Interleukin-1beta Converting Enzyme Research Articles

Identification of NRF2, a member of the NF-E2 family of transcription factors, as a substrate for caspase-3(-like) proteases.

Apoptosis is mediated by members of the interleukin-1beta converting enzyme (ICE) family of proteases (caspases), which are activated by diverse stimuli, although the downstream molecular targets of caspases are still poorly understood. Using the modified yeast two-hybrid system, which we recently established to clone genes for caspase substrates, we identified NRF2 as a novel caspase substrate. NRF2 is a member of the NF-E2 family of basic region leucine-zipper transcription factors and has been shown to induce phase II detoxifying enzymes through anti-oxidant response elements. NRF2 was cleaved at two sites by recombinant caspase-3 in vitro as well as in HeLa cells during TNFalpha-mediated apoptosis. Overexpression of the C-terminal cleavage fragment containing the DNA binding and leucine-zipper domains induced apoptosis in HeLa cells. These observations suggest that NRF2 might have some role in the induction of apoptosis after cleavage by caspases.

Overexpression of IL-1ra gene up-regulates interleukin-1beta converting enzyme (ICE) gene expression: possible mechanism underlying IL-1beta-resistance of cancer cells.

We investigated the interaction of endogenous interleukin (IL)-1β, IL-1ra, and interleukin-1β converting enzyme (ICE) in four human urological cancer cell lines, KU-19-19, KU-1, KU-2 and KU-19-20. Northern blot analysis showed that IL-1β gene was expressed in all cell lines. On the other hand, in KU-19-19 and KU-19-20, the gene expressions of both IL-1ra and ICE were suppressed. MTT assay revealed that IL-1β (10 ng ml−1) promoted cell growth in KU-19-19 and KU-19-20, while it inhibited in KU-1 and KU-2. An ICE inhibitor, Acetyl-Tyr-Val-Ala-Asp-CHO (YVAD-CHO) blocked IL-1β-induced growth inhibition in KU-1 and KU-2. Overexpression of the secretory type IL-1ra with adenovirus vector (AxIL-1ra) enhanced ICE gene expression, while exogenous IL-1ra (100 ng ml–1) did not enhance it. Furthermore, AxIL-1ra treatment promoted endogenous IL-1β secretion and induced significant growth inhibition and apoptotic cell death on KU-19-19 and KU-19-20. Treatment with either IL-1ra (100 ng ml−1), IL-1β antibody (100 μg ml−1), or YVAD-CHO blocked AxIL-1ra-induced cell death in KU-19-19 and KU-19-20. These results suggest that IL-1β-sensitivity depends on the level of ICE gene expression, which is regulated by the level of endogenous sIL-1ra expression. This is a first report on the intracellular function of sIL-1ra and these findings may provide key insights into the mechanism underlying the viability of cancer cells. © 1999 Cancer Research Campaign

Apoptotic pathways mobilized in microglia and neurones as a consequence of chromogranin A-induced microglial activation.

Senile plaques of Alzheimer's brain are characterized by activated microglia and immunoreactivity for the peptide chromogranin A. We have investigated the mechanisms by which chromogranin A activates microglia, producing modulators of neuronal survival. Primary cultures of rat brain-derived microglia display a reactive phenotype within 24 h of exposure to 10 nM chromogranin A, culminating in microglial death via apoptotic mechanisms mediated by interleukin-1beta converting enzyme. The signalling cascade initiated by chromogranin A triggers nitric oxide production followed by enhanced microglial glutamate release, inhibition of which prevents microglial death. The plasma membrane carrier inhibitor aminoadipate and the type II/III metabotropic glutamate receptor antagonist (RS)-alpha-methyl-4-sulphonophenylglycine are equally protective. A significant amount of the released glutamate occurs from bafilomycin-sensitive stores, suggesting a vesicular mode of release. Inhibition of this component of release affords significant microglial protection. Conditioned medium from activated microglia kills cerebellar granule cells by inducing caspase-3-dependent neuronal apoptosis. Brain-derived neurotrophic factor is partially neuroprotective, as are ionotropic glutamate receptor antagonists, and, when combined with boiling of conditioned medium, full protection is achieved; nitric oxide synthase inhibitors are ineffective.

Insulin-like growth factor I protects oligodendrocytes from tumor necrosis factor-alpha-induced injury.

Tumor necrosis factor-alpha (TNF-alpha) has been causally implicated in several demyelinating disorders, including multiple sclerosis. Because insulin-like growth factor I (IGF-I) is a potent stimulator of myelination, we investigated whether it can protect oligodendrocytes and myelination from TNF-alpha-induced damage using mouse glial cultures as a model. Compared with controls, TNF-alpha decreased oligodendrocyte number by approximately 40% and doubled the number of apoptotic oligodendrocytes and their precursors. Addition of Boc-aspartyl(Ome)-fluoromethyl ketone (BAF), an inhibitor of interleukin-1beta converting enzyme (ICE)/caspase proteases, blocked TNF-alpha-induced reductions in oligodendrocytes, indicating that the TNF-alpha-induced reduction in oligodendrocytes is, at least in part, due to apoptosis, and that ICE/caspases are one of TNF-alpha action mediators. Simultaneous addition of IGF-I to TNF-alpha-treated cultures negated these TNF-alpha effects nearly completely. Furthermore, IGF-I promoted oligodendrocyte precursor proliferation and/or differentiation in TNF-alpha-treated cultures. To analyze TNF-alpha and IGF-I actions on oligodendrocyte function, we measured the abundance of messenger RNAs (mRNAs) for two major myelin-specific proteins, myelin basic protein (MBP) and proteolipid protein (PLP). While TNF-alpha decreased MBP and PLP mRNA abundance by 5- to 6-fold, IGF-I abrogated TNF-alpha-induced reductions in a dose- and time-dependent manner. The changes in MBP and PLP mRNA abundance could not be completely explained by the changes in oligodendrocyte number, indicating that myelin protein gene expression is regulated by both TNF-alpha and IGF-I. These data support the hypothesis that TNF-alpha can mediate oligodendrocyte and myelin damage, and indicate that IGF-I protects oligodendrocytes from TNF-alpha insults by blocking TNF-alpha-induced apoptosis, and by promoting oligodendrocyte and precursor proliferation/differentiation and myelin protein gene expression.

Modulatory in vitro effects of interleukin-1 receptor antagonist (IL-1Ra) or antisense oligonucleotide to interleukin-1 beta converting enzyme (ICE) on acute myeloid leukaemia (AML) cell growth.

We investigated the effects of interleukin-1 receptor antagonist (IL-1Ra) on the spontaneous proliferation and AML colony forming unit (CFU-AML) formation of bone marrow and peripheral blood cells in 50 acute myeloid leukaemia (AML) patients. Exposure to IL-1Ra (10 micrograms/ml) caused either decreased, unaltered or increased AML cell proliferation, as well as of CFU-AML colony formation, depending on the individual patient, but the inhibitory effects were dominant. To evaluate the involvement of IL-1 beta converting enzyme (ICE) in the autonomous AML cell growth, the effects of an antisense oligonucleotide on ICE were examined in 19 of these patients. In a majority of patients, antisense ICE suppressed both AML cell proliferation and CFU-AML although a stimulatory effect was sometimes evident. The proportion of AML patients with suppression obtained by antisense ICE was higher than with IL-1Ra, suggesting the involvement of additional ICE-dependent cytokine(s) in AML cell growth besides IL-1. The presence of IL-1Ra or antisense ICE also suppressed the endogenous IL-1 beta production of AML cells, at both the level of pro-IL-1 beta and mature IL-1 beta. Although inhibition by IL-1Ra or antisense ICE on growth parameters of AML cells in vitro prevailed, indicating the importance of IL-1 activity in autonomous AML cell growth, stimulatory effects on the cells of some patients suggest that AML is a heterogenous disorder regarding IL-1 beta regulation.

Cytokine expression and induction of acinar cell apoptosis after pancreatic duct ligation in mice.

To clarify the role of cytokines and acinar cell apoptosis in the pathogenesis of acute pancreatitis, we investigated the expression of intrapancreatic cytokines and apoptosis-related molecules in mice after pancreatic duct ligation (PDL). From day 1 or 3 after PDL, the expression of interleukin-1alpha (IL-1alpha), IL-1beta, IL-1 receptor antagonist, IL-6, IL-10, and tumor necrosis factor (TNF-alpha) mRNA were up-regulated in the pancreas, suggesting that these cytokines may be involved in the development of pancreatitis after PDL. Acinar cell apoptosis was observed in the pancreas at rates of 0.13 +/- 0.03, 1.32 +/- 0.38, and 0.86 +/- 0.23% on days 1, 3, and 7 after PDL, respectively. Significant increases in intrapancreatic mRNA levels of TNF-alpha, Fas ligand (FasL), and IL-1beta-converting enzyme (ICE) were observed from day 3 after PDL with the appearance of acinar cell apoptosis. The serum amylase activity peaked on day 1 after PDL and gradually decreased on days 3 and 7 after PDL. These results suggest that acinar cell apoptosis induced after PDL may modulate the progression of acute pancreatitis by reducing the release of digestive enzymes and may therefore be a host defense mechanism, and that acinar cell apoptosis after PDL may be mediated by the TNF-alpha and/or Fas/FasL and ICE system.

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.

Enhancement of lipopolysaccharide-stimulated PGE2 and IL-1beta production in gingival fibroblast cells from old rats.

The effect of aging on gingival fibroblasts in response to bacterial infection was studied. Rat gingival fibroblast (rGF) cells were cultured from gingival tissue removed from young (6 weeks old) and old (20 months old) rats. Both types of rGF cells were challenged with lipopolysaccharide (LPS) from the periodontal pathogen Campylobacter rectus. The levels of prostaglandin E2 (PGE2) and interleukin 1beta (IL-1beta) released into the cultured medium were measured by a specific radioimmunoassay. LPS stimulated PGE2 and IL-1beta production in a dose-and time-dependent manner in rGF cells from both young and old rats was seen. Production of PGE2 and IL-1beta by rGF cells from the old rats was higher than those from the young in response to LPS. This greater ability from the older rGF cells to produce PGE2 and IL-1beta was due to higher mRNA levels of cyclooxygenase 2 and IL-1beta, respectively. In contrast, cyclooxygenase-1 and IL-1beta converting enzyme gene mRNA levels remained unchanged. Because LPS-stimulated PGE2 and IL-1beta production was enhanced by in vivo cellular aging, aging of GF may affect the severity of inflammation and bone resorption by producing a large amount of PGE2 and IL-1beta in response to bacterial infection.

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.

Impairment of TNF-Receptor-1 Signaling but not Fas Signaling Diminishes T-Cell Apoptosis in Myelin Oligodendrocyte Glycoprotein Peptide-Induced Chronic Demyelinating Autoimmune Encephalomyelitis in Mice

T-cell apoptosis in inflammatory demyelinating lesions of chronic myelin oligodendrocyte glycoprotein peptide35-55 induced autoimmune encephalomyelitis was studied in several different gene knockout mice as well as their wild-type counterparts. The gene deletions included tumor necrosis factor (TNF) alpha, lymphotoxin, TNF receptor 1 or 2, Fas-L, inducible nitric oxide synthase, perforin, and interleukin1beta-converting enzyme. Impairment of the TNF receptor 1 pathway led to a 50% reduction of T-cell apoptosis in the central nervous system lesions, whereas the other genetic deletions showed no significant effect. Our study thus identified the TNF receptor 1 signaling pathway as one mechanism responsible for the removal of T lymphocytes from inflammatory demyelinating lesions of the central nervous system.

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.

Apoptosis in batch cultures of Chinese hamster ovary cells.

One of the main problems in the culture of Chinese Hamster Ovary (CHO) cells continues to be the inability to maintain the viability of the cultures over an extended period of time. The rapid decline in viability at the end of the culture is exacerbated by the absence of serum. In trying to reduce the extent of death in these cultures, we first tried to determine the mode of death. We found that more than 80% of the cells in a standard serum-free batch culture of CHO cells in suspension died via apoptosis--as evidenced by condensed chromatin and the appearance of a characteristic DNA ladder. Furthermore, when protein synthesis was inhibited using cycloheximide, the cells underwent rapid apoptosis indicating that death proteins were present in greater abundance than survival proteins in our CHO cells. Cell lysate from CHO cells showed evidence of cysteine protease (caspase) activity. Caspases of the Interleukin-1-beta-Converting Enzyme (ICE) family, e.g., CPP32, Mch-1, etc., have been implicated in the apoptotic process. Surprisingly, a caspase peptide inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoro-methyl-ketone (z-VAD.fmk), was unable to substantially extend the life of a serum-free batch culture of CHO cells. In addition, z-VAD.fmk was only marginally able to extend viability in response to withdrawal of growth and survival factors, insulin and transferrin. In both these instances, z-VAD.fmk was able to prevent cleavage of caspase substrates, but not protect cells from death. However, we found that bcl-2 expression was able to significantly extend viabilities in CHO batch culture. Bcl-2 expression also substantially extended the viability of cultures in response to insulin and transferrin withdrawal. These results provide interesting insights into the pathways of death in a CHO cell.

Nitric oxide-releasing NSAIDs inhibit interleukin-1beta converting enzyme-like cysteine proteases and protect endothelial cells from apoptosis induced by TNFalpha.

Nitric oxide (NO)-releasing NSAIDs are a new class of NSAID derivatives with markedly reduced gastrointestinal toxicity. Although it has been demonstrated that NO-NSAIDs spare gastric mucosal blood flow, molecular determinants involved in this effect are unknown. To investigate the effect of aspirin, naproxen and flurbiprofen, and their NO-derivatives, on gastric apoptosis and endothelial cell damage induced by tumour necrosis factor-alpha (TNFalpha). In other systems, TNFalpha-induced apoptosis is mediated by caspases, a growing family of cysteine proteases similar to the IL-1beta converting enzyme (ICE), and so we have investigated whether NO-NSAIDs modulate ICE-like endopeptidases. Rats were treated orally with aspirin, naproxen and flurbiprofen, or their NO-releasing derivatives in equimolar doses, and were killed 3 h later to assess mucosal damage and caspase activity. Endothelial cells (HUVECs) were obtained from human umbilical cord by enzymatic digestion. Caspase 1 and 3 activities were measured by a fluorimetric assay using selective peptides as substrates and inhibitors. Apoptosis was quantified by ELISA specific for histone-associated DNA fragments and by the terminal transferase nick-end translation method (TUNEL). In vivo NSAID administration caused a time-dependent increase in gastric mucosal damage and caspase activity. NCX-4016, NO-naproxen and NO-flurbiprofen did not cause any mucosal damage and prevented cysteine protease activation. NSAIDs and NO-NSAIDs stimulated TNFalpha release. Exposure to TNFalpha resulted in a time- and concentration-dependent HUVEC apoptosis, an effect that was prevented by pretreating the cells with NCX-4016, NO-naproxen, NO-flurbiprofen, SNP or Z-VAD.FMK, a pan-caspase inhibitor. The activation of ICE-like cysteine proteases was required to mediate TNFalpha-induced apoptosis of HUVECs. Exogenous NO donors inhibited TNFalpha-induced cysteine protease activation. Inhibition of caspase activity was due to S-nitrosylation of ICE/CPP32-like proteases. NO-NSAIDs prevented IL-1beta release from endotoxin-stimulated macrophages. NO-releasing NSAIDs are a new class of non-peptide caspase inhibitors. Inhibition of ICE-like cysteine proteases prevents endothelial cell damage induced by pro-inflammatory agents and might contribute to the gastro-protective effects of NO-NSAIDs.

Recruitment of apoptotic cysteine proteases (caspases) in influenza virus-induced cell death.

Influenza virus infection induces apoptosis in cultured cells with an augmented expression of Fas (APO-1/CD95). Caspases, a family of cysteine proteases structurally related to interleukin-1-beta-converting enzyme (ICE), play crucial roles in apoptosis induced by various stimuli, including Fas. However, activation of the caspase-cascade seems to be different in various pathways of apoptotic stimuli. We therefore examined the involvement of caspases in influenza virus-induced apoptosis using caspase inhibitors. We found that z-VAD-fmk and z-IETD-fmk effectively inhibited virus-induced apoptosis, whereas Ac-DEVD-CHO and Ac-YVAD-CHO showed partial and little effect on virus-induced cell death, respectively. Consistently, caspase-3-like activity, but not caspase-1-like activity, was increased in the virus-infected cells. The transfection of plasmids encoding viral inhibitors of caspase (v-FLIP or crmA) into HeLa cells inhibited apoptosis by virus infection. The peptide inhibitors of caspases used in this study did not inhibit viral replication. We conclude that influenza virus infection activates some caspases, and that this activation may be downstream of viral replication.

Nitric oxide as a bifunctional regulator of apoptosis.

It was inevitable that important relationships between two of the most intensely studied topics in biomedical research, apoptosis and nitric oxide (NO), would become apparent. Apoptosis is essential to normal development as well as physiological cell turnover. Although apoptosis in excess can manifest as tissue damage, a failure to undergo apoptosis constitutes pathological cellular overgrowth. It is now evident that NO and its reaction products can either promote or prevent apoptosis in a multitude of settings. The ubiquitous distribution of the NO synthases and the remarkable diffusibility and diverse chemical reactivity of NO in biological systems make this molecule unique among the regulators of apoptosis. Understanding the factors that govern the consequences of NO exposure on cell viability and identifying the conditions in which NO regulation of apoptosis contribute to pathology are topics of considerable interest and potential importance. In this article, we will review the recent observations on NO as a regulator of apoptosis. Apoptosis, or programmed cell death, is distinguished from lytic or necrotic cell death by specific biochemical and structural events (see recent review in Reference 11 ). Apoptogenic signals trigger cell-specific signaling pathways, including protease activation, followed by the appearance of morphological changes characteristic of cells undergoing apoptosis, including condensation of nuclei and cytoplasm, blebbing of the cytoplasmic membranes, and finally fragmentation into apoptotic bodies that are phagocytosed by neighboring cells. The elucidation of the signaling events in apoptosis is occurring at a rapid pace and includes the identification of the key roles of cysteine proteases (known as caspases), Bcl-2 family members, and mitochondria. Caspases, the mammalian counterpart of ced-3 in Caenorhabditis elegans , are a family of cysteine proteases now known to contain at least 14 homologs. Ectopic expression of any of the caspase family proteases can cause apoptosis; however, not all caspase family …

Induction of pituitary cytokine transcripts by peripheral lipopolysaccharide.

Systemically administered lipopolysaccharide (LPS) elicits profound changes in pituitary hormone secretion. Pro-inflammatory cytokines have been proposed as mediators of these responses. In this study, we used in-situ hybridization histochemistry to investigate LPS-induced cytokine gene expression in the rat pituitary. After i.p. or i.v. injection of various doses of LPS, mRNA for the immediate-early gene IkappaBu (an inhibitor of NF-kappaB, a transcription factor that regulates the expression of many pro-inflammatory cytokines) was induced in the anterior lobe as early as 0.5 h. The induced IkappaBalpha mRNA expression peaked at 1 h. In the posterior lobe, IkappaBalpha mRNA was first induced at 0.5 h and peaked at 2 h. A similar spatiotemporal pattern of interleukin-1b (IL-1) mRNA induction was observed. In addition, at 2 h after injection, TNFalpha, IL-1beta converting enzyme (ICE), and IL-1 receptor antagonist (IL-1RA) mRNAs were induced in both anterior and posterior lobes. Type 1 IL-1 receptor (IL-1R1) mRNA was constitutively expressed in the pituitary, and its expression level did not change after the LPS injection. Interestingly, the mRNA coding for glial fibrillary acidic protein (GFAP), an astrocyte marker, was selectively induced in the posterior lobe at 2 h after LPS injection, suggesting that LPS affects pituicyte function. Together, these results suggest that LPS acts directly on the pituitary to rapidly induce cytokine expression. Locally synthesized cytokines may activate cytokine receptor bearing cells to modulate the endocrine activities of the pituitary.

Transgenic mice expressing a dominant negative mutant interleukin-1beta converting enzyme show resistance to MPTP neurotoxicity.

Increasing evidence implicates apoptosis as a major mechanism of cell death in neurodegenerative diseases. Recent evidence has demonstrated that chronic administration of MPTP can lead to apoptotic cell death. In the present study we examined whether transgenic mice expressing a dominant negative inhibitor of interleukin-1beta convertase enzyme (ICE) are resistant to MPTP induced neurotoxicity. MPTP resulted in a significant depletion of dopamine, DOPAC and HVA in littermate control mice which were completely inhibited in the mutant interleukin-1beta converting enzyme mice. There was also significant protection against MPTP-induced depletion of tyrosine hydroxylase-immunoreactive neurons. There was no alteration in MPTP uptake or metabolism. These results provide further evidence that apoptotic cell death as well as ICE may play an important role in the neurotoxicity of MPTP.

Bcl-2 interferes with the execution phase, but not upstream events, in glucocorticoid-induced leukemia apoptosis.

Due to their growth arrest- and apoptosis-inducing ability, glucocorticoids (GC) are widely used in the therapy of various lymphoid malignancies. Cell death is associated with activation of members of the interleukin-1beta-converting enzyme (ICE) protease/caspase family and, is presumably prevented by the anti-apoptotic protein Bcl-2. To further address the role of Bcl-2 in GC-mediated cytotoxicity, we generated subclones of the GC-sensitive human T-cell acute lymphoblastic leukemia line CCRF-CEM, in which transgenic Bcl-2 expression is regulated by tetracycline. Up to about 48 h, exogenous Bcl-2 almost completely protected these cells from apoptosis, digestion of poly-ADP ribose polymerase (PARP) and generation of Asp-Glu-Val-Asp cleaving (DEVDase) activity. However, when the cells were cultured for another 24 h in the continuous presence of GC, they underwent massive apoptosis that was associated with DEVDase activity and PARP cleavage. Bcl-2 did not markedly affect GC-mediated growth arrest, thereby separating the anti-proliferative from the apoptosis-inducing effect of GC. Moreover, Bcl-2 did not prevent the dramatic reduction in the levels of several mRNAs observed during GC treatment, including the transgenic Bcl-2 mRNA. Thus, Bcl-2 can be placed upstream of effector caspase activation, but downstream of other GC-regulated events, such as growth arrest and the potentially critical repression of steady state levels of multiple mRNA.

Expression of IL 1betaconverting enzyme in 5-FU induced apoptosis in esophageal carcinoma cells.

AIM:To study the role of interleukin 1beta converting enzyme(ICE)in antitumor drug induced apoptosis in tumor cells.METHODS:Morphological changes in human esophageal carcinoma Eca-109 cells after treated with 5-fluorouracil (5-FU) were observed under light and electron microscope. Expression of ICE in the tumor cells exposed to 5-FU was examined by the immunocytochemical method.RESULTS:The cells treated with 5-FU displayed disappearance of nucleoli, chromatin gathering under nuclear envelope, karyorrhexis, budding and the formation of apoptotic bodies.The expression of ICE was negative in control cells, and 5-FU could induce the ICE expression in Eca-109 cells undergoing apoptosis. The number and the staining intensity of positive cells increased with the extension of action time.CONCLUSION: 5-FU may induce apoptosis in human esophageal carcinoma Eca-109 cells; ICE gene may be involved in the regulation of 5-FU induced apoptosis; and ICE protein may mediate apoptosis induced by 5-FU.

Peptide based interleukin-1 beta converting enzyme (ICE) inhibitors: synthesis, structure activity relationships and crystallographic study of the ICE-inhibitor complex.

Based on the X-ray structure of the complex of Ac-Tyr-Val-Ala-Asp-H (L-709049) and interleukin-1 beta converting enzyme (ICE), we synthesized compounds which were derived from 2-NapCO-Val-Pro-Asp-CH2OPh (1) to obtain a potent inhibitor in the cell assay. Among these compounds, (3S)-N-methanesulfonyl-3-[[1-[N-(2-naphthoyl)-L-valyl]-L-prolyl]amino]- 4-oxobutanamide (27c) showed high potency not only in the enzyme assay but also cell assay with IC50 values of 38 nM and 0.23 microM, respectively. Compound 27c, with a c log P value of 1.76, had a more hydrophilic character compared with 1. Compound 27c also dose dependently inhibited LPS-primed ATP-induced IL-1 beta release in mice. The crystal structure of the complex of compound 27c and ICE revealed that compound 27c had further interactions with ICE in the naphthoyl group at the P4 position and in the methyl group of the methanesulfonamidecarbonyl group at the P1 position, compared with L-709049. To our knowledge, compound 27c is the first example that shows a strong inhibitory activity without the carboxyl group at the P1 position.