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PubMed2006

Loss of caspase-9 reveals its essential role for caspase-2 activation and mitochondrial membrane depolarization.

Caspase-9 plays an important role in apoptosis induced by genotoxic stress. Irradiation and anticancer drugs trigger mitochondrial outer membrane permeabilization, resulting in cytochrome c release and caspase-9 activation. Two highly contentious issues, however, remain: It is unclear whether the loss of the mitochondrial membrane potential DeltaPsi(M) contributes to cytochrome c release and whether caspases are involved. Moreover, an unresolved question is whether caspase-2 functions as an initiator in genotoxic stress-induced apoptosis. In the present study, we have identified a mutant Jurkat T-cell line that is deficient in caspase-9 and resistant to apoptosis. Anticancer drugs, however, could activate proapoptotic Bcl-2 proteins and cytochrome c release, similarly as in caspase-9-proficient cells. Interestingly, despite these alterations, the cells retained DeltaPsi(M). Furthermore, processing and enzyme activity of caspase-2 were not observed in the absence of caspase-9. Reconstitution of caspase-9 expression restored not only apoptosis but also the loss of DeltaPsi(M) and caspase-2 activity. Thus, we provide genetic evidence that caspase-9 is indispensable for drug-induced apoptosis in cancer cells. Moreover, loss of DeltaPsi(M) can be functionally separated from cytochrome c release. Caspase-9 is not only required for DeltaPsi(M) loss but also for caspase-2 activation, suggesting that these two events are downstream of the apoptosome.

Antineoplastic Agents↗

PubMed2003

Z-FA-fmk inhibits effector caspases but not initiator caspases 8 and 10, and demonstrates that novel anticancer retinoid-related molecules induce apoptosis via the intrinsic pathway.

Synthetic retinoid-related molecules (RRMs) have been described that show strong antiproliferative activity and induce apoptosis in cancer cells. These RRMs induce caspase activity independently of the retinoid receptors in Jurkat T cells. We observed that the inhibitor of cathepsins B and L Z-FA-fmk blocks the induction of DEVDase activity, DNA fragmentation, and externalization of phosphatidylserine by selective RRMs. Z-FA-fmk can inhibit caspase activity in vitro and selectively inhibits recombinant effector caspases 2, -3, -6, and -7. In contrast, purified initiator caspases 8 and 10 are not affected, whereas the apoptosome-associated caspase 9 is only partially inhibited by Z-FA-fmk in vitro. These data correlate with the covalent binding of biotinylated Z-FA-fmk to the active large subunit of effector caspases. This selective targeting of effector caspases is also observed in Jurkat cells and has been used to demonstrate that RRMs induce apoptosis through the mitochondrial pathway and activate caspase 8 in a Z-FA-fmk-sensitive manner. Thus, Z-FA-fmk fails to inhibit Fas-mediated activation of caspase 8, but completely inhibits RRM-induced processing of caspase 8. Z-FA-fmk does not prevent the autoproteolytic cleavage of caspase 9 in Jurkat cells and partially inhibits the processing and full maturation of effector caspases induced by the RRMs. Moreover, Z-VAD-fmk and Z-FA-fmk have no effect on the release of cytochrome c induced by the RRMs. Other cathepsin inhibitors elicit no effect on RRM-induced apoptosis in Jurkat cells, suggesting that caspases are the major effectors of RRM action.

Amino Acid Chloromethyl Ketones↗

PubMed2004

Caspase-8 and caspase-3 small interfering RNA decreases ischemia/reperfusion injury to the liver in mice.

BACKGROUND: Ischemia/reperfusion injury (I/R injury) of the liver remains a significant problem during liver surgery and transplantation. I/R injury is associated with liver apoptosis, which is mediated by death receptors such as Fas and tumor necrosis factor alpha (TNF-alpha), and/or mitochondrial dysfunction induced by cellular stress. Caspase-8 is presumed to be the apex of the death-mediated apoptosis pathway, whereas caspase-3 belongs to the "effector" proteases in the apoptosis cascade. Synthetic small interfering RNAs (siRNAs) specifically suppress gene expression by RNA interference. Therefore, we evaluated the therapeutic efficacy of caspase-8 and caspase-3 siRNA in a murine model of liver I/R injury. METHODS: In C57BL/6 mice, 45% or 70% of the liver mass was clamped for 90 minutes. For survival analysis, total hepatic ischemia was induced for 45 minutes. In vivo delivery of siRNA was performed via the portal vein by high-volume injection (0.5 nmol of siRNA in 1 mL containing 10% lipiodol) 60 minutes before ischemia. As a control, animals received either vehicle or non-sense siRNA (siRNA-scrambled). RESULTS: Liver uptake of siRNA was analyzed in transgenic mice who express beta-galactosidase (beta-gal) (C57BL/6J-TgN(MTn-LacZ)204Bri) after administration of siRNA-LacZ. A 3- to 4-fold decrease in beta-gal activity was accomplished at 0.5 nmol. No significant change in beta-gal activity was demonstrated in mice receiving non-sense siRNA. Immunohistochemical studies found that 60% of the liver cells efficiently took up siRNA. Significant reduction in serum aspartate transaminase was found in animals treated with siRNA caspase-8 or caspase-3 compared with siRNA-scrambed or vehicle-treated controls. More than a 60% reduction in caspase-8 and caspase-3 gene expression and activities was accomplished after siRNA administration. Animals treated with siRNA presented lower infiltration of polymorphonuclear leukocytes and better preservation of the liver architecture compared with controls. All of the control mice subjected to total liver ischemia died within 5 days. In contrast, 30% of the animals given siRNA caspase-8 and 50% of those treated with siRNA caspase-3 survived indefinitely (>30 days). CONCLUSIONS: Small interfering RNA targeted to caspase-8 and caspase-3 provided significant protection against I/R injury to the liver. This approach could be therapeutic in liver transplantation and other conditions associated with I/R injury to the liver.

Animals↗

PubMed2005

In situ trapping of activated initiator caspases reveals a role for caspase-2 in heat shock-induced apoptosis.

Activation of 'initiator' (or 'apical') caspases-2, -8 or -9 (refs 1-3) is crucial for induction of apoptosis. These caspases function to activate executioner caspapses that, in turn, orchestrate apoptotic cell death. Here, we show that a cell-permeable, biotinylated pan-caspase inhibitor (bVAD-fmk) both inhibited and 'trapped' the apical caspase activated when apoptosis was triggered. As expected, only caspase-8 was trapped in response to ligation of death receptors, whereas only caspase-9 was trapped in response to a variety of other apoptosis-inducing agents. Caspase-2 was exclusively activated in heat shock-induced apoptosis. This activation of caspase-2 was also observed in cells protected from heat-shock-induced apoptosis by Bcl-2 or Bcl-xL. Reduced sensitivity to heat-shock-induced death was observed in caspase-2(-/-) cells. Furthermore, cells lacking the adapter molecule RAIDD failed to activate caspase-2 after heat shock treatment and showed resistance to apoptosis in this setting. This approach unambiguously identifies the apical caspase activated in response to apoptotic stimuli, and establishes caspase-2 as a proximal mediator of heat shock-induced apoptosis.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Zinc-mediated regulation of caspases activity: dose-dependent inhibition or activation of caspase-3 in the human Burkitt lymphoma B cells (Ramos).

Divalent cations, including Zinc and Manganese ions, are important modulators of cell activation. We investigated the ability of these two divalent cations to modulate apoptosis in human Burkitt lymphoma B cells line (Ramos). We found that Zinc (from 10 to 50 microM) inhibited Manganese-induced caspase-3 activation and apoptosis of Ramos cells. Higher concentration of Zinc (50 to 100 microM) did not prevent Manganese-mediated apoptosis but rather increased cell death among Ramos cells. This Zinc-mediated cell death was associated with apoptotic features such as cell shrinkage, the presence of phosphatidylserine residues on the outer leaflet of the cells, chromatin condensation, DNA fragmentation and decrease of mitochondrial transmembrane potential. Zinc-mediated apoptosis was associated with caspase-9 and caspase-3 activation as revealed by the appearance of active p35 fragment of caspase-9 and p19 and p17 of caspase-3 as well as in vivo cleavage of PARP and of a cell-permeable fluorogenic caspase-3 substrate (Phiphilux-G(1)D(2)). Both Zinc-mediated apoptosis and caspase-3 activation were prevented by the cell-permeable, broad-spectrum inhibitor of caspases (zVAD-fmk) or overexpression of bcl-2. In addition, we show that Zinc-induced loss of transmembrane mitochondrial potential is a caspase-independent event, since it is not modified by the presence of zVAD-fmk, which is inhibited by overexpression of bcl-2. These results indicate that depending on its concentration, Zinc can exert opposite effects on caspase-3 activation and apoptosis in human B lymphoma cells: concentrations below 50 microM inhibit caspase-3 activation and apoptosis whereas higher concentrations of Zinc activate a death pathway associated with apoptotic-like features and caspase-3 activation.

Amino Acid Chloromethyl Ketones↗

PubMed2006

Prevention of renal ischemic injury by silencing the expression of renal caspase 3 and caspase 8.

BACKGROUND: Apoptotic pathways mediated by caspases play a critical role in renal ischemia-reperfusion injury (IRI). Downregulation of the caspase cascade, using small interfering RNA (siRNA) to silence the expression of caspase 3 and caspase 8, may have substantial therapeutic potential for limiting renal injury. METHODS: IRI was induced in mice by clamping of the renal vein and artery for 25 or 35 min at 37 degrees C. Caspase 3 and caspase 8 (caspase 3/8) siRNA was administrated by hydrodynamic injection. Quantitative polymerase chain reaction (PCR) and immunohistochemistry were used to analyze the gene silencing efficacy, and the therapeutic effects of siRNA were evaluated by renal function analysis, histological examination, and overall survival of mice suffering from IRI. RESULTS: In this study, we have shown, using quantitative PCR, that IRI is associated with increased levels of renal caspase 3/8 mRNA. Mice treated with caspase 3/8 siRNA showed a significant down-regulation in kidney expression of caspase 3/8 at both, transcriptional and protein levels. Kidney function in IRI was protected by siRNA therapy, as levels of blood urea nitrogen and creatinine were significantly reduced in mice treated with siRNA. Histological examination demonstrated that tissue injury caused by IRI was significantly reduced as a result of caspase 3/8 siRNA treatment. Furthermore, survival data showed that more than 70% of mice in siRNA-treated groups survived until the end of the eight-day observation period. CONCLUSION: Herein, we have demonstrated the therapeutic potential of using siRNA to knock down the expression of caspases and prevent acute renal injury.

Animals↗

PubMed2004

Association of active caspase 8 with the mitochondrial membrane during apoptosis: potential roles in cleaving BAP31 and caspase 3 and mediating mitochondrion-endoplasmic reticulum cross talk in etoposide-induced cell death.

It was recently demonstrated that during apoptosis, active caspase 9 and caspase 3 rapidly accumulate in the mitochondrion-enriched membrane fraction (D. Chandra and D. G. Tang, J. Biol. Chem.278:17408-17420, 2003). We now show that active caspase 8 also becomes associated with the membranes in apoptosis caused by multiple stimuli. In MDA-MB231 breast cancer cells treated with etoposide (VP16), active caspase 8 is detected only in the membrane fraction, which contains both mitochondria and endoplasmic reticulum (ER), as revealed by fractionation studies. Immunofluorescence microscopy, however, shows that procaspase 8 and active caspase 8 predominantly colocalize with the mitochondria. Biochemical analysis demonstrates that both procaspase 8 and active caspase 8 are localized mainly on the outer mitochondrial membrane (OMM) as integral proteins. Functional analyses with dominant-negative mutants, small interfering RNAs, peptide inhibitors, and Fas-associated death domain (FADD)- and caspase 8-deficient Jurkat T cells establish that the mitochondrion-localized active caspase 8 results mainly from the FADD-dependent and tumor necrosis factor receptor-associated death domain-dependent mechanisms and that caspase 8 activation plays a causal role in VP16-induced caspase 3 activation and cell death. Finally, we present evidence that the OMM-localized active caspase 8 can activate cytosolic caspase 3 and ER-localized BAP31. Cleavage of BAP31 leads to the generation of ER- localized, proapoptotic BAP20, which may mediate mitochondrion-ER cross talk through a Ca(2+)-dependent mechanism.

Antineoplastic Agents, Phytogenic↗

PubMed2000

Identification of a caspase-2 isoform that behaves as an endogenous inhibitor of the caspase cascade.

Procaspase-2 is one of the aspartate-specific cysteine proteases that are activated in response to various apoptotic stimuli. Two isoforms of human procaspase-2 have been described initially. Overexpression of the long isoform (caspase-2L) promotes cell death whereas the short isoform (caspase-2S) antagonizes some apoptotic pathways. In the present study, we identified two additional CASP-2 mRNAs, designated CASP-2L-Pro and CASP-2s-Pro. The proteins encoded by these isoforms corresponded to the prodomain of procaspase-2L and -2S, in which the last alpha-helix of their caspase recruitment domains was deleted. Caspase-2L-Pro mRNA and protein were detected in a series of human tissues and cell lines. Yeast 2-hybrid assays and immunoprecipitation studies indicated that caspase-2L-Pro can interact with procaspase-2L and the adaptor protein RAIDD/CRADD, but not with FADD/MORT1 or APAF-1 adaptor proteins. The addition of recombinant caspase-2L-Pro negatively interfered with cytochrome c/dATP-mediated activation of the caspase cascade in a cell-free system. In transient expression studies of human B lymphoma Namalwa cells, overexpression of caspase-2L-Pro weakly induced apoptosis, which was prevented by a D83A/E87A double mutation. In stable selected CASP-2L-Pro-transfected Namalwa cells, overexpression of caspase-2L-Pro delayed apoptotic DNA fragmentation induced by death receptor agonists (anti-Fas antibodies, tumor necrosis factor-alpha) and DNA topoisomerase I- (camptothecin) and II- (etoposide) inhibitors, and prevented etoposide-induced activation of the caspase cascade. These inhibitory effects were not observed in stable transfected cells expressing the D83A/E87A double mutant. Altogether, these data indicated that the caspase-2L-Pro isoform functions as an endogenous apoptosis inhibitory protein that antagonizes caspase activation and cell death.

Amino Acid Sequence↗

PubMed2000

Caspase-3 is activated following axotomy of neonatal facial motoneurons and caspase-3 gene deletion delays axotomy-induced cell death in rodents.

In this report, we examined the possible functions of the cell death protease, caspase-3, in the axotomy-induced apoptosis of facial motoneurons in newborn rodents. Using in situ hybridization and Western blot, we found higher levels of caspase-3 mRNA and pro-caspase-3 protein expression in motoneurons of neonatal and 2-week-old rats than adult rats. Following facial motoneuron axotomy, caspase-3 mRNA and protein expression increased in motoneurons of both neonatal and adult rats. However, using an antibody directed to the activated form of the caspase-3 protease, we found that catalytically active caspase-3 was present only in axotomized neonatal motoneurons. As motoneurons in neonatal but not adult rodents are susceptible to axotomy-induced apoptosis, we hypothesized that caspase-3 may play a role in their demise. To determine the necessity of caspase-3 activation in axotomy-induced apoptosis, we counted the number of surviving motoneurons at 4 and 7 days following axotomy in wild type mice and caspase-3 gene-deleted mice. There were nearly three times more surviving motoneurons in caspase-3 gene-deleted mice than in wild type mice at both 4 days (mean 1074 vs. 464, P<0.005) and 7 days (mean 469 vs. 190, P<0.005) following injury, indicating a slower rate of death. Examination of the dying motoneurons using TUNEL staining (for fragmented DNA) and bisbenzimide staining (for nuclear morphology) revealed incomplete nuclear condensation in caspase-3-deficient motoneurons. These results demonstrate that caspase-3 activation plays important roles in the rapid demise of axotomized neonatal motoneurons.

Age Factors↗

PubMed1999

A cloning method to identify caspases and their regulators in yeast: identification of Drosophila IAP1 as an inhibitor of the Drosophila caspase DCP-1.

Site-specific proteases play critical roles in regulating many cellular processes. To identify novel site-specific proteases, their regulators, and substrates, we have designed a general reporter system in Saccharomyces cerevisiae in which a transcription factor is linked to the intracellular domain of a transmembrane protein by protease cleavage sites. Here, we explore the efficacy of this approach by using caspases, a family of aspartate-specific cysteine proteases, as a model. Introduction of an active caspase into cells that express a caspase-cleavable reporter results in the release of the transcription factor from the membrane and subsequent activation of a nuclear reporter. We show that known caspases activate the reporter, that an activator of caspase activity stimulates reporter activation in the presence of an otherwise inactive caspase, and that caspase inhibitors suppress caspase-dependent reporter activity. We also find that, although low or moderate levels of active caspase expression do not compromise yeast cell growth, higher level expression leads to lethality. We have exploited this observation to isolate clones from a Drosophila embryo cDNA library that block DCP-1 caspase-dependent yeast cell death. Among these clones, we identified the known cell death inhibitor DIAP1. We showed, by using bacterially synthesized proteins, that glutathione S-transferase-DIAP1 directly inhibits DCP-1 caspase activity but that it had minimal effect on the activity of a predomainless version of a second Drosophila caspase, drICE.

Animals↗

PubMed2000

Regulation of apoptosis by phosphatidylinositol 4,5-bisphosphate inhibition of caspases, and caspase inactivation of phosphatidylinositol phosphate 5-kinases.

Phosphoinositides such as phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate promote cell survival and protect against apoptosis by activating Akt/PKB, which phosphorylates components of the apoptotic machinery. We now report that another phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2) is a direct inhibitor of initiator caspases 8 and 9, and their common effector caspase 3. PIP2 inhibited procaspase 9 processing in cell extracts and in a reconstituted procaspase 9/Apaf1 apoptosome system. It inhibited purified caspase 3 and 8 activity, at physiologically attainable PIP2 levels in mixed lipid vesicles. Caspase 3 binding to PIP2 was confirmed by cosedimentation with mixed lipid vesicles. Overexpression of phosphatidylinositol phosphate 5-kinase alpha (PIP5KIalpha), which synthesizes PIP2, suppressed apoptosis, whereas a kinase-deficient mutant did not. Protection by the wild-type PIP5KIalpha was accompanied by decreases in the generation of activated caspases and of caspase 3-cleaved PARP. Protection was not mediated through PIP3 or Akt activation. An anti-apoptotic role for PIP(2) is further substantiated by our finding that PIP5KIalpha was cleaved by caspase 3 during apoptosis, and cleavage inactivated PIP5KIalpha in vitro. Mutation of the P(4) position (D279A) of the PIP5KIalpha caspase 3 cleavage consensus prevented cleavage in vitro, and during apoptosis in vivo. Significantly, the caspase 3-resistant PIP5KIalpha mutant was more effective in suppressing apoptosis than the wild-type kinase. These results show that PIP2 is a direct regulator of apical and effector caspases in the death receptor and mitochondrial pathways, and that PIP5KIalpha inactivation contributes to the progression of apoptosis. This novel feedforward amplification mechanism for maintaining the balance between life and death of a cell works through phosphoinositide regulation of caspases and caspase regulation of phosphoinositide synthesis.

Apoptosis↗

PubMed1999

Accumulation of caspase-3 messenger ribonucleic acid and induction of caspase activity in the ovine corpus luteum following prostaglandin F2alpha treatment in vivo.

Caspase-3, a vertebrate homologue of the protein encoded by the Caenorhabditis elegans cell death gene, ced-3, induces apoptosis when overexpressed in eukaryotic cells. Since apoptosis occurs during corpus luteum (CL) regression in many species, including the ewe, these studies were conducted to 1) isolate a cDNA encoding ovine caspase-3, 2) measure steady state amounts of caspase-3 mRNA in the CL during luteolysis induced by prostaglandin F2alpha (PGF2alpha) and during the time of maternal recognition of pregnancy, and 3) measure changes in caspase activity during PGF2alpha-initiated luteal regression. Oligonucleotide primers corresponding to a human caspase-3 cDNA sequence were combined with total RNA from ovine CL in a reverse transcription-polymerase chain reaction-based procedure to amplify a 640-base pair partial cDNA with a nucleotide sequence 86% and 81% identical to the human and rat caspase-3 cDNAs, respectively. CL were collected from ewes at 0, 12, or 24 h after treatment with PGF2alpha on Day 10 of the estrous cycle and from nonpregnant and pregnant ewes on Day 12 or Day 14 of the cycle. Northern blot analysis of total cellular RNA from ovine CL and a radiolabeled ovine caspase-3 cRNA probe indicated the presence of a single mRNA transcript of approximately 2.5 kilobases. Levels of caspase-3 mRNA were approximately 3-fold higher (p < 0.05) in CL at 12 h and 24 h after PGF2alpha in comparison to those levels measured in matched CL from untreated ewes. There were no differences (p > 0.05) in amounts of caspase-3 mRNA in CL on Day 12 or Day 14 of the estrous cycle compared to Day 12 or Day 14 of pregnancy, respectively. Caspase activity in CL (measured by the ability of CL lysates to cleave an artificial caspase substrate) was also significantly (p < 0.05) increased in CL collected after treatment with PGF2alpha compared to CL collected from nontreated ewes. We conclude that physiological cell death during PGF2alpha-induced luteal regression in the ewe is mediated, at least in part, via increased expression and activity of the caspase family of pro-apoptotic proteases.

Animals↗

PubMed2003

Restoration of TRAIL-induced apoptosis in a caspase-8-deficient neuroblastoma cell line by stable re-expression of caspase-8.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in most tumor cells, a process sometimes potentiated by chemotherapeutic drugs or cycloheximide (CHX). Childhood neuroblastoma (NB) is a clinically and biologically heterogeneous neoplasm whose behavior can be explained by differential regulation of apoptosis. The non-invasive S-type NB cell lines are sensitive to TRAIL, whereas the invasive N-type NB cell lines are resistant. We have reported the silencing of caspase-8 expression in N-type cells as a possible mechanism of death receptor-mediated resistance to apoptosis in NB. The recently observed deregulation of caspase-10 in these cells prompted us to investigate the particular contribution of caspase-8 silencing in the resistance to TRAIL in N-type cells. Stable caspase-8 expression was therefore restored in the IGR-N91 cell line by retroviral infection. The IGR-N91-C8 cells became sensitive to TRAIL-mediated apoptosis, whereas the control vector-infected IGR-N91-M cells remained resistant. Interestingly, the apoptotic response to TRAIL was enhanced by co-treatment of SH-EP and IGR-N91-C8 cells with CHX or with sub-toxic concentration of doxorubicin (DOX) in a caspase-dependent manner, as cells could be protected from death by specific caspase-8 or pan-caspase inhibitors. CHX or DOX was shown to enhance TRAIL-induced caspase-8 activation and loss of mitochondrial transmembrane potential. In conclusion, restoration of active caspase-8 expression in caspase-8- and caspase-10-deficient IGR-N-91 cell line is necessary and sufficient to fully restore TRAIL-mediated cell death. Moreover, DOX and CHX were able to sensitize NB cell lines to TRAIL-induced apoptosis in a caspase-8-dependent manner by engaging death receptor and mitochondrial signaling pathways.

Animals↗

PubMed2003

Requirement of caspase-3 for efficient apoptosis induction and caspase-7 activation but not viral replication or cell rounding in cells infected with vesicular stomatitis virus.

Infection with vesicular stomatitis virus (VSV), a rhabdovirus and economically significant animal pathogen, was previously demonstrated to induce apoptosis. The mechanism of induction and the role of apoptosis in the VSV-host response have not been completely elucidated. Previous data from our laboratory have suggested that caspase-3 is required for the induction of apoptosis but not viral replication in VSV-infected cells. However, these studies used inhibitors that are selective but not specific for caspase-3. To circumvent this difficulty, we infected both MCF-7 cells which do not express caspases-3 (null), and stable transfectants which express caspase-3 (C3+). When caspase-3 null cells were infected, significant PARP cleavage did not occur, but when C3+ cells were infected, PARP cleavage did occur efficiently. Studies in null and C3+ also suggest that: (1) caspases-3 and -7 are activated sequentially after VSV infection; (2) cell shrinkage and detachment are caspase-3 dependent, but cell rounding is not; and (3) the viral titers were similar between caspase-3 null and C3+ cells suggesting that activation of caspases-3 and -7 are not required for viral replication. Taken together, these results strongly support that the activation of caspase-3 by VSV infection is required for efficient apoptosis induction but not viral replication in vitro. Apoptosis mediated by caspase-3, then, is likely either a host cell response to viral replication or perhaps may be required for in vivo viral replication and spread.

Apoptosis↗

PubMed2001

Transfection of caspase-3 in the caspase-3-deficient Hodgkin's disease cell line, KMH2, results in enhanced sensitivity to CD95-, TRAIL-, and ARA-C-induced apoptosis.

BACKGROUND: CD95(Fas/apo-1) is a cell surface protein member of the tumor necrosis factor receptor family that serves an important role in the induction of apoptosis in several cell types. Although expression of CD95 has been detected on Hodgkin/Reed Sternberg (HRS) cells in situ, our understanding of the biological significance of this molecule in Hodgkin's disease (HD) is limited. DESIGN: We analyzed both CD95-related apoptotic signaling and its effects on the expression of several factors involved in the regulation of apoptotic mechanisms including: caspase-3, caspase-8, bcl-2, bcl-x, and Bax in HD cell lines (L-428, L-540, HDLM-2, HS-445, and KM-H2). RESULTS: HD cell lines showed similar expression levels of CD95 and all but KM-H2 demonstrated variable increases in apoptosis after CD95 stimulation by the agonistic monoclonal antibody, CH11. There was no significant correlation between CD95 sensitivity and constitutive expression levels of caspase-8, bcl-2, bcl-x, and Bax. Caspase-3 transcript was demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) in all cell lines but protein was at low to nearly undetectable levels in KM-H2 cells. Transfection of KM-H2 cells with pro-caspase-3 resulted in a markedly enhanced apoptotic response to CD95 stimulation that was blocked by pretreatment with the caspase-3 inhibitor, DEVD-FMK. In addition, pro-caspase-3-transfected KM-H2 cells showed significantly increased sensitivity to other caspase-3-dependent apoptotic stimuli, including the death-inducing ligand, TRAIL, and the chemotherapeutic agent, Ara-C. CONCLUSION: These data demonstrate that caspase-3 expression plays an important role in CD95-mediated apoptosis in HD cell lines. Furthermore, lack of or decreased expression of caspase-3 in HD cells impairs their apoptotic response not only to CD95 but also to other caspase-3-dependent apoptotic stimuli.

Antibodies, Monoclonal↗

PubMed2000

Caspase-8 activation and bid cleavage contribute to MCF7 cellular execution in a caspase-3-dependent manner during staurosporine-mediated apoptosis.

There are at least two distinct classes of caspases, initiators (e.g. caspases-8, -9, and -10) and effectors (e.g. caspase-3). Furthermore, it is believed that there are two distinct primary apoptotic signaling pathways, one of which is mediated by death receptors controlled by caspases-8/10, and the other by the release of cytochrome c and activation of a caspase-9/Apaf1/cytochrome c apoptosome. However, several recent reports have demonstrated that caspase-8, and its substrate Bid, are frequently activated in response to certain apoptotic stimuli in a death receptor-independent manner. These results suggest that significant cross-talk may exist between these two distinct signaling arms, allowing each to take advantage of elements unique to the other. Here we provide evidence that activation of caspase-8, and subsequent Bid cleavage, does indeed participate in cytochrome c-mediated apoptosis, at least in certain circumstances and cell types. Furthermore, the participation of activated caspase-3 is essential for activation of caspase-8 and Bid processing to occur. Although caspase-8 activation is not required for the execution of a cytochrome c-mediated death signal, we found that it greatly shortens the execution time. Thus, caspase-8 involvement in cytochrome c-mediated cell death may help to amplify weaker death signals and ensure that apoptosis occurs within a certain time frame.

Apoptosis↗

PubMed1999

Sequential activation of caspase-1 and caspase-3-like proteases during apoptosis in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a group of hematopoietic disorders characterized by the concomitant presence of peripheral cytopenias and normocellular to hypercellular BM. This paradox has been proposed to be due to the presence of excessive proliferation matched by excessive intramedullary apoptosis of hematopoietic cells. When cultured in vitro MDS BM mononuclear cells (BMMC) undergo apoptosis within 4 h. We measured caspase-1-like and caspase-3-like activity in 22 MDS and 4 normal BM immediately following cell separation or after 4 h culture. When cultured in vitro, MDS BMMC demonstrated an increased apoptotic index within 4 h as measured by in situ end-labeling of fragmented DNA that was matched by a concurrent increase in caspase-3-like specific activity, and the two were significantly correlated. During the 4 h culture, a sequential activation of caspase-1-like and caspase-3-like activities was detected. Caspase-1-like specific activity was detected early and transiently at approximately 15 min, followed by a gradual increase in caspase-3-like-specific activity peaking at 2 h. When the broad-spectrum caspase inhibitor, Z-VAD.FMK, was included in the MDS BM aspirate 4 h culture, apoptosis was attenuated. We conclude that sequential activation of caspase-1-like and caspase-3-like activities may form the central biochemical pathway of apoptosis in BMMC from some MDS patients, and prevention of this process by caspase inhibitors may be of significant therapeutic value for these patients, in whom supportive care continues to be the mainstay of therapy.

Amino Acid Chloromethyl Ketones↗

PubMed1998

Characterization of the chicken interleukin-1beta converting enzyme (caspase-1) cDNA and expression of caspase-1 mRNA in the hen.

We have cloned and sequenced a chicken homolog to the mammalian interleukin-1beta (IL-1beta)-converting enzyme (caspase-1) cDNA and have evaluated caspase-1 mRNA expression in various tissues from the domestic hen, including ovarian follicle granulosa and theca layers. The deduced amino acid (aa) sequence of chicken caspase-1 is 44.9% identical to human caspase-1, and contains an active site pentapeptide that is characteristic of the caspase family of cysteine proteases. Of interest, however, is that the putative chicken caspase-1 cDNA is predicted to encode a comparatively short (19aa) N-terminal prodomain, as well as two Cys residues within the active pentapeptide (QC162C163RG) as compared to the QACRG pentapeptide found in the mammalian caspase-1 sequence. While the chicken caspase-1 mRNA transcript is widely expressed among different tissues, levels are particularly high in the bursa of Fabricius and comparatively low in ovarian follicles at all stages of development. Finally, treatment of granulosa cells with IL-1beta, the primary if not sole product of caspase-1 activity, fails to either promote apoptotic cell death or enhance viability in granulosa cells. Considering the relatively low levels of caspase-1 mRNA expression in ovarian follicle tissues plus the inability of IL-1beta to alter granulosa cell viability, in vitro, it is concluded that caspase-1 is not an integral part of the apoptotic pathway in granulosa cells. However, the pattern of mRNA expression is consistent with a requirement for caspase-1 mediated IL-1beta production in chicken immune tissues.

Amino Acid Sequence↗