Altered proteasomal function due to the expression of polyglutamine-expanded truncated N-terminal huntingtin induces apoptosis by caspase activation through mitochondrial cytochrome c release

Expansion of CAG repeats within the coding region of target genes is the cause of several autosomal dominant neurodegenerative diseases including Huntington's disease (HD). A hallmark of HD is the proteolytic production of N-terminal fragments of huntingtin containing polyglutamine repeats that form ubiquitinated aggregates in the nucleus and cytoplasm of the affected neurons. In this study, we used an ecdysone-inducible stable mouse neuro2a cell line that expresses truncated N-terminal huntingtin (tNhtt) with different polyglutamine length, along with mice transgenic for HD exon 1, to demonstrate that the ubiquitin-proteasome pathway is involved in the pathogenesis of HD. Proteasomal 20S core catalytic component was redistributed to the polyglutamine aggregates in both the cellular and transgenic mouse models. Proteasome inhibitor dramatically increased the rate of aggregate formation caused by tNhtt protein with 60 glutamine (60Q) repeats, but had very little influence on aggregate formation by tNhtt protein with 150Q repeats. Both normal and polyglutamine-expanded tNhtt proteins were degraded by proteasome, but the rate of degradation was inversely proportional to the repeat length. The shift of the proteasomal components from the total cellular environment to the aggregates, as well as the comparatively slower degradation of tNhtt with longer polyglutamine, decreased the proteasome's availability for degrading other key target proteins, such as p53. This altered proteasomal function was associated with disrupted mitochondrial membrane potential, released cytochrome c from mitochondria into the cytosol and activated caspase-9- and caspase-3-like proteases. These results suggest that the impaired proteasomal function plays an important role in polyglutamine protein-induced cell death.     

Bax, reactive oxygen, and cytochrome c release in neuronal apoptosis

Half of all neurons produced during embryogenesis undergo apoptotic death shortly before birth or soon thereafter. Two cell culture models have been used extensively to investigate the cellular and molecular mechanisms underlying apoptosis during neuronal development: (a) sympathetic neurons deprived of their required neurotrophic factor, nerve growth factor, and (b) cerebellar granule neurons deprived of serum in low-potassium medium. A dramatic increase in mitochondrial-derived reactive oxygen species (ROS) occurs during the apoptotic death of both of these cell types. These ROS lie downstream from the proapoptotic protein, Bax. Bax normally resides in the cytoplasm, but translocates to the outer mitochondrial membrane during apoptosis. Once associated with mitochondria, Bax causes release of apoptogenic factors from the mitochondria into the cytoplasm, thus inducing or augmenting the apoptotic cascade. Although there is much controversy about the exact mechanism by which Bax causes release of these factors, recent evidence suggests that the Bax-induced ROS are critical for this release to occur in both sympathetic and cerebellar granule neurons. Because Bax is critical for the apoptotic death of many other types of neurons, it is likely that increased ROS is important for the death of these cells as well.     

Mitochondrial cytochrome c release in radiation-induced apoptosis of human peripheral T cells

We examined sequential changes in post-irradiated peripheral blood T cells taken from normal volunteers, using a microscopy-video system, mitochondrial membrane potential assay, annexin V, propidium iodide, and cytochrome c ELISA kit. After 5 Gy irradiation with 10 MV X-ray from a linear accelerator, the percentages of apoptotic T cells were estimated as approximately 5, 10, 20, 35, and 70%, at 0, 3, 6, 10, and 20 h after irradiation, respectively, as observed with the microscopy-video system. Using a CCD camera-equipped fluorescence microscope and MitoCapture, a mitochondrial membrane potential indicator, approximately half of the T cells showed dysfunction of mitochondrial membrane potential at 10 h after 5 Gy irradiation. With regard to annexin V and propidium iodide, approximately 40 and 5% of the human peripheral T cells showed positivity against annexin V and propidium iodide at that time, respectively. Mitochondrial cytochrome c release from the mitochondria to the cytosol was confirmed to start at 10 h and to reach a maximum at 20 h after 5 Gy of irradiation. These results demonstrated that mitochondrial cytochrome c release occurred following dysfunction of mitochondrial membrane potential in radiation-induced T cell apoptosis.     

Apoptosis via the B cell antigen receptor requires Bax translocation and involves mitochondrial depolarization, cytochrome C release, and caspase-9 activation

Various routes to apoptosis can be active during B cell development. In a model system of mature B cells, differences in caspase-3 processing have suggested that antigen receptor (BCR)-mediated apoptosis may involve a zVAD-insensitive initiator protease(s). In search of the events leading to caspase-3 activation, we now establish that both CD95- and BCR-mediated apoptosis depend on Bax activation and cytochrome C (cytC) release. Nevertheless, the timing and caspase-dependence of mitochondrial membrane depolarization differed considerably after CD95- or BCR-triggering. To delineate events subsequent to cytC release, we compared apoptosis induced via BCR triggering and via direct mitochondrial depolarization by CCCP. In both cases, partial processing of caspase-3 was observed in the presence of zVAD. By expression in 293 cells we addressed the potential of candidate initiator caspases to function in the presence of zVAD, and found that caspase-9 efficiently processed caspase-3, while caspase-2 or -8 were inactive. Finally, retroviral expression of dominant-negative caspase-9 inhibited both CD95- and BCR-mediated apoptosis. In conclusion, we obtained no evidence for involvement of a BCR-specific protease. Instead, our data show for the first time that the BCR-signal causes Bax translocation, followed by mitochondrial depolarization, and cytC release. Subsequent caspase-9 activation can solely account for events further downstream.     

Polyalanine tracts directly induce the release of cytochrome c, independently of the mitochondrial permeability transition pore, leading to apoptosis

In recent years, several novel types of disorder caused by the expansion of triplet repeats in specific genes have been characterized; in the polyalanine diseases, these expanded repeats result in proteins with aberrantly elongated polyalanine tracts. In this study, we fused expanded polyalanine tracts to yellow fluorescent protein to examine their physical interaction with mitochondria. Tracts containing more than 23 alanine repeats were found to physically associate with mitochondria, strongly suggesting that an interaction between polyalanine tracts and mitochondria is a contributing factor in the pathology of polyalanine diseases. Furthermore, in in vitro experiments, polyalanine tracts induced release of cytochrome c from mitochondria and caspase-3 activation, independently of the mitochondrial permeability transition pore. These results suggest that oligomerized polyalanine tracts might induce the rupture of the mitochondrial membrane, the subsequent release of cytochrome c , and apoptosis. This novel mechanism for polyalanine tract cytotoxicity might be common to the pathogenesis of all polyalanine diseases. Further investigation of this mechanism might aid the development of therapies for these diseases.     

Mechanism of triptolide-induced apoptosis: effect on caspase activation and Bid cleavage and essentiality of the hydroxyl group of triptolide

Triptolide is a compound extracted from the Chinese herb Tripterygium wilfordii Hook. f. Triptolide has potent anticancer activity. However, the mechanisms by which triptolide exerts its anticancer activities remain unclear. To explore the molecular mechanisms involved in the anticancer activity of triptolide, we have examined the effect of triptolide on the growth of pancreatic carcinoma PANC-1 and cervical adenocarcinoma HeLa cells. We found that treatment of both HeLa and PANC-1 cells with triptolide potently suppressed cell growth and induced apoptosis, indicated by nuclear fragmentation and blebbing. In both HeLa and PANC-1 cells, apoptosis induced by triptolide was associated with activation of both caspase-3 and caspase-8, and cleavage of poly(ADP-ribose) polymerase and Bid. Moreover, in HeLa cells, caspase-9 is also significantly activated in response to triptolide. Overexpression of Bcl-2 in HeLa cells substantially attenuated triptolide-induced apoptosis. Interestingly, substitution of the 14-OH of triptolide with an acetyl group abrogated both its anticancer and its antiinflammatory activities. Our studies suggest that triptolide may exert its anticancer effects by initiating apoptosis through both death-receptor- and mitochondria-mediated pathways. Our results indicate that both the apoptosis-promoting and the antiinflammatory activities of triptolide depend on the 14-OH group.     

The role of calpain in caspase activation during etoposide induced apoptosis in T cells.

T cells treated with the drug etoposide undergo apoptotic death characterized by early evidence of nuclear damage followed by loss of mitochondrial integrity and cell lysis. Calpains and caspases are cytoplasmic proteases and there is increasing evidence of cross-talk between these proteases in death pathways. In this study we have investigated the role of calpain, in etoposide-triggered apoptosis in the 2B4 murine T cell hybridoma. Cell permeable inhibitors of calpain, ALLnM, E64 and calpeptin that block Fas ligand-Fas-mediated death in T cells, blocked etoposide-induced nuclear damage, loss of mitochondrial integrity and cell lysis. A broad spectrum peptide inhibitor of caspases, ZVAD-fmk, partially blocked nuclear damage but poorly inhibited mitochondrial damage or cell lysis triggered by etoposide. Etoposide-induced expression of the cleaved, proteolytically active form of caspase 3, and DEVD-ase activity, detected prior to nuclear damage, were blocked in the presence of calpain inhibitors. Collectively, these data describe a role for calpain in regulating etoposide-induced apoptosis via a caspase-dependent pathway in T cells.     

Evidence for caspase effects on release of cytochrome c and AIF in a model of ischemia in cortical neurons

Neuronal apoptosis following ischemia can be mediated by a caspase-dependent pathway, which involves the mitochondrial release of cytochrome c that initiates a cascade of caspase activation. In addition, there is a caspase-independent pathway, which is mediated by the release of apoptosis-inducing factor (AIF). Using caspase inhibitor gene therapy, we investigated the roles of caspases on the mitochondrial release of cyt c and the release of AIF. Specifically, we used herpes simplex virus-1 amplicon vectors to ectopically express a viral caspase inhibitor (crmA or p35) in mixed cortical cultures exposed to oxygen/glucose deprivation. Overexpression of either crmA or p35 (but not the caspase-3 inhibitor DEVD) inhibited the release of AIF; this suggests that there can be cross-talk between the caspase-dependent and the ostensibly caspase-independent pathway. In addition, both crmA overexpression and DEVD inhibited cyt c release, suggesting a positive feedback loop involving activated caspases stimulating cyt c release.     

Coxiella burnetii inhibits activation of host cell apoptosis through a mechanism that involves preventing cytochrome c release from mitochondria

Coxiella burnetii is an obligate intracellular pathogen and the etiological agent of the human disease Q fever. C. burnetii infects mammalian cells and then remodels the membrane-bound compartment in which it resides into a unique lysosome-derived organelle that supports bacterial multiplication. To gain insight into the mechanisms by which C. burnetii is able to multiply intracellularly, we examined the ability of host cells to respond to signals that normally induce apoptosis. Our data show that mammalian cells infected with C. burnetii are resistant to apoptosis induced by staurosporine and UV light. C. burnetii infection prevented caspase 3/7 activation and limited fragmentation of the host cell nucleus in response to agonists that induce apoptosis. Inhibition of bacterial protein synthesis reduced the antiapoptotic effect that C. burnetii exerted on infected host cells. Inhibition of apoptosis in C. burnetii-infected cells did not correlate with the degradation of proapoptotic BH3-only proteins involved in activation of the intrinsic cell death pathway; however, cytochrome c release from mitochondria was diminished in cells infected with C. burnetii upon induction of apoptosis. These data indicate that C. burnetii can interfere with the intrinsic cell death pathway during infection by producing proteins that either directly or indirectly prevent release of cytochrome c from mitochondria. It is likely that inhibition of apoptosis by C. burnetii represents an important virulence property that allows this obligate intracellular pathogen to maintain host cell viability despite inducing stress that would normally activate the intrinsic death pathway.     

Caspase activation by granzyme B is indirect,and caspase autoprocessing requires the release of proapoptotic mitochondrial factors

Apoptosis in response to granzyme B involves activation of caspase-dependent target cell death pathways. Herein, we show that granzyme B initiates caspase processing but cannot fully process procaspase-3 in intact Jurkat T leukemia or NT2 neuronal cells. Rather, the release from mitochondria of proapoptotic mediators cytochrome c, Smac/Diablo, and HtrA2/Omi facilitates full activation of caspases that results from autoprocessing. Bcl-2 overexpression in mitochondria suppresses the release of these proapoptotic molecules, resulting in cell survival despite partial procaspase processing by granzyme B. We propose that binding of inhibitor of apoptosis (IAP) proteins to partially processed procaspases inhibits cell death unless mitochondrial disruption also occurs in response to granzyme B or activated BH3-domain proteins such as truncated Bid.     

Cytotoxicity of tubeimoside I in human choriocarcinoma JEG-3 cells by induction of cytochrome c release and apoptosis via the mitochondrial-related signaling pathway

Mitochondria play important roles in the intrinsic pathways that trigger apoptosis. Anticancer chemotherapies eliminate cancer cells mainly through the induction of apoptosis. In the present study, we investigated the mechanism of the cytotoxic effects of tubeimoside I (TBMS1) on the human choriocarcinoma cell line (JEG-3). Choriocarcinoma is one of the most common malignant tumors in the reproductive system. TBMS1, a triterpenoid saponin, isolated from the tubers of Bolbostemma paniculatum (Maxim) Franquet (Cucurbitaceae), showed potent antitumor effects. However, the potential roles of TBMS1 in the treatment of choriocarcinoma remain unknown. In the present study, we examined the effects of TBMS1 on JEG-3 cells. TBMS1 displayed strong growth inhibitory effects on JEG-3 cell growth. In addition, TBMS1 treatment with TBMS1 led to marked cell apoptosis, significant cell cycle arrest at G2 phase and decrease in mitochondrial transmembrane potential (ΔΨm). Cytochrome c was released from the mitochondria and caspase-3 expression was enhanced. Furthermore, TBMS1 induced the up-regulation of Bcl-2 associated X protein (Bax) expression, down-regulation of Bcl-2 expression, inhibition of nuclear factor-κ-B (NF-κB) function and impacted the phosphorylation of p38 mitogen-activated protein kinase (p38/MAPK), extracellular signal-regulated kinases (ERK)1/2 and protein kinase B (Akt). Taken together, our findings suggest that TBMS1 is an efficient apoptosis-inducing agent for choriocarcinoma cells, which exerts its effects, at least partially, by the induction of mitochondrial dysfunction and regulation of the p38/MAPK, ERK1/2 and PI3K/Akt signaling pathways.     

Creating conditions similar to those that occur during exposure of cells to microgravity induces apoptosis in human lymphocytes by 5-lipoxygenase-mediated mitochondrial uncoupling and cytochrome c release

Creating conditions similar to those that occur during exposure of cells to microgravity induced a sixfold increase of apoptotic bodies and DNA fragments in human lymphocytes, paralleled by an early (within 2 h) fourfold increase in 5-lipoxygenase (5-LOX) activity and a fivefold decrease in mitochondrial membrane potential and increase in cytochrome c release (within 4 and 8 h, respectively). Similar membrane potential and cytochrome c release were observed in isolated mitochondria treated with physiological amounts of 5-LOX and were enhanced by creating conditions similar to those that occur during exposure of cells to microgravity. 5-LOX inhibitors, 5,8,11,14-eicosatetraynoic acid and caffeic acid, completely prevented apoptosis, whereas the phospholipase A(2) inhibitor methyl-arachidonoyl fluorophosphonate and the 5-LOX activating protein inhibitor MK886 reduced it to 65-70%. The intracellular calcium chelator EGTA-acetoxymethylester reduced 5-LOX activity and apoptosis to 30-40% of controls, whereas the p38 mitogen-activated protein kinase inhibitor SB203580 was ineffective. The caspase-3 and caspase-9 inhibitors Z-Asp(OCH(3))-Glu(OCH(3))-Val-Asp(OCH(3))-fluoromethylketone (FMK) and Z-Leu-Glu(OCH(3))-His-Asp(OCH(3))-FMK reduced apoptotic bodies to 25-30% of the control cells. Finally, creating conditions similar to those that occur during exposure of cells to microgravity did not induce apoptosis in human lymphoma U937 cells, which did not express an active 5-LOX.     

Inhibition of apoptosis by Escherichia coli K1 is accompanied by increased expression of BclXL and blockade of mitochondrial cytochrome c release in macrophages

Escherichia coli K1 survival in the blood is a critical step for the onset of meningitis in neonates. Therefore, the circulating bacteria are impelled to avoid host defense mechanisms by finding a niche to survive and multiply. Our recent studies have shown that E. coli K1 enters and survives in both monocytes and macrophages in the newborn rat model of meningitis as well as in macrophage cell lines. Here we demonstrate that E. coli K1 not only extends the survival of human and murine infected macrophage cell lines but also renders them resistant to apoptosis induced by staurosporine. Macrophages infected with wild-type E. coli expressing outer membrane protein A (OmpA), but not with OmpA- E. coli, are resistant to DNA fragmentation and phosphatidylserine exposure induced by staurosporine. Infection with OmpA+ E. coli induces the expression of Bcl(XL), an antiapoptotic protein, both at the mRNA level as assessed by gene array analysis and at the protein level as evaluated by immunoblotting. OmpA- E. coli infection of macrophages induced the release of cytochrome c from mitochondria into the cytosol and the activation of caspases 3, 6, and 9, events that were significantly blocked in OmpA+ E. coli-infected macrophages. In addition, OmpA+ E. coli-infected cells were resistant to a decrease in the transmembrane potential of mitochondria induced by staurosporine as measured by the MitoCapture fluorescence technique. Complementation of OmpA- E. coli with a plasmid containing the ompA gene restored the ability of OmpA- E. coli to inhibit the apoptosis of infected macrophages, further demonstrating that E. coli OmpA expression is critical for inducing macrophage survival and thereby finding a safe haven for its growth.     

Early release of mitochondrial cytochrome c and expression of mitochondrial epitope 7A6 with a porphyrin-derived photosensitizer: Bcl-2 and Bcl-xL overexpression do not prevent early mitochondrial events but still depress caspase activity.

Certain nonmetallic porphyrins have potent antitumor activity upon visible light irradiation. Treatment of HeLa cells with nanomolar amounts of the photochemo therapeutic agent verteporfin and red light mobilized caspases 2, 3, 6, 7, 8, and 9, caused degradation of specific caspase substrates, and resulted in morphologic changes consistent with apoptosis. Caspase processing was detectable by 1 hour after light irradiation. The mitochondrial 7A6 epitope, recognized by monoclonal antibody APO2.7, became accessible, and cytochrome c was detectable within the cytosolic fraction of cells treated with verteporfin immediately after light irradiation. The general caspase inhibitor benzyloxycarboyl-Val-Ala-Asp-fluoromethylketone did not prevent 7A6 expression produced by photosensitization at peptide concentrations which completely prevented caspase activation and cleavage of caspase-specific substrates. Enforced overexpression of Bcl-2 or Bcl-xL prevented cytochrome c release and 7A6 expression produced by ultraviolet B light treatment, but did not prevent cytochrome c release or 7A6 expression elicited by verteporfin photosensitization. Bcl-2 or Bcl-xL overexpression delayed morphologic changes, depressed caspase activation, and limited substrate degradation, but did not protect against loss of viability after verteporfin photosensitization. This observation indicates that cells overexpressing Bcl-2 or Bcl-xL exhibit resistance to caspase activation even after the appearance of cytochrome c in the cytosol. Porphyrin photosensitizers are effective chemotherapeutic agents that elicit primary proapoptotic mitochondrial events even in the setting of heightened Bcl-2 or Bcl-xL expression.     

Advanced glycation end products induce T cell apoptosis: Involvement of oxidative stress, caspase and the mitochondrial pathway

Accumulation of advanced glycation end products (AGEs) is a hallmark in aged people. T cells play important roles in maintaining homeostasis of immune function. This study investigated the effects of AGEs-bovine serum albumin (AGEs) in human T cells. Incubation of Jurkat and several immortalized T cell lines with AGEs resulted in cell death dose-dependently. AGEs-induced cell death was partially but significantly blocked by neutralizing antibodies recognizing receptor of AGEs. In addition to detecting DNA nick, simultaneous stainings of annexin V with 7-amino-actinomycin D further confirmed the apoptotic nature of cell death. AGEs also caused apoptosis in purified T cells. Although AGEs-induced apoptosis could be blocked by the pan-caspase inhibitor, Ala-Asp-fluomethyl ketone (Z-VAD-fmk), there was no activation of caspase-3, -5, -8 and -9. AGEs caused mitochondrial outer membrane permeabilization and this process was prevented by an antioxidant or Z-VAD-fmk. Furthermore, AGEs treatment led to translocation of apoptosis inducing factor (AIF) from the mitochondria into the nucleus. Altogether, this report demonstrated that AGEs induced T cell apoptosis in an oxidative stress-associated and caspase-dependent manner with involvement of the mitochondrial pathway. It is likely that AGEs-induced T cell apoptosis may play a role in T cell homeostasis in ageing.     

Visilizumab induces apoptosis of mucosal T lymphocytes in ulcerative colitis through activation of caspase 3 and 8 dependent pathways

Visilizumab, a humanized low-Fc receptor binding anti-CD3 antibody, induces rapid clinical response in patients with steroid-refractory ulcerative colitis (UC). Several effective treatments in IBD have been linked to the induction of mucosal T cell apoptosis. The aim of the present study was to evaluate the effect of visilizumab on the apoptosis of lamina propria (LP) and peripheral blood (PB) lymphocytes isolated from patients with UC. Visilizumab induced dose- and time-dependent apoptosis of LP T cells isolated from non-IBD individuals, UC or CD patients. Maximal effect was seen at a concentration of 100 ng/ml and it was 33% for normal, 34% for UC and 23% for CD LP T cells following 24 h stimulation. Visilizumab induced apoptosis predominantly of CD4⁺ LP T cells, whereas CD8⁺ LP T cells were relatively resistant to apoptosis. Visilizumab did not induce apoptosis of PB T cells from UC patients. Visilizumab-induced apoptosis of LP T cells was dependent on caspase 3 and 8, but not caspase 9 activation and did not involve the Fas/FasL pathway. Low-Fc receptor binding Abs such as visilizumab may be highly effective for the treatment of UC through induction of apoptosis of LP T cells and rapid elimination of lesional pathogenic T cells in the gut mucosa.     

Bongkrekic acid ameliorates ischemic neuronal death in the cortex by preventing cytochrome c release and inhibiting astrocyte activation

Mitochondrial release of cytochrome c (cyt-c) plays a critical role in initiating cell death after cerebral ischemia. The objective of this study was to determine whether bongkrekic acid (BKA) ameliorates ischemic neuronal damage by inhibiting the release of cyt-c. These results showed that a 10min period of global ischemia caused neuronal death, increased the release of cyt-c and activated astrocytes in the cortex and CA1. BKA treatment reduced ischemic-induced neuronal death, prevented cyt-c release and inhibited astrocyte activation in the cortex, but not in the CA1. These results suggest that the neuroprotective effect of BKA is associated with its ability to prevent cyt-c release and to inhibit astrocyte activation.     

Induction of apoptosis by Se-MSC in U937 human leukemia cells through release of cytochrome c and activation of caspases and PKC-delta: mutual regulation between caspases and PKC-delta via a positive feedback mechanism

Se-methylselenocysteine (Se-MSC) has been shown to possess potent chemopreventive and anti-tumor properties. However, its exact mechanism of action is still not well understood. The present study investigated the mechanism of Se-MSC on the induction of apoptosis using U937 human leukemia cells. Se-MSC induced dose- and time-dependent apoptosis of U937 cells as assessed by flow cytometry analysis, DNA fragmentation, and proteolytic cleavage of poly-(ADP-ribose) polymerase (PARP). Se-MSC increased time- and dose-dependent cytochrome c accumulation in the cytosol, which was greatly inhibited by overexpression of Bcl-2, suggesting that the apoptotic effect by Se-MSC in U937 cells is mitochondrial-dependent. Se-MSC also induced activation of caspases, followed by proteolytic cleavage of PKC-delta. The Se-MSC-induced apoptosis required activities of caspases since pretreatment of a pan-caspase inhibitor z-VAD-fmk greatly suppressed the Se-MSC-induced apoptosis as well as proteolytic cleavage of PKC-delta, suggesting activation of caspases is critical for the Se-MSC-induced apoptosis, and caspases lie upstream of PKC-delta. The Se-MSC-induced apoptosis of U937 cells also required activity of PKC-delta because pretreatment of rottlerin, a specific PKC-delta inhibitor greatly blocked the Se-MSC-induced apoptosis as well as processing and activities of caspases, suggesting activation of PKC-delta is also important for the Se-MSC-induced apoptosis of U937 cells, and PKC-delta lies upstream of caspases. Together, our data suggest the apoptotic mechanism by Se-MSC in U937 cells may be related to cytochrome c release from the mitochondria, and mutual activation between caspases and PKC-delta via a positive feedback mechanism, which may potentiate the apoptotic action by Se-MSC in U937 cells.     

Cytochrome c release, mitochondrial membrane depolarization, caspase-3 activation, and Bax-alpha cleavage during IFN-alpha-induced apoptosis in Daudi B lymphoma cells.

Interferon-alpha (IFN-alpha) displays antitumor action by inducing direct cytotoxicity against tumor cells in addition to generation of cytotoxic cells. The IFN-alpha-induced direct cytotoxicity is at least partly due to induction of apoptosis. In the present study, we examined signaling pathways implicated in IFN-alpha-induced apoptosis in Daudi cells. Release of cytochrome c from mitochondria to cytosol was found after 12 h incubation with IFN-alpha, followed by a decline in mitochondrial membrane potential (Delta psi(m)) and procaspase-3 activation at 24 and 36 h, respectively. Cleavage of endogenous Bax-alpha (21 kDa), generating an 18-kDa fragment (p18 Bax-alpha), was found at 36 h. Although the endogenous p21 Bax-alpha was located in both cytosol and mitochondrial membranes, the p18 Bax-alpha resided only on mitochondrial membranes. IFN-alpha-induced apoptosis occurred 48 h after stimulation, with a further increase in proportion up to 72 h. Pretreatment with pancaspase inhibitor Z-VAD-fmk substantially inhibited the IFN-alpha-mediated Bax-alpha cleavage and apoptosis, but not the decline in Delta psi(m), suggesting the possibility that caspase-3 activation is implicated in the Bax-alpha cleavage, probably leading to amplification of the apoptotic processes. Our results suggest that modulation of endogenous p21 Bax-alpha is implicated in IFN-alpha-induced apoptosis.