Sendai virus infection induces apoptosis through activation of caspase-8 (FLICE) and caspase-3 (CPP32)

Sendai virus (SV) infection and replication lead to a strong cytopathic effect with subsequent death of host cells. We now show that SV infection triggers an apoptotic program in target cells. Incubation of infected cells with the peptide inhibitor z-VAD-fmk abrogated SV-induced apoptosis, indicating that proteases of the caspase family were involved. Moreover, proteolytic activation of two distinct caspases, CPP32/caspase-3 and, as shown for the first time in virus-infected cells, FLICE/caspase-8, could be detected. So far, activation of FLICE/caspase-8 has been described in apoptosis triggered by death receptors, including CD95 and tumor necrosis factor (TNF)-R1. In contrast, we could show that SV-induced apoptosis did not require TNF or CD95 ligand. We further found that apoptosis of infected cells did not influence the maturation and budding of SV progeny. In conclusion, SV-induced cell injury is mediated by CD95- and TNF-R1-independent activation of caspases, leading to the death of host cells without impairment of the viral life cycle.     

Dimerization and autoprocessing of the Nedd2 (caspase-2) precursor requires both the prodomain and the carboxyl-terminal regions

Nedd2 (caspase-2) is a cysteine protease of the caspase family that has been demonstrated to play a role in the apoptotic pathway. The 51-kDa precursor of Nedd2 undergoes cleavage into two subunits following various apoptotic stimuli. In this study, we have investigated the dimerization of the Nedd2 precursor (pro-Nedd2) in Saccharomyces cerevisiae and its self-processing activity in vivo. We demonstrate that the expression of pro-Nedd2 in yeast cells results in processing of the precursor. A catalytically inactive pro-Nedd2 mutant dimerized in yeast, and the dimerization required both the prodomain and the carboxyl-terminal residues. Aspartate mutants that block the removal of the p14/p12 subunits, but not the wild-type Nedd2, were shown to dimerize in yeast cells, suggesting that dimerization occurs prior to processing. In vitro processing of pro-Nedd2 by recombinant active Nedd2 defined the aspartate residues that are crucial for processing to occur. Both the in vivo and in vitro processing of pro-Nedd2 directly correlated with its ability to induce cell death in transient overexpression experiments.     

Interleukin 1beta-converting enzyme (caspase-1) is overexpressed in adenocarcinoma of the pancreas

We investigated the expression of interleukin 1beta-converting enzyme (ICE; caspase-1) in human adenocarcinomas of the pancreas. Immunohistochemistry and Western blot analyses revealed an overexpression of ICE in 71 and 80% of tumor cells, respectively. Also, on a mRNA level, ICE mRNA was overexpressed in 45% of the cases, as compared to normal pancreatic tissue. Interestingly, the overexpression of ICE in tumor cells correlated significantly with the overexpression of cyclin D1, epidermal growth factor, and epidermal growth factor receptor (P < 0.0005, P < 0.05, and P < 0.002, respectively), which are involved in cell cycle progression and proliferation in human pancreatic carcinoma. This is the first report concerning ICE expression in human carcinomas; however, the exact mechanism underlying these close correlations warrant further research.     

A caspase-9 variant missing the catalytic site is an endogenous inhibitor of apoptosis

It is likely that endogenous inhibitors of the apical caspases such as caspase-9 exist to prevent undesirable activation of caspase cascades. A naturally occurring variant of caspase-9 named caspase-9S was cloned from human liver. Caspase-9S is missing most of the large subunit of caspase-9, including the catalytic site, but has the intact prodomain and small subunit. Caspase-9S did not show apoptotic activity in transfection analysis. Overexpression of caspase-9S inhibited apoptosis induced by caspase-9, indicating that caspase-9S is an endogenous dominant-negative of caspase-9. Moreover, caspase-9S inhibited apoptosis induced by tumor necrosis factor(TNF)-alpha, TNF factor-related apoptosis-inducing ligand (TRAIL), Bax, or Fas-associated death domain-containing protein (FADD) as well as the combination of Apaf-1 and caspase-9. In vitro binding assays demonstrated that caspase-9S binds to Apaf-1 and blocks the binding of caspase-9 to Apaf-1. Coexpression of caspase-9 and caspase-9S mRNA was identified in various cell lines. Thus, caspase-9S acts as a dominant-negative inhibitor of caspase-9 activation, at least in part, by blocking Apaf-1-caspase-9 interaction.     

Defects in regulation of apoptosis in caspase-2-deficient mice

During embryonic development, a large number of cells die naturally to shape the new organism. Members of the caspase family of proteases are essential intracellular death effectors. Herein, we generated caspase-2-deficient mice to evaluate the requirement for this enzyme in various paradigms of apoptosis. Excess numbers of germ cells were endowed in ovaries of mutant mice and the oocytes were found to be resistant to cell death following exposure to chemotherapeutic drugs. Apoptosis mediated by granzyme B and perforin was defective in caspase-2-deficient B lymphoblasts. In contrast, cell death of motor neurons during development was accelerated in caspase-2-deficient mice. In addition, caspase-2-deficient sympathetic neurons underwent apoptosis more effectively than wild-type neurons when deprived of NGF. Thus, caspase-2 acts both as a positive and negative cell death effector, depending upon cell lineage and stage of development.     

Caspase activation is required for nitric oxide-mediated, CD95(APO-1/Fas)-dependent and independent apoptosis in human neoplastic lymphoid cells

Nitric oxide (NO), an important effector molecule involved in immune regulation and host defense, was shown to induce apoptosis in lymphoma cells. In the present report the NO donor glycerol trinitrate was found to induce apoptosis in Jurkat cells that are sensitive to CD95-mediated kill. In contrast, a CD95-resistant Jurkat subclone showed substantial protection from apoptosis after exposure to NO. NO induced mRNA expression of CD95 (APO-1/Fas) and TRAIL/APO-2 ligands. Moreover, NO triggered apoptosis in freshly isolated human leukemic lymphocytes which were also sensitive to anti-CD95 treatment. The ability of NO to induce apoptosis was completely blocked by a broad-spectrum ICE (interleukin-1beta converting enzyme)-protease/caspase inhibitor and correlated with FLICE/caspase-8 activation. This activation was abrogated in some neoplastic lymphoid cells but not in others by the inhibitor of protein synthesis cycloheximide. Our results were confirmed using an in vitro experimental model of coculture of human lymphoid target cells with activated bovine endothelial cells generating NO as effectors. Furthermore, the inhibition of endogenous NO production with the inducible NO synthase inhibitor NG-monomethyl-L-arginine caused a complete abrogation of the apoptotic effect. Our data provide evidence that NO-induced apoptosis in human neoplastic lymphoid cells strictly requires activation of caspases, in particular FLICE, the most CD95 receptor-proximal caspase. Depending on the cell line tested this activation required or was independent of the CD95 receptor/ligand system.     

p53 is involved in but not required for ionizing radiation-induced caspase-3 activation and apoptosis in human lymphoblast cell lines

The caspase-3 has been shown to be involved in mediating apoptosis induced by different stimuli. However, it is still unclear whether p53 is required for the ionizing radiation (IR)-induced caspase-3 activation. In the present study, we examined IR-induced apoptosis in three closely related human lymphoblast cell lines that differ in p53 status. Irradiation of TK6 cells (wild-type p53) with 4 Gy gamma-rays resulted in rapid apoptosis, whereas the apoptotic response was delayed and reduced in WTK1 cells (mutant p53) and the TK6 derivative line expressing HPV16 E6 (abrogated p53). The differential apoptotic responses in these cell lines correlated with caspase-3 activation. IR induced an early as well as a late phase of caspase-3 activation in TK6 but only a delayed onset in WTK1 and TK6-E6-5E cells. The early phase of caspase-3 activation coincided with an elevation of p53 and bax protein levels. Pretreatment of all three cell lines with a caspases inhibitor z-VAD-FMK inhibited apoptosis. These results suggest that IR-induced apoptosis is mediated by a mechanism involving the caspase-3 cascade, which is shared by both p53-dependent and -independent pathways. The activation of caspase-3 by IR may thus engage at least two separate mechanisms, one through the regulation of the bcl-2 family members by p53, whereas the other yet-to-be-identified one involves neither p53 nor bax.     

Butyrate-induced differentiation of Caco-2 cells is associated with apoptosis and early induction of p21Waf1/Cip1 and p27Kip1

BACKGROUND: Intestinal mucosal turnover is a process of proliferation, differentiation, and apoptosis; the mechanisms remain largely undefined. The purpose of our study was to (1) assess the relationship between apoptosis and enterocyte differentiation and (2) determine whether the cell-cycle inhibitors, p21Waf1/Cip1 and p27Kip1, or the apoptosis inhibitors, Bcl-2 and Bcl-XL, may be involved. METHODS: Gut-derived Caco-2 cells were treated with sodium butyrate. Apoptosis was assessed by Hoechst stain, DNA laddering, and annexin V assay; differentiation was determined by alkaline phosphatase and sucrase activity. RNA and protein were analyzed for expression of p21Waf1/Cip1, p27Kip1, and members of the Bcl-2 family. RESULTS: Treatment of Caco-2 cells with sodium butyrate resulted in the concomitant induction of both differentiation (increased alkaline phosphatase and sucrase activity) and apoptosis. Increased levels of p21Waf1/Cip1 and p27Kip1 mRNA and protein were detected at 24 hours, occurring before apoptosis or differentiation; decreased mRNA levels of Bcl-2 and Bcl-XL were noted at 24 hours. CONCLUSIONS: Differentiation and apoptosis occurred simultaneously in Caco-2 cells, suggesting that apoptosis may be linked to enterocyte differentiation. The induction of p21Waf1/Cip1 and p27Kip1 and the down-regulation of Bcl-2 and Bcl-XL further suggest a link between the cell-cycle mechanisms regulating enterocyte differentiation and apoptosis.     

Synergistic activation of MAP kinase (ERK1/2) by erythropoietin and stem cell factor is essential for expanded erythropoiesis

Stem cell factor (SCF) and erythropoietin (EPO) work synergistically to support erythropoiesis, but the mechanism for this synergism is unknown. By using purified human erythroid colony-forming cells (ECFC), we have found that SCF and EPO synergistically activate MAP kinase (MAPK, ERK1/2), which correlates with the cell growth and thus may be responsible for the synergistic effects. Treatment of the cells with PD98059 and wortmannin, inhibitors of MEK and PI-3 kinase, respectively, inhibited the synergistic activation of MAPK and also the cell growth, further supporting this conclusion. Wortmannin only inhibits MAPK activation induced by EPO but not that by SCF, suggesting that SCF and EPO may activate MAPK through different pathways, which would facilitate synergy. Furthermore, EPO, but not SCF, led to activation of STAT5, whereas SCF and wortmannin had no effect on the EPO-induced STAT5 activation, suggesting that STAT5 is not involved in the synergistic action of SCF and EPO. Together, the data suggest that synergistic activation of MAPK by SCF and EPO is essential for expanded erythropoiesis.     

Calpain activation is upstream of caspases in radiation-induced apoptosis

The molecular events involved in apoptosis induced by ionizing radiation remain unresolved. In this paper we show that the cleavage of fodrin to a 150 kDa fragment is an early proteolytic event in radiation-induced apoptosis in the Burkitts' Lymphoma cell line BL30A and requires 100 microM zVAD-fmk for inhibition. Caspases-1, -3, -6 and -7 were shown to cleave fodrin to the 150 kDa fragment in vitro and all were inhibited by 10 microM zVAD-fmk. We also show that the in vitro cleavage of fodrin by calpain is inhibited by 100 microM zVAD-fmk as was the calpain-mediated hydrolysis of casein. We demonstrate that calpain is activated within 15 min after radiation exposure, concomitant with the cleavage of fodrin to the 150 kDa fragment whereas caspase-3 is activated at 2 h correlating with the cleavage of fodrin to the 120 kDa fragment. These results support a role for calpain in the early phases of the radiation-induced apoptosis pathway, upstream of the caspases.     

Delta9-tetrahydrocannabinol induces apoptosis in macrophages and lymphocytes: involvement of Bcl-2 and caspase-1

Apoptosis is programed cell death characterized by certain cellular changes and regulated by various gene products including Bcl-2 and caspase-1. The marijuana cannabinoid, Delta9tetrahydrocannabinol (THC), has been reported to suppress in culture the proliferation of splenocytes and increase the release of IL-1 from macrophages; however, the mechanisms of these effects remain unclear. Because cannabinoids have also been reported to induce apoptosis and because the release of IL-1 and suppression of lymphoproliferation are related to apoptosis, we tested for the induction of apoptosis by THC in murine immune cell cultures. Splenocytes cultured with Con A for up to 24 hr showed evidence of DNA fragmentation determined by gel electrophoresis, terminal deoxynucleotide transferase-mediated dUTP-fluorescein nick end labeling and 3H-thymidine labeling and THC (15-30 microM) treatment increased fragmentation under these conditions. Resident peritoneal macrophages cultured with lipopolysaccharides showed no obvious fragmentation unless they were also treated with THC. Time course studies examining DNA fragmentation and cell membrane integrity (assessed by dye exclusion) showed that fragmentation preceded membrane damage indicating that THC induced apoptosis rather than cell necrosis. In addition, THC treatment of splenocytes resulted in a decrease of Bcl-2 mRNA and protein as measured by Northern and Western blotting, respectively, and the drug induced apoptosis was blocked by the caspase inhibitor, Ac-Tyr-Val-Ala-L-aspartic acid aldehyde. These data suggest that THC treatment of cultured immune cells induces apoptosis through the regulation of Bcl-2 and caspase activity.     

Phosphatidylinositol 3,4,5-trisphosphate-dependent stimulation of phospholipase C-gamma2 is an early key event in FcgammaRIIA-mediated activation of human platelets

Platelets express a single class of Fcgamma receptor (FcgammaRIIA), which is involved in heparin-associated thrombocytopenia and possibly in inflammation. FcgammaRIIA cross-linking induces platelet secretion and aggregation, together with a number of cellular events such as tyrosine phosphorylation, activation of phospholipase C-gamma2 (PLC-gamma2), and calcium signaling. Here, we show that in response to FcgammaRIIA cross-linking, phosphatidylinositol (3,4, 5)-trisphosphate (PtdIns(3,4,5)P3) is rapidly produced, whereas phosphatidylinositol (3,4)-bisphosphate accumulates more slowly, demonstrating a marked activation of phosphoinositide 3-kinase (PI 3-kinase). Inhibition of PI 3-kinase by wortmannin or LY294002 abolished platelet secretion and aggregation, as well as phospholipase C (PLC) activation, indicating a role of this lipid kinase in the early phase of platelet activation. Inhibition of PLCgamma2 was not related to its tyrosine phosphorylation state, since wortmannin actually suppressed its dephosphorylation, which requires platelet aggregation and integrin alphaIIb/beta3 engagement. In contrast, the stable association of PLCgamma2 to the membrane/cytoskeleton interface observed at early stage of platelet activation was fully abolished upon inhibition of PI 3-kinase. In addition, PLCgamma2 was able to preferentially interact in vitro with PtdIns(3,4,5)P3. Finally, exogenous PtdIns(3,4,5)P3 restored PLC activation in permeabilized platelets treated with wortmannin. We propose that PI 3-kinase and its product PtdIns(3,4,5)P3 play a key role in the activation and adequate location of PLCgamma2 induced by FcgammaRIIA cross-linking.     

Events in apoptosis. Acidification is downstream of protease activation and BCL-2 protection

Cytoplasmic acidification is now recognized as a feature of apoptosis in a variety of systems. However, its relation to other events in the process of apoptosis is not yet characterized. In this work, we examined the effect of BCL-2 overexpression on acidification mediated by cycloheximide treatment or Fas ligation in Jurkat T-lymphoblasts. We find that BCL-2 overexpression attenuates cytoplasmic acidification and apoptosis detected by annexin V labeling. Acidification and phosphatidylserine externalization were found to occur concurrently. We also examined the requirement for protease activation for cytoplasmic acidification to occur and found that inhibition of interleukin-1beta converting enzyme/CED-3 family proteases (using carbobenzoxy-Val-Ala-Asp-fluoromethylketone, an inhibitor of these proteases) prevents acidification and apoptosis mediated by Fas ligation. These studies suggest that BCL-2 acts at a point upstream of acidification and that protease activation is also upstream of acidification.     

Intracellular IL-1beta is an inhibitor of Fas-mediated apoptosis

Fas-mediated apoptosis has been shown to be mediated by the IL-1beta converting enzyme (ICE) pathway. To determine the relationship between ICE and its substrate IL-1beta, we examined six human cell lines for susceptibility to Fas-mediated apoptosis and Fas induction of ICE-like activity. The human B lymphoblastoid cell line SKW6.4 and the human T lymphoma cell lines Jurkat, CEM-6, H-9, and MOLT4 were susceptible to Fas-mediated apoptosis, whereas the human promyelocytic leukemia cell line HL-60 was resistant to Fas-mediated apoptosis. ICE mRNA was highly expressed in SKW6.4, H-9, and HL-60 cells, and ICE-like activity increased during Fas-mediated apoptosis in SKW6.4 cells. In contrast, IL-1beta mRNA was highly expressed only in HL-60 cells. Acetyl-Tyr-Val-Ala-Asp-chloromethylketone, a tetrapeptidyl inhibitor of ICE, prevented Fas-mediated apoptosis strongly in SKW6.4 and H-9 cells but weakly or marginally in other cells. To examine whether intracellular IL-1beta is a proteolytic substrate or an endogenous competitive inhibitor against other substrates for Fas-ICE-mediated apoptosis in SKW6.4 cells, we established precursor IL-1beta transfectant clones using SKW6.4 cells. We demonstrated that stably transfected SKW6.4 cells expressing precursor IL-1beta, but not cells transfected with the empty vector, exhibited resistance to Fas-mediated apoptosis due to competitive inhibition of ICE-like activity, which was associated with increased cleavage of precursor IL-1beta to mature IL-1beta. These results suggest that Fas-mediated apoptosis is mediated by ICE cleavage of proteolytic substrates other than IL-1beta and that IL-1beta is an endogenous inhibitor of Fas-mediated apoptosis.     

Sp1 is required for the early response of alpha2(I) collagen to transforming growth factor-beta1

It is currently debated whether AP1 or Sp1 is the factor that mediates transforming growth factor beta1 (TGF-beta) stimulation of the human alpha2(I) collagen (COL1A2) gene by binding to an upstream promoter element (TbRE). The present study was designed to resolve this controversy by correlating expression of COL1A2, AP1, and Sp1 in the same cell line and under different experimental conditions. The results strongly indicate that Sp1 is required for the immediate early response of COL1A2 to TGF-beta and AP1 is not. The Sp1 inhibitor mithramycin blocked stimulation of alpha2(I) collagen mRNA accumulation by TGF-beta, whereas the AP1 inhibitor curcumin had no effect. Furthermore, antibodies against Jun-B and c-Jun failed to identify immunologically related proteins in the TbRE-bound complex, irrespective of whether they were purified from untreated or TGF-beta-treated cells. AP1 did bind to the TbRE probe in vitro, but only in the absence of the upstream Sp1 recognition sequence. Based on this finding and DNA transfection results, we conclude that the AP1 sequence of the TbRE represents a cryptic site used under experimental conditions that either eliminate the more favorable Sp1 binding site or force the balance toward the less probable. Finally, a combination of cell transfections and DNA-binding assays excluded that COL1A2 transactivation involves the retinoblastoma gene product (pRb), an activator of Sp1, the pRb-related protein p107, an inhibitor of Sp1, or the Sp1-related repressor, Sp3.     

Processing/activation of CPP32-like proteases is involved in transforming growth factor beta1-induced apoptosis in rat hepatocytes

Apoptosis induced in rat hepatocytes by transforming growth factor beta1 (TGF-beta1) was accompanied by the activation of interleukin-1beta converting enzyme (ICE)-like proteases. Cell lysates were isolated at various times after TGF-beta1 treatment and analyzed for ICE and CPP32-like activity, using N-acetyl-Tyr-Val-Ala-Asp-7-amino-4-methylcoumarin (Ac-YVAD.AMC) and benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (Z-DEVD.AFC), respectively. CPP32-like but not ICE protease activity increased in a time dependent manner and preceded the onset of apoptosis. Kinetic studies in cell lysates indicated that more than one CPP32-like protease was being activated. This was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting of TGF-beta1-treated cells, which showed limited processing of CPP32 as shown by the appearance of the catalytically active p17 subunit. Loss of pro-Mch3alpha was also observed but the catalytically active p19 subunit was not detected. Staurosporine, which induced a much greater level of hepatocyte apoptosis, produced a concomitant increase in CPP32/Mch3alpha processing as shown by the appearance of the p17/p19 subunits and the corresponding increase in CPP32-like protease activity. Apoptosis, CPP32/Mch3alpha processing and the increase in CPP32-like protease activity induced by TGF-beta1 and staurosporine were abolished in hepatocytes pretreated with Z-Asp-Glu-Val-Asp (OMe) fluoromethylketone (Z-DEVD.FMK) or Z-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK). These peptide analogues were potent inhibitors of CPP32-like protease activity in lysates. Pretreatment of hepatocytes with cycloheximide also blocked TGF-beta1-induced apoptosis and the increase in CPP32-like activity. Unlike Z-VAD.FMK and Z-DEVD.FMK, cycloheximide did not inhibit CPP32-like protease activity in cell lysates. Thus, cycloheximide may block apoptosis by inhibiting the synthesis of a protein, which is involved in the upstream events responsible for the activation of the CPP32-like protease activity. Our studies have identified two of the CPP32-like proteases, namely CPP32 and Mch3alpha, which are activated during the execution phase of hepatocyte apoptosis.     

Alteration of proteins regulating apoptosis, Bcl-2, Bcl-x, Bax, Bak, Bad, ICH-1 and CPP32, in Alzheimer's disease

Recently, apoptosis has been implicated in the selective neuronal loss of Alzheimer's disease (AD). Apoptosis is regulated by the B cell leukemia-2 gene product (Bcl-2) family (Bcl-2, Bcl-x, Bax, Bak and Bad) and the caspase family (ICH-1 and CPP32), with apoptosis being prevented by Bcl-2 and Bcl-x, and promoted by Bax, Bak, Bad, ICH-1 and CPP32. In the present study, we examined the levels of these proteins in the membranous and cytosolic fractions of temporal cortex in AD and control brain. In the membranous fraction, the levels of Bcl-2 alpha, Bcl-xL, Bcl-x beta, Bak and Bad were increased in AD. In the cytosolic fractions, the level of Bcl-x beta was increased, while Bcl-xL, Bax, Bak, and Bad and ICH-1L were unchanged. CPP32 was not detected in AD or control brain. These findings demonstrate a differential involvement of cell death-regulatory proteins in AD and suggest that Bak, Bad, Bcl-2 and Bcl-x are upregulated in AD brains.