Manganese induces apoptosis of human B cells: caspase-dependent cell death blocked by bcl-2.

Manganese ions block apoptosis of phagocytes induced by various agents. The prevention of apoptosis was attributed to the activation of manganous superoxide dismutase (Mn-SOD) and to the antioxidant function of free Mn2+ cations. However, the effect of Mn2+ on B cell apoptosis is not documented. In this study, we investigated the effects of Mn2+ on the apoptotic process in human B cells. We observed that Mn2+ but not Mg2+ or Ca2+, inhibited cell growth and induced apoptosis of activated tonsilar B cells, Epstein Barr virus (EBV)-negative Burkitt's lymphoma cell lines (BL-CL) and EBV-transformed B cell lines (EBV-BCL). In the same conditions, no apoptosis was observed in U937, a monoblastic cell line. Induction of B cell apoptosis by Mn2+ was time- and dose-dependent. The cell permeable tripeptide inhibitor of ICE family cysteine proteases, zVAD-fmk, suppressed Mn2+-induced apoptosis. Furthermore, Mn2+ triggered the activation of interleukin-1beta converting enzyme (ICE/caspase 1), followed by the activation of CPP32/Yama/Apopain/caspase-3. In addition, poly-(ADP-ribose) polymerase (PARP), a cellular substrate for CPP32 protease was degraded to generate apoptotic fragments in Mn2+-treated B cell lines. The inhibitor, zVAD-fmk suppressed Mn2+-triggered CPP32 activation and PARP cleavage and apoptosis. These results indicate that the activation of caspase family proteases is required for the apoptotic process induced by Mn2+ treatment of B cells. While the caspase-1 inhibitor YVAD was unable to block apoptosis, the caspase-3 specific inhibitor DEVD-cmk, partially inhibited Mn2+-induced CPP32 activation, PARP cleavage and apoptosis of cells. Moreover, Bcl-2 overexpression in BL-CL effectively protected cells from apoptosis and cell death induced by manganese. This is the first report showing the involvement of Mn2+ in the regulation of B lymphocyte death presumably via a caspase-dependent process with a death-protective effect of Bcl-2.     

Processing/Activation of At Least Four Interleukin-1β Converting Enzyme–like Proteases Occurs during the Execution Phase of Apoptosis in Human Monocytic Tumor Cells

Identification of the processing/activation of multiple interleukin-1β converting enzyme (ICE)–like proteases and their target substrates in the intact cell is critical to our understanding of the apoptotic process. In this study we demonstrate processing/activation of at least four ICE-like proteases during the execution phase of apoptosis in human monocytic tumor THP.1 cells. Apoptosis was accompanied by processing of Ich-1, CPP32, and Mch3α to their catalytically active subunits, and lysates from these cells displayed a proteolytic activity with kinetics, characteristic of CPP32/Mch3α but not of ICE. Fluorescence-activated cell sorting was used to obtain pure populations of normal and apoptotic cells. In apoptotic cells, extensive cleavage of Ich-1, CPP32, and Mch3α was observed together with proteolysis of the ICE-like protease substrates, poly (ADP-ribose) polymerase (PARP), the 70-kD protein component of U1 small nuclear ribonucleoprotein (U170K), and lamins A/B. In contrast, no cleavage of CPP32, Mch3α or the substrates was observed in normal cells. In cells exposed to an apoptotic stimulus, some processing of Ich-1 was detected in morphologically normal cells, suggesting that cleavage of Ich-1 may occur early in the apoptotic process. The ICE-like protease inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK), inhibited apoptosis and cleavage of Ich-1, CPP32, Mch3α, Mch2α, PARP, U1-70K, and lamins. These results suggest that Z-VAD.FMK inhibits apoptosis by inhibiting a key effector protease upstream of Ich-1, CPP32, Mch3α, and Mch2α. Together these observations demonstrate that processing/activation of Ich-1, CPP32, Mch3α, and Mch2α accompanies the execution phase of apoptosis in THP.1 cells. This is the first demonstration of the activation of at least four ICE-like proteases in apoptotic cells, providing further evidence for a requirement for the activation of multiple ICE-like proteases during apoptosis.     

Role of Ced-3/ICE-family proteases in staurosporine-induced programmed cell death

In the accompanying paper by Weil et al. (1996) we show that staurosporine (STS), in the presence of cycloheximide (CHX) to inhibit protein synthesis, induces apoptotic cell death in a large variety of nucleated mammalian cell types, suggesting that all nucleated mammalian cells constitutively express all of the proteins required to undergo programmed cell death (PCD). The reliability of that conclusion depends on the evidence that STS-induced, and (STS + CHS)-induced, cell deaths are bona fide examples of PCD. There is rapidly accumulating evidence that some members of the Ced-3/Interleukin-1 beta converting enzyme (ICE) family of cysteine proteases are part of the basic machinery of PCD. Here we show that Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a cell-permeable, irreversible, tripeptide inhibitor of some of these proteases, suppresses STS-induced and (STS + CHX)-induced cell death in a wide variety of mammalian cell types, including anucleate cytoplasts, providing strong evidence that these are all bona fide examples of PCD. We show that the Ced-3/ICE family member CPP32 becomes activated in STS- induced PCD, and that Bcl-2 inhibits this activation. Most important, we show that, in some cells at least, one or more CPP32-family members, but not ICE itself, is required for STS-induced PCD. Finally, we show that zVAD-fmk suppresses PCD in the interdigital webs in developing mouse paws and blocks the removal of web tissue during digit development, suggesting that this inhibition will be a useful tool for investigating the roles of PCD in various developmental processes.     

ICE/CED-3 Family Executes Oligodendrocyte Apoptosis by Tumor Necrosis Factor

Abstract: Tumor necrosis factor (TNF) is thought to be one of the mediators responsible for the damage of oligodendrocytes (OLGs) in multiple sclerosis (MS). We report here the involvement of the interleukin 1β-converting enzyme (ICE)/ Caenorhabditis elegans gene ced-3 (CED-3) family in TNF-mediated cell death of OLGs. The addition of TNF-α to primary cultures of OLGs that express ice and cpp32 significantly decreased the number of live OLGs in 72 h. DNA fragmentation was detected in TNF-treated OLGs at 36 h with the terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. Benzyloxycarbonyl-Asp-CH₂OC(O)-2,6-dichlorobenzene, an inhibitor of the ICE/CED-3 family that shows p35 -like inhibitory specificity, protected against the TNF-induced cell death of OLGs. Furthermore, acetyl-YVAD-CHO (a specific inhibitor of ICE-like proteases) as well as acetyl-DEVD-CHO (a specific inhibitor of CPP32-like proteases) enhanced the survival of OLGs treated with TNF-α, indicating that ICE- and the CPP32-mediated cell death pathways are activated in TNF-induced OLG cell death. Our results suggest that the inhibition of ICE/CED-3 proteases may be a novel approach to treat neurodegenerative diseases such as MS.     

Characterization of CPP32-Like Protease Activity Following Apoptotic Challenge in SH-SY5Y Neuroblastoma Cells

Abstract: We characterized the activation of interleukin-1β-converting enzyme (ICE)-like proteases (caspases) in human neuroblastoma cells (SH-SY5Y) following challenge with staurosporine, an established agent known to induce apoptosis. Time course analyses of lactate dehydrogenase release detected a significant increase in cell death as early as 6 h that continued at least until 24 h following staurosporine treatment. Western blot analyses using anti-poly(ADP-ribose) polymerase (anti-PARP) and anti-CPP32 antibodies revealed proteolytic processing of CPP32 (an ICE homologue) as well as fragmentation of PARP as early as 3 h following staurosporine challenge. Furthermore, the hydrolysis of the CPP32 substrate acetyl-DEVD-7-amido-4-methylcoumarin was detected as early as 3 h and became maximal at 6 h after staurosporine challenge, suggesting a delayed and sustained period of CPP32-like activation. In addition, we used the first immunohistochemical examination of CPP32 and PARP in cells following an apoptotic challenge. The localization of CPP32 in untreated SH-SY5Y cells was exclusively restricted to the cytoplasm. Following staurosporine challenge there was a condensing of CPP32 immunofluorescence from the cytoplasm to a region adjacent to the plasma membrane. In contrast, PARP immunofluorescence was evenly distributed in the nucleus in untreated SH-SY5Y cells and on staurosporine challenge was found to be associated with condensed chromatin. It is important that a pan ICE inhibitor [carbobenzoxy-Asp-CH₂OC(O)-2,6-dichlorobenzene] was able to attenuate lactate dehydrogenase release and PARP and CPP32 cleavage and altered immunohistochemical staining patterns for PARP and CPP32 following staurosporine challenge.     

Photodynamic therapy induces caspase-3 activation in HL-60 cells

Caspases have been shown to play a crucial role in apoptosis induced by various deleterious and physiologic stimuli. In this study, we show for the first time that photodynamic therapy (PDT), using benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) as the photosensitizer, induces the complete cleavage and subsequent activation of caspase-3 (CPP32/Yama/Apopain) but not caspase-1 (ICE) in human promyelocytic leukemia HL-60 cells. Poly(ADP-ribose) polymerase (PARP) and the catalytic subunit of DNA dependent protein kinase (DNA PK(CS)) were cleaved within 60 min of light activation of BPD-MA. The general caspase inhibitor Z-Asp-2,6 dichlorobenzoyloxymethylketone (Z-Asp-DCB) blocked PARP cleavage while the serine protease inhibitors 3,4-dichloroisocoumarin (DCI) and N-tosyl-lysyl chloromethyl ketone (TLCK) blocked the cleavage of caspase-3 suggesting that they act upstream of caspase-3 activation. All three inhibitors were able to block DNA fragmentation that was induced by treatment with BPD-MA followed by light application. These studies demonstrate that protease activity, particularly that of caspase-3, is triggered in HL-60 cells treated with lethal levels of BPD-MA and visible light.     

人肺癌细胞CPP32基因的克隆及表达

蛋白酶尤其是ＩＣＥ家族的蛋白酶是细胞死亡机制的核心成分．ＩＣＥ蛋白酶家族中,ＣＰＰ３２（又称Ｙａｍａ,ａｐｏｐａｉｎ）在不同形式的凋亡途径中起核心作用．为深入研究ＣＰＰ３２的结构与功能,克隆了ＣＰＰ３２基因,并在大肠杆菌中进行了表达．采用ＲＴ－ＰＣＲ技术从人肺癌细胞株中获得了ＣＰＰ３２蛋白酶基因．ＤＮＡ序列分析表明,该基因由已报道的编码ＣＰＰ３２αｐ２０亚单位和ＣＰＰ３２βｐ１０亚单位的核苷酸组成,提示ＩＣＥ家族蛋白酶寡聚化可能受ＤＮＡ水平调控．将获得的ＣＰＰ３２基因分别重组到ｐＢＶ３２１和ｐＥＸ３１Ｂ载体上,并分别转化到大肠杆菌中,均获得了ＣＰＰ３２基因的较高表达,表达产物主要以包涵体形式存在．     

Zinc inhibits apoptosis upstream of ICE/CED-3 proteases rather than at the level of an endonuclease

Apoptosis is commonly associated with DNA digestion, but it remains controversial as to which endonuclease is involved. The ability of zinc to inhibit DNA digestion in intact cells, and inhibit a Ca2+/Mg2+-dependent endonuclease in cell lysates, has been used frequently to suggest this is the endonuclease involved. However, zinc has many other effects on cells, and here it is shown that zinc also prevents many upstream events in apoptosis. These studies were performed in human ML-1 cells following incubation with etoposide. During apoptosis, these cells undergo intracellular acidification, increased accumulation of Hoechst 33342, DNA digestion and chromatin condensation. Zinc inhibited all of these events. An upstream event in apoptosis is activation of ICE/CED-3 proteases which is commonly observed as proteolysis of a substrate protein, poly(ADP-ribose) polymerase (PARP). The ICE/CED-3 proteases are themselves activated by proteolysis, and this was detected here by cleavage of one family member CPP32. Zinc prevented cleavage of both CPP32 and PARP. We recently demonstrated that dephosphorylation of the retinoblastoma susceptibility protein Rb was a marker of an event even further upstream in apoptosis; zinc was also found to inhibit Rb dephosphorylation. Therefore, zinc must protect cells at a very early step in the apoptotic pathway, and not as a direct inhibitor of an endonuclease.     

Bcl-2 acts upstream of the PARP protease and prevents its activation

Apoptosis has recently been extensively studied and multiple factors have been implicated in its regulation. It remains unclear how these factors are ordered in the cell death pathway. Here we investigate the relationship between the inhibitor of apoptosis, bcl-2, and the PARP protease, prlCE/CPP32, recently implicated in apoptosis. Using PARP proteolysis as an indicator of the activation of the PARP protease, we find that the chemotherapeutic agent, etoposide, induces apoptosis and PARP proteolysis in Molt4 cells as early as 4 h with cell death lagging behind this event. In contrast, Molt4 cells that over-express bcl-2 show no PARP proteolysis or cell death. In order to determine if bcl-2 inhibits the PARP protease or its activation, we developed a cell-free system. Using this system with extracts from etoposide-treated cells and purified bovine PARP, we demonstrate that extracts from bcl-2 over-expressing cells cause little or no PARP proteolysis. Whereas, extracts from control vector cells contain an active PARP protease. This protease is inhibited by the tetrapeptide ICE-like protease inhibitor, YVAD-chloromethylketone. Interestingly, this protease is not inhibited by the addition of purified bcl-2 protein. These results rule out that bcl-2 directly inhibits the active protease or that it has an effect downstream of prlCE/CPP32 such as preventing access to the PARP substrate. These results also demonstrate a role of bcl-2 in interfering with an upstream signal required to activate the PARP protease and allow us to begin to order the components in the apoptotic pathway.     

Increases in Bcl-2 and cleavage of caspase-1 and caspase-3 in human brain after head injury.

The bcl-2 and caspase families are important regulators of programmed cell death in experimental models of ischemic, excitotoxic, and traumatic brain injury. The Bcl-2 family members Bcl-2 and Bcl-xL suppress programmed cell death, whereas Bax promotes programmed cell death. Activated caspase-1 (interleukin-1beta converting enzyme) and caspase-3 (Yama/Apopain/Cpp32) cleave proteins that are important in maintaining cytoskeletal integrity and DNA repair, and activate deoxyribonucleases, producing cell death with morphological features of apoptosis. To address the question of whether these Bcl-2 and caspase family members participate in the process of delayed neuronal death in humans, we examined brain tissue samples removed from adult patients during surgical decompression for intracranial hypertension in the acute phase after traumatic brain injury (n=8) and compared these samples to brain tissue obtained at autopsy from non-trauma patients (n=6). An increase in Bcl-2 but not Bcl-xL or Bax, cleavage of caspase-1, up-regulation and cleavage of caspase-3, and evidence for DNA fragmentation with both apoptotic and necrotic morphologies were found in tissue from traumatic brain injury patients compared with controls. These findings are the first to demonstrate that programmed cell death occurs in human brain after acute injury, and identify potential pharmacological and molecular targets for the treatment of human head injury.     

Genetic and metabolic status of NGF-deprived sympathetic neurons saved by an inhibitor of ICE family proteases

Sympathetic neurons undergo programmed cell death (PCD) when deprived of NGF. We used an inhibitor to examine the function of interleukin-1 beta-converting enzyme (ICE) family proteases during sympathetic neuronal death and to assess the metabolic and genetic status of neurons saved by such inhibition. Bocaspartyl(OMe)-fluoromethylketone (BAF), a cell-permeable inhibitor of the ICE family of cysteine proteases, inhibited ICE and CPP32 (IC50 approximately 4 microM) in vitro and blocked Fas-mediated apoptosis in thymocytes (EC50 approximately 10 microM). At similar concentrations, BAF also blocked the NGF deprivation-induced death of rat sympathetic neurons in culture. Compared to NGF-maintained neurons, BAF-saved neurons had markedly smaller somas and maintained only basal levels of protein synthesis; readdition of NGF restored growth and metabolism. Although BAF blocked apoptosis in sympathetic neurons, it did not prevent the fall in protein synthesis or the increase in the expression of c-jun, c- fos, and other mRNAs that occur during neuronal PCD, implying that the ICE-family proteases function downstream of these events during PCD.NGF and BAF rescued sympathetic neurons with an identical time course, suggesting that NGF, in addition to inhibiting metabolic and genetic events associated with neuronal PCD, can act posttranslationally to abort apoptosis at a time point indistinguishable from the activation of cysteine proteases. Both poly-(ADP ribose) polymerase and pro-ICE and Ced-3 homolog-1 (ICH-1) appear to be cleaved in a BAF-inhibitable manner, although the majority of pro-CPP32 appears unchanged, suggesting that ICH-1 is activated during neuronal PCD. Potential implications of these findings for anti-apoptotic therapies are discussed.     

Macromolecular substrates for the ICE-like proteases during apoptosis

The interleukin-1β-converting enzyme (ICE) family of proteases is an important component of the mechanism of the apoptotic process, but the physiologic roles of the different homologs during apoptosis remain unclear. Significant information about the roles of proteolysis in apoptosis will be gained through identification of the distal substrates through which these proteases achieve their pro-apoptotic effects. Identification of these substrates therefore remains an important challenge. A subset of autoantibodies from patients with systemic lupus erythematosus (SLE) recognize molecules that are specifically cleaved early during apoptosis. Several of the identified autoantigens are nuclear proteins (PARP, U1-70 kDa, and DNA-PK_CS) that are substrates for CPP32 in vitro and in apoptotic cells. Of note, these substrates are catalytic proteins involved in homeostatic pathways, suggesting that abolition of homeostasis is one fundamental feature ensuring the rapid irreversibility of the apoptotic process. Identification of the other substrates for this protease family will provide the tools to assess the roles of the different proteases in apoptotic death. J. Cell. Biochem. 64:50–54. © 1997 Wiley-Liss, Inc.     

Sequential activation of three distinct ICE-like activities in Fas-ligated Jurkat cells

ICE family proteases have been implicated as important effectors of the apoptotic pathway, perhaps acting hierarchically in a protease cascade. Using cleavage of endogenous protease substrates as probes, three distinct tiers of ICE-like activity were observed after Fas ligation in Jurkat cells. The earliest cleavage detected (30 min) was of fodrin, and produced a 150 kDa fragment. The second phase of cleavage (50 min) involved PARP, U1-70kDa and DNA-PK_cs, all substrates of the CPP32-like proteases. Lamin B cleavage was observed during the third cleavage phase (90 min). Distinct inhibition profiles obtained using a panel of peptide-based inhibitors of ICE-like proteases clearly distinguished the three different cleavage phases. These studies provide evidence for a sequence of ICE-like proteolytic activity during apoptosis. The early fodrin cleavage, producing a 150 kDa fragment, identifies an ICE-like activity proximal to CPP32 in Fas-induced Jurkat cell apoptosis.     

Involvement of the proteasome in the programmed cell death of NGF-deprived sympathetic neurons.

Sympathetic neurons undergo programmed cell death (PCD) upon deprivation of nerve growth factor (NGF). PCD of neurons is blocked by inhibitors of the interleukin-1beta converting enzyme (ICE)/Ced-3-like cysteine protease, indicating involvement of this class of proteases in the cell death programme. Here we demonstrate that the proteolytic activities of the proteasome are also essential in PCD of neurons. Nanomolar concentrations of several proteasome inhibitors, including the highly selective inhibitor lactacystin, not only prolonged survival of NGF-deprived neurons but also prevented processing of poly(ADP-ribose) polymerase which is known to be cleaved by an ICE/Ced-3 family member during PCD. These results demonstrate that the proteasome is a key regulator of neuronal PCD and that, within this process, it is involved upstream of proteases of the ICE/Ced-3 family. This order of events was confirmed in macrophages where lactacystin inhibited the proteolytic activation of precursor ICE and the subsequent generation of active interleukin-1beta.     

Pivotal role of a DEVD-sensitive step in etoposide-induced and Fas-mediated apoptotic pathways.

We investigated the role of proteases in the pathway that leads from specific DNA damage induced by etoposide (VP-16), a topoisomerase II inhibitor, to apoptotic DNA fragmentation in the U937 human leukemic cell line. In a reconstituted cell-free system, Triton-soluble extracts from VP-16-treated cells induced internucleosomal DNA fragmentation in nuclei from untreated cells. This effect was inhibited by the tetrapeptide Ac-DEVD-CHO, a competitive inhibitor of the interleukin-1 beta-converting enzyme (ICE)-related protease CPP32, but was not influenced by Ac-YVAD-CHO and Ac-YVAD-CMK, two specific inhibitors of ICE. The three tetrapeptides inhibited Fas-mediated apoptotic DNA fragmentation in the cell-free system. Internucleosomal DNA fragmentation, triggered by either VP-16 or an anti-Fas antibody, was associated with proteolytic cleavage of the poly(ADP-ribose)polymerase (PARP), a decrease in the level of 32 kDa CPP32 proenzyme and the appearance of the CPP32 p17 active subunit. Conversely, the expression of Ich-1L, another ICE-like protease, remained stable in apoptotic U937 cells. Several cysteine and serine protease inhibitors prevented apoptotic DNA fragmentation by acting either upstream or downstream of the DEVD-sensitive protease(s) activation and PARP cleavage. We conclude that a DEVD-sensitive step, which could involve CPP32, plays a central role in the proteolytic pathway that mediates apoptotic DNA fragmentation in VP-16-treated leukemic cells at the crossing with Fas-mediated pathway.     

Targeted expression of ced-3 and Ice induces programmed cell death in Drosophila

CED-3 is a cysteine protease required for programmed cell death in the nematode, Caenorhabditis elegans, and shares a sequence similarity with mammalian ICE (interleukin-1beta converting enzyme) family proteases. Both CED-3 and ICE family proteases can induce programmed cell death in mammalian cells. Structural and functional similarities between CED-3 and ICE family proteases indicate that the mechanism of cell death is evolutionarily conserved, suggesting the presence of a similar mechanism involving CED-3/ICE-like proteases in Drosophila. Here we determined whether CED-3 or ICE functions to induce programmed cell death in Drosophila. We have generated transformant lines in which ced-3 or Ice is ectopically expressed using the GAL4-UAS system. Expression of CED-3 and ICE can elicit cell death in Drosophila and the cell death was blocked by coexpressing the p35 gene which encodes a viral inhibitor of CED-3/ICE proteases. Results support the idea that the mechanism of programmed cell death controlled by CED-3/ICE is conserved among widely divergent animal species including Drosophila, and the system described provides a tool to dissect cell death mechanism downstream of CED-3/ICE proteases.     

Fas Induces Cytoplasmic Apoptotic Responses and Activation of the MKK7-JNK/SAPK and MKK6-p38 Pathways Independent of CPP32-like Proteases

IL-1β converting enzyme (ICE) family cysteine proteases are subdivided into three groups; ICE-, CPP32-, and Ich-1–like proteases. In Fas-induced apoptosis, activation of ICE-like proteases is followed by activation of CPP32-like proteases which is thought to be essential for execution of the cell death. It was recently reported that two subfamily members of the mitogen-activated protein kinase superfamily, JNK/SAPK and p38, are activated during Fas-induced apoptosis. Here, we have shown that MKK7, but not SEK1/ MKK4, is activated by Fas as an activator for JNK/ SAPK and that MKK6 is a major activator for p38 in Fas signaling. Then, to dissect various cellular responses induced by Fas, we used several peptide inhibitors for ICE family proteases in Fas-treated Jurkat cells and KB cells. While Z-VAD-FK which inhibited almost all the Fas-induced cellular responses blocked the activation of JNK/SAPK and p38, Ac-DEVD-CHO and Z-DEVD-FK, specific inhibitors for CPP32-like proteases, which inhibited the Fas-induced chromatin condensation and DNA fragmentation did not block the activation of JNK/SAPK and p38. Interestingly, these DEVD-type inhibitors did not block the Fas-induced morphological changes (cell shrinkage and surface blebbing), induction of Apo2.7 antigen, or the cell death (as assessed by the dye exclusion ability). These results suggest that the Fas-induced activation of the JNK/SAPK and p38 signaling pathways does not require CPP32-like proteases and that CPP32-like proteases, although essential for apoptotic nuclear events (such as chromatin condensation and DNA fragmentation), are not required for other apoptotic events in the cytoplasm or the cell death itself. Thus, the Fas signaling pathway diverges into multiple, separate processes, each of which may be responsible for part of the apoptotic cellular responses.     

The cytotoxic cell protease granzyme B initiates apoptosis in a cell-free system by proteolytic processing and activation of the ICE/CED-3 family protease, CPP32, via a novel two-step mechanism.

The major mechanism of cytotoxic lymphocyte killing involves the directed release of granules containing perforin and a number of proteases onto the target cell membrane. One of these proteases, granzyme B, has an unusual substrate site preference for Asp residues, a property that it shares with members of the emerging interleukin-1beta-converting enzyme (ICE)/CED-3 family of proteases. Here we show that granzyme B is sufficient to reproduce rapidly all of the key features of apoptosis, including the degradation of several protein substrates, when introduced into Jurkat cell-free extracts. Granzyme B-induced apoptosis was neutralized by a tetrapeptide inhibitor of the ICE/CED-3 family protease, CPP32, whereas a similar inhibitor of ICE had no effect. Granzyme B was found to convert CPP32, but not ICE, to its active form by cleaving between the large and small subunits of the CPP32 proenzyme, resulting in removal of the prodomain via an autocatalytic step. The cowpox virus protein CrmA, a known inhibitor of ICE family proteases as well as granzyme B, inhibited granzyme B-mediated CPP32 processing and apoptosis. These data demonstrate that CPP32 activation is a key event during apoptosis initiated by granzyme B.     

A Role for Caspases in Lens Fiber Differentiation

There is increasing evidence that programmed cell death (PCD) depends on a novel family of intracellular cysteine proteases, called caspases, that includes the Ced-3 protease in the nematode Caenorhabditis elegans and the interleukin-1β–converting enzyme (ICE)-like proteases in mammals. Some developing cells, including lens epithelial cells, erythroblasts, and keratinocytes, lose their nucleus and other organelles when they terminally differentiate, but it is not known whether the enzymatic machinery of PCD is involved in any of these normal differentiation events. We show here that at least one CPP32 (caspase-3)-like member of the caspase family becomes activated when rodent lens epithelial cells terminally differentiate into anucleate lens fibers in vivo, and that a peptide inhibitor of these proteases blocks the denucleation process in an in vitro model of lens fiber differentiation. These findings suggest that at least part of the machinery of PCD is involved in lens fiber differentiation.     

Caspase-3/CPP32-like activity is not sufficient to mediate apoptosis in an IL-2 dependent T cell line

CTLL cells undergo apoptosis when cultured in the absence of IL-2. The IL-1-converting-enzyme (ICE)/ caspase family has been implicated as an integral component of some forms of apoptosis. Numerous members of the caspase family have been identified, and it appears as if caspase-3/CPP32 plays a critical role. Previously we demonstrated that ICE/caspase-1 expression increases in CTLL cells during apoptosis; however, inhibition of ICE activity did not abrogate apoptotic death. The purpose of this report is to determine if other members of the caspase family are involved in T cell apoptosis induced by growth factor starvation. We show that cytosolic CPP32-like activity, as measured by the cleavage of DEVD-pNA and poly(ADP-ribose) polymerase (PARP), increases during apoptosis following growth factor deprivation. Cytosolic CPP32-like activity is inhibited in cells treated with the broad spectrum ICE family inhibitor boc-aspartyl(OMe)-fluoromethylketone (D-FMK) and by VAD-FMK and DEVD-FMK which have greater specificity for CPP32-like ICE homologs; however, only the broad spectrum ICE inhibitor D-FMK inhibited apoptosis. Our results suggest that apoptosis induced by growth factor deprivation involves the caspase family, but increased CPP32-like activity is not sufficient to mediate apoptosis induced by IL-2 starvation.