Yersinia enterocolitica YopP-induced apoptosis of macrophages involves the apoptotic signaling cascade upstream of bid.
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Yersinia enterocolitica induces apoptosis in macrophages by injecting the plasmid-encoded YopP (YopJ in other Yersinia species). Recently it was reported that YopP/J is a member of an ubiquitin-like protein cysteine protease family and that the catalytic core of YopP/J is required for its inhibition of the MAPK and NF-kappaB pathways. Here we analyzed the YopP/J-induced apoptotic signaling pathway. YopP-mediated cell death could be inhibited by addition of the zVAD caspase inhibitor, but not by DEVD or YVAD. Generation of truncated Bid (tBid) was the first apoptosis-related event that we observed. The subsequent translocation of tBid to the mitochondria induced the release of cytochrome c, leading to the activation of procaspase-9 and the executioner procaspases-3 and -7. Inhibition of the postmitochondrial executioner caspases-3 and -7 did not affect Bid cleavage. Bid cleavage could not be observed in a yopP-deficient Y. enterocolitica strain, showing that this event requires YopP. Disruption of the catalytic core of YopP abolished the rapid generation of tBid, thereby hampering induction of apoptosis by Y. enterocolitica. This finding supports the idea that YopP/J induces apoptosis by directly acting on cell death pathways, rather than being the mere consequence of gene induction inhibition in combination with microbial stimulation of the macrophage. (+info)
Tumor necrosis factor alpha induces BID cleavage and bypasses antiapoptotic signals in prostate cancer LNCaP cells.
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Survival of cancer cells in response to therapy, immune response, or metastasis depends on interactions between pro- and antiapoptotic signals. Two major proapoptotic pathways have been described: (a) a death receptor pathway; and (b) a mitochondrial pathway. We reported previously that Akt and the epidermal growth factor (EGF) receptor send separate, redundant survival signals that act to inhibit the mitochondrial proapoptotic pathway in prostate cancer LNCaP cells. However, it was unclear at what level the pro- and antiapoptotic signals interact in these cells, and it was also unclear whether these signals would inhibit the death receptor pathway. We found that EGF can protect LNCaP cells from apoptosis induced by LY294002 but not from tumor necrosis factor a (TNF-alpha)-induced apoptosis. Furthermore, TNF-alpha induced apoptosis under conditions in which Akt was active. Treatment with TNF-alpha resulted in activation of caspase 8 and cleavage of BID, which in turn induced cytochrome c release and caspase 9-dependent activation of effector caspases. Thus, proapoptotic signals induced by both TNF-alpha and LY294002 converge on mitochondria and trigger cytochrome c release. Because EGF can inhibit cytochrome c release induced by LY294002 but not cytochrome c release induced by TNF-alpha, we suggest that the EGF survival mechanism operates on the mitochondrial pathway at a site upstream of cytochrome c release. The ability of TNF-alpha to bypass survival signals from activated EGF receptor and Akt in prostate cancer cells makes death receptor signaling a promising avenue for therapeutic intervention. (+info)
Apoptotic signaling pathway activated by Helicobacter pylori infection and increase of apoptosis-inducing activity under serum-starved conditions.
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The enhanced gastric epithelial cell apoptosis observed during infection with Helicobacter pylori has been suggested to be of significance in the etiology of gastritis, peptic ulcers, and neoplasia. To investigate the cell death signaling induced by H. pylori infection, human gastric epithelial cells were incubated with H. pylori for up to 72 h. H. pylori infection induced the activation of caspase -8, -9, and -3 and the expression of the proapoptotic Bcl-2 family proteins Bad and Bid. The peak of the activity of the caspases occurred at 24 h. At this time, the inhibition of caspase-8 or -9 almost completely suppressed H. pylori-induced apoptosis. Inhibition of caspase-8 suppressed the expression of Bad and Bid and the subsequent activation of caspase-9 and -3. These observations indicate that H. pylori induces apoptosis through a pathway involving the sequential induction of apical caspase-8 activity, the proapoptotic proteins Bad and Bid, caspase-9 activity, and effector caspase-3 activity. Activation of the pathway was independent of CagA or vacuolating toxin. A membrane fraction of H. pylori was sufficient to activate this pathway, and treatment with proteinase K eliminated the activity. Apoptotic activity of the membrane fraction was significantly increased by incubating the bacteria under serum-starved conditions for 24 h. These observations suggest that environmental conditions in the human stomach could induce H. pylori-mediated pathogenesis, leading to a variety of clinical outcomes. (+info)
Cell type specific involvement of death receptor and mitochondrial pathways in drug-induced apoptosis.
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Apoptosis in response to cellular stress such as treatment with cytotoxic drugs is mediated by effector caspases (caspase-3) which can be activated by different initiator pathways. Here, we report on a cell type specific triggering of death receptor and/or mitochondrial pathways upon drug treatment. In type I cells (BJAB), both the receptor and the mitochondrial pathway were activated upon drug treatment, since blockade of either the receptor pathway by overexpression of dominant negative FADD (FADD-DN) or of the mitochondrial pathway by overexpression of Bcl-X(L) only partially inhibited apoptosis. Drug treatment induced formation of a FADD- and caspase-8-containing CD95 death-inducing signaling complex (DISC) in type I cells resulting in activation of caspase-8 as the most apical caspase. In contrast, in type II cells (Jurkat), apoptosis was predominantly controlled by mitochondria, since overexpression of Bcl-2 completely blocked drug-induced apoptosis, while overexpression of FADD-DN had no protective effect. In these cells, caspases including caspase-8 were activated by mitochondria-driven signaling events and no DISC was detected despite expression levels of CD95, FADD and caspase-8 proteins comparable to type I cells. Likewise, drug-induced CD95 aggregation was predominantly found in type I cells. Bid was cleaved prior to mitochondrial alterations in type I cells providing a molecular link between caspase-8 activation and mitochondrial perturbations, whereas in type II cells, Bid was cleaved downstream of mitochondria. Our findings of a cell type specific response to cytotoxic drugs have implications for the identification of molecular parameters for chemosensitivity or resistance in different tumor cells. (+info)
Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death.
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Multiple death signals influence mitochondria during apoptosis, yet the critical initiating event for mitochondrial dysfunction in vivo has been unclear. tBID, the caspase-activated form of a "BH3-domain-only" BCL-2 family member, triggers the homooligomerization of "multidomain" conserved proapoptotic family members BAK or BAX, resulting in the release of cytochrome c from mitochondria. We find that cells lacking both Bax and Bak, but not cells lacking only one of these components, are completely resistant to tBID-induced cytochrome c release and apoptosis. Moreover, doubly deficient cells are resistant to multiple apoptotic stimuli that act through disruption of mitochondrial function: staurosporine, ultraviolet radiation, growth factor deprivation, etoposide, and the endoplasmic reticulum stress stimuli thapsigargin and tunicamycin. Thus, activation of a "multidomain" proapoptotic member, BAX or BAK, appears to be an essential gateway to mitochondrial dysfunction required for cell death in response to diverse stimuli. (+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)
Activation of pro-death Bcl-2 family proteins and mitochondria apoptosis pathway in tumor necrosis factor-alpha-induced liver injury.
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Tumor necrosis factor-alpha (TNFalpha)-induced cytotoxicity contributes to the pathogenesis in inflammatory and immune responses. Here, we studied the role of pro-death Bcl-2 family proteins and the mitochondria apoptosis pathway in the development of TNFalpha-induced hepatic injury during endotoxemia. After treating mice with lipopolysaccharide or TNFalpha in the presence of d-galactosamine, Bid was cleaved and translocated to mitochondria in hepatocytes. Independently, Bax was also activated by the death receptor engagement and translocated to mitochondria. However, its subsequent insertion into the mitochondrial membrane depends on Bid. Nevertheless, Bid was required, but Bax could be dispensed for the mitochondrial release of cytochrome c from mitochondria, suggesting that Bid could activate additional downstream molecules other than Bax. The lack of this Bid-dependent mitochondria activation and cytochrome c release in the bid-deficient mice was responsible for the significantly delayed effector caspase activation and hepatocyte injury upon endotoxin treatment, culminating in a prolonged survival of the bid-deficient mice. Additional genetic factor(s) could further modify the dependence of TNFalpha toxicity on the mitochondria pathway as the bid-deficient 129/SvJ mice manifested an even higher resistance than the same type of mice in C57BL/6 background. The functional significance of the mitochondria apoptosis pathway was thus elucidated in the TNFalpha-mediated pathogenesis in vivo. (+info)
Coexpression of mutant p53 and p193 renders embryonic stem cell-derived cardiomyocytes responsive to the growth-promoting activities of adenoviral E1A.
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Expression of adenoviral E1A in cardiomyocytes results in the activation of DNA synthesis followed by apoptosis. In contrast, expression of simian virus 40 large T antigen induces sustained cardiomyocyte proliferation. Previous studies have shown that T antigen binds to 2 proapoptotic proteins in cardiomyocytes, namely the p53 tumor suppressor and p193 (a new member of the BH3-only proapoptosis subfamily). Structure-function analyses identified a p193 C-terminal truncation mutant that encodes prosurvival activity. This mutant was used to test the role of p193 in E1A-induced cardiomyocyte apoptosis. E1A induced apoptosis in cardiomyocytes derived from differentiating embryonic stem cells. Expression of the prosurvival p193 mutant alone or a mutant p53 alone did not block E1A-induced apoptosis. In contrast, combinatorial expression of mutant p193 and mutant p53 blocked E1A-induced apoptosis, resulting in a proliferative response indistinguishable from that seen with T antigen. These results confirm the hypothesis that there are 2 proapoptotic pathways, encoded by p53 and p193, respectively, which restrict cardiomyocyte cell cycle activity in differentiating embryonic stem cell cultures. Furthermore, these results explain in molecular terms the phenotypic differences of E1A versus T-antigen gene transfer in cardiomyocytes. (+info)