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)

Apoptosis, the cellular mechanism of ovarian follicular atresia and luteal regression, is triggered by the activation of a proteolytic cascade of cysteine aspartate-specific proteases (caspases). The principle downstream effector of cell death is caspase-3, but little is known about the role or regulation of this enzyme in ovarian apoptosis. Two substrates of caspase-3, actin and poly(ADP-ribose) polymerase (PARP), are inhibitors of DNase I, which is the endonuclease responsible for ovarian apoptotic DNA degradation. We therefore investigated the proteolytic cleavage of actin and PARP as well as the localization of caspase-3 during follicular atresia (induced by gonadotropin withdrawal) and luteal regression (induced by prostaglandin F2alpha) in the rat ovary. Apoptotic DNA degradation was evident during both follicular atresia and luteal regression, but cleavage of PARP and actin was observed only during luteal regression. Caspase-3 was localized in luteal cells of healthy corpora lutea (CL) and in theca, but not in granulosa cells of healthy follicles. However, caspase-3 immunostaining was evident in granulosa cells of atretic follicles in a pattern similar to that of the localization of granulosa cell death. There was no difference between healthy and apoptotic CL in the distribution or intensity of caspase-3 staining. These results demonstrate that the cleavage of actin and PARP are not necessary for activation of apoptotic DNA degradation during ovarian apoptosis. In addition, the presence of caspase-3 in granulosa cells of atretic, but not healthy, follicles suggests that the expression of this enzyme is regulated by gonadotropin and may be up-regulated as part of the apoptotic process in granulosa cells.
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PMID:Caspase-3 in the rat ovary: localization and possible role in follicular atresia and luteal regression. 962 16

Apoptosis is a programmed form of cell death characterized by biochemical and morphological changes affecting the nucleus, cytoplasm, and plasma membrane. These changes in various cellular compartments are widely regarded as mechanistically linked events in a single "program" in which activation of caspases and proteolysis of intracellular substrates represent a final common pathway leading to cell death. To date there has been very limited exploration of the linkage of this program to the plasma membrane changes, which bring about swift recognition, uptake, and safe degradation of apoptotic cells by phagocytes. Using the mitochondrial inhibitors antimycin A and oligomycin in human monocytic THP.1 cells triggered into apoptosis, we report the uncoupling of plasma membrane changes from other features of apoptosis. These inhibitors blocked increased plasma membrane permeability, externalization of phosphatidylserine, and recognition by two classes of phagocytes but not activation of caspase-3, cleavage of poly(ADP-ribose) polymerase and DNA fragmentation. Externalization of phosphatidylserine in apoptotic human leukemic U937 cells was also dissociated from caspase activation. Thus changes governing safe clearance of apoptotic cells may be regulated by an independent pathway to those bringing about caspase activation. This finding could have important consequences for attempts to manipulate cell death for therapeutic gain in vivo.
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PMID:Dissociation of phagocyte recognition of cells undergoing apoptosis from other features of the apoptotic program. 962 55

Apoptotic changes occurred specifically in a macrophage-like cell line, J774.1, treated with lipopolysaccharide (LPS) and cycloheximide (CHX) prior to the release of lactate dehydrogenase (LDH). The addition of 100 ng/ml LPS and 10 microg/ml CHX induced both the formation of DNA nicks and elevation of caspase-3-like activity (DEVDase) after 75 min, and then the cleavage of poly(ADP-ribose) polymerase (PARP) into 28-kDa fragments, formation of apoptotic bodies, and DNA ladder formation. These apoptotic changes were reversible until 60 min, however, later than 75 min after LPS and CHX addition, the apoptosis proceeded normally even on extensive washing of the macrophages, which removed the LPS and CHX. These results suggest that there is a "point of no return" in the apoptotic processes in macrophages induced by LPS and CHX and that DNA nicks and activation of DEVDase are critical for these processes.
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PMID:Apoptotic changes preceding necrosis in lipopolysaccharide-treated macrophages in the presence of cycloheximide. 963 79

The polyamine analogue, N1-ethyl-N11-[(cycloheptyl)methyl]-4,8-diazaundecane (CHENSpm)-induced programmed cell death in NCI H157 cells is accompanied by cytochrome c release, the loss of mitochondrial membrane potential, activation of caspase-3, caspase-mediated poly(ADP-ribose) polymerase cleavage, G2-M arrest, and DNA and nuclear fragmentation. Overexpression of Bcl-2 completely inhibits CHENSpm-induced cytochrome c release, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage. However, Bcl-2 does not abrogate CHENSpm-induced programmed cell death. These results suggest that although cytochrome c release and activation of the caspase-3 protease cascade contribute to the rapid and efficient execution of apoptosis, a caspase cascade-independent pathway also exists and can be activated by CHENSpm treatment.
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PMID:Unsymmetrically substituted polyamine analogue induces caspase-independent programmed cell death in Bcl-2-overexpressing cells. 966 78

Sulfur mustard (SM) induces vesication via poorly understood pathways. The blisters that are formed result primarily from the detachment of the epidermis from the dermis at the level of the basement membrane. In addition, there is toxicity to the basal cells, although no careful study has been performed to determine the precise mode of cell death biochemically. We describe here two potential mechanisms by which SM causes basal cell death and detachment: namely, induction of terminal differentiation and apoptosis. In the presence of 100 microM SM, terminal differentiation was rapidly induced in primary human keratinocytes that included the expression of the differentiation-specific markers K1 and K10 and the cross-linking of the cornified envelope precursor protein involucrin. The expression of the attachment protein, fibronectin, was also reduced in a time- and dose-dependent fashion. Features common to both differentiation and apoptosis were also induced in 100 microM SM, including the rapid induction of p53 and the reduction of Bcl-2. At higher concentrations of SM (i.e., 300 microM), formation of the characteristic nucleosome-sized DNA ladders, TUNEL-positive staining of cells, activation of the cysteine protease caspase-3/apopain, and cleavage of the death substrate poly(ADP-ribose) polymerase, were observed both in vivo and in vitro. Both the differentiation and the apoptotic processes appeared to be calmodulin dependent, because the calmodulin inhibitor W-7 blocked the expression of the differentiation-specific markers, as well as the apoptotic response, in a concentration-dependent fashion. In addition, the intracellular Ca2+ chelator, BAPTA-AM, blocked the differentiation response and attenuated the apoptotic response. These results suggest a strategy for designing inhibitors of SM vesication via the Ca2+-calmodulin or caspase-3/PARP pathway.
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PMID:Sulfur mustard induces markers of terminal differentiation and apoptosis in keratinocytes via a Ca2+-calmodulin and caspase-dependent pathway. 966 88

Nitric oxide (NO) promotes apoptotic cell death in the mouse macrophage cell line RAW 264.7 and in the human promyelocytic leukaemia cell line U937, which exemplifies p53-dependent and p53-independent executive death pathways. Here, we followed the cleavage of two caspase substrates during NO-intoxication, assaying poly(ADP-ribose) polymerase and U1-70kDa small ribonucleoprotein (U1-70kDa) degradation. By using pharmacological inhibitors, we found that Z-aspartyl-2,6-dichlorobenzoyloxymethylketone (Z-Asp-CH2-DCB; 100 microM), a caspase-like protease inhibitor, completely blocked S-nitrosoglutathione (GSNO)-induced apoptosis in both RAW 264.7 and U937 cells (IC50 = 50 microM for RAW 264.7 macrophages vs. IC50 = 33 microM for U937 cells). Notably, a characterized caspase-3 (Ac-DEVD-CHO) inhibitor left NO-induced DNA fragmentation and the appearance of an apoptotic morphology unaltered, although completely blocking caspase-3 activity. However, Z-Asp-CH2-DCB suppressed protease-mediated U1-70kDa cleavage and DNA fragmentation in parallel. In contrast, poly(ADP-ribose) polymerase cleavage in U937 cells was only delayed by Z-Asp-CH2-DCB, while poly(ADP-ribose) polymerase digestion in RAW 264.7 macrophages proceeded unaltered. We further compared U1-70kDa and poly(ADP-ribose) polymerase cleavage in stably Bcl-2 transfected RAW 264.7 macrophages. Rbcl2-2, a Bcl-2 overexpressing clone, suppressed DNA fragmentation and U1-70kDa digestion in response to GSNO, although allowing delayed but complete poly(ADP-ribose) polymerase degradation. Conclusively, poly(ADP-ribose) polymerase cleavage not causatively coincided with the appearance of other apoptotic parameters. Our results suggest that NO-induced apoptosis demands a Z-Asp-CH2-DCB inhibitable caspase activity, most likely distinct from caspase-3 and caspase-1. NO-mediated executive apoptotic signaling results in U1-70kDa and poly(ADP-ribose) polymerase cleavage. Whereas U1-70kDa digestion closely correlates to the occurrence of apoptotic parameters such as DNA fragmentation or an apoptotic morphology, poly(ADP-ribose) polymerase-breakdown does not.
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PMID:Protease activation during nitric oxide-induced apoptosis: comparison between poly(ADP-ribose) polymerase and U1-70kDa cleavage. 967 Nov 15

Caspases are aspartate-specific cysteine proteases that play a pivotal role in drug-induced cell death. We designed RT-PCR assays to analyse the expression of CASP-3, CASP-4, CASP-6 and the long and short isoforms of CASP-2 genes in human cells. These genes heterogeneously coexpress in leukemic cell lines and bone marrow samples from patients with de novo acute myelogenous leukemia at diagnosis. Treatment of U937 and HL60 leukemic cells and HT29 colon carcinoma cells with the topoisomerase II inhibitor etoposide upregulates CASP-2 and CASP-3 genes in these cells before inducing their apoptosis. This effect of etoposide is not observed in K562 cells and bcl-2-transfected U937 cells which are less sensitive to drug-induced apoptosis. Nuclear run-on experiments demonstrate that etoposide increases CASP gene transcription in U937 cells, an effect that is prevented by Bcl-2 overexpression. Upregulation of CASP genes is associated with an enhanced synthesis of related procaspases that precedes the appearance of apoptosis markers including caspase-3 activation, poly(ADP-ribose) polymerase cleavage and internucleosomal DNA fragmentation. These results suggest that the ability of tumor cells to upregulate CASP-2 and CASP-3 genes in response to cytotoxic drugs could be predictive of their sensitivity to drug-induced apoptosis.
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PMID:Upregulation of CASP genes in human tumor cells undergoing etoposide-induced apoptosis. 967 9

Cells expressing the R273H mutant of p53, which lacks sequence specific DNA binding capacity, do not undergo cell cycle arrest in G1 following exposure to ionizing or UV radiation because of their inability to induce p21Waf1/Cip1, a cyclin-dependent kinase inhibitor and downstream mediator of p53-dependent DNA damage-induced growth arrest. Following UV-irradiation or treatment with an inhibitor of RNA pol II, we observed a rapid induction of the apoptotic process, as evidenced by DNA fragmentation and the proteolytic cleavage of poly(ADP-ribose) polymerase. Using mimosine, a p21Waf1/Cip1 inducer that bypasses the requirement for transcriptional transactivation by p53, we demonstrated that a G1 cell cycle arrest can prevent apoptosis following UV-irradiation or treatment with an RNA polymerase 11 inhibitor. Serum starvation, which also synchronized cells in G1 but did not induce p21Waf1/Cip1, did not protect cells from apoptosis. These results demonstrate that restoring a late G1 checkpoint by inducing p21Waf1/Cip1 expression can protect cells from DNA damage induced apoptosis. Our results suggest that p21Waf1/Cip1 can interrupt the apoptotic process at a point downstream from p53 accumulation but upstream from caspase-3 activation.
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PMID:p21-induced cycle arrest in G1 protects cells from apoptosis induced by UV-irradiation or RNA polymerase II blockage. 969 54

Detachment-induced cell death (DICD) is considered to be one of the means by which intestinal epithelial cells (IEC) die of apoptosis as they reach the lumen and are shed. Caspases, a family of cysteine proteases, play a central role in initiating, amplifying, and executing apoptosis; however, the pattern of caspase activation in response to distinct apoptotic stimuli remains unknown. We investigated the kinetics of caspase activation during DICD in freshly isolated human IEC. DNA fragmentation is observed 90 min after detachment and is preceded by the sequential activation of preformed members of the CPP32 family of caspases. Activation of caspase 6 and cleavage of the endogenous caspase substrate poly(ADP-ribose) polymerase (EC 2.4.2.30) are detected within 15 min of detachment, 30-45 min before caspase 3 activation. Caspase 1 and caspase 10 are present as proenzymes, yet they remain inactive in response to this trigger of apoptosis. Human IEC are primed to rapidly undergo detachment-induced apoptosis involving the selective and sequential activation of preformed caspases. This study may enhance our understanding of physiological events occurring as IEC are shed. Their rapid apoptotic response to detachment may facilitate the high turnover of cells and ensure homeostasis in the intestinal epithelium.
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PMID:Sequential and rapid activation of select caspases during apoptosis of normal intestinal epithelial cells. 969 13

Taxol, 1-beta-D-arabinofuranosylcytosine (ara-C), and etoposide induce apoptosis in HL-60 cells that is blocked by overexpression of Bcl-2 or Bcl-xL.A 60-amino acid "loop" domain of Bcl-2 and Bcl-xL that contains phosphorylation sites is known to negatively regulate their antiapoptotic function. In the present studies, Taxol-, ara-C-, or etoposide-induced apoptosis was examined in HL-60/Bcl-2delta and HL-60/Bcl-xLdelta cells that express the loop-deletional mutant cDNA constructs p19Bcl-2delta32-80 and p18Bcl-xLdelta26-83, respectively. This was compared with control HL-60/neo cells as well as HL-60/Bcl-2 and HL-60/Bcl-xL cells. The latter two cell lines overexpress full-length Bcl-2 and Bcl-xL, respectively. Immunoblot analyses showed that HL-60/neo and HL-60/Bcl-2delta cells express similar levels of p26Bcl-2. In contrast, as compared with HL-60/neo, HL-60/Bcl-xLdelta cells expressed significantly lower levels of p26Bcl-2. p29Bcl-xL and p21Bax levels were similar in all cell types. Exposure to etoposide (50 microM) or ara-C (100 microM) for 4 h induced apoptosis in HL-60/neo cells, but not in HL-60/Bcl-2, HL-60/Bcl-xL, HL-60/Bcl-2delta, or HL-60/Bcl-xLdelta cells. In contrast, Taxol treatment (500 nM for 24 h) triggered the molecular cascade of apoptosis, represented by the cytosolic increase of cytochrome c and poly(ADP-ribose) polymerase or the DNA fragmentation factor cleavage activity of caspase-3 in HL-60/neo cells as well as in HL-60/Bcl-xLdelta and HL-60/Bcl-2delta cells, but not in their counterparts overexpressing full-length Bcl-2 and Bcl-xL. Equal amounts of p26Bcl-2 were coimmunoprecipitated with apoptosis protease-activating factor 1 (APAF-1) in HL-60/neo and HL-60/Bcl-2delta cells, whereas a markedly higher level of p26Bcl-2 coimmunoprecipitated with APAF-1 in HL-60/Bcl-2 cells. In association with Taxol-induced apoptosis, the levels of Bcl-2 that were coimmunoprecipitated with APAF-1 declined in HL-60/neo and HL-60/Bcl-2delta cells. This was not observed in HL-60/Bcl-2 cells, in which Taxol-induced apoptosis was blocked. Previous studies have demonstrated that Taxol induces phosphorylation of Bcl-2 in association with Taxol-induced apoptosis of HL-60/neo cells. Immunoblot analysis demonstrated a Taxol-induced mobility shift of Bcl-2 but not p19Bcl-2delta. Taxol also increased [32P]Pi incorporation in p26Bcl-2, but not in p19Bcl-2delta or p18Bcl-xL. These findings indicate that the loop domain is necessary for the Taxol-induced mobility shift and phosphorylation of Bcl-2. Loop domain also seems to be necessary for the antiapoptotic effect of Bcl-2 against Taxol-induced apoptosis but not ara-C- or etoposide-induced apoptosis.
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PMID:"Loop" domain is necessary for taxol-induced mobility shift and phosphorylation of Bcl-2 as well as for inhibiting taxol-induced cytosolic accumulation of cytochrome c and apoptosis. 969 42


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