Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.22.56 (caspase-3)
35,750 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We induced apoptosis in cultured rat hippocampal neurons by exposure to the protein kinase inhibitor staurosporine (30 nM, 24 hr). Treatment with the antioxidant (+/-)-alpha-tocopherol (100 microM) or the superoxide dismutase-mimetic manganese tetrakis (4-benzoyl acid) porphyrin (1 microM) significantly reduced staurosporine-induced cell death. Using hydroethidine-based digital videomicroscopy, we observed a significant increase in intracellular superoxide production that peaked 6-8 hr into the staurosporine exposure. This increase occurred in the absence of gross mitochondrial depolarization monitored with the voltage-sensitive probe tetramethylrhodamine ethyl ester. We then prepared extracts from staurosporine-treated hippocampal neurons and monitored cleavage of acetyl-Tyr-Val-Ala-Asp-aminomethyl-coumarin and acetyl-Asp-Glu-Val-Asp-AMC, fluorogenic substrates for caspase-1-like and caspase-3-like proteases, respectively. Staurosporine caused a significant increase in caspase-1-like activity that preceded intracellular superoxide production and reached a maximum after 30 min. Caspase-3-like activity paralleled intracellular superoxide production, with peak activity seen after 8 hr. Treatment with the corresponding caspase-3-like protease inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde (10 microM) prevented the increase in caspase-3-like activity and staurosporine-induced nuclear fragmentation, but failed to prevent the rise in superoxide production and subsequent cell death. In contrast, treatment with caspase-1-like protease inhibitors reduced both superoxide production and cell death. Of note, antioxidants prevented superoxide production, caspase-3-like protease activity, and cell death even when added 4 hr after the onset of the staurosporine exposure. These results suggest a scenario of an early, caspase-1-like activity followed by a delayed intracellular superoxide production that mediates staurosporine-induced cell death of cultured rat hippocampal neurons.
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PMID:Staurosporine-induced apoptosis of cultured rat hippocampal neurons involves caspase-1-like proteases as upstream initiators and increased production of superoxide as a main downstream effector. 976 65

Interleukin-1beta-converting enzyme (ICE) is believed to be one of the key proteases involved in apoptosis. Since the precursor form of interleukin-1beta (pre-IL-1beta) is one of the well known substrates for ICE, and a potassium/proton ionophore, nigericin, enhances IL-1beta processing, the authors hypothesized that nigericin induces apoptosis through the activation of ICE. In a lipopolysaccharide (LPS)-stimulated and nigericin-treated human monocytic cell line, THP-1, apoptosis was induced, as assessed as to a decrease in cell size, chromatin condensation, exposure of phosphatidylserine and DNA fragmentation. Under exactly the same conditions, nigericin also induced IL-1beta processing in these cells, which was significantly inhibited by an ICE inhibitor, acetyl-Tyr-Val-Ala-Asp-CHO. On the contrary, treatment with this inhibitor at the same concentration did not inhibit nigericin-induced apoptosis, assessed as to the decrease in cell size, chromatin condensation and DNA fragmentation. Although apoptosis induced by nigericin was also observed for LPS-stimulated human peripheral blood mononuclear cells and a mouse T lymphoma cell line, EL-4, the ICE inhibitor did not inhibit the apoptosis in the cells. These results suggest that activated ICE is not involved in the apoptosis induced by nigericin. Since apopain activity was not augmented under the same conditions, neither ICE nor apopain may play any role in the nigericin-induced apoptosis.
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PMID:Activation of interleukin-1beta-converting enzyme by nigericin is independent of apoptosis. 977 Mar 25

p21-activated protein kinase gamma-PAK (Pak2, PAK I) is cleaved by CPP32 (caspase 3) during apoptosis and plays a key role in regulation of cell death. In vitro, CPP32 cleaves recombinant gamma-PAK into two peptides; 1-212 contains the majority of the regulatory domain whereas 213-524 contains 34 amino acids of the regulatory domain plus the entire catalytic domain. Following cleavage, both peptides become autophosphorylated with [gamma-32P]ATP. Peptide 1-212 migrates at 27,000 daltons (p27) upon SDS-polyacrylamide gel electrophoresis and at 32,000 daltons following autophosphorylation on serine (p27P); the catalytic subunit migrates at 34,000 daltons (p34) before and after autophosphorylation on threonine. Following caspase cleavage, a significant lag (approximately 5 min) is observed before autophosphorylation and activity are detected. When gamma-PAK is autophosphorylated with ATP(Mg) alone and then cleaved, only p27 contains phosphate, and the enzyme is inactive with exogenous substrate. After autophosphorylation of gamma-PAK in the presence of Cdc42(GTPgammaS) or histone 4, both cleavage products contain phosphate and gamma-PAK is catalytically active. Mutation of the conserved Thr-402 to alanine greatly reduces autophosphorylation and protein kinase activity following cleavage. Thus activation of gamma-PAK via cleavage by CPP32 is a two-step mechanism wherein autophosphorylation of the regulatory domain is a priming step, and activation coincides with autophosphorylation of the catalytic domain.
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PMID:Cleavage and activation of p21-activated protein kinase gamma-PAK by CPP32 (caspase 3). Effects of autophosphorylation on activity. 978 69

Brefeldin A (BFA) has recently been shown to induce apoptosis in human tumor cells in a p53-independent fashion. In this study, BFA-induced apoptosis was analyzed in the human Jurkat T-cell line. Apoptosis occurred 8 h after treatment with BFA and at concentrations as low as 10 ng/ml and increased with the duration of BFA exposure. Forskolin, an inhibitor of BFA-induced deaggregation of the Golgi-microtubular complex in some cell lines, failed to reverse BFA-mediated apoptosis. Further study of the mechanism of BFA-induced apoptosis was conducted by using a series of peptide protease inhibitors. Complete inhibition of cell death was achieved with benzyloxycarbonyl-Val-Ala-Asp-fluromethylketone, a peptide inhibitor of the caspase protease family, and Z-Asp-Glu-Val-Asp-FMK, a specific inhibitor of caspase-3. Both Acetyl-Tyr-Val-Ala-Asp-chloromethylketone and Acetyl-Tyr-Val-Ala-Asp-aldehyde, selective caspase-1 (interleukin-1beta converting enzyme) inhibitors, exerted only partial protection of cells from apoptosis at higher concentrations. Z-Phe-Ala-FMK, a cysteine protease inhibitor lacking aspartate at the P1 position, did not have any impact on BFA-induced apoptosis. Furthermore, Jurkat cells transfected with the proto-oncoprotein Bcl-2, which is able to block various apoptotic conditions, showed remarkable resistance to the apoptotic effect of BFA. Thus, the data indicate that BFA-induced apoptosis requires caspase(s) activation, primarily the activation of caspase-3, and is inhibited by overexpression of Bcl-2.
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PMID:Brefeldin A-mediated apoptosis requires the activation of caspases and is inhibited by Bcl-2. 982 1

Induction of apoptosis in human monocytic THP.1 cells by etoposide or N-tosyl-L-phenylalanyl chloromethyl ketone resulted in release of mitochondrial cytochrome c, formation of ultracondensed mitochondria, development of outer mitochondrial membrane discontinuities and a reduction in mitochondrial membrane potential (delta psi m), as well as externalisation of phosphatidylserine, caspase-3 and -7 activation, proteolysis of poly(ADP-ribose) polymerase and lamin B1. The caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone inhibited all these ultrastructural and biochemical characteristics of apoptosis except for the release of cytochrome c. Release of mitochondrial cytochrome c was a late event in non-apoptotic cell death occurring after commitment to cell death and without caspase activation. Thus apoptosis is characterised by release of mitochondrial cytochrome c prior to formation of ultracondensed mitochondria and a reduction in delta psi m and by a mechanism independent of rupture of the outer mitochondrial membrane.
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PMID:Apoptosis, in human monocytic THP.1 cells, results in the release of cytochrome c from mitochondria prior to their ultracondensation, formation of outer membrane discontinuities and reduction in inner membrane potential. 984 82

Different classes of anticancer drugs may trigger apoptosis by acting on different subcellular targets and by activating distinct signaling pathways. Here, we report that betulinic acid (BetA) is a prototype cytotoxic agent that triggers apoptosis by a direct effect on mitochondria. In isolated mitochondria, BetA directly induces loss of transmembrane potential independent of a benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone-inhibitable caspase. This is inhibited by bongkrekic acid, an agent that stabilizes the permeability transition pore complex. Mitochondria undergoing BetA-induced permeability transition mediate cleavage of caspase-8 (FLICE/MACH/Mch5) and caspase-3 (CPP32/Yama) in a cell-free system. Soluble factors such as cytochrome c or apoptosis-inducing factor released from BetA-treated mitochondria are sufficient for cleavage of caspases and nuclear fragmentation. Addition of cytochrome c to cytosolic extracts results in cleavage of caspase-3, but not of caspase-8. However, supernatants of mitochondria, which have undergone permeability transition, and partially purified apoptosis-inducing factor activate both caspase-8 and caspase-3 in cytosolic extracts and suffice to activate recombinant caspase-8. These findings show that induction of mitochondrial permeability transition alone is sufficient to trigger the full apoptosis program and that some cytotoxic drugs such as BetA may induce apoptosis via a direct effect on mitochondria.
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PMID:Activation of mitochondria and release of mitochondrial apoptogenic factors by betulinic acid. 985 46

We previously demonstrated that treatment with cycloheximide (CHX) converted the phenotype of Fas-resistant human prostatic carcinoma cell lines to Fas-sensitive and that resistance to Fas-mediated apoptosis was due to a dominant-negative protein(s). In this study, we investigated the sequential activation of caspase family members, to gain insight into the likely site of action of the suppressor protein(s). We did not find Tyr-Val-Ala-Aspase activity in any of the cell lines examined. Time-dependent Asp-Glu-Val-Aspase activity was detected during Fas-mediated apoptosis in Fas-sensitive cell lines PC3 and ALVA31. Asp-Glu-Val-Aspase activity in Fas-resistant cell lines DU145 and JCA1, was detected only under combined treatment with CHX and anti-Fas agonistic mAb. In experiments with caspase inhibitors we show that Fas-mediated apoptosis in PC3 is mainly executed by the caspase-3 subfamily, but another member(s) of the caspase family may be involved in Fas-mediated apoptosis in ALVA31, DU145, and JCA1. Western blot analysis revealed that Fas-ligation activated caspase-7, but not caspase-3. The activated form of caspase-8 was detected in DU145 only after 4 h of simultaneous treatment with CHX and anti-Fas mAb, whereas in PC3 caspase-8 was found to be activated after 1 h of Fas-ligation. We have also found that treatment with staurosporin did not activate caspase-8, whereas staurosporin induced apoptosis at the same levels in both Fas-resistant and Fas-sensitive cell lines. These results suggest that an inhibitory protein(s), which suppresses apoptosis in Fas-resistant cell lines, presumably acts at the apex of apoptotic cascade by preventing the activation of caspase-8.
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PMID:Fas-mediated apoptosis in human prostatic carcinoma cell lines occurs via activation of caspase-8 and caspase-7. 986 48

Glucocorticoids (GCs) are essential therapeutic reagents for the treatment of lymphomas and leukemias. GCs cause cell death in certain types of lymphoid cells mediated by the process known as apoptosis. This cell death is completely inhibited by Bcl-2. Here we report that Bcl-2 and benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), a broad spectrum caspase inhibitor, prevent loss of mitochondrial membrane potential (delta psi m) and the production of reactive oxygen species (ROS) caused by GC, while acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), an inhibitor of the caspase-3 family proteases, does not. This suggests that the inhibition by Bcl-2 and activation of some initiator caspases are upstream events of mitochondrial damage, whereas the activation of caspase-3 family proteases occurs downstream of mitochondrial changes. We also demonstrate that caspase-6 but not caspase-3 is cleaved and activated during GC-mediated apoptosis and that poly(ADP-ribose) polymerase (PARP), a substrate of caspases, also undergoes proteolysis. In addition, we provide the evidence that DNA fragmentation is markedly inhibited by Ac-DEVD-CHO, while cell death, assessed by the damage of the plasma membrane, is marginally inhibited or merely delayed.
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PMID:Investigation of glucocorticoid-induced apoptotic pathway: processing of caspase-6 but not caspase-3. 989 10

It is now well established that the caspases, a family of cysteine proteases, play a key role in apoptosis. Although overexpressing each of the caspases in cells triggered apoptosis, the precise role and contribution of individual caspases are still unclear. Caspase-1, the first caspase discovered, was initially implicated in mammalian apoptosis because of its similarity to the gene product ced-3. Using whole cells as well as an in vitro system to study apoptosis, the role of caspase-1 in Fas-mediated apoptosis in Jurkat T cells was examined in greater detail. Using various peptide-based caspase inhibitors, our results showed that N-acetyl-Tyr-Val-Ala-Asp chloromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone efficiently blocked Fas-mediated apoptosis in Jurkat T cells, whereas N-acetyl-Tyr-Val-Ala-Asp aldehyde, which is more specific for caspase-1, had little effect. Cell lysates derived from anti-Fas-stimulated cells, which readily induced apoptotic nuclei morphology and DNA fragmentation in isolated thymocyte nuclei, had no caspase-1 activity using proIL-1beta as a substrate. Time-course studies showed no caspase-1 activity during the activation of apoptosis in Jurkat cells by agonistic Fas antibodies. Furthermore, no pro-caspase-1 protein nor activated form of the protein was detected in normal or apoptotic Jurkat cells. In contrast, both caspase-2 and caspase-3 were readily detected as proenzymes in control cells and their activated forms were detected in apoptotic cells. Incubation of recombinant active caspase-1 with control cell lysates did not activate the apoptotic cascade as shown by the lack of detectable apoptotic nuclei promoting activity using isolated nuclei as substrate. However, under similar conditions proIL-1beta was readily processed into the mature cytokine, indicating that the recombinant caspase-1 remained active in the presence of control cell lysates. Taken together our results demonstrate that caspase-1 is not required for the induction of apoptosis in Jurkat T cells mediated by the Fas antigen.
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PMID:Caspase-1 is not involved in CD95/Fas-induced apoptosis in Jurkat T cells. 992 65

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


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