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)

Cellular cholesterol homeostasis is controlled by sterol-regulated proteolysis of membrane-bound transcription factors called sterol-regulatory element binding proteins (SREBPs). CPP32, a cysteine protease, was shown previously to cleave SREBP-1 and SREBP-2 in vitro at an aspartic acid between the basic helix-loop-helix leucine zipper domain and the first trans-membrane domain, liberating a transcriptionally active fragment. Here, we show that CPP32 exists in an inactive 32 kDa form in Chinese hamster ovary (CHO) cells. When apoptosis was induced with the protein kinase inhibitor staurosporine, CPP32 was cleaved to subunits of 20 and 10 kDa to form the active protease. Under these conditions membrane-bound SREBP-1 and SREBP-2 were both cleaved, and the transcriptionally active N-terminal fragments were found in nuclear extracts. Similar results were obtained in human U937 cells induced to undergo apoptosis by anti-Fas and etoposide. The apoptosis-induced cleavage of SREBPs was not suppressed by sterols, indicating that apoptosis-induced cleavage and sterol-regulated cleavage are mediated by different proteases. CHO cells expressing a mutant SREBP-2 with an Asp--> Ala mutation at the CPP32 cleavage site showed sterol-regulated cleavage but no apoptosis-induced cleavage. These data are consistent with the emerging concept that CPP32 is a central mediator in apoptosis. They also indicate that SREBPs, like poly (ADP) ribose polymerase, are cleaved by CPP32 during programmed cell death.
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PMID:Cleavage of sterol regulatory element binding proteins (SREBPs) by CPP32 during apoptosis. 860 70

Fas (Apo1/CD95) is a member of the tumour necrosis factor/nerve growth factor receptor superfamily and mediates apoptosis in various cell types (for review sec [1]). Although this apoptotic activity has been clearly related to homeostasis in the immune system and pathological situations in non-lymphoid organs, the Fas signaling pathway remains mostly elusive. We and others previously showed that Fas-induced apoptosis of primary culture hepatocytes requires either an inhibitor of translation or a protein kinase inhibitor, suggesting that two distinct pathways of Fas signaling exist in hepatocytes. We report here that activation of ICE-like and CPP32-like cysteine proteases are required for Fas-mediated apoptosis, but that these pathways involve different subclasses of serine proteases and are selectively modulated by inhibitors of protein tyrosine kinases. These results confirm that distinct pathways can lead to Fas-induced apoptosis in hepatocytes. Further understanding of these pathways could facilitate the rational design of anti-apoptotic drugs in liver diseases associated with massive Fas-mediated hepatocyte apoptosis, including fulminant hepatitis.
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PMID:Multiple pathways of Fas-induced apoptosis in primary culture of hepatocytes. 895 79

We examined the effects of the cell-permeable, broad spectrum peptide caspase inhibitors, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD.fmk), and BOC-Asp(OMe)-fluoromethyl ketone (BOC-D.fmk), on apoptosis induced by anti-CD2, anti-Fas, and the protein kinase inhibitor staurosporine in activated human peripheral T lymphocytes. We monitored ultrastructural, flow cytometric, and biochemical apoptotic changes, including externalization of phosphatidylserine, cleavage of poly(ADP-ribose) polymerase (PARP) and lamins, activation of caspase-3 and caspase-7, decrease in mitochondrial membrane potential, and DNA fragmentation. Z-VAD.fmk and BOC-D.fmk completely inhibited all the biochemical and ultrastructural changes of apoptosis in anti-Fas-treated cells. In marked contrast, neither Z-VAD.fmk nor BOC-D.fmk inhibited CD2- or staurosporine-mediated cell shrinkage, dilatation of the endoplasmic reticulum (seen in anti-CD2-treated cells), externalization of phosphatidylserine, and loss of mitochondrial membrane potential that accompanied cell death. However, these inhibitors did inhibit the cleavage of PARP and lamins and the formation of hypodiploid cells, and partially inhibited chromatin condensation. These results demonstrate that in activated T cells, anti-CD2 and staurosporine induce a caspase-independent cell death pathway that exhibits prominent cytoplasmic features of apoptosis. However, caspase activation is required for the proteolytic degradation of nuclear substrates such as PARP and lamins together with the DNA fragmentation and extreme chromatin condensation that occur in apoptotic cells.
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PMID:Caspase-independent cell death induced by anti-CD2 or staurosporine in activated human peripheral T lymphocytes. 975 54

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

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

Mitochondria are sites of cellular energy production but may also influence life and death decisions by initiating or inhibiting cell death. Mitochondrial depolarization and the subsequent release of pro-apoptotic factors have been suggested to be required for the activation of a cell death program in some forms of neuronal apoptosis. We induced apoptosis in cultured rat hippocampal neurons by exposure to the protein kinase inhibitor staurosporine (STS) (300 nM). The time course of mitochondrial membrane potential (DeltaPsi(m)) during apoptosis was examined using the probe tetramethylrhodamine ethyl ester (TMRE). Cells exhibited no decrease in TMRE fluorescence, indicative of mitochondrial depolarization, up to 8 hr after STS exposure. Rather, baseline TMRE fluorescence remained unchanged up to 2 hr and thereafter actually increased significantly. Throughout this time period, the mitochondria could also be depolarized with the protonophore carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP, 0.1 microM), exhibiting the same relative magnitude of fluorescence release (unquenching) as controls. Even after 16 hr of staurosporine treatment, neurons that showed signs of nuclear apoptosis maintained DeltaPsi(m) and could be depolarized with FCCP. In contrast, caspase-3-like activity had increased roughly sevenfold by 2 hr and >20-fold by 8 hr. Double-labeling of hippocampal neurons with the potential-sensitive probe Mitotracker Red Chloromethyl X-Rosamine and an antibody to cytochrome c demonstrated at the subcellular level that mitochondrial cytochrome c release also occurred in the absence of mitochondrial depolarization. These data suggest that mitochondrial depolarization is not a decisive event in neuronal apoptosis.
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PMID:Mitochondrial depolarization is not required for neuronal apoptosis. 1046 Feb 46

Apoptosis occurs as a physiologic process in the ovarian life cycle. Staurosporine, a protein kinase inhibitor, is reported to induce apoptosis. Here, we hypothesize that staurosporine will induce apoptosis in human luteinized granulosa cells and that mitochondria and the caspase cascade participate in this process. Luteinized granulosa cells isolated from in vitro fertilization patients were treated with staurosporine. Microscopy revealed that staurosporine treatment resulted in cells exhibiting evidence of apoptosis, including cell detachment, loss of cell processes, membrane shrinkage, and formation of apoptotic bodies. In the staurosporine-treated cells, flow cytometry and confocal microscopy showed a decrease in the mitochondrial cardiolipin levels. Western analysis showed cleavage of caspase-9, an initiator caspase, of caspase-3, an executioner caspase, and of a caspase substrate, poly-(ADP-ribose)-polymerase (PARP) in staurosporine-treated cells. These data support our hypothesis and that this is the first demonstration of the involvement of mitochondria and of cleavage of caspases in human luteinized granulosa cell apoptosis. This may serve as a useful model to delineate the mechanism of apoptosis in the ovary, such as corpus luteum regression.
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PMID:Mitochondria and caspases in induced apoptosis in human luteinized granulosa cells. 1070 90

The ability of two different Jurkat sublines, termed standard and JM, to form DNA ladders was investigated after various apoptotic stimuli. Exposure to a broad spectrum of drugs interfering with signal transduction or cellular metabolism revealed distinct differences between both Jurkat sublines with regard to the pattern of DNA degradation. In standard Jurkat cells, internucleosomal DNA cleavage occurred only after treatment with the protein kinase inhibitor staurosporine. In contrast, the JM subline responded with internucleosomal DNA fragmentation to exposure to gemcitabine, cycloheximide or staurosporine. All drugs induced the formation of DNA fragments of about 50 kb in both sublines, as revealed by pulse field electrophoresis, except H2O2, which caused unspecific DNA degradation. The staurosporine-induced DNA ladder formation was accompanied by an increase in caspase-3 activity in both lines which, however, was considerably lower in Jurkat JM cells after gemcitabine or cycloheximide exposure. When the analysis of internucleosomal DNA degradation was carried out after mycoplasma infection, both Jurkat lines responded with DNA ladder formation after exposure to all drugs used (here only shown for the standard subline). Employing the zymogram technique, nuclease activities of 47 kDa and 54 kDa were detected in culture supernatants, cell homogenates and nuclear extracts only when mycoplasma-infected, whereas the samples obtained from mycoplasma-free sublines were nuclease-negative using this technique, indicating that these endonucleases were of mycoplasmal origin. After drug exposure, the mycoplasmal nucleases must have gained access to the cytoplasm and nuclei of their host cells by an unknown mechanism.
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PMID:Different sublines of Jurkat cells respond with varying susceptibility of internucleosomal DNA degradation to different mediators of apoptosis. 1095 23

We have investigated cell killing in cultured rat embryonic cortical neurons exposed to the protein kinase inhibitor staurosporine, the excitatory amino acid N-methyl-D-aspartate (NMDA), or a combination thereof. Our data indicate that there are several populations of neurons that differ in their response to these agents. Cultures exposed to NMDA undergo cell death typified by lactate dehydrogenase (LDH) leakage which is likely primarily necrotic in that little caspase-3 activation or oligonucleosome formation is observed even when followed for 48 h. Cells exposed to staurosporine (STS) exhibit rapid, extensive activation of caspase-3 with coincident LDH leakage, oligonucleosome formation and TUNEL staining. Both LDH leakage and oligonucleosome content were significantly more elevated at 48 h than at 20 h with STS treatment while caspase-3 activity peaked early (8-20 h) and declined markedly by 48 h. Deletion of NMDA-responsive neurons by pre-treatment of the cultures with NMDA for 4 days prevented the late phase (20-48 h) increases in LDH leakage and oligonucleosomes in the remaining neuronal population. Caspase-3 activity was also completely abolished by NMDA pre-treatment. These results indicate that cells susceptible to acute NMDA-induced toxicity can be killed by non-apoptotic means when exposed to NMDA; however, they undergo a delayed, apoptotic death when exposed to STS. Interestingly, removal of NMDA-responsive cells prevents the processing of procaspase-3; thus, STS-induced apoptosis in cells resistant to NMDA-mediated killing proceeds independent of caspase-3 activation. The data indicate that nearly all neurons in these mixed cultures can undergo apoptosis in response to appropriate stimuli such as STS but that the temporal nature, and the pathways activated in response to STS, vary amongst the subpopulations of neurons. These findings may help to explain the simultaneous appearance of features of both apoptosis and necrosis observed in vivo following cerebral ischemia.
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PMID:NMDA-sensitive neurons profoundly influence delayed staurosporine-induced apoptosis in rat mixed cortical neuronal cultures. 1108 98

We previously demonstrated a loss in Ca(2+)/Calmodulin-dependent protein kinase (CaM kinase) activity in SH-SY5Y undergoing thapsigargin-mediated apoptosis. To extend that finding we report that CaM kinase inhibition potentiates thapsigargin-mediated cell death. CaM kinase inhibitor KN93 on its own exhibits little toxicity up to 10 mM, as measured by release of lactate dehydrogenase (LDH) into the culture medium. In SH-SY5Y cells pretreated with KN93 and the non-selective protein kinase inhibitor k252a and then treated with 2 mM thapsigargin, loss of viability is significantly greater than in cells treated with thapsigargin alone. Pretreatment with the pan-caspase inhibitor Z-D-DCB prevented the thapsigargin-mediated increase in LDH release. Furthermore, thapsigargin-induced caspase-3-like activation, demonstrated by poly(ADP)ribose polymerase cleavage and pro-caspase-3 processing, was elevated in the presence of KN93.
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PMID:Calcium/calmodulin-dependent protein kinase inhibition potentiates thapsigargin-mediated cell death in SH-SY5Y human neuroblastoma cells. 1124 32


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