Interleukin I J Converting Enzyme-like Proteases Are Essential for p53-mediated Transcriptionally Dependent Apoptosis1

p53-mediated apoptosis in baby rat kidney (BRK) cell lines transformed by EIA and p53(vaIl35) requires a transcriptionally functional p53. Coexpression of the El B 19K protein in BRK cell lines transformed by EIA and p53(vaIl35) rescues cells from p53-mediated apoptosis, and this is paralleled by the absence of both lamin and poly(ADP-nbose) polymerase cleavage. Therefore, the role of interleukin I 13 converting enzyme (ICE)-Iike proteases in p53-mediated, transcriptionally dependent apoptosis was investigated. The ICE-like protease CPP32 was proteolytically activated during p53-mediated apoptosis in BRK cells, and this required a transcriptionally competent p53. Substitution of the p53 transactivation domain with the transactivation domain of herpes simplex virus VPI6 (VPI6/p53) resulted in accelerated kinetics of both apoptosis and Bax induction. Moreover, apoptosis induced by p53, VPI6/p53, and Bax was abrogated by Z-VAD.FMK, an inhibitor of ICE-like proteases. These results indicate that all apoptotic pathways downstream of p53mediated transcription converge upon the activation of ICE-like proteases. Introduction Apoptosis or programmed cell death is a genetically programmed cell suicide response that serves a critical role in normal development and homeostasis (1 , 2). Apoptosis can be triggered by a variety of stimuli including viral infection (3, 4), growth factor withdrawal (5-10), and DNA damage resulting from radiation (1 1-1 3) and chemotherapeutic drugs (14). The hallmark signs of apoptosis include chromatin condenReceived 2/1 1/97; accepted 2/26/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to mdicate this fact. 1 This work was supported by Grants CA 60088 and CA 53370 from NIH (to E. W.). P. S. was supported in part by a Center for Advanced Biotechnology and Medicine predoctoral fellowship. 2 To whom requests for reprints should be addressed, at Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, NJ 08854. Phone: (908) 235-5329; Fax: (908) 235-5318. sation, internucleosomal cleavage of DNA, cell shrinkage, and the formation of apoptotic bodies, which are ultimately engulfed by phagocytic cells (1 , 2, 15). The entire process constitutes a tightly controlled series of events that can have dire consequences if deregulated at any point. Inappropriate apoptosis can contribute to the pathogenicity of diseases such as AIDS and to neurodegenerative disorders such as Alzheimer’s disease and spinal muscular atrophy. The perpetual inhibition of apoptosis, however, can facilitate the development of cancer (1 , 2). In certain cases of follicular B-cell lymphoma, the antiapoptotic protein Bcl-2 becomes overexpressed as a result of a translocation event that juxtaposes the bc!-2 gene next to the immunoglobulin heavy chain gene promoter. The subsequent overexpression of Bcl-2 contributes to the development of neoplasia by preventing apoptosis induced by activated c-myc (1 6, 17). Therefore, understanding the mechanisms of apoptosis would contribute greatly to our concept of how neoplastic transformation is perpetuated. The p53 tumor suppressor protein is a potent mediator of apoptosis and a transcription factor capable of both transactivation and transcriptional repression (1 0, 18-23). It has been established that in certain cell types, p53-mediated apoptosis can occur independently of new protein synthesis (9, 21 , 23). The mechanism(s) by which p53-transcriptionally independent apoptosis occurs is, however, uncertain but may depend upon either p53-mediated transcriptional repression of survival factors or the ability of p53 to interact with the DNA repair machinery (9, 24-26). However, in other cell types a transcriptionally competent p53 is essential for p53-mediated apoptosis (27, 28). Indeed, we have demonstrated that p53-mediated transcription is essential for El Ainduced apoptosis in BRK3 cells (29). Therefore, in BRK cells, the downstream targets of p53-mediated transcription are the principle effectors of E1A-induced apoptosis. We have demonstrated that BRK cell lines transformed by E1A and p53(vaIl 35) undergo p53-mediated apoptosis only at the permissive temperature (1 8). Therefore, E1A-induced apoptosis in BRK cells is strictly p53 dependent. p53-mediated apoptosis in BRK cells is accompanied by a progressive increase in the p53-inducible protein Bax (30). Moreover, the ectopic expression of Bax in E1A and p53(vall 35) transformed cell lines at the restrictive temperature is sufficient to induce apoptosis (30). Both the El B 1 9K protein and Bcl-2 bind to and antagonize the apoptotic function of Bax (30, 31). In addition, the stable expression of either Bcl-2 or the El B 19K protein in BRK cell lines transformed by E1A and 3 The abbreviations used are: BRK, baby rat kidney; ICE, interieukin 1 f3 converting enzyme; HSV, herpes simplex virus; TNF, tumor necrosis factor; PARP, poly(ADP-ribose) polymerase. 644 ICE-like Proteases and Apoptosis p53(vall 35) abrogates p53-mediated apoptosis at the permissive temperature (32, 33). These results implicate Bax as a pivotal trigger of the downstream events associated with p53-mediated transcriptionally dependent apoptosis. Recent evidence indicates that there are tumor-derived p53 mutants that are defective for p53-mediated tumor suppression, apoptosis, and the ability to transactivate bax (30, 34-38). Interestingly, these p53 mutants are still competent for both the transactivation of the cyclin/cyclin-dependent kinase inhibitor protein p21/WAF-l and for growth arrest. These results demonstrate the importance of apoptosis as a potential mechanism of p53-mediated tumor suppression. In addition, these results indicate that Bax may mediate the tumor suppressor function of p53. Indeed, Bax antagonizes the ability of the El B 19K protein to cooperate with E1A in the transformation of primary rodent cells in culture (39). Therefore, determining the downstream events associated with Bax-mediated apoptosis could potentially advance our understanding of how p53-mediated tumor suppression is implemented. ICE-like proteases are a class of enzymes that have been implicated as key mediators of many different forms of apoptosis. This is due in large part to the fact that biological and chemical inhibitors of ICE-like proteases effectively abrogate apoptosis induced by a variety of stimuli. The baculovirus p35 protein prevents TNF-cr and Fas/APO-l -mediated apoptosis (40), and developmentally regulated programmed cell death in the nematode Caenortiabditis elegans and in Drosophila (41 , 42). The cowpox virus CrmA protein has been shown to be an effective inhibitor of Fas/APO-l -mediated apoptosis (43-45), aswell as apoptosis in neuronal cells induced by growth factor withdrawal (46). In addition, p35 and CrmA are capable of inhibiting apoptosis induced by the ectopic expression of ICE-like proteases (47). Both p35 and CrmA serve as cleavable substrates for ICE-like proteases and thereby act as competitive inhibitors (42, 47). Likewise, synthetic tetrapeptide inhibitors of ICE-like proteases, such as Z-VAD.FMK, which are designed to mimic substrate cleavage sites, are also potent inhibitors of apoptosis both in vivo and in vitro (43, 48-54). Collectively, these results offer conceptual proof that activated ICE-like proteases are the key downstream effectors of most if not all forms of apoptosis. We and others have demonstrated recently that ICE-like proteases are activated during p53-mediated apoptosis (55, 56). In addition, we have demonstrated that BRK cells rescued from p53-mediated apoptosis by the El B 19K protein are devoid of ICE-like protease activation (56). These results implicate a functional role for ICE-like proteases in p53mediated apoptosis. However, these results do not rule out the contribution of other p53-dependent mechanisms to regulate apoptosis. In the present study, we unequivocally demonstrate that all apoptotic mechanisms that are triggered downstream of p53-mediated transcription are funneled through activated ICE-like proteases. BRK cell lines transformed by E1A and p53(vaIl 35) at the restrictive temperature are completely rescued from p53-mediated apoptosis at the permissive ternperature in the presence of Z-VAD.FMK. In addition, we demonstrate that when the p53 transactivation domain is replaced with the transactivation domain of HSV VP1 6, the kinetics of p53-mediated apoptosis are accelerated, and Bax levels are dramatically up-regulated. Moreover, Bax-mediated apoptosis is inhibitable by Z-VAD.FMK. These results indicate that p53-mediated transcription is sufficient for both apoptosis and the activation of ICE-like proteases and implicate Bax asakey downstream effector of p53-mediated ICE-like protease activation. In addition, these results implicate a direct correlation between the tumor suppressor function of p53 and the Bax-mediated activation of ICE-like proteases. Results ICE-like Proteases Are the Key Effectors of p53-mediated, Transcriptionally Dependent Apoptosis. The Ani cell line was generated by the transformation of primary BRK cells with E1A and p53(vall35) (18). This cell line is transformed at the restrictive temperature for p53 (38.5#{176}C) but undergoes p53-mediated apoptosis at the permissive ternperature (32#{176}C; Refs. 18, 29, 32, and 56). We have dernonstrated recently that p53-mediated apoptosis in the Mi cell line is paralleled by the proteolytic cleavage of nuclear lamins and the DNA repair enzyrne PARP (56). In contrast, BAK cell lines that are protected from p53-mediated apoptosis by the El B 19K protein are devoid of both lamin and PARP cleavage activity (56). These results implicate a functional role for ICE-like proteases in p53-mediated apoptosis. However, inhibiting the proteolytic cleavage of nuclear lamins attenuates p53-mediated apoptosis but does not abrogate it entirely (56). Therefore, there may be mechanisms in addition to the activation of ICE-like proteases that contribute to p53-mediated apoptosis in the Ani cell line. To directly address this issue, the Anl cell line was incubated at the permissive temperature for 24 and 48 h in the presence of either ZVAD.FMK, a specific inhibitor of ICE-like proteases, or a control peptide, and cell viability was subsequently assessed. Anl cells treated with the control peptide underwent extensive p53-mediated apoptosis after incubation for 24 and 48 h at the permissive temperature (Fig. la). In contrast, Mi cells treated with Z-VAD.FMK were completely protected from p53-mediated apoptosis up to 24 h at the permissive temperature. The protective effect of Z-VAD.FMK was transient, because cells eventually succumbed to p53-mediated apoptosis by 48 h (Fig. la). Readdition of Z-VAD.FMK at the 24 h time point prevented p53-mediated apoptosis at the 48-h time point (Fig. 1 , b and c). Thus, transitory inhibition of p53-dependent apoptosis can be attributed to degradation of Z-VAD.FMK owing, probably, to the labile fluoromethylketone. Moreover, cells protected from p53-mediated apoptosis by Z-VAD.FMK showed no significant increases in cell viability (Fig. ib). These results are consistent with the ability of Z-VAD.FMK to inhibit p53-mediated apoptosis but not growth arrest, which is what occurs when p53-dependent apoptosis is inhibited by the El B 19K protein. Indeed, the El B 19K protein functions downstream of p53-mediated transcription to specifically abrogate p53-mediated apoptosis while leaving p53-mediated growth arrest functionally