c-Myc in fibroblasts deficient in both Bax and Bak. We conclude that c-Myc triggers a proapoptotic mitochondrial destabilizing activity that cooperates with proapoptotic members of the Bcl-2 family.