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)

Multidrug resistance mediated by the drug efflux protein, P-glycoprotein (P-gp), is one mechanism that tumor cells use to escape death induced by chemotherapeutic agents. However, the mechanism by which P-gp confers resistance to a large variety of structurally diverse molecules has remained elusive. In this study, classical multidrug resistant human CEM and K562 tumor cell lines expressing high levels of P-gp were less sensitive to multiple forms of caspase-dependent cell death, including that mediated by cytotoxic drugs and ligation of Fas. The DNA fragmentation and membrane damage inflicted by these stimuli were defined as caspase dependent by various soluble peptide fluoromethylketone caspase inhibitors. Inhibition of P-gp function by the anti-P-gp mAb MRK-16 or verapamil could reverse resistance to these forms of cell death. Inhibition of P-gp function also enhanced drug or Fas-mediated activation of caspase-3 in drug-resistant CEM cells. By contrast, caspase-independent cell death events in the same cells, including those mediated by pore-forming proteins or intact NK cells, were not affected by P-gp expression. These observations suggest that, in addition to effluxing drugs, P-gp may play a specific role in regulating some caspase-dependent apoptotic pathways.
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PMID:The drug efflux protein, P-glycoprotein, additionally protects drug-resistant tumor cells from multiple forms of caspase-dependent apoptosis. 961 32

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

Fas-mediated apoptosis is one of the major death processes of hepatocytes in liver diseases. Although compensatory regeneration occurs during liver injury, it has not been determined whether regenerating hepatocytes die by the same apoptotic process as quiescent hepatocytes. To clarify this issue, the hepatocyte apoptotic process, after injection of agonistic anti-mouse Fas, was compared between sham-operated mice and two-thirds partially hepatectomized mice. The onset of hepatocyte apoptosis was retarded in hepatectomized mice, as evidenced by both morphological and biochemical observations, resulting in significantly prolonged animal survival. Flow cytometric analysis revealed similar levels of Fas expression on hepatocytes between hepatectomized mice and sham-operated mice; however, the activation of liver caspase-3-like protease after Fas stimulation was suppressed in hepatectomized mice, whereas pro-caspase-3 expression did not change with or without hepatectomy. Anti-tumor necrosis factor alfa (TNF alpha), when administered before hepatectomy, partially reversed suppression of caspase-3-like activity after Fas stimulation. Furthermore, the injection of TNF alpha into untreated mice suppressed caspase-3-like activity and prolonged animal survival after Fas stimulation. These results indicate that Fas-signaling events at the level or upstream of caspase-3-like protease are suppressed during liver regeneration, resulting in delayed hepatocyte apoptosis, and also that TNF alpha acts as one of the protective factors against Fas-mediated hepatocyte apoptosis.
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PMID:Delayed Fas-mediated hepatocyte apoptosis during liver regeneration in mice: hepatoprotective role of TNF alpha. 962 Mar 38

Dysregulation of apoptosis leading to reduced DNA repair capacity, increased DNA mutation, and chromosomal instability is one of the pathological mechanisms associated with aging. Rothmund-Thomson syndrome (RTS) is a human genetic disease characterized by several features of premature aging. Although the genetic defect has not been identified, defects in DNA repair capacity have been implicated in the pathogenesis of this disease. To determine whether dysregulation of apoptosis is associated with the pathogenesis of RTS symptoms, we investigated the sensitivity of a lymphoblastoid cell line--derived from a young (10-year-old) individual with RTS--to cell death induced by anti-Fas antibody (clone: CH-11). Cell lines derived from a normal young (14-year-old) individual and a normal aged (79-year-old) individual were used as controls. Treatment with CH-11 (500 ng/ml) resulted in significantly decreased cell viability in the RTS cell line (42.4% +/- 4.2%) and that derived from the aged individual (47.3% +/- 9.2%) as compared to the normal young cell line (66.9% +/- 7.0%). The concentrations of CH-11 required to induce 50% cell death in the RTS (IC50, 890 ng/ml) and that derived from the aged individual (IC50, 3640 ng/ml) were lower than that of the control young cell line (IC50 > 10(5) ng/ml). The lower viability was due to increased susceptibility to apoptosis to CH-11 in the RTS (59.0% +/- 2.0%) compared to that in the normal young cell line (40.9% +/- 0.9%) as shown by 7-amino-actinomycin D (7-AAD) staining (p < .005). Treatment of the RTS cell line with acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), a specific inhibitor of caspase-3, significantly increased the cell viability after CH-11 treatment (75.9% +/- 2.2%). Taken together, these results provide the first evidence to show that RTS lymphoblastoid has an increased sensitivity to cell death mediated by Fas and that inhibition of caspase-3 activity may be a potential target in reversing the sensitivity of RTS cells to Fas-mediated apoptosis in vitro.
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PMID:Increased susceptibility to apoptosis induced by anti-Fas antibody in a Rothmund-Thomson syndrome lymphoblastoid cell line. 962 28

To dissect intracellular pathways involved in B cell Ag receptor (BCR)-mediated and Fas-induced human B cell death, we isolated clones of the Burkitt lymphoma cell line Ramos with different apoptosis sensitivities. Selection for sensitivity to Fas-induced apoptosis also selected for clones with enhanced BCR death sensitivity and vice versa. In contrast, clones resistant to Fas-mediated apoptosis could still undergo BCR-induced cell death. Based on the functional phenotypes of these clones, we hypothesized that both receptor-induced apoptosis pathways are initially distinct but may eventually converge. Indeed, ligation of both Fas and BCR resulted in cleavage of the IL-1beta-converting enzyme/Ced-3-like protease caspase 3 and its substrates Ac-Asp-Glu-Val-Asp-aldehyde and poly(ADP-ribose) polymerase. Markedly, qualitative differences in the caspase 3 cleavage pattern induced by Fas or BCR ligation were observed; whereas Fas ligation generated caspase 3 cleavage products of 19/20 and 17 kDa, only the latter cleavage product was found upon BCR cross-linking. The caspase inhibitor Val-Ala-Asp-fluoromethylketone blocked both Fas- and BCR-mediated apoptosis, but differentially affected caspase 3 cleavage induced by either stimulus. Finally, overexpression of a Fas-associated death domain (FADD) dominant-negative mutant protein was found to inhibit Fas-induced apoptosis but not BCR-induced apoptosis. Together our findings imply that Fas and BCR couple, via FADD-dependent and FADD-independent mechanisms, respectively, to distinct proteases upstream of caspase 3.
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PMID:Dissection of pathways leading to antigen receptor-induced and Fas/CD95-induced apoptosis in human B cells. 963 25

A20 zinc finger protein is a product of a cytokine-induced primary response gene. It functions as a negative regulator of the tumor necrosis factor (TNF) inhibiting both TNF-mediated apoptosis and activation of transcription factors. We demonstrated that A20 overexpression blocks early TNF-induced signaling events including the generation of free radicals, the fall in mitochondrial transmembrane potential (delta psi(m)), and the activation of caspase-3-like apoptotic proteases. General inhibitor of caspases, cow pox virus-derived CrmA, also inhibited TNF-induced mitochondrial changes indicating that early caspase activation occurs upstream from mitochondrial changes. Interestingly, changes in mitochondrial function or induction of caspase-3-like activity induced by anti-Fas or doxorubicin were not inhibited by A20. The data show that A20 is a specific inhibitor of TNF signaling and acts upstream of INF-induced free radical formation, fall in mitochondrial transmembrane potential (delta psi(m)), and activation of caspase-3-like proteases.
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PMID:TNF-induced mitochondrial changes and activation of apoptotic proteases are inhibited by A20. 965 22

Upon transforming growth factor-beta (TGF-beta) treatment, Ramos cells, a B-cell lymphoma cell line, undergo apoptosis, as measured by annexin V labeling, DNA fragmentation, and propidium iodide staining. Apoptosis could be observed by 24 h after TGF-beta exposure and occurred before the development of a significant blockage of cell cycle progression. TGF-beta-mediated apoptosis was also accompanied by a strong induction of caspase-3 subfamily activity. Incubation of cells with the caspase inhibitor Z-VAD.FMK at 20 microM, but not at 10 microM, prevented TGF-beta-induced apoptosis from occurring. By comparison, caspase-3 subfamily activity was 87% inhibited at 10 microM Z-VAD.FMK and completely inhibited at 20 microM. Because of TGF-beta's well-established role of regulating gene transcription, the mRNA levels for proteins associated with apoptosis (Fas- and Fas-associated proteins, Bcl-2 family members, IAP proteins, and I kappa B) were also studied. After 24 h of TGF-beta treatment, the most significant mRNA changes occurred with Bcl-XL (two-fold decrease) and Bik (twofold increase). TGF-beta treatment also resulted after 48 h in a fivefold decrease in Bcl-XL protein levels, based on immunoblotting analysis. Therefore, TGF-beta-mediated apoptosis involves the activation of caspases. In addition, TGF-beta transcriptionally regulates Bcl-2 family members, Bcl-XL and Bik, to further influence the apoptosis process.
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PMID:Transforming growth factor-beta-mediated apoptosis in the Ramos B-lymphoma cell line is accompanied by caspase activation and Bcl-XL downregulation. 966 22

Fas-mediated apoptosis is an important regulatory mechanism for the development of T-cells and prevention of oncogenesis. Here, we establish Chinese hamster ovary (CHO) cell lines which stably express Fas antigen, and analyzed apoptosis induced by anti-Fas IgM. While Fas-transfected hamster cells did not undergo apoptosis when stimulated with anti-Fas antibody in the presence of medium containing 10% serum, in reduced serum concentrations, anti-Fas antibody caused these cells to round up and detach from the culture dish. Analysis of the DNA content by a flow cytometry demonstrated a significant increase of cells with sub-G1 amount of DNA upon Fas stimulation in the low serum concentrations. The increase in the number of apoptosis cells was inhibited by an apopain (CPP32, caspase 3) inhibitor or insulin-like growth factor-I. In contrast, apoptosis in a Fas-transfected mouse T-cell line occurred in the presence of 10% serum. these results suggest that factors including insulin-like growth factor-I in fetal bovine serum protect CHO cells from apopain-dependent apoptosis mediated by Fas-antigen stimulation.
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PMID:Alleviation of apoptosis by serum in Chinese hamster ovary cells ectopically expressing human Fas antigen. 966 63

Caspase-mediated proteolysis is a critical and central element of the apoptotic process; therefore, it is important to identify the downstream molecular targets of caspases. We established a method for cloning the genes of caspase substrates by two major modifications of the yeast two-hybrid system: (i) both large and small subunits of active caspases were expressed in yeast under ADH1 promoters and the small subunit was fused to the LexA DNA-binding domain; and (ii) a point mutation was introduced that substituted serine for the active site cysteine and thereby prevented proteolytic cleavage of the substrates, possibly stabilizing the enzyme-substrate complexes in yeast. After screening a mouse embryo cDNA expression library by using the bait plasmid for caspase-3, we obtained 13 clones that encoded proteins binding to caspase-3, and showed that 10 clones including gelsolin, an actin-regulatory protein implicated in apoptosis, were cleaved by recombinant caspase-3 in vitro. Using the same bait, we also isolated human gelsolin cDNA from a human thymus cDNA expression library. We showed that human gelsolin was cleaved during Fas-mediated apoptosis in vivo and that the caspase-3 cleavage site of human gelsolin was at D352 of DQTD352G, findings consistent with previous observations on murine gelsolin. In addition, we ascribed the antiapoptotic activity of gelsolin (which we previously reported) to prevention of a step leading to cytochrome c release from the mitochondria into the cytosol. Our results indicate that this cloning method is useful for identification of the substrates of caspases and possibly also of other enzymes.
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PMID:A cloning method for caspase substrates that uses the yeast two-hybrid system: cloning of the antiapoptotic gene gelsolin. 967 12

Cytototoxic T lymphocyte-induced apoptosis can occur either through the directed exocytosis of granzyme B and perforin or via ligation of Fas. Both pathways involve the activation of a family of cysteine proteinases, the caspases, that cleave substrates at aspartic acid and are themselves activated by cleavage at internal aspartate residues. Fas recruits caspase 8, which initiates the death program through the subsequent activation of caspase 3. Granzyme B can process both caspase 8 and 3 in vitro, suggesting that both Fas and granzyme B access the apoptotic program in the same way. Here we demonstrate that although the two mechanisms are similar, the events that lead to activation of caspase 3 can be distinguished in vivo on the basis of their sensitivities to both pharmacological and virus-encoded caspase inhibitors. In cytotoxic T lymphocytes-mediated death the initial cleavage event on caspase 3 is insensitive to benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (zVAD-fmk) inhibition in both mouse and human systems. During Fas-mediated death, however, activation of caspase 3 is completely inhibited to zVAD-fmk. In addition, the viral serpin SPI-2, a homologue of cytokine response modifier A (crmA), is an effective inhibitor of the Fas but not the granzyme pathway. Our results demonstrate that whereas Fas-mediated activation of caspase 3 requires an upstream caspase activity that is zVAD-fmk-sensitive, the initial cleavage of caspase 3 during granule-mediated cell death is insensitive to zVAD-fmk, suggesting that caspase 3 is cleaved directly by granzyme B in vivo.
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PMID:Cytotoxic T lymphocyte-assisted suicide. Caspase 3 activation is primarily the result of the direct action of granzyme B. 969 85


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