Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P42574 (caspase-3)
 45,978 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The goals of this work were to establish a reproducible and effective model of apoptosis in a cell line derived from advanced prostate cancer and to study the role of the caspase family of proteases in mediating apoptosis in this system. The study involved the use of the prostate cancer cell line LNCaP. Apoptosis was induced using the hydroxymethyl glutaryl CoA reductase inhibitor, lovastatin, and was evaluated by agarose gel electrophoresis of genomic DNA, morphological criteria, and terminal deoxynucleotidyl transferase-mediated nick end labeling. Caspases were studied by catalytic activity, mRNA induction, and protein processing. Lovastatin (30 microM) was an effective inducer of apoptosis, causing changes that were evident after 48 h and essentially complete after 96-120 h of treatment. These effects were prevented by the simultaneous addition of mevalonate (300 microM) to the culture medium. Lovastatin induced a proteolytic activity that was able to cleave the enzyme poly(ADP-ribose) polymerase and the substrate Z-DEVD-AFC, which is modeled after the P1-P4 amino acids of the poly(ADP-ribose) polymerase cleavage site. Caspase-7, but not caspase-3, underwent proteolytic activation during lovastatin-induced apoptosis, an effect prevented by mevalonate. Caspase-7 was the only detected interleukin 1beta converting enzyme family protease with DEVD cleavage activity that exhibited lovastatin-induced mRNA up-regulation. Again, mevalonate blocked this effect. Lovastatin-induced apoptosis also was prevented when the caspase inhibitors Z-DEVD-CH2F or Z-VAD-CH2F (100 microM) where added to the medium. These studies have identified lovastatin as a powerful inducer of apoptosis in the cell line LNCaP. Caspase activation was a necessary event for LNCaP cells to undergo apoptosis during treatment with lovastatin. Of the caspases tested, only caspase-7 underwent proteolytic activation after stimulation with lovastatin. Identification of caspase-7 as a potential mediator of lovastatin-induced apoptosis broadens our knowledge of the molecular events associated with programmed cell death in a cell line derived from prostatic epithelium.
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PMID:Caspase-7 is activated during lovastatin-induced apoptosis of the prostate cancer cell line LNCaP. 942 61

Ceramide, a sphingolipid generated by the hydrolysis of membrane-associated sphingomyelin, appears to play a role as a gauge of apoptosis. A further metabolite of ceramide, sphingosine 1-phosphate (SPP), prevents ceramide-mediated apoptosis, and it has been suggested that the balance between intracellular ceramide and SPP levels may determine the cell fate (Cuvillier, O., Pirianov, G, Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, J. S., and Spiegel, S. (1996) Nature 381, 800-803). Here, we investigated the role of SPP and the protein kinase C activator, phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), in the caspase cascade leading to the proteolysis of poly(ADP-ribose) polymerase (PARP) and lamins. In Jurkat T cells, Fas ligation or addition of exogenous C2-ceramide induced activations of caspase-3/CPP32 and caspase-7/Mch3 followed by PARP cleavage, effects that can be blocked either by SPP or TPA. Furthermore, both SPP and TPA inhibit the activation of caspase-6/Mch2 and subsequent lamin B cleavage. Ceramide, in contrast to Fas ligation, did not induce activation of caspase-8/FLICE and neither SPP nor TPA were able to prevent this activation. Thus, SPP, likely generated via protein kinase C-mediated activation of sphingosine kinase, suppresses the apoptotic pathway downstream of FLICE but upstream of the executioner caspases, caspase-3, -6, and -7.
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PMID:Sphingosine 1-phosphate inhibits activation of caspases that cleave poly(ADP-ribose) polymerase and lamins during Fas- and ceramide-mediated apoptosis in Jurkat T lymphocytes. 944 2

The mechanism of Fas antigen-induced hepatocyte apoptosis was investigated. Using a monoclonal antibody directed against the Fas antigen, apoptosis was induced in freshly isolated murine hepatocytes within 90 minutes of antibody addition as assessed by plasma membrane bleb formation, chromatin condensation, and DNA fragmentation. Pretreatment of the cells with the caspase inhibitors, N-acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO), benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK), or Z-Asp-2,6-dichlorobenzoyloxymethylketone inhibited anti-Fas-mediated apoptosis. Likewise, the serine protease inhibitors, N-tosyl-L-phenyl chloromethyl ketone (TPCK) and 3,4-dichloroisocoumarin (DCI), prevented apoptosis, whereas N-tosyl-L-lysine chloromethyl ketone (TLCK), Ac-Leu-Leu-L-norleucinal, Ac-Leu-Leu-L-methional, and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane were without effect. Examination of CED-3/caspase-3-related caspases revealed that pro-caspases-3 (CPP32) and -7 (Mch-3alpha) were rapidly processed after Fas antigen stimulation. Caspase-7 was further cleaved to form the catalytically active subunits. In contrast, the p17 subunit of caspase-3 was not detected, indicating slow formation or rapid degradation. The activation of CED-3-related caspases was further confirmed by an increase in the rate of Z-DEVD-7-amino-4-trifluoromethylcoumarin (Z-DEVD-AFC) hydrolysis that was sensitive to Ac-DEVD-CHO and was inhibited by pretreatment of the cells with TPCK but not by DCI. In contrast, no increase in the rates of hydrolysis of Z-YVAD-AFC, a substrate for caspase-1, was detected. Investigation of the in situ proteolytic cleavage of the CED-3 related caspases substrate, poly(ADP-ribose) polymerase, revealed that this protein was not degraded in hepatocytes undergoing Fas-mediated apoptosis. Taken together, our results show that processing of caspases, in particular, caspases-7 and -3, occurs during Fas-induced apoptosis of mouse hepatocytes and suggest a role of these proteases as well as serine protease(s) in the apoptotic response.
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PMID:Fas-mediated apoptosis in mouse hepatocytes involves the processing and activation of caspases. 962 Mar 37

Granzyme B is a protease involved in the induction of rapid target cell death by cytotoxic lymphocytes. Definition of the substrate specificity of granzyme B allows for the identification of in vivo substrates in this process. By using the combinatorial methods of synthetic substrate libraries and substrate-phage display, an optimal substrate for granzyme B that spans over six subsites was determined to be Ile-Glu-Xaa-(Asp downward arrowXaa)-Gly, with cleavage of the Asp downward arrowXaa peptide bond. Granzyme B proteolysis was shown to be highly dependent on the length and sequence of the substrate, supporting the role of granzyme B as a regulatory protease. Arginine 192 was identified as a determinant of P3-Glu and P1-Asp substrate specificity. Mutagenesis of arginine 192 to glutamate reversed the preference for negatively charged amino acids at P3 to positively charged amino acids. The preferred substrate sequence matches the activation sites of caspase 3 and caspase 7 and thus is consistent with the role of granzyme B in activation of these proteases during apoptosis. The caspase substrate poly(ADP)-ribose polymerase is cleaved by granzyme B in a cell-free assay at two sites that resemble the granzyme B specificity determined by the combinatorial methods. Many caspase substrates contain granzyme B cleavage sites and are proposed as potential granzyme B targets, suggesting a redundant function with certain caspases.
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PMID:Definition and redesign of the extended substrate specificity of granzyme B. 976 64

The cytosolic factor Cif (cytochrome c-efflux inducing factor) was activated by the apoptosis inducers staurosporine and anti-Fas antibodies and rapidly induced the efflux of cytochrome c from purified human mitochondria. HL-60 cells that stably overexpressed a bcl-2 cDNA transgene (Bcl-2:HL-60 cells) contained mitochondria and a cytosol that were resistant to exogenous Cif and that lacked detectable endogenous Cif activity, respectively. Therefore, Bcl-2 overexpression negated Cif activity and suggested that the requirement for Cif resides upstream of Bcl-2 on the apoptotic signal transduction pathway. The addition of purified caspase 3, caspase 7, or caspase 8 to the cytosolic extract from Bcl-2:HL-60 cells, however, restored Cif activity, demonstrating that the inhibition of Cif by Bcl-2 overexpression could be overcome by activated caspases. Moreover, the addition of purified caspases to cytosolic extracts prepared from parental HL-60 cells was also sufficient to cause Cif activation, suggesting that caspases might be required for Cif activation. Consistent with these observations, Fas-induced apoptosis in Jurkat cells resulted in caspase 8 activation and subsequently in activation of Cif. Finally, we demonstrate that the activation of Cif correlated with the activation of the Bcl-2 family member Bid by caspases and that Cif activity was selectively neutralized by anti-Bid antibodies. Taken together, these results indicate that Cif is identical to Bid and that it can be inhibited by Bcl-2 and activated by caspases. Thus, Cif (Bid) is an important biological regulator for the transduction of apoptotic signals.
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PMID:Cif (Cytochrome c efflux-inducing factor) activity is regulated by Bcl-2 and caspases and correlates with the activation of Bid. 989 Oct 71

We studied the molecular mechanisms of apoptosis in the prostate cancer cell line LNCaP and whether overexpression of caspase activity could force this cell line to undergo apoptosis. The inhibitor of phosphomevalonate decarboxylase, sodium phenylacetate, and the protein kinase inhibitor staurosporine induced (a) release of cytochrome c from the mitochondria to the cytosol; (b) reduction in mitochondrial transmembrane potential; (c) proteolytic processing of caspase-3 and -7 but not -2; (d) cleavage of the DEVD substrate and the death substrates poly(ADP-ribose) polymerase and DNA fragmentation factor; and (e) apoptosis. The panspecific inhibitor of caspase activation N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-FMK) prevented all of these events except release of mitochondrial cytochrome c into the cytosol. None of these apoptotic signaling events were elicited by staurosporine or sodium phenylacetate treatment of LNCaP-Bcl-2 cells that overexpress the oncoprotein Bcl-2. Because caspase-7 is activated in every model of apoptosis that we have characterized thus far, we wished to learn whether overexpression of this protease could directly cause apoptosis of LNCaP cells. By using a replication-defective adenovirus, overexpression of caspase-7 protein in both LNCaP and LNCaP-Bcl-2 cells was accompanied by induction of cleavage of the DEVD substrate and TUNEL. These studies have demonstrated that caspase-7 and -3 are critical mediators of apoptosis in LNCaP cells. Caspase-7 was proteolytically activated in every model of apoptosis that we have developed, and the overexpression of it induced apoptosis of LNCaP and LNCaP-Bcl-2 cells. Thus, adenoviral-mediated transfer of caspase-7 may offer a new effective approach for the treatment of prostate cancer.
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PMID:Signaling pathway activated during apoptosis of the prostate cancer cell line LNCaP: overexpression of caspase-7 as a new gene therapy strategy for prostate cancer. 992 51

Apoptosis is instrumental in the processes generating the diversity of the B-cell repertoire. Autoreactive B-cells are eliminated by anti-IgM crosslinking after encountering self-antigens, but precise mechanisms leading to B-cell apoptosis are still not well understood. We report here the cleavage of the transcription factor SP1 in the human Burkitt lymphoma cell line BL60 during anti-IgM-induced apoptosis. Western blot analysis revealed two cleavage products of approximately 68 kDa and 45 kDa after induction of apoptosis. Cleavage could be completely inhibited by zDEVD-fmk, an inhibitor specific for caspase 3-like proteases. In-vitro cleavage of recombinant SP1 by recombinant caspase 3 (CPP32) or caspase 7 (Mch 3) results in similar cleavage products as those observed in vivo. Recombinant caspase 6 (Mch 2) primarily generates a 68-kDa cleavage product, as observed after calcium ionophore (CaI) induced B-cell apoptosis. In contrast, caspase 1 (ICE) did not cleave SP1 in vitro. The time course of SP1 cleavage during anti-IgM-induced apoptosis is paralleled by an increase of caspase activity measured by DEVD-p-nitroanilide (DEVD-pNA) cleavage. DNA band-shift assays revealed a decrease in the intensity of the full length SP1/DNA complex and an increase in the intensity of a smaller complex due to the binding of one SP1 cleavage product. By Edman sequencing we could identify a caspase 3 cleavage site after Asp584 (D584AQPQAGR), generating a 22-kDa C-terminal SP1 protein fragment which still contains the DNA binding site. Our results show the cleavage of the human transcription factor SP1 in vivo and in vitro, underlining the central role of caspase 3-like proteases during the process of anti-IgM-induced apoptosis.
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PMID:Cleavage of transcription factor SP1 by caspases during anti-IgM-induced B-cell apoptosis. 1010 59

The B cell lymphoma WEHI-231 has been used as a model to study immature B cell tolerance, based on its capacity to undergo growth arrest and programmed cell death on B cell receptor (BCR) cross-linking. Using this model to identify the molecular mechanisms underlying these processes, we found that BCR cross-linking results in the selective activation of caspase 7/Mch3, but not of the other two members of the CPP32 family, caspase 2/Nedd2 and caspase 3/CPP32. This was evidenced by the induction of proteolytic activity against the substrate for the CPP32 subfamily of caspases (z-DVED-AMC) in vitro, as well as PARP proteolysis in vivo and by the processing of the 35 kDa Mch3 into a 32 kDa species, which was later further proteolyzed. The general caspase inhibitor z-VAD-fmk, but not the CPP32 family inhibitor Ac-DEVD-CHO, blocked anti- micro-induced apoptosis, indicating that a caspase not belonging to the CPP32-like family is also implicated in anti- micro-triggered apoptosis. In contrast, z-VAD-fmk was not able to counteract growth arrest induced by anti- micro treatment, suggesting that caspase activation is not necessary for induction of growth arrest. Neither of the inhibitors prevented Mch3 processing; however, z-VAD-fmk prevented proteolysis of the p32 subunit, suggesting that further processing of this subunit is associated with apoptosis. Bcl-2 overexpression prevented anti- micro induction of CPP32-like activity and apoptosis, and blocked further processing of the Mch3 p32 subunit. In contrast, CD40 stimulation completely blocked the appearance of the p32 subunit in addition to blocking CPP32-like activity and apoptosis induced by BCR cross-linking. Moreover, only CD40 stimulation was able to prevent anti- micro-induced growth arrest, which was correlated with inhibition of retinoblastoma and of cyclin A down-regulation. In splenic B cells, Mch3 is also specifically proteolyzed ex vivo after induction of apoptosis by BCR cross-linking, demonstrating the specific involvement of caspase-7/Mch3 in apoptosis induced in B cell tolerance.
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PMID:Caspase activation by BCR cross-linking in immature B cells: differential effects on growth arrest and apoptosis. 1022 36

Our study examines the effect of apoptosis on prothymosin alpha, an abundant, nuclear protein intimately involved with proliferation of all mammalian cells. When HeLa cells were treated with actinomycin D, with etoposide, or with staurosporine following synchronization with hydroxyurea, they underwent apoptosis based on several specific criteria, including fragmentation of DNA and activation of specific caspases. Similarly treated NIH3T3 cells arrested and displayed no indicators of apoptosis. In HeLa, but not in NIH3T3 cells, prothymosin alpha levels declined precipitously and a truncated version of the protein was formed. The following observations implicate caspase activity: (1) The truncated polypeptide arose only in the treated HeLa cell cultures. (2) The appearance of the truncated polypeptide coincided with the activation of caspase 3 and the cleavage of poly(ADP-ribose) polymerase, a known caspase substrate. (3) Carbobenzoxy-DEVD-fluoromethylketone, a cell-permeable caspase 3 inhibitor, blocked cleavage and degradation of prothymosin alpha. (4) The same inhibitor, when added to mixed extracts of apoptotic and normal cells, prevented cleavage of intact prothymosin alpha. (5) Recombinant caspase 3 and, to a much lesser extent, caspase 7 truncated purified prothymosin alpha. (6) In HeLa cells, cleavage occurred at three overlapping caspase 3-like sites with the consensus sequence D-X-X-D and released 10 to 14 residues from the carboxyl terminus, including the core nuclear localization signal. Two immediate consequences of the cleavage were observed: truncated prothymosin alpha was no longer confined to the nucleus and it was deficient in phosphate. These data suggest that the disabling of prothymosin alpha is a significant event in apoptosis. J. Cell. Physiol. 182:256-268, 2000. Published 2000 Wiley-Liss, Inc.
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PMID:Functional discontinuities in prothymosin alpha caused by caspase cleavage in apoptotic cells. 1062 90

Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced apoptosis in human hematopoietic U937 cells by itself and in a synergistic manner with tumor necrosis factor (TNF). GM-CSF-induced apoptosis was not inhibited by caspase inhibitors YVAD-CMK, DEVD-CHO and z-VAD-FMK, under the condition that these inhibitors potently suppressed TNF-induced apoptosis. Both GM-CSF and TNF induced caspase 3-like activity in this cell line though the time course was distinct between two cytokines, and combined stimulation of cells with GM-CSF plus TNF induced additive or synergistic activation of caspase 3-like activity. Amount of immunoreactive cleaved forms of caspase 3 recognized by specific antibody was completely dissociated with its enzymatic activity when the cells were stimulated with GM-CSF, but not with TNF. These results indicate that GM-CSF induces apoptosis of U937 cells via unknown pathway, which seems to be mediated by caspase 3-like activity, yet not caspase 3 itself, resistant to the caspase inhibitors, and synergistically interacts with conventional caspase 3 pathway of TNF. Possible involvement of caspases 1 and 8 (-like activity) but not caspase 7 in this pathway was also suggested.
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PMID:Induction of apoptosis in human hematopoietic U937 cells by granulocyte-macrophage colony-stimulating factor: possible existence of caspase 3-like pathway. 1076 46


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