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1.
Exposure of cells to hyperthermia is known to induce apoptosis, although the underlying mechanisms are only partially understood. Here, we examine the molecular requirements necessary for heat-induced apoptosis using genetically modified Jurkat T-lymphocytes. Cells stably overexpressing Bcl-2/Bcl-x(L) or stably depleted of Apaf-1 were completely resistant to heat-induced apoptosis, implicating the involvement of the mitochondria-mediated pathway. Pretreatment of wild-type cells with the cell-permeable biotinylated general caspase inhibitor b-VAD-fmk (biotin-Val-Ala-Asp(OMe)-CH(2)F) both inhibited heat-induced apoptosis and affinity-labeled activated initiator caspase-2, -8, and -9. Despite this finding, however, cells engineered to be deficient in caspase-8, caspase-2, or the caspase-2 adaptor protein RAIDD (receptor-interacting protein (RIP)-associated Ich-1/CED homologous protein with death domain) remained susceptible to heat-induced apoptosis. Additionally, b-VAD-fmk failed to label any activated initiator caspase in Apaf-1-deficient cells exposed to hyperthermia. Cells lacking Apaf-1 or the pro-apoptotic BH3-only protein Bid exhibited lower levels of heat-induced Bak activation, cytochrome c release, and loss of mitochondrial membrane potential, although cleavage of Bid to truncated Bid (tBid) occurred downstream of caspase-9 activation. Combined, the data suggest that caspase-9 is the critical initiator caspase activated during heat-induced apoptosis and that tBid may function to promote cytochrome c release during this process as part of a feed-forward amplification loop. 相似文献
2.
Mary E. Shawgo Shary N. Shelton John D. Robertson 《The Journal of biological chemistry》2009,284(48):33447-33455
Activation of executioner caspases during receptor-mediated apoptosis in type II cells requires the engagement of the mitochondrial apoptotic pathway. Although it is well established that recruitment of mitochondria in this context involves the cleavage of Bid to truncated Bid (tBid), the precise post-mitochondrial signaling responsible for executioner caspase activation is controversial. Here, we used distinct clones of type II Jurkat T-lymphocytes in which the mitochondrial apoptotic pathway had been inhibited to investigate the molecular requirements necessary for Fas-induced apoptosis. Cells overexpressing either Bcl-2 or Bcl-xL were protected from apoptosis induced by agonistic anti-Fas antibody. By comparison, Apaf-1-deficient Jurkat cells were sensitive to anti-Fas, exhibiting Bid cleavage, Bak activation, the release of cytochrome c and Smac, and activation of executioner caspase-3. Inhibiting downstream caspase activation with the pharmacological inhibitor Z-DEVD-fmk or by expressing the BIR1/BIR2 domains of X-linked inhibitor of apoptosis protein (XIAP) decreased all anti-Fas-induced apoptotic changes. Additionally, pretreatment of Bcl-xL-overexpressing cells with a Smac mimetic sensitized these cells to Fas-induced apoptosis. Combined, our findings strongly suggest that Fas-mediated activation of executioner caspases and induction of apoptosis do not depend on apoptosome-mediated caspase-9 activation in prototypical type II cells. 相似文献
3.
Mitochondrial outer membrane permeabilization and the release of intermembrane space proteins, such as cytochrome c, are early events during intrinsic (mitochondria-mediated) apoptotic signaling. Although this process is generally accepted to require the activation of Bak or Bax, the underlying mechanism responsible for their activation during true intrinsic apoptosis is not well understood. In the current study, we investigated the molecular requirements necessary for Bak activation using distinct clones of Bax-deficient Jurkat T-lymphocytes in which the intrinsic pathway had been inhibited. Cells stably overexpressing Bcl-2/Bcl-x(L) or stably depleted of Apaf-1 were equally resistant to apoptosis induced by the DNA-damaging anticancer drug etoposide as determined by phosphatidylserine externalization and caspase activation. Strikingly, characterization of mitochondrial apoptotic events in all three drug-resistant cell lines revealed that, without exception, resistance to apoptosis was associated with an absence of Bak activation, cytochrome c release, and mitochondrial membrane depolarization. Furthermore, we found that etoposide-induced apoptosis and mitochondrial events were inhibited in cells stably overexpressing either full-length X-linked inhibitor of apoptosis protein (XIAP) or the BIR1/BIR2 domains of XIAP. Combined, our findings suggest that caspase-mediated positive amplification of initial mitochondrial changes can determine the threshold for irreversible activation of the intrinsic apoptotic pathway. 相似文献
4.
Bcr-Abl-mediated protection from apoptosis downstream of mitochondrial cytochrome c release 下载免费PDF全文
Deming PB Schafer ZT Tashker JS Potts MB Deshmukh M Kornbluth S 《Molecular and cellular biology》2004,24(23):10289-10299
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. 相似文献
5.
《Cell cycle (Georgetown, Tex.)》2013,12(23):4128-4137
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. 相似文献
6.
Preservation of mitochondrial structure and function after Bid- or Bax-mediated cytochrome c release 总被引:17,自引:0,他引:17
von Ahsen O Renken C Perkins G Kluck RM Bossy-Wetzel E Newmeyer DD 《The Journal of cell biology》2000,150(5):1027-1036
Proapoptotic members of the Bcl-2 protein family, including Bid and Bax, can activate apoptosis by directly interacting with mitochondria to cause cytochrome c translocation from the intermembrane space into the cytoplasm, thereby triggering Apaf-1-mediated caspase activation. Under some circumstances, when caspase activation is blocked, cells can recover from cytochrome c translocation; this suggests that apoptotic mitochondria may not always suffer catastrophic damage arising from the process of cytochrome c release. We now show that recombinant Bid and Bax cause complete cytochrome c loss from isolated mitochondria in vitro, but preserve the ultrastructure and protein import function of mitochondria, which depend on inner membrane polarization. We also demonstrate that, if caspases are inhibited, mitochondrial protein import function is retained in UV-irradiated or staurosporine-treated cells, despite the complete translocation of cytochrome c. Thus, Bid and Bax act only on the outer membrane, and lesions in the inner membrane occurring during apoptosis are shown to be secondary caspase-dependent events. 相似文献
7.
Ting Li Linlin Zeng Wei Gao Mei-Zhen Cui Xueqi Fu Xuemin Xu 《生物化学与生物物理学报:疾病的分子基础》2013,1832(3):453-474
Presenilin-associated protein (PSAP) has been identified as a mitochondrial proapoptotic protein. However, the mechanism by which PSAP induces apoptosis remains unknown. To this end, we have established an inducible expression system. Using this system, we have examined the roles of B-cell lymphoma 2 (Bcl-2) family proteins, cytochrome c, Smac (Smac/Diablo, second mitochondria-derived activator of caspases/direct IAP binding protein with low PI), and Apaf-1 (apoptotic protease-activating factor) in PSAP-induced apoptosis. Our results demonstrate that knockdown of Apaf-1 abolished PSAP-induced caspase activation and poly(ADP ribose) polymerase (PARP) cleavage, indicating that the apoptosome formation triggered by cytochrome c is crucial for PSAP-induced apoptosis. Our data also demonstrate that knockdown of Smac abolished PSAP-induced caspase activation and PARP cleavage, indicating that, in addition to Apaf-1 or apoptosome formation, Smac is also essential for PSAP-induced apoptosis. However, interestingly, our data demonstrate that overexpression of Bcl-2 and Bcl-xL did not protect cells from PSAP-induced apoptosis, and that knockdown of Bid, Bax, and Bak had no effect on PSAP-induced cytochrome c and Smac release, indicating that PSAP-induced apoptosis is not regulated by Bcl-2 family proteins. These results strongly suggest that PSAP evokes mitochondrial apoptotic cascades via a novel mechanism that is not regulated by Bcl-2 family proteins, but that both the formation of cytochrome c-Apaf-1 apoptosome and the presence of Smac are absolutely required for PSAP-induced apoptosis. 相似文献
8.
Caspase-dependent cytosolic release of cytochrome c and membrane translocation of Bax in p53-induced apoptosis 总被引:6,自引:0,他引:6
Gao CF Ren S Zhang L Nakajima T Ichinose S Hara T Koike K Tsuchida N 《Experimental cell research》2001,265(1):145-151
Activation of p53 induces apoptosis in various cell types. However, the mechanism by which p53 induces apoptosis is still unclear. We reported previously that the activation of a temperature-sensitive mutant p53 (p53(138Val)) induced activation of caspase 3 and apoptosis in Jurkat cells. To elucidate the pathway linking p53 and downstream caspases, we examined the activation of caspases 8 and 9 in apoptotic cells. The results showed that both caspases were activated during apoptosis as judged by the appearance of cleavage products from procaspases and the caspase activities to cleave specific fluorogenic substrates. The significant inhibition of apoptosis by a tetrapeptide inhibitor of caspase 8 and caspase 9 suggested that both caspases are required for apoptosis induction. In addition, the membrane translocation of Bax and cytosolic release of cytochrome c, but not loss of mitochondrial membrane potential, were detected at an early stage of apoptosis. Moreover, Bax translocation, cytochrome c release, and caspase 9 activation were blocked by the broad-spectrum caspase inhibitor, Z-VAD-fmk and the caspase 8-preferential inhibitor, Ac-IETD-CHO, suggesting that the mitochondria might participate in apoptosis by amplifying the upstream death signals. In conclusion, our results indicated that activation of caspase 8 or other caspase(s) by p53 triggered the membrane translocation of Bax and cytosolic release of cytochrome c, which might amplify the apoptotic signal by activating caspase 9 and its downstream caspases. 相似文献
9.
Raghu Gogada Michael Amadori Honghao Zhang Anthony Jones Alissa Verone Jason Pitarresi Sirisha Jandhyam Varun Prabhu Jennifer D Black Dhyan Chandra 《Cell cycle (Georgetown, Tex.)》2011,10(23):4128-4137
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 相似文献
10.
Caspase-9-induced mitochondrial disruption through cleavage of anti-apoptotic BCL-2 family members 总被引:6,自引:0,他引:6
Chen M Guerrero AD Huang L Shabier Z Pan M Tan TH Wang J 《The Journal of biological chemistry》2007,282(46):33888-33895
Mitochondrial disruption during apoptosis results in the release of cytochrome c that forms apoptosomes with Apaf-1 and caspase-9. Activation of caspase-9 by dimerization in apoptosomes then triggers a caspase signaling cascade. In addition, other apoptosis signaling molecules released from the mitochondrion, such as apoptosis-inducing factor and endonuclease G, may induce caspase-9-independent apoptosis. To determine the signaling events induced by caspase-9, we used chemically induced dimerization for specific activation of caspase-9. We observed that caspase-9 dimerization resulted in the loss of mitochondrial membrane potential and the cleavage of anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1. Moreover, cleavage-resistant Bcl-2, Bcl-xL, or Mcl-1 potently inhibited caspase-9-dependent loss of mitochondrial membrane potential and the release of cytochrome c. Our data suggest that a caspase-9 signaling cascade induces feedback disruption of the mitochondrion through cleavage of anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1. 相似文献
11.
Interactions between the histone deacetylase inhibitor sodium butyrate (SB) and phorbol 12-myristate 13-acetate (PMA) were examined in human myeloid leukemia cells (U937 and HL-60). Exposure of U937 cells to 1 mM SB and 1 nM PMA (24 h) markedly induced caspase activation and apoptosis, events accompanied by impaired differentiation induction (e.g., reduced plastic adherence and diminished expression of CD11b) as well as reduced clonogenic survival. The PKC inhibitor GF109203X blocked SB-/PMA-mediated apoptosis. Comparable results were obtained in HL-60 cells. Apoptosis was associated with early procaspase 8 activation and Bid cleavage, accompanied by pronounced mitochondrial damage (e.g., loss of mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c release). Neutralization of endogenous TNFalpha by a human soluble TNF receptor substantially blocked SB-/PMA-induced cytochrome c release and apoptosis. Consistent with this, ectopic expression of a mutant dominant-negative caspase 8 or CrmA resulted in a significant decrease in SB-/PMA-induced apoptosis, whereas Bcl-2 overexpression did not. SB/PMA treatment also triggered a decline in the S and G(2)M populations, and dephosphorylation of p34(cdc2). These results indicate that SB interacts with low concentrations of PMA to induce apoptosis in human leukemia cells and that this process proceeds through a PKC-/TNFalpha-dependent pathway in which procaspase 8 and Bid activation play key roles. 相似文献
12.
Doyle BT O'Neill AJ Fitzpatrick JM Watson RW 《Apoptosis : an international journal on programmed cell death》2004,9(3):345-352
HL-60 cell differentiation into neutrophil like cells is associated with their induction of apoptosis. We investigated the cellular events that occur pre and post mitochondrial permeability transition to determine the role of the mitochondria in the induction of differentiation induced apoptosis. Pro-apoptotic Bax was translocated to and cleaved at the mitochondrial membrane in addition to t-Bid activation. These processes contributed to mitochondrial membrane disruption and the release of cytochrome c and Smac/DIABLO. The release of cytochrome c was caspase independent, as the caspase inhibitor Z-VAD.fmk, which inhibited apoptosis, did not block the release of cytochrome c. In contrast, the release of Smac/DIABLO was partially inhibited by caspase inhibition indicating differential release pathways for these mitochondrial pro-apoptotic factors. In addition to caspase inhibition we assessed the effects of the Bcl-2 anti-apoptotic family on differentiation induced apoptosis. BH4-Bcl-xl-TAT recombinant protein did not delay apoptosis, but did block the release of cytochrome c and Smac/DIABLO. Bcl-2 over-expression also inhibited differentiation induced apoptosis but was associated with the inhibition of the differentiation process. Differentiation mediated mitochondrial release of cytochrome c and Smac/DIABLO, may not trigger the induction of apoptosis, as BH4-Bclxl-TAT blocks the release of pro-apoptotic factors from the mitochondria, but does not prevent apoptosis. 相似文献
13.
Increased mitochondrial cytochrome c levels and mitochondrial hyperpolarization precede camptothecin-induced apoptosis in Jurkat cells 总被引:4,自引:0,他引:4
Sánchez-Alcázar JA Ault JG Khodjakov A Schneider E 《Cell death and differentiation》2000,7(11):1090-1100
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. 相似文献
14.
Newmeyer DD Bossy-Wetzel E Kluck RM Wolf BB Beere HM Green DR 《Cell death and differentiation》2000,7(4):402-407
Bcl-2 and its relative, Bcl-xL, inhibit apoptotic cell death primarily by controlling the activation of caspase proteases. Previous reports have suggested at least two distinct mechanisms: Bcl-2 and Bcl-xL may inhibit either the formation of the cytochrome c/Apaf-1/caspase-9 apoptosome complex (by preventing cytochrome c release from mitochondria) or the function of this apoptosome (through a direct interaction of Bcl-2 or Bcl-xL with Apaf-1). To evaluate this latter possibility, we added recombinant Bcl-xL protein to cell-free apoptotic systems derived from Jurkat cells and Xenopus eggs. At low concentrations (50 nM), Bcl-xL was able to block the release of cytochrome c from mitochondria. However, although Bcl-xL did associate with Apaf-1, it was unable to inhibit caspase activation induced by the addition of cytochrome c, even at much higher concentrations (1-5 microM). These observations, together with previous results obtained with Bcl-2, argue that Bcl-xL and Bcl-2 cannot block the apoptosome-mediated activation of caspase-9. 相似文献
15.
16.
The adapter protein apoptotic protease-activating factor-1 (Apaf-1) is proteolytically processed during apoptosis 总被引:12,自引:0,他引:12
Lauber K Appel HA Schlosser SF Gregor M Schulze-Osthoff K Wesselborg S 《The Journal of biological chemistry》2001,276(32):29772-29781
Apoptotic protease-activating factor-1 (Apaf-1), a key regulator of the mitochondrial apoptosis pathway, consists of three functional regions: (i) an N-terminal caspase recruitment domain (CARD) that can bind to procaspase-9, (ii) a CED-4-like region enabling self-oligomerization, and (iii) a regulatory C terminus with WD-40 repeats masking the CARD and CED-4 region. During apoptosis, cytochrome c and dATP can relieve the inhibitory action of the WD-40 repeats and thus enable the oligomerization of Apaf-1 and the subsequent recruitment and activation of procaspase-9. Here, we report that different apoptotic stimuli induced the caspase-mediated cleavage of Apaf-1 into an 84-kDa fragment. The same Apaf-1 fragment was obtained in vitro by incubation of cell lysates with either cytochrome c/dATP or caspase-3 but not with caspase-6 or caspase-8. Apaf-1 was cleaved at the N terminus, leading to the removal of its CARD H1 helix. An additional cleavage site was located within the WD-40 repeats and enabled the oligomerization of p84 into a approximately 440-kDa Apaf-1 multimer even in the absence of cytochrome c. Due to the partial loss of its CARD, the p84 multimer was devoid of caspase-9 or other caspase activity. Thus, our data indicate that Apaf-1 cleavage causes the release of caspases from the apoptosome in the course of apoptosis. 相似文献
17.
This study examined the signaling events induced by shikonin that lead to the induction of apoptosis in Bcr/ Abl-positive chronic myelogenous leukemia (CML) cells (e.g., K562, LAMA84). Treatment of K562 cells with shikonin (e.g., 0.5 pM) resulted in profound induction of apoptosis accompanied by rapid generation of reactive oxygen species (ROS), striking activation of c-Jun-N-terminal kinase (JNK) and p38, marked release of the mitochondrial proteins cytochrome c and Smac/DIABLO, activation of caspase-9 and -3, and cleavage of PARP. Scavenging of ROS completely blocked all of the above-mentioned events (i.e., JNK and p38 phosphorylation, cytochrome c and Smac/DIABLO release, caspase and PARP cleavage, as well as the induction of apoptosis) following shikonin treatment. Inhibition of JNK and knock-down of JNK1 significantly attenuated cytochrome c release, caspase cleavage and apoptosis, but did not affect shikonin-mediated ROS production. Additionally, inhibition of caspase activation completely blocked shikonin-induced apoptosis, but did not appreciably modify shikonin-mediated cytochrome c release or ROS generation. Altogether, these findings demonstrate that shikonin-induced oxidative injury operates at a proximal point in apoptotic signaling cascades, and subsequently activates the stress-related JNK pathway, triggers mitochondrial dysfunction, cytochrome c release, and caspase activation, and leads to apoptosis. Our data also suggest that shikonin may be a promising agent for the treatment of CML, as a generator of ROS. 相似文献
18.
Shary N. Shelton Mary E. Shawgo John D. Robertson 《The Journal of biological chemistry》2009,284(17):11247-11255
The extent to which the BH3-only protein Bid is important for intrinsic
(mitochondria-mediated) apoptotic cell death induced by genotoxic stress
remains controversial. In the present study, we examine this issue using a
panel of gene-manipulated Bax-deficient Jurkat T-lymphocytes. Cells stably
depleted of Bid were far less sensitive than control-transfected cells to
etoposide-induced apoptosis. In particular, drug-induced Bak activation,
cytochrome c release, loss of mitochondrial membrane potential, and
caspase activation were all decreased in cells lacking Bid. Reconstitution
experiments using recombinant proteins and permeabilized Bid-deficient cells
demonstrated that truncated Bid (tBid), but not full-length Bid, potently
induced Bak activation and the release of cytochrome c. Further,
caspase-8-deficient Jurkat cells efficiently cleaved Bid and were sensitive to
drug-induced apoptosis. By comparison, Apaf-1-deficient cells, as well as
cells overexpressing full-length X-linked inhibitor of apoptosis protein
(XIAP) or the BIR1/BIR2 domains of XIAP, failed to cleave Bid in response to
genotoxic stress. These data suggest that tBid plays an important regulatory
role in the execution of DNA damage-induced cytochrome c release and
apoptosis. However, the fact that cleavage of Bid to tBid is mediated by
executioner caspases suggests that a self-amplifying feed forward loop
involving caspases, Bid, and mitochondria may help determine irreversible
commitment to apoptosis.Apoptosis is an active form of cell death that plays an essential role
during normal embryonic development and in the maintenance of tissue
homeostasis in the adult organism
(1). Consequently,
dysregulation of apoptosis has been implicated as a contributing factor to the
onset of different pathological conditions, including cancer. In addition, it
is now generally accepted that many genotoxic anticancer drugs are effective
against tumor cells for their ability to induce mitochondria-mediated
apoptosis (2). Similarly,
mutations or the altered expression of pro- and anti-apoptotic proteins can
contribute to the development of drug resistance.Execution of apoptosis is mediated by a family of cysteine-dependent
aspartate-specific proteases (caspases). During true mitochondria-mediated
apoptosis, members of the Bcl-2 family of proteins are the primary regulators
of caspase activation for their role in controlling mitochondrial outer
membrane permeabilization
(MOMP)2
(3). The process of MOMP
results in the release of cytochrome c, second mitochondria-derived
activator of caspase (Smac, also known as DIABLO), and Omi (also known as
HtrA2) into the cytosol where they converge to promote the activation of
caspase-9 within the apoptotic protease-activating factor-1 (Apaf-1)
apoptosome complex. The Bcl-2 family contains proteins with opposing
functions, and it is generally thought that the induction of MOMP requires the
activation of either Bak or Bax triggered by a Bcl-2 homology 3 (BH3)-only
protein
(4–6).
Indeed, evidence in the literature indicates that cells lacking either Bak or
Bax exhibit only subtle defects in MOMP, whereas doubly deficient cells are
often found to be highly resistant to mitochondria-mediated apoptosis
(7,
8).At present, there are two models for the activation of Bax or Bak by
BH3-only proteins. One model argues that BH3-only proteins function as either
“sensitizer” (e.g. Bad and Noxa) or
“activator” proteins (e.g. truncated Bid (tBid), Bim, and
perhaps Puma) (9). In this
scenario, a sensitizer protein is needed to displace an activator protein from
a prosurvival protein (e.g. Bcl-2, Bcl-xL, or Mcl-1) to
activate Bak or Bax. The second model argues that BH3-only proteins bind and
inhibit the function of prosurvival Bcl-2 proteins, which normally bind to and
inhibit Bak and Bax (10,
11). Of the seven or so known
BH3-only proteins (6), Bid is
unique in that it requires post-translational modification for activation,
most notably involving caspase-8-mediated cleavage to tBid
(12–14).
Bid normally resides in the cytosol and possibly the nucleus
(15). Upon being cleaved, the
C-terminal fragment (tBid) is myristoylated at its newly exposed N terminus,
translocates to the outer mitochondrial membrane (OMM), and/or activates Bak
or Bax protein (16). Recently,
it was shown that the N-terminal cleavage fragment of Bid is quickly
ubiquitinated for degradation and that this degradation is necessary for the
pro-apoptotic function of tBid
(17). The same study also
concluded that, although full-length Bid is capable of translocating to the
OMM, it is not able to induce MOMP on its own
(17). A well characterized
example of tBid involvement during apoptosis is in the engagement of the
mitochondrial apoptotic pathway in so-called type II cells upon activation of
the extrinsic pathway
(18).Here, we have investigated whether Bid plays a functional role in the
induction of MOMP during apoptosis in response to the genotoxic anticancer
drug etoposide. To that end, we used Bax-deficient Jurkat cells that are
stably depleted of Bid and evaluated the extent to which these cells underwent
drug-induced MOMP. In addition, Jurkat clones in which the intrinsic pathway
had been inhibited due to the stable knockdown of Apaf-1 or the overexpression
of full-length XIAP or the baculoviral IAP repeats 1 and 2 (BIR1/BIR2) of XIAP
were used to gain insight into the molecular requirements necessary for
cleavage of Bid to tBid during drug-induced apoptosis. Strikingly, the data
showed that etoposide-induced apoptosis was decreased in Bid-deficient Jurkat
cells. In particular, cells lacking Bid expression exhibited decreased Bak
activation, cytochrome c release, loss of mitochondrial membrane
potential (ΔΨ), and caspase activation. Further, incubation of
permeabilized Bid-deficient cells with recombinant tBid, but not full-length
Bid, induced Bak dimerization and cytochrome c release.
Significantly, we also found that cleavage of Bid to tBid occurred strictly
downstream of Apaf-1 by a mechanism that required active executioner
caspases. 相似文献
19.
Hyun Ju Woo In Taek Oh Jung Yi Lee Do Youn Jun Myung Chul Seu Koan Sik Woo Min Hee Nam Young Ho Kim 《Process Biochemistry》2012,47(12):1861-1871
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 (Δψm) 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, Δψm 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. 相似文献
20.
Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition 总被引:1,自引:0,他引:1
We report here the identification of a novel protein, Smac, which promotes caspase activation in the cytochrome c/Apaf-1/caspase-9 pathway. Smac promotes caspase-9 activation by binding to inhibitor of apoptosis proteins, IAPs, and removing their inhibitory activity. Smac is normally a mitochondrial protein but is released into the cytosol when cells undergo apoptosis. Mitochondrial import and cleavage of its signal peptide are required for Smac to gain its apoptotic activity. Overexpression of Smac increases cells' sensitivity to apoptotic stimuli. Smac is the second mitochondrial protein, along with cytochrome c, that promotes apoptosis by activating caspases. 相似文献