Interleukin-1beta Converting Enzyme Family Research Articles

Lactacystin, a specific inhibitor of the proteasome, induces apoptosis and activates caspase-3 in cultured cerebellar granule cells.

The multicatalytic protease complex or proteasome is a fundamental nonlysosomal tool that the cell uses to process or degrade proteins at a fast rate through the ubiquitin and ATP-dependent proteolytic pathway. Examples of these important proteins include the tumor suppressor protein p53, various cyclins, the cyclin-dependent kinase inhibitor p27, NFkappaB, IkappaB, c-fos, and c-jun. The activation of proteolytic enzymes, including certain cystein-proteases of the ced-3/ICE (interleukin-1beta-converting enzyme) family, is a characteristic feature of the apoptotic program. However, the role of the multicatalytic protease complex in apoptosis is not well known. In order to obtain further information regarding the participation of the ubiquitin-mediated pathway in the decision of the cell to execute the cell death program, we have used a specific inhibitor of the multicatalytic protease complex, lactacystin, in cultured cerebellar granule cells. Cells were obtained from the cerebellum of 6- to 8-day-old Wistar rats and cultured in Neurobasal medium supplemented with B-27. Addition of lactacystin to the cultures induced apoptosis of the granule cells in a time-dependent fashion. The morphological changes produced by the proteasome inhibitor included nuclear condensation and DNA fragmentation measured by the diphenylamine test, as well as a positive labeling by the TUNEL (terminal deoxynucleotidyltransferase mediated-dUTP nick end labeling) assay, all of them typical features of apoptosis. Concomitant with apoptosis, there were changes in the expression of the ubiquitin mRNA, a progressive depletion in the free ubiquitin pool, and an increase in the high molecular weight ubiquitin-protein conjugates. Caspase-3, a member of the ced-3/ICE family of cystein-proteases, showed a marked increase in activity in the lactacystin-treated cells. In flow cytometry studies, the amount of cells in the S phase of the cell cycle was smaller in the lactacystin-treated cells than in controls, suggesting that apoptosis could be due, in part, to an alteration of the cell cycle.

Inhibition of interleukin-1beta converting enzyme family proteases (caspases) reduces cold injury-induced brain trauma and DNA fragmentation in mice.

The authors examined the effect of z-VAD.FMK, an inhibitor that blocks caspase family proteases, on cold injury-induced brain trauma, in which apoptosis as well as necrosis is assumed to play a role. A vehicle alone or with z-VAD.FMK was administered into the cerebral ventricles of mice 15 minutes before and 24 and 48 hours after cold injury. At 24 hours after cold injury, infarction volumes in the z-VAD.FMK-treated animals were significantly smaller than infarction volumes in the vehicle-treated animals, and were further decreased at 72 hours (0.92 +/- 1.80 mm3, z-VAD.FMK-treated animals; 7.46 +/- 3.53 mm3, vehicle-treated animals; mean +/- SD, n = 7 to 8). The amount of DNA fragmentation was significantly decreased in the z-VAD.FMK-treated animals compared with the vehicle-treated animals, as shown by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling staining and DNA gel electrophoresis. By Western blot analysis, both the proform and activated form of interleukin-1beta converting enzyme (caspase 1) were detected in the control brain, and the activated form showed moderate reduction after cold injury-induced brain trauma. These results indicate that caspase inhibitors could reduce cold injury-induced brain trauma by preventing neuronal cell death by DNA damage. The caspase family proteases appear to contribute to the mechanisms of cell death in cold injury-induced brain trauma and to provide therapeutic targets for traumatic brain injury.

Partial Purification and Characterization of Two Distinct Types of Caspases from Human Epidermis

Recent observations demonstrated that interleukin-1beta converting enzyme family proteases, now referred to as caspase family, play central roles in apoptosis, or programmed cell death. In this study, we tried to isolate and characterize epidermal caspases. By DEAE-Sephacel anion-exchange chromatography, human cornified cell extract showed two caspase-like fractions (F-I and F-II) with different substrate specificities. These were further purified by Sephacryl S-200, Mono Q ion exchange and Superose 6 gel chromatography. F-I showed a molecular weight of 30 kDa and specifically hydrolyzed acetyl-Asp-Glu-Val-Asp-methylcoumarinamide, a fluorogenic substrate for caspase-3 (CPP32) with a Km value of 13.8 microM. F-I generated a characteristic 85 kDa fragment from poly(ADP-ribose) polymerase. Inhibitor susceptibility of F-I was very similar to that of caspase-3, further confirming the caspase-3-like properties of F-I. In contrast, the molecular weight of F-II was estimated to be 110 kDa, which was much higher than the other caspases. F-II equally hydrolyzed acetyl-Asp-Glu-Val-Asp-methylcoumarinamide, and acetyl-Tyr-Val-Ala-Asp-methylcoumarinamide, caspase-1 (interleukin-1beta converting enzyme)-specific substrate, and was inhibited by acetyl-Tyr-Val-Ala-Asp-aldehyde and acetyl-Tyr-Val-Ala-Asp-aldehyde. Affinity labeling using biotinylated YVAD-cmk demonstrated several positive bands ranging from 25 to 35 kDa, supporting the hypothesis that F-II is a complex of multiple caspases. Reverse transcriptase-polymerase chain reaction analysis demonstrated that among known caspases tested, caspase-1, -2, -3, -4, and -7 were expressed in cultured human keratinocytes. These results suggest that multiple caspases are synthesized in human keratinocytes and are involved in terminal differentiation.

ICE family proteases in inflammation and apoptosis.

The interleukin-1 beta converting enzyme (ICE) was first identified as a unique cysteine protease that processes the inactive precursor of the pro-inflammatory cytokine IL-1 beta to its mature active form. Subsequent revelation that a C. elegans cell death gene ced-3 bears sequence homology to ICE has led to rapid identification of at least nine other members of this gene family in humans, some of which are involved in apoptosis. Analyses of ICE-deficient mice generated by gene targeting technology reveal that this enzyme is important in maturation of several cytokines. The ICE deficient mice are resistant in several models of localized and systemic inflammation. ICE itself, however, is not required for Fas-mediated apoptosis, a physiological process for elimination of activated lymphocytes. Selective inhibitors of ICE would be novel therapeutic agents for treatment of diseases where excess inflammation contributes to pathological processes.

Lysophosphatidic acid induces necrosis and apoptosis in hippocampal neurons.

A diverse body of evidence indicates a role for the lipid biomediator lysophosphatidic acid (LPA) in the CNS. This study identifies and characterizes the induction of neuronal death by LPA. Treatment of cultured hippocampal neurons from embryonic rat brains with 50 microM LPA resulted in neuronal necrosis, as determined morphologically and by the release of lactate dehydrogenase. A concentration of LPA as low as 10 microM led to the release of lactate dehydrogenase. In contrast, treatment of neurons with 0.1 or 1.0 microM LPA resulted in apoptosis, as determined by chromatin condensation. In addition, neuronal death induced by 1 microM LPA was characterized as apoptotic on the basis of terminal dUTP nick end-labeling (TUNEL) staining, externalization of phosphatidylserine, and protection against chromatin condensation, TUNEL staining, and phosphatidylserine externalization by treatment with N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a broad-spectrum inhibitor of caspases, i.e., members of the interleukin-1beta converting enzyme family. Studies with antagonists of ionotropic glutamate receptors did not indicate a significant role for these receptors in apoptosis induced by 1 microM LPA. LPA (1 microM) also induced a decrease in mitochondrial membrane potential. Moreover, pretreatment of neurons with cyclosporin A protected against the LPA-induced decrease in mitochondrial membrane potential and neuronal apoptosis. Thus, LPA, at pathophysiological levels, can induce neuronal apoptosis and could thereby participate in neurodegenerative disorders.

Caenorhabditis elegans CED-9 protein is a bifunctional cell-death inhibitor.

The Caenorhabditis elegans gene ced-9 prevents cells from undergoing programmed cell death and encodes a protein similar to the mammalian cell-death inhibitor Bcl-2. We show here that the CED-9 protein is a substrate for the C. elegans cell-death protease CED-3, which is a member of a family of cysteine proteases first defined by CED-3 and human interleukin-1beta converting enzyme (ICE). CED-9 can be cleaved by CED-3 at two sites near its amino terminus, and the presence of at least one of these sites is important for complete protection by CED-9 against cell death. Cleavage of CED-9 by CED-3 generates a carboxy-terminal product that resembles Bcl-2 in sequence and in function. Bcl-2 and the baculovirus protein p35, which inhibits cell death in different species through a mechanism that depends on the presence of its cleavage site for the CED-3/ICE family of proteases, inhibit cell death additively in C. elegans. Our results indicate that CED-9 prevents programmed cell death in C. elegans through two distinct mechanisms: first, CED-9 may, by analogy with p35, directly inhibit the CED-3 protease by an interaction involving the CED-3 cleavage sites in CED-9; second, CED-9 may directly or indirectly inhibit CED-3 by means of a protective mechanism similar to that used by mammalian Bcl-2.

Induction of in vitro nuclear apoptosis activity coincides with the production of 50 kDa cytosolic protein.

Human monocytic leukemia U937 cells undergo apoptosis when cells are treated with the anticancer drug etoposide. To study the mechanism of drug-induced apoptosis, we used an in vitro apoptosis system with cytosol from etoposide-treated U937 cells. The cytosol from apoptotic U937 cells showed activity to induce morphologic changes and oligonucleosomal DNA fragmentation in isolated nuclei in vitro; both are typical features of apoptosis. We generated monoclonal antibodies to the proteins in the etoposide-treated U937 cytosol. We found that a 50 kDa protein, recognized by SN-1 monoclonal antibody, appeared in the cytosol of U937 cells, in accordance with its cell-free apoptosis activity. Z-Asp, an inhibitor of interleukin-1beta converting enzyme (ICE) family proteases, inhibited the appearance of the 50 kDa protein and the emergence of the cell-free apoptosis activity in the etoposide-treated U937 cytosol. These results indicate that the 50 kDa protein is produced by the activation of ICE family protease during apoptosis and suggest some roles of the protein in the development of apoptosis.

Impaired apoptotic death signaling in inflammatory lung neutrophils is associated with decreased expression of interleukin-1beta converting enzyme family proteases (caspases)

Background. Fas and tumor necrosis factor receptor 1 (TNFR1) are membrane proteins that signal for apoptotic cell death by downstream activation of proteins of the interleukin-1β converting enzyme (ICE) family. Spontaneous apoptosis is delayed in neutrophils activated by transmigration into an inflammatory focus. In this study we evaluated the effects of transmigration on Fas and TNFR1-induced apoptosis and apoptotic gene expression. Methods. Sprague-Dawley rats were killed 4 hours after intratracheal challenge with 500 μg lipopolysaccharide (LPS). Neutrophils isolated from the systemic circulation (circulation) or bronchoalveolar lavage fluid (lung) were incubated with or without an agonistic antibody to Fas (clone CH-11, 100 ng/ml) or TNF (10 ng/ml) for 24 hours. Apoptosis and Fas expression were assessed by flow cytometry. Expression of the antiapoptotic protein Bcl-2 and proapoptotic proteins ICE and CPP32 were measured by Western blots. Results. Neutrophils transmigrating into the lung in response to LPS showed delayed apoptosis compared with circulating neutrophils and failed to undergo apoptosis in response to anti-Fas antibody or TNF-α. Fas expression was unaltered; however, TNFR1 expression was reduced. Bcl-2 was not detected in either group; both the pro- and active forms of ICE and active CPP32 were significantly decreased in lung neutrophils. The specific ICE inhibitor, YVAD-CMK, partially blocked the increased rates of apoptosis resulting from engagement of Fas or TNFR1. Conclusions. Neutrophil transmigration retards apoptosis through engagement of the death receptors Fas and TNFR1. This refractory state is associated with reduced levels of proapoptotic proteins. Blunted responsiveness to physiologic apoptotic stimuli prolongs neutrophil functional survival during acute inflammation and may contribute to the tissue injury associated with acute respiratory distress syndrome.

Activation of Caspase-2 in Apoptosis

Members of the CED-3/interleukin-1beta-converting enzyme (ICE) protease (caspase) family are synthesized as proforms, which are proteolytically cleaved and activated during apoptosis. We report here that caspase-2 (ICH-1/NEDD-2), a member of the ICE family, is activated during apoptosis by another ICE member, a caspase-3 (CPP32)-like protease(s). When cells are induced to undergo apoptosis, endogenous caspase-2 is first cleaved into three fragments of 32-33 kDa and 14 kDa, which are then further processed into 18- and 12-kDa active subunits. Up to 50 microM N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), a caspase-3-preferred peptide inhibitor, inhibits caspase-2 activation and DNA fragmentation in vivo, but does not prevent loss of mitochondrial function, while higher concentrations of DEVD-CHO (>50 microM) inhibit both. In comparison, although the activity of caspase-3 is very sensitive to the inhibition of DEVD-CHO (<50 nM), inhibition of caspase-3 activation as marked by processing of the proform requires more than 100 microM DEVD-CHO. Our results suggest that the first cleavage of caspase-2 is accomplished by a caspase-3-like activity, and other ICE-like proteases less sensitive to DEVD-CHO may be responsible for activation of caspase-3 and loss of mitochondrial function.

Suppression of interleukin-1beta converting enzyme (ICE)-induced apoptosis by SV40 large T antigen.

The Interleukin-1beta converting enzyme (ICE) family of proteins, homologs of the C elegans cell death gene product CED-3, play important roles in controlling vertebrate programmed cell death. Because inhibition of apoptosis may be an essential step in tumorigenesis, we investigated the interaction of the simian virus 40 large T antigen (T ag) with the ICE family. COS-1 cells which were transformed by the simian virus 40 do not die when transfected with expression constructs of Ice or Ich-1(L). We found that expression of T ag alone significantly prevents the ICE-induced apoptosis. p53, but not pRb or p107, antagonizes the effect of T ag on the suppression of ICE-induced cell death, but not on ICH-1(L)-mediated cell death. Thus, wild type p53 may potentiate ICE-induced apoptosis. Expression of a temperature sensitive mutant p53Val(135) sensitizes COS-1 cells to apoptosis induced by ICE at permissive but not at non-permissive temperature. While induction of bax, p21(WAF1/CIP), or cyclin D1 gene expression is observed in the COS-1 p53Val(135) cells at the permissive temperature, overexpression of bax, but not p21(WAF1/CIP) or cyclin D1, potentiates ICE-induced COS-1 cell death. Taken together, these results suggest that T ag may modulate the cells' susceptibility to death by suppressing activity of the ICE family through inhibiting p53.

Actin cleavage by CPP-32/apopain during the development of apoptosis.

Interleukin-1beta-converting enzyme (ICE)/ced-3 family proteases play key roles in apoptosis. However, cellular substrates for ICE family proteases involved in apoptosis are not well understood. We previously showed that actin is cleaved in vitro by an ICE family protease, distinct from ICE itself, which is activated during VP-16-induced apoptosis. In this report, we demonstrate that the actin-cleaving ICE-family protease in the apoptotic cell extract is the activated CPP-32/apopain. CPP-32 effectively cleaves actin protein to 15 kDa and 31 kDa fragments. Studies with an antibody raised against Gly-Gln-Val-Ile-Thr peptide, the N-terminal sequence of the cleaved 15 kDa actin fragment, showed that actin is also cleaved in vivo during the development of apoptosis. Moreover, Benzyloxycarbonyl-Glu-Val-Asp-CH2OC(O)-2,6,-dichlorobenzene (Z-EVD-CH2-DCB), a selective inhibitor of CPP-32(-like) protease, efficiently inhibited the cleavage of actin and the apoptosis of VP-16-treated U937 cells. Our present results indicate that actin is the substrate of CPP-32/apopain(-like) protease both in vitro and in vivo and suggest the role of actin in the control of cell growth and apoptosis.

Interaction of CED-4 with CED-3 and CED-9: a molecular framework for cell death.

Previous genetic studies of the nematode Caenorhabditis elegans identified three important components of the cell death machinery. CED-3 and CED-4 function to kill cells, whereas CED-9 protects cells from death. Here CED-9 and its mammalian homolog Bcl-xL (a member of the Bcl-2 family of cell death regulators) were both found to interact with and inhibit the function of CED-4. In addition, analysis revealed that CED-4 can simultaneously interact with CED-3 and its mammalian counterparts interleukin-1beta-converting enzyme (ICE) and FLICE. Thus, CED-4 plays a central role in the cell death pathway, biochemically linking CED-9 and the Bcl-2 family to CED-3 and the ICE family of pro-apoptotic cysteine proteases.

Fas activation of the p38 mitogen-activated protein kinase signalling pathway requires ICE/CED-3 family proteases.

The Fas receptor mediates a signalling cascade resulting in programmed cell death (apoptosis) within hours of receptor cross-linking. In this study Fas activated the stress-responsive mitogen-activated protein kinases, p38 and JNK, within 2 h in Jurkat T lymphocytes but not the mitogen-responsive kinase ERK1 or pp70S6k. Fas activation of p38 correlated temporally with the onset of apoptosis, and transfection of constitutively active MKK3 (glu), an upstream regulator of p38, potentiated Fas-induced cell death, suggesting a potential involvement of the MKK3/p38 activation pathway in Fas-mediated apoptosis. Fas has been shown to require ICE (interleukin-1 beta-converting enzyme) family proteases to induce apoptosis from studies utilizing the cowpox ICE inhibitor protein CrmA, the synthetic tetrapeptide ICE inhibitor YVAD-CMK, and the tripeptide pan-ICE inhibitor Z-VAD-FMK. In this study, crmA antagonized, and YVAD-CMK and Z-VAD-FMK completely inhibited, Fas activation of p38 kinase activity, demonstrating that Fas-dependent activation of p38 requires ICE/CED-3 family members and conversely that the MKK3/p38 activation cascade represents a downstream target for the ICE/CED-3 family proteases. Intriguingly, p38 activation by sorbitol and etoposide was resistant to YVAD-CMK and Z-VAD-FMK, suggesting the existence of an additional mechanism(s) of p38 regulation. The ICE/CED-3 family-p38 regulatory relationship described in the current work indicates that in addition to the previously described destructive cleavage of substrates such as poly(ADP ribose) polymerase, lamins, and topoisomerase, the apoptotic cysteine proteases also function to regulate stress kinase signalling cascades.

Increased bax and interleukin-1beta-converting enzyme messenger ribonucleic acid levels coincide with apoptosis in the bovine corpus luteum during structural regression.

Recent reports have demonstrated that prostaglandin F2alpha-induced generation of reactive oxygen species or their intermediates inhibits progesterone synthesis and may also serve as a trigger for apoptosis in the corpus luteum (CL). BCL-2, an inhibitor of apoptosis in a wide variety of cell types, has been reported to prevent oxidative stress-induced cell death. Thus, the present studies were conducted to determine whether levels of mRNA encoding BCL-2 and related members of this gene family (BAX and BCL-Xshort, which induce apoptosis; BCL-Xlong, a BCL-2 homologue that prevents apoptosis) differed in functional (Day 21 of pregnancy) versus regressed (Day 21 of the estrous cycle) CL in the bovine ovary. Levels of mRNAs encoding p53, a transcriptional regulator of the bcl-2 and bax genes, and interleukin-1beta-converting enzyme (ICE), a protein recently implicated in the induction of apoptosis whose expression may be enhanced by oxidative stress, were also assessed. Partial cDNA clones encoding bovine bax, bcl-x, p53, and Ice were isolated using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique with total RNA prepared from functional or regressed CL. A bovine bcl-2 cDNA could not be isolated from luteal tissue RNA despite the use of several primer pairs for amplification. Total RNA was then extracted from functional or regressed CL and analyzed by Northern blot analysis. The occurrence of apoptosis in regressed CL, as evidenced by the presence of internucleosomal DNA cleavage, was associated with a significant increase in both bax and Ice mRNA levels as compared with levels of bax and Ice expression in functional CL (p < 0.05, n = 3). There were no significant differences in bcl-x or p53 mRNA levels in functional versus regressed CL. Analysis of bcl-x mRNA by RT-PCR revealed that the long form was the primary, if not only, mRNA expressed in functional and regressed bovine luteal tissue. On the basis of data that increased expression of bax is associated with, and may be required for, apoptosis in ovarian granulosa cells and germ cells, we propose that BAX may play a similar role in apoptosis induction during luteal regression. Moreover, the increased Ice mRNA levels in regressed CL provides the first evidence that the ICE family of death proteases may be involved in luteolysis.

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.

Involvement of ICE family proteases in apoptosis induced by reoxygenation of hypoxic hepatocytes.

Cell death due to reoxygenation after hypoxia was characterized in primary cultured hepatocytes. Fluorescence and electron microscopic analyses of reoxygenated hepatocytes revealed morphological characteristics of apoptosis, including chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies. Few necrotic hepatocytes, defined by loss of plasma membrane integrity, mitochondrial swelling, and formation of large vacuoles, were observed. Activation of interleukin-1 beta-converting enzyme (ICE)-like and CPP32/Yama-like proteases, which are known to drive apoptosis, was observed during reoxygenation, and addition of their respective inhibitors inhibited the induction of apoptosis, indicating the involvement of ICE family proteases in apoptosis by reoxygenation. Production of oxygen radicals was enhanced by reoxygenation of hypoxic cells, and reoxygenation-induced apoptosis was inhibited by oxygen radical scavengers, suggesting a role for reactive oxygen species as a triggering factor in cell death. Electrophoretic analysis revealed the presence of 50-kb DNA fragments but not oligonucleosomal DNA fragments in reoxygenation-induced apoptotic hepatocytes.

Functional Characterization of the Prodomain of Interleukin1β-converting Enzyme

Interleukin-1beta-converting enzyme (ICE) has been identified as the main protease responsible for maturation of the prodomain of interleukin-1beta. Recently, it was shown to belong to a larger gene family, members of which play an important role in programmed cell death. A common feature of the ICE family proteases is the presence of a prodomain that has been hypothesized to keep the enzyme in an inactive form. Expression analysis in yeast revealed autocatalytic degradation of p45ICE, but not of p30ICE lacking a prodomain. We further demonstrate that p45ICE, in which the critical cysteine has been mutated, is still able to dimerize in vivo. Dimerization requires the prodomain and occurs prior to autoprocessing. These results provide evidence for a regulatory role of the prodomain of ICE.

Induction of apoptosis by baculovirus transactivator IE1.

Apoptosis is induced upon infection of SF-21 cells by mutants of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) lacking a functional p35 gene which encodes a stoichiometric inhibitor of members of the interleukin-1beta converting enzyme family of cysteine proteases (N.J. Bump et al, Science 269:1885-1888, 1995; R.J. Clem, M. Fechheimer, and L.K. Miller, Science 254:1388-1390, 1991). We found that transfection of SF-21 cells with the AcMNPV ie-1 gene was sufficient to induce apoptosis, which was characterized by fragmentation of cellular DNA into oligonucleosomal fragments and apoptotic body formation. No signs of apoptosis were observed in Trichoplusia ni TN-368 cells transfected with ie-1, a result which is consistent with the observation that p35 mutants of AcMNPV do not induce apoptosis in this cell line. Cotransfection of SF-21 cells with p35 blocked ie-1-induced apoptosis, indicating that expression of ie-1 activates apoptosis through a p35-inhibitable cysteine protease pathway. Cotransfection with Cp-iap, an active member of another family of antiapoptotic inhibitors of apoptosis (iaps), also inhibited IE1-induced apoptosis. Thus, ie-1 may participate in inducing apoptosis in AcMNPV-infected cells, although the dependence of induction on DNA replication suggests that ie-1 is not the direct apoptotic signal during infection. The ie-1 gene product, IE1, is known to be a potent transactivator of baculovirus gene expression that interacts with specific palindromic sequences which can act as transcriptional enhancers and as origins of DNA replication in transient assays.

Proteases in apoptosis

The interleukin-1 beta-converting enzyme (ICE)-like family proteases have recently been identified as key enzymes in apoptotic cell death. Among these proteases one can identify specific activities which may be involved in cytokine production or in resident protein cleavage. Several factors influence the constitutive apoptotic mechanism and may provide insight into the role of protease(s) in apoptosis. Although it appears that ICE family members play a most important role in promoting apoptotic cell death, evidence has been advanced that other proteases are also involved in sequential or parallel steps of apoptosis. Activation of a particular protease can lead to processing molecules either of the same or different proteases, leading to an activation of a protease cascade. Here we attempt to summarize the current thinking concerning these proteases and their involvement in apoptosis.

Calcium-dependent, Interleukin 1β-converting Enzyme Inhibitor-insensitive Degradation of Lamin B1 and DNA Fragmentation in Isolated Thymocyte Nuclei

Recent work suggests that the proteolytic degradation of the nuclear lamins is a common event in apoptosis, although the nature of the proteases involved is still not clear. Our previous work showed that the degradation of lamin B1 in glucocorticoid-treated thymocytes occurs via a Ca2+-sensitive mechanism and that exogenous Ca2+ promotes lamin degradation in isolated thymocyte nuclei from untreated cells. Here we demonstrate that peptide-based inhibitors of the interleukin 1beta-converting enzyme family of cysteine proteases (Tyr-Val-Ala-Asp fluoromethyl ketone) and of the nuclear scaffold multicatalytic proteinase (Ala-Pro-Phe chloromethyl ketone) block the degradation of lamin B1 to a 21-kDa fragment in thymocytes treated with glucocorticoid, the Ca2+-mobilizing agent thapsigargin, or antibodies to the T cell receptor. However, among a panel of inhibitors specific for several different proteases implicated in apoptosis, only tosylphenylalanyl chloromethyl ketone and the nuclear scaffold protease inhibitor block lamin degradation, histone H1 cleavage, and DNA fragmentation in isolated thymocyte nuclei incubated with Ca2+. Overexpression of human BCL-2 in nuclei by stable transfection resulted in an inhibition of Ca2+-stimulated lamin degradation and DNA fragmentation, suggesting that endogenous nuclear BCL-2 regulates activation of the nuclear scaffold protease. The results demonstrate the existence of an alternative pathway of lamin degradation and DNA fragmentation mediated by a resident Ca2+-stimulated nuclear protease that is not directly dependent upon activation of the interleukin 1beta-converting enzyme family of cell death regulators.