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Until recently, the p53 transcriptional target p21, a member of the Cip/Kip family, was almost solely viewed as a nuclear protein with a principal function of inhibiting cyclin-dependent kinase (CDK) activity and hence, cell cycle progression. However, emerging evidence now suggests additional functions for p21 in diverse cellular processes including a role as a modulator of apoptosis. Several mechanisms are suggested by which p21 interferes with the apoptotic machinery that encompass either CDK-independent events such as transcriptional regulation and direct binding to pro-apoptotic gene products in the cytoplasm, or that were based on inhibition of CDKs that in several studies were required downstream of caspases for the generation of characteristic apoptotic alterations. Very recently, we have shown that p21 protects cells from irradiation-induced apoptosis by suppression of CDK activity that appears to be required downstream of the mitochondria for an efficient activation of the caspase cascade. Together with other reports, our results not only demonstrate the close connection of events leading to either cell cycle arrest or apoptosis, but also indicate that the CDK inhibitor p21 battles apoptosis at multiple frontiers.

Proteases of the caspase family are thought to be activated by proteolytic processing of their inactive zymogens. However, although proteolytic cleavage is sufficient for executioner caspases, a different mechanism has been recently proposed for initiator caspases, such as caspase-8, which are believed to be activated by proximity-induced dimerization. According to this model, dimerization rather than proteolytic processing is considered as the critical event for caspase-8 activation. Such a mechanism would suggest that in the absence of a dimerization platform such as the death-inducing signaling complex, caspase-8 proteolytic cleavage would result in an inactive enzyme. As several studies have described caspase-8 cleavage during mitochondrial apoptosis, we now investigated whether caspase-8 becomes indeed catalytically active in this pathway. Using an in vivo affinity labeling approach, we demonstrate that caspase-8 is activated in etoposide-treated cells in vivo in the absence of the receptor-induced death-inducing signaling complex formation. Furthermore, we show that both caspase-3 and -6 are required for the efficient activation of caspase-8. Our data therefore indicate that interchain cleavage of caspase-8 in the mitochondrial pathway is sufficient to produce an active enzyme even in the absence of receptor-driven procaspase-8 dimerization.

Although caspase-2 represents the most conserved caspase across species and was the second caspase identified, its precise function remains enigmatic. In several cell types we show that knockdown of caspase-2 specifically impaired DNA damage-induced p21 expression, whereas overexpression of a caspase-2 mutant increased p21 levels. Caspase-2 did not influence p21 mRNA transcription; moreover, various inhibitors targeting proteasomal or non-proteasomal proteases, including caspases, could not restore p21 protein levels following knockdown of caspase-2. As, however, silencing of caspase-2 impaired exogenous expression of p21 constructs containing 3′-UTR sequences, our results strongly indicate that caspase-2 regulates p21 expression at the translational level. Intriguingly, unlike depletion of caspase-2, which prevented p21 expression and thereby reverted the γ-IR-induced senescent phenotype of wild-type HCT116 colon carcinoma cells into apoptosis, knockdown of none of the caspase-2-interacting components RAIDD, RIP or DNA-PKcs was able to mimic these processes. Together, our data suggest that this novel role of caspase-2 as a translational regulator of p21 expression occurs not only independently of its enzymatic activity but also does not require known caspase-2-activating platforms.

Although initially described as a regulator of cell cycle progression, the cyclin-dependent kinase inhibitor p21 is now known to also modulate various other biological processes including transcription, differentiation and apoptosis. These versatile activities of p21 are mainly mediated via direct binding to various transcription factors, pro-apoptotic proteins and kinases that are usually inhibited by this interaction. Here we provide in vitro evidence that p21 not only inhibits, but also activates certain kinases in a remarkable substrate-dependent manner. Whereas phosphorylation of the tumor suppressor p53 by several isoforms of the cJun N-terminal kinases (JNKs) was greatly attenuated in the presence of p21, phosphorylation of cJun remained either unaffected or was even enhanced. Furthermore, p21 strongly increased phosphorylation of cFos and MBP by ERK1 and ERK2, while p53 phosphorylation was increased and inhibited, respectively. Also p38α and glycogen synthase kinase-3 beta (GSK-3β) were found differentially regulated by p21 in a substrate-dependent manner, while casein kinase-1 epsilon (CK1e) was not affected. Together with our finding that the stress-induced p53 phosphorylation pattern differs greatly between p21-proficient and -deficient HCT116 colon carcinoma cells, our results suggest that p21 is able to influence kinase activities both in a negative and positive manner.

Pifithrin-alpha (PFT-alpha) was shown to specifically block transcriptional activity of the tumor suppressor p53 and was therefore proposed to be useful in preventing the severe side effects often associated with chemo- and radiotherapy. We report here that although PFT-alpha efficiently protected different cell types from DNA damage-induced apoptosis, it mediated this effect regardless of the presence or absence of p53. Interestingly, PFT-alpha blocked the apoptosome-mediated processing and activation of caspase-9 and -3 without interfering with the activation of mitochondria. Neither the DNA damage-induced activation of Bax or Bak nor the loss of the mitochondrial membrane potential or the final release of cytochrome c were inhibited by this compound. Instead, the ability of PFT-alpha to protect p53-deficient cells from DNA damage-induced caspase activation and apoptosis was greatly diminished after siRNA-mediated downregulation of cyclin-D1 expression. In contrast, downregulation of other proteins involved in cell-cycle progression, such as the retinoblastoma protein, cyclin D3, as well as the cyclin-dependent kinases, 2, 4 and 6, could not abolish this protection. Thus, our data show that PFT-alpha protects cells from DNA damage-induced apoptosis also by a p53-independent mechanism that takes place downstream of mitochondria and that might involve cyclin D1.

The role of the cyclin-dependent kinase (CDK) inhibitor p21 as a mediator of p53-induced growth arrest is well established. In addition, recent data provide strong evidence for new emerging functions of p21, including a role as a modulator of apoptosis. The mechanisms, however, by which p21 interferes with the death machinery, especially following ionizing radiation (IR), are largely unknown. Here, we report that IR induced caspase-9 and caspase-3 activation and subsequent apoptosis only in p21-deficient colon carcinoma cells, whereas similar treated wild-type cells were permanently arrested in the G2-M phase, correlating with the induction of cellular senescence. Interestingly, activation of the mitochondrial pathway, including caspase-2 processing, depolarization of the outer mitochondrial membrane, and cytochrome c release, was achieved by IR in both cell lines, indicating that p21 inhibits an event downstream of mitochondria but preceding caspase-9 activation. IR-induced p21 protein expression was restricted to the nucleus, and no evidence for a mitochondrial or cytoplasmic association was found. In addition, p21 did neither interact with caspase-3 or caspase-9, suggesting that these events are not required for the observed protection. Consistent with this assumption, we found that CDK inhibitors potently abrogated IR-induced caspase processing and activation without affecting mitochondrial events. In addition, in vitro caspase activation assays yielded higher caspase-3 activities in extracts of irradiated p21-deficient cells compared with extracts of similar treated wild-type cells. Thus, our results strongly indicate that p21 protects cells from IR-induced apoptosis by suppression of CDK activity that seems to be required for activation of the caspase cascade downstream of the mitochondria. (Cancer Res 2006; 66(23): 11254-62)

Caspase-9 plays an important role in apoptosis induced by genotoxic stress. Irradiation and anticancer drugs trigger mitochondrial outer membrane permeabilization, resulting in cytochrome c release and caspase-9 activation. Two highly contentious issues, however, remain: It is unclear whether the loss of the mitochondrial membrane potential ΔΨMcontributes to cytochrome c release and whether caspases are involved. Moreover, an unresolved question is whether caspase-2 functions as an initiator in genotoxic stress-induced apoptosis. In the present study, we have identified a mutant Jurkat T-cell line that is deficient in caspase-9 and resistant to apoptosis. Anticancer drugs, however, could activate proapoptotic Bcl-2 proteins and cytochrome c release, similarly as in caspase-9–proficient cells. Interestingly, despite these alterations, the cells retained ΔΨM. Furthermore, processing and enzyme activity of caspase-2 were not observed in the absence of caspase-9. Reconstitution of caspase-9 expression restored not only apoptosis but also the loss of ΔΨMand caspase-2 activity. Thus, we provide genetic evidence that caspase-9 is indispensable for drug-induced apoptosis in cancer cells. Moreover, loss of ΔΨMcan be functionally separated from cytochrome c release. Caspase-9 is not only required for ΔΨMloss but also for caspase-2 activation, suggesting that these two events are downstream of the apoptosome.

Due to their tremendous apoptosis-inducing potential, proteasomal inhibitors (PIs) have recently entered clinical trials. Here we show, however, that various PIs rescued proliferating tumor cells from death receptor-induced apoptosis. This protection correlated with the stabilization of X-linked IAP (XIAP) and c-FLIP and the inhibition of caspase activation. Together with the observation that PIs could not protect cells expressing XIAP or c-FLIP short interfering RNAs (siRNAs) from death receptor-induced apoptosis, our results demonstrate that PIs mediate their protective effect via the stabilization of these antiapoptotic proteins. Furthermore, we show that once these proteins were eliminated, either by long-term treatment with death receptor ligands or by siRNA-mediated suppression, active caspases accumulated to an even larger extent in the presence of PIs. Together, our data support a biphasic role for the proteasome in apoptosis, as they show that its constitutive activity is crucial for the rapid initiation of the death program by eliminating antiapoptotic proteins, whereas at later stages, the proteasome acts in an antiapoptotic manner due to the proteolysis of caspases. Thus, for a successful PI-based tumor therapy, it is crucial to carefully evaluate basal proteasomal activity and the status of antiapoptotic proteins, as their PI-mediated prolonged stability might even cause adverse effects, leading to the survival of a tumor.

The family of aspartate-specific and cysteine-dependent proteases, called caspases, is crucial not only for apoptosis but also for differentiation and cell cycle progression. Several research groups have recently published data ruling in or out the participation of caspase-10, an initiator caspase