p53 induces apoptosis by caspase activation through mitochondrial cytochrome c release

The p53 tumor suppressor gene is critically involved in cell cycle regulation, DNA repair, and programmed cell death. Several lines of evidence suggest that p53 death signals lead to caspase activation; however, the mechanism of caspase activation by p53 still is unclear. Expressing wild type p53 by means of an adenoviral expression vector, we were able to induce apoptotic cell death, as characterized by morphological changes, phosphatidylserine externalization, and internucleosomal DNA fragmentation, in p53(null) Saos-2 cells. This cell death was accompanied by caspase activation as well as by cleavage of caspase substrates and was preceded by mitochondrial cytochrome c release. The addition of the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) directly after transduction almost completely prevented p53-induced apoptotic cell death but did not inhibit mitochondrial cytochrome c release. In contrast, N-acetylcysteine, even at high concentrations, could not prevent induction of programmed cell death by p53 expression. Cytosolic extracts from Saos-2 cells transduced with p53, but not from Saos-2 cells transduced with the empty adenoviral vector, contained a cytochrome c-releasing activity in vitro, which was still active in the presence of zVAD-fmk. When Bax was immunodepleted from the cytosolic extracts of p53-expressing cells before incubation with isolated mitochondria, the in vitro cytochrome c release was abolished. Thus, we could demonstrate in cells and in vitro that p53 activates the apoptotic machinery through induction of the release of cytochrome c from the mitochondrial intermembrane space. Furthermore, we provide in vitro evidence for the requirement of cytosolic Bax for this cytochrome c-releasing activity of p53 in Saos-2 cells.     

A cytosolic factor is required for mitochondrial cytochrome c efflux during apoptosis

Treatment of HL-60 cells with staurosporine (STS) induced mitochondrial cytochrome c efflux into the cytosol, which was followed by caspase-3 activation and apoptosis. Consistent with these observations, in vitro experiments demonstrated that, except for cytochrome c, the cytosol of HL-60 cells contained sufficient amounts of all factors required for caspase-3 activation. In contrast, treatment of HCW-2 cells (an apoptotic-resistant HL-60 subclone) with STS failed to induce significant amounts of mitochondrial cytochrome c efflux, caspase-3 activation, and apoptosis. In vitro assays strongly suggested that a lack of cytochrome c in the cytosol was the primary limiting factor for caspase-3 activation in HCW-2 cells. To explore the mechanism which regulates mitochondrial cytochrome c efflux, we developed an in vitro assay which showed that cytosolic extracts from STS-treated, but not untreated, HL-60 cells contained an activity, which we designated 'CIF' (cytochrome c-efflux inducing factor), which rapidly induced cytochrome c efflux from HL-60 mitochondria. In contrast, there was no detectable CIF activity in STS-treated HCW-2 cells although the mitochondria from HCW-2 cells were responsive to the CIF activity from STS-treated HL-60 cells. These experiments have identified a novel activity, CIF, which is required for cytochrome c efflux and they indicate that the absence of CIF is the biochemical explanation for the impaired ability of HCW-2 cells to activate caspase-3 and undergo apoptosis.     

A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis.

The mechanism during apoptosis by which cytochrome c is rapidly and completely released in the absence of mitochondrial swelling is uncertain. Here, we show that two distinct pathways are involved. One mediates release of cytochrome c across the outer mitochondrial membrane, and another, characterized in this study, is responsible for the redistribution of cytochrome c stored in intramitochondrial cristae. We have found that the "BH3-only" molecule tBID induces a striking remodeling of mitochondrial structure with mobilization of the cytochrome c stores (approximately 85%) in cristae. This reorganization does not require tBID's BH3 domain and is independent of BAK, but is inhibited by CsA. During this process, individual cristae become fused and the junctions between the cristae and the intermembrane space are opened.     

Aspirin induces apoptosis through mitochondrial cytochrome c release

Aspirin and other non-steroidal anti-inflammatory drugs induce apoptosis in many cell types. Although the involvement of caspases has been demonstrated, the mechanism leading to caspase activation remains unknown. We have studied the role of the mitochondrial pathway in aspirin-induced apoptosis. The apoptotic effect of aspirin was analyzed in different cell lines (Jurkat, MOLT-4, Raji and HL-60) showing induction of mitochondrial cytochrome c release and caspases 9, 3 and 8 processing. Furthermore, early aspirin-induced cytochrome c release was not affected by the caspase inhibitor Z-VAD·fmk and preceded loss of mitochondrial membrane potential. Therefore, aspirin-induced apoptosis involves caspase activation through cytochrome c release.     

Nitrosylation of cytochrome c during apoptosis

Cytochrome c released from mitochondria into the cytoplasm plays a critical role in many forms of apoptosis by stimulating apoptosome formation and subsequent caspase activation. However, the mechanisms regulating cytochrome c apoptotic activity are not understood. Here we demonstrate that cytochrome c is nitrosylated on its heme iron during apoptosis. Nitrosylated cytochrome c is found predominantly in the cytoplasm in control cells. In contrast, when cytochrome c release from mitochondria is inhibited by overexpression of the anti-apoptotic proteins B cell lymphoma/leukemia (Bcl)-2 or Bcl-X(L), nitrosylated cytochrome c is found in the mitochondria. These data suggest that during apoptosis, cytochrome c is nitrosylated in mitochondria and then rapidly released into the cytoplasm in the absence of Bcl-2 or Bcl-X(L) overexpression. In vitro nitrosylation of cytochrome c increases caspase-3 activation in cell lysates. Moreover, the inhibition of intracellular cytochrome c nitrosylation is associated with a decrease in apoptosis, suggesting that cytochrome c nitrosylation is a proapoptotic modification. We conclude that nitrosylation of the heme iron of cytochrome c may be a novel mechanism of apoptosis regulation.     

Bax activation and mitochondrial insertion during apoptosis

The mitochondrial apoptotic pathway is a highly regulated biological mechanism which determines cell fate. It is defined as a cascade of events, going from an apoptotic stimulus to the MOM permeabilization, resulting in the activation of the so-called executive phase. This pathway is very often altered in cancer cells.The mitochondrial permeabilization is under the control of the Bcl-2 family of proteins (pBcls). These proteins share one to four homology domains (designed BH1-4) with Bcl-2, and are susceptible of homo- and/or hetero-dimerization. In spite of a poor amino-acid sequence homology, these proteins exhibit very similar tertiary structures. Strikingly, while some of these proteins are anti-apoptotic, the others are pro-apoptotic. Pro-apoptotic proteins are further divided in two sub-classes: multi-domains proteins, among which Bax and Bak, which exhibit BH1-3 domains, and BH3-only proteins (or BOPs). Schematically, BOPs and anti-apoptotic proteins antagonistically regulate the activation of the multi-domain proteins Bax and Bak and their oligomerization in the MOM, the latter process being responsible for the apoptotic mitochondrial permeabilization.Considering the critical role of Bax in cancer cells apoptosis, we focus in this review on the molecular events of Bax activation through its interaction with the other proteins from the Bcl-2 family. The mechanism by which Bax triggers the MOM permeabilization once activated will be discussed in some other reviews in this special issue.     

Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis

Here we report that in staurosporine-induced apoptosis of HeLa cells, Bid, a BH3 domain containing protein, translocates from the cytosol to mitochondria. This event is associated with a change in conformation of Bax which leads to the unmasking of its NH2-terminal domain and is accompanied by the release of cytochrome c from mitochondria. A similar finding is reported for cerebellar granule cells undergoing apoptosis induced by serum and potassium deprivation. The Bax-conformational change is prevented by Bcl-2 and Bcl-xL but not by caspase inhibitors. Using isolated mitochondria and various BH3 mutants of Bid, we demonstrate that direct binding of Bid to Bax is a prerequisite for Bax structural change and cytochrome c release. Bcl-xL can inhibit the effect of Bid by interacting directly with Bax. Moreover, using mitochondria from Bax-deficient tumor cell lines, we show that Bid- induced release of cytochrome c is negligible when Bid is added alone, but dramatically increased when Bid and Bax are added together. Taken together, our results suggest that, during certain types of apoptosis, Bid translocates to mitochondria and binds to Bax, leading to a change in conformation of Bax and to cytochrome c release from mitochondria.     

Nuclear translocation of cytochrome c during apoptosis

Release of cytochrome c from mitochondria is a major event during apoptosis. Released cytochrome c has been shown to activate caspase-dependent apoptotic signals. In this report, we provide evidence for a novel role of cytochrome c in caspase-independent nuclear apoptosis. We showed that cytochrome c, released from mitochondria upon apoptosis induction, gradually accumulates in the nucleus as evidenced by both immunofluorescence and subcellular fractionation. Parallel to nuclear accumulation of cytochrome c, acetylated histone H2A, but not unmodified H2A, was released from the nucleus to the cytoplasm. Addition of purified cytochrome c to isolated nuclei recapitulated the preferential release of acetylated, but not deacetylated, histone H2A. Cytochrome c was also found to induce chromatin condensation. These results suggest that the nuclear accumulation of cytochrome c may be directly involved in the remodeling of chromatin. Our results provide evidence of a novel role for cytochrome c in inducing nuclear apoptosis.     

The temporal relationship between protein phosphatase, mitochondrial cytochrome c release, and caspase activation in apoptosis

Apoptosis is mediated by members of the caspase family of proteases which can be activated by release of mitochondrial cytochrome c. Additional members of the caspase family are activated at the cell surface in response to direct stimulus from the external environment such as by activation of the Fas receptor. It has been suggested that these upstream caspases directly activate the downstream caspases which would obviate a role for cytochrome c in apoptosis induced by the Fas receptor. We demonstrate that cytochrome c is released from mitochondria of Jurkat cells in response to both staurosporine and an agonistic anti-Fas antibody and that only the latter is inhibited by the caspase inhibitor z-VAD-FMK. This suggests that an upstream caspase such as caspase-8 is required for the Fas-mediated release of mitochondrial cytochrome c. The protein phosphatase inhibitor calyculin A prevented cytochrome c release and apoptosis induced by both agents, suggesting that release of cytochrome c is required in both models. Zinc, once thought of as an endonuclease inhibitor, has previously been shown to prevent the activation of caspase-3. We show that zinc prevents the activation of downstream caspases and apoptosis induced by both insults, yet does not prevent release of mitochondrial cytochrome c. The ability of calyculin A and zinc to prevent DNA digestion implies that the mitochondrial pathway is important for induction of apoptosis by both agents. These results do not support an alternative pathway in which caspase-8 directly activates caspase-3. These results also demonstrate that a critical protein phosphatase regulates the release of cytochrome c and apoptosis induced by both insults.     

Ultrastructural localization of cytochrome c in apoptosis demonstrates mitochondrial heterogeneity

Release of apoptogenic factors into the cytosol including cytochrome c is triggering the execution phase of apoptosis through activation of cytoplasmic effector caspases. How loss of function of the electron transport chain can be reconciled with an adequate energy supply necessary for executing the apoptotic program was studied in granulosa cell (GC) sheets cultured up to 72 h without gonadotrophic support. Cytochrome c was localized ultrastructurally by oxidation of diaminobenzidine tetrahydrochloride both in living and fixed cells. In uncultured GC sheets all cells show staining over their entire mitochondrial population. In 72 h cultured sheets in the absence of FSH pre-apoptotic GC's display two subsets of mitochondria: normal sized stained mitochondria and small orthodox mitochondria without demonstrable cytochrome function. Apoptotic cells contain several mitochondria with preservation of respiratory function besides unstained orthodox mitochondria. The cytochrome c containing mitochondria typically display dilated intracristal spaces, a mitochondrial conformation related to increased ATP production. Cytochrome c release was confirmed by Western blotting. In 72 h cultures supplemented with FSH, GC's displayed staining over their entire mitochondrial population. In cultures lacking FSH, but partially protected from apoptosis through caspase inhibition, the cytochrome c release was not inhibited. Thus in the present studied model dysfunction of only a subset of mitochondria is instrumental to initiate the apoptotic program while a functional electron transport chain is maintained until the degradation phase in a subset of respiring mitochondria.     

Bcr-Abl-mediated protection from apoptosis downstream of mitochondrial cytochrome c release

Bcr-Abl, activated in chronic myelogenous leukemias, is a potent cell death inhibitor. Previous reports have shown that Bcr-Abl prevents apoptosis through inhibition of mitochondrial cytochrome c release. We report here that Bcr-Abl also inhibits caspase activation after the release of cytochrome c. Bcr-Abl inhibited caspase activation by cytochrome c added to cell-free lysates and prevented apoptosis when cytochrome c was microinjected into intact cells. Bcr-Abl acted posttranslationally to prevent the cytochrome c-induced binding of Apaf-1 to procaspase 9. Although Bcr-Abl prevented interaction of endogenous Apaf-1 with the recombinant prodomain of caspase 9, it did not affect the association of endogenous caspase 9 with the isolated Apaf-1 caspase recruitment domain (CARD) or Apaf-1 lacking WD-40 repeats. These data suggest that Apaf-1 recruitment of caspase 9 is faulty in the presence of Bcr-Abl and that cytochrome c/dATP-induced exposure of the Apaf-1 CARD is likely defective. These data provide a novel locus of Bcr-Abl antiapoptotic action and suggest a distinct mechanism of apoptosomal inhibition.     

Increased mitochondrial cytochrome c levels and mitochondrial hyperpolarization precede camptothecin-induced apoptosis in Jurkat cells

Mitochondria play a central role in apoptosis through release of cytochrome c and activation of caspases. In the present study, we showed that, in Jurkat human T cells, camptothecin-induced apoptosis is preceded by (i) an increase in cytochrome c and subunit IV of cytochrome c oxidase (COX IV) levels in mitochondria; and (ii) an elevation of the mitochondrial membrane potential (Delta(Psi)m). These events are followed by cytochrome c release into the cytosol, cytochrome c and COX IV depletion from mitochondria, externalization of phosphatidylserine (PS), disruption of Delta(Psi)m, caspase activation, poly(ADP-ribose)polymerase cleavage and DNA fragmentation. The pan-caspase inhibitor z-VAD.fmk blocked camptothecin-induced PS externalization, disruption of Delta(Psi)m and DNA fragmentation, suggesting that these events are mediated by caspase activation. In contrast, z-VAD did not prevent cytochrome c release, despite preventing cytochrome c and COX IV depletion from mitochondria. Together, these data suggest that mitochondrial cytochrome c and COX IV enrichment are early events preceding the onset of apoptosis and that cytochrome c release is upstream of caspase activation and loss of Delta(Psi)m. Furthermore, prevention by z-VAD of cytochrome c and COX IV depletion in mitochondria suggests the possibility that a caspase-like activity in mitochondria is involved in the proteolytic depletion of respiratory chain proteins. Activation of this activity may play an important role in drug-induced apoptosis.     

Phosphatidyl serine exposure during apoptosis precedes release of cytochrome c and decrease in mitochondrial transmembrane potential

Time kinetics of phosphatidyl serine (PS) exposure were compared to other apoptotic parameters following different apoptotic stimuli. Our data indicate that anti-Fas treatment of L929sAhFas cells results in rapid exposure of PS, which precedes decrease in mitochondrial transmembrane potential (DeltaPsi(m)) and release of cytochrome c, indicating that PS exposure occurs independently of these mitochondrial events. Also during TNF-, etoposide- or staurosporine-mediated apoptosis in PC60 RI/RII cells, PS-positive cells were observed before they had a decreased DeltaPsi(m). However, during growth factor depletion-induced death of 32D cells, both phenomena seemed to occur at the same time.     

Inhibiting Drp1-mediated mitochondrial fission selectively prevents the release of cytochrome c during apoptosis

Most cell death stimuli trigger the mitochondrial release of cytochrome c and other cofactors that induce caspase activation and ensuing apoptosis. Apoptosis is also associated with massive mitochondrial fragmentation and cristae remodeling. Dynamin-related protein 1 (Drp1), a protein of the mitochondrial fission machinery, has been reported to participate in apoptotic mitochondrial fragmentation. Several theories explaining the mechanisms of cytochrome c release have been proposed. One suggests that it relies on the activation of Drp1-mediated mitochondrial fission. Here, we report that downregulation of Drp1 inhibits fragmentation of the mitochondrial network and partially prevents the release of cytochrome c but fails to prevent the release of other mitochondrial factors such as second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI, Omi/HtrA2, adenylate kinase 2 and deafness dystonia peptide/TIMM8a. An explanation for the prevention of cytochrome c release is provided by our observation that inhibiting Drp1-mediated mitochondrial fission prevents the mitochondrial release of soluble OPA1 that was proposed to regulate cristae remodeling and complete cytochrome c release during apoptosis. Finally, we observed that downregulation of Drp1 delays but does not inhibit apoptosis, suggesting that mitochondrial fragmentation is not a prerequisite for apoptosis.     

Bax and the mitochondrial permeability transition cooperate in the release of cytochrome c during endoplasmic reticulum-stress-induced apoptosis

Endoplasmic reticulum (ER) stress induces apoptosis by mechanisms that are not fully clear. Here we show that ER stress induced by the Ca(2+)-ATPase inhibitor thapsigargin (THG) activates cytochrome c-dependent apoptosis through cooperation between Bax and the mitochondrial permeability transition (MPT) in human leukemic CEM cells. Pharmacological inhibition of the MPT as well as small interfering RNA (siRNA) knockdown of the MPT core component cyclophilin D blocked cytochrome c release and caspase-dependent apoptosis but did not prevent Bax activation, translocation or N-terminal exposure in mitochondria. siRNA knockdown of Bax also blocked THG-mediated cytochrome c release and apoptosis, but did not prevent MPT activation and resulted in caspase-independent cell death. Our results show that ER-stress-induced cell death involves a caspase and Bax-dependent pathway as well as a caspase-independent MPT-directed pathway.     

Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization.

Mitochondrial cytochrome c, which functions as an electron carrier in the respiratory chain, translocates to the cytosol in cells undergoing apoptosis, where it participates in the activation of DEVD-specific caspases. The apoptosis inhibitors Bcl-2 or Bcl-xL prevent the efflux of cytochrome c from mitochondria. The mechanism responsible for the release of cytochrome c from mitochondria during apoptosis is unknown. Here, we report that cytochrome c release from mitochondria is an early event in the apoptotic process induced by UVB irradiation or staurosporine treatment in CEM or HeLa cells, preceding or at the time of DEVD-specific caspase activation and substrate cleavage. A reduction in mitochondrial transmembrane potential (Deltapsim) occurred considerably later than cytochrome c translocation and caspase activation, and was not necessary for DNA fragmentation. Although zVAD-fmk substantially blocked caspase activity, a reduction in Deltapsim and cell death, it failed to prevent the passage of cytochrome c from mitochondria to the cytosol. Thus the translocation of cytochrome c from mitochondria to cytosol does not require a mitochondrial transmembrane depolarization.     

The course of etoposide-induced apoptosis from damage to DNA and p53 activation to mitochondrial release of cytochrome c

Treatment of L929 fibroblasts by the topoisomerase II inhibitor etoposide killed 50% of the cells within 72 h. The cell killing was preceded by the release of cytochrome c from the mitochondria. Simultaneous treatment of the cells with wortmannin, cycloheximide, furosemide, cyclosporin A, or decylubiquinone prevented the release of cytochrome c and significantly reduced the loss of viability. Etoposide caused the phosphorylation of p53 within 6 h, an effect prevented by wortmannin, an inhibitor of DNA-dependent protein kinase (DNA-PK). The activation of p53 by etoposide resulted in the up-regulation of the pro-apoptotic protein Bax, a result that was prevented by the protein synthesis inhibitor cycloheximide. The increase in the content of Bax was followed by the translocation of this protein from the cytosol to the mitochondria, an event that was inhibited by furosemide, a chloride channel inhibitor. Stably transfected L929 fibroblasts that overexpress Akt were resistant to etoposide and did not translocate Bax to the mitochondria or release cytochrome c. Bax levels in these transfected cells were comparable with the wild-type cells. The release of cytochrome c upon translocation of Bax has been attributed to induction of the mitochondrial permeability transition (MPT). Cyclosporin A and decylubiquinone, inhibitors of MPT, prevented the release of cytochrome c without affecting Bax translocation. These data define a sequence of biochemical events that mediates the apoptosis induced by etoposide. This cascade proceeds by coupling DNA damage to p53 phosphorylation through the action of DNA-PK. The activation of p53 increases Bax synthesis. The translocation of Bax to the mitochondria induces the MPT, the event that releases cytochrome c and culminates in the death of the cells.     

The late increase in intracellular free radical oxygen species during apoptosis is associated with cytochrome c release,caspase activation,and mitochondrial dysfunction

Mitochondria play central roles in cellular metabolism and apoptosis and are a major source of reactive oxygen species (ROS). We investigated the role of ROS and mitochondria in radiation-induced apoptosis in multiple myeloma cells. Two distinct levels of ROS were generated following irradiation: a small increase observed early, and a pronounced late increase, associated with depletion of reduced glutathione (GSH) and collapse of mitochondrial membrane potential (deltapsi(m)). Exogenous ROS and caspase-3 induced deltapsi(m) drop and cytochrome c release from mitochondria, which could be prevented by molecular (dominant-negative caspase-9) and pharmacologic (zVAD-fmk) caspase inhibitors and overexpression of Bcl-2. Exogenous ROS also induced mitochondrial permeability transition (PT) pore opening and cytochrome c release in isolated mitochondria, which could be blocked by inhibition of PT with cyclosporin A. These results indicate that the late ROS production is associated with increased PT pore opening and decreased deltapsi(m), and GSH, events associated with caspase activation and cytochrome c release.     

Abrin induces HeLa cell apoptosis by cytochrome c release and caspase activation

We identified apoptosis as being a significant mechanism of toxicity following the exposure of HeLa cell cultures to abrin holotoxin, which is in addition to its inhibition of protein biosynthesis by N-glycosidase activity. The treatment of HeLa cell cultures with abrin resulted in apoptotic cell death, as characterized by morphological and biochemical changes, i.e., cell shrinkage, internucleosomal DNA fragmentation, the occurrence of hypodiploid DNA, chromatin condensation, nuclear breakdown, DNA single strand breaks by TUNEL assay, and phosphatidylserine (PS) externalization. This apoptotic cell death was accompanied by caspase-9 and caspase-3 activation, as indicated by the cleavage of caspase substrates, which was preceded by mitochondrial cytochrome c release. The broad-spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVADfmk), prevented abrin-triggered caspase activation and partially abolished apoptotic cell death, but did not affect mitochondrial cytochrome c release. These results suggest that the release of mitochondrial cytochrome c, and the sequential caspase-9 and caspase-3 activations are important events in the signal transduction pathway of abrin-induced apoptotic cell death in the HeLa cell line.