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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

CD95 antigen (also known as Fas or Apo-1) and Fas ligand play key roles in apoptosis of cells of the immune system, function as effector molecules of cytotoxic T lymphocytes, and function in the elimination of activated lymphocytes during the downregulation of the immune response. The critical roles of the Fas-Fas ligand system in apoptosis suggest that its inactivation may be involved in malignant transformation. We analyzed the expression of Fas antigen on adult T-cell leukemia (ATL) cells by flow cytometry and found that Fas antigen expression was absent in a case of ATL and markedly decreased in another case among 47 cases examined. Apoptosis could not be induced in the Fas-negative ATL cells by antibody against Fas antigen. Sequencing of reverse transcription-polymerase chain reaction products of the Fas genes in the Fas negative cells showed two types of aberrant transcripts: one had a 5-bp deletion and a 1-bp insertion in exon 2, and the other transcript lacked exon 4. These mutations caused the premature termination of both alleles, resulting in the loss of expression of surface Fas antigen. These aberrant transcripts were not detected in a nonleukemic B-cell line from the same patient. An RNase protection assay of the Fas gene showed mutations in 2 additional cases with Fas-positive ATL cells of 35 cases examined: 1 case lacked exon 4 and the other was a silent mutation. In the Fas antigen-negative case, leukemic cells were resistant to anticancer drugs in vivo, indicating that the loss of expression of Fas antigen may be associated with a poor response to anticancer drugs. Indeed, Fas-negative ATL cells were resistant to adriamycin-induced apoptosis in vitro, which is consistent with the finding that ATL in this case was resistant to chemotherapy. These findings indicate that mutation of the Fas gene may be associated with the progression of ATL and with resistance to anticancer drugs.
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PMID:Mutation of CD95 (Fas/Apo-1) gene in adult T-cell leukemia cells. 957 32

Cytokine-driven activation of hepatic stellate cells (HSC) in tissue injury and inflammation is a key pathogenetic event in liver fibrogenesis leading to an expanded pool of matrix producing myofibroblasts (MFB) which represent the transformed counterpart of HSC. We hypothesize that expansion of the pool of MFB might also be accomplished by modulation of apoptosis, which plays an opposite and complementary role to mitosis in the cellular homeostasis. We characterized the susceptibility of HSC in primary culture and of MFB in secondary culture to apoptosis induced by the soluble Fas ligand (sFasL) and related the effects to the expression levels of Fas (APO-1/CD95) and some major proapoptotic and contra-apoptotic protooncogenes. MFB showed a dose-dependent apoptotic reaction upon exposure to sFasL as evidenced by a strong increase of nucleosomal DNA fragments, loss of cellular DNA, positive TUNEL reaction, and annexin staining. The effect was found only if protein synthesis (cycloheximide) or RNA synthesis (actinomycin D) were arrested. HSC maintained for various times in primary culture were completely resistant to sFasL in combination with cycloheximide, but in late primary cultures (day 7 onward) an increasing susceptibility to sFasL-mediated apoptosis was developed. By semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis and alkaline phosphatase-anti-alkaline phosphatase staining Fas receptor was identified both in HSC and MFB at comparable expression levels. The expression of the contra-apoptotic protooncogenes bcl-2 and bcl-xl was found to be much stronger in early HSC than in late HSC and MFB as shown by ribonuclease protection assay. The expression of bcl-2 was additionally confirmed by semiquantitative RT-PCR and immunoblotting. Proapoptotic bax was found in comparable quantities at the RNA level in HSC and MFB but at the protein level MFB showed increased bax expression. It is concluded that transformation of HSC to MFB is paralleled by an increasing sensitivity to sFasL-mediated apoptosis, which might be related to a strong decrease of bcl-2 and bcl-xl expression, leading to a preponderance of proapoptotic gene expression in MFB. Modulation of apoptotic susceptibility of transforming HSC could be an important complementary pathway in the pathogenesis of fibrosis.
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PMID:Transformation-dependent susceptibility of rat hepatic stellate cells to apoptosis induced by soluble Fas ligand. 969 16

Interferon gamma (IFNgamma) induces apoptosis in purified human erythroid colony-forming cells (ECFC) and inhibits cell growth. Fas (APO-1; CD95) and Fas ligand (FasL) mediate apoptosis induced by IFNgamma, because Fas is significantly upregulated by IFNgamma, whereas Fas ligand is constitutively present in the ECFC and neutralization of FasL greatly reduces the apoptosis. Because conversion of caspases from their dormant proenzyme forms to active enzymes has a critical role in transducing a cascade leading to apoptosis, we performed further studies of the expression and activation of caspases in normal human and IFNgamma-treated day-6 ECFC to better understand the mechanism of IFNgamma action in producing this cell death. RNase protection assays showed that the caspase-1, -2, -6, -8, and -9 mRNAs were upregulated by IFNgamma, whereas the caspase-5 and -7 mRNAs were not increased. Western blots showed that FLICE/caspase-8 was upregulated and activated by 24 hours of incubation with IFNgamma. FADD was not similarly altered by incubation with IFNgamma. Western blots of ICE/caspase-1, which might be required for amplification of the initial FLICE activation signal, showed that pro-ICE expression significantly increased after treatment with IFNgamma for 24 hours and cleavage of pro-ICE also increased. CPP32/apopain/caspase-3, responsible for the proteolytic cleavage of poly (ADP) ribose polymerase (PARP), was also studied and treatment of ECFC with IFNgamma resulted in an increased concentration of caspase-3 by 24 hours and a clear induction of enzyme activation by 48 hours, which was identified by the appearance of its p17-kD peptide fragment. The cleavage of PARP was demonstrated by an obvious increase of the 89-kD PARP cleavage product, which was observed at almost the same time as caspase-3 activation in the IFNgamma-treated cells, whereas untreated ECFC showed little change. Peptide inhibitors of the caspase proteins, DEVD-fmk, DEVD-cho, YVAD-cho, and IETD-fmk, were incubated with the ECFC to obtain further evidence for the involvement of caspases in IFNgamma-induced apoptosis. The activation of FLICE/caspase-8 and CPP32/caspase-3 and cleavage of PARP clearly were inhibited, but the reduction of cell growth due to apoptosis, induced by IFNgamma, was only partially blocked by the presence of the inhibitors. These results indicate that IFNgamma acts on ECFC not only to upregulate Fas, but also to selectively upregulate caspases-1, -3, and -8, which are activated and produce apoptosis, whereas the concentrations of FasL and FADD are not demonstrably changed.
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PMID:Interferon gamma induces upregulation and activation of caspases 1, 3, and 8 to produce apoptosis in human erythroid progenitor cells. 1023 83

The Fas/FasL system mediates apoptosis in several different cell types, including T lymphocytes. Fas ligand (FasL), a 40-kD type II membrane protein also expressed in activated T cells, belongs to the tumor necrosis factor ligand family. We describe a new alternative splicing of mouse FasL, named FasL short (FasLs), cloned by reverse transcriptase-polymerase chain reaction. FasLs is encoded by part of exon 1 and part of exon 4 of FasL gene. The protein encoded by FasLs mRNA has a putative initiation code at position 756 and preserves the same reading frame as FasL, resulting in a short molecule lacking the intracellular, the transmembrane, and part of the extracellular domains. RNase protection and immunoprecipitation analysis showed that FasLs is expressed in nonactivated normal spleen cells and in hybridoma T cells and that it is upregulated upon activation by anti-CD3 monoclonal antibody (MoAb). Moreover, FasLs-transfected cells expressed soluble FasLs in the supernatant and became resistant to apoptosis induced by agonist anti-Fas MoAb. Thus, FasLs, a new alternative splicing of FasL, is involved in the regulation of Fas/FasL-mediated cell death.
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PMID:Cloning and expression of a short Fas ligand: A new alternatively spliced product of the mouse Fas ligand gene. 1055 56

Cytotoxic endoribonucleases (RNases) possess a potential for use in cancer therapy. However, the molecular determinants of RNase-induced cell death are not well understood. In this work, we identify such determinants of the cytotoxicity induced by onconase, an amphibian cytotoxic RNase. Onconase displayed a remarkable specificity for tRNA in vivo, leaving rRNA and mRNA apparently undamaged. Onconase-treated cells displayed apoptosis-associated cell blebbing, nuclear pyknosis and fragmentation (karyorrhexis), DNA fragmentation, and activation of caspase-3-like activity. The cytotoxic action of onconase correlated with inhibition of protein synthesis; however, we present evidence for the existence of a mechanism of onconase-induced apoptosis that is independent of inhibition of protein synthesis. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone (zVADfmk), at concentrations that completely prevent apoptosis and caspase activation induced by ligation of the death receptor Fas, had only a partial protective effect on onconase-induced cell death. The proapoptotic activity of the p53 tumor suppressor protein and the Fas ligand/Fas/Fas-associating protein with death domain (FADD)/caspase-8 proapoptotic cascade were not required for onconase-induced apoptosis. Procaspases-9, -3, and -7 were processed in onconase-treated cells, suggesting the involvement of the mitochondrial apoptotic machinery in onconase-induced apoptosis. However, the onconase-induced activation of the caspase-9/caspase-3 cascade correlated with atypically little release of cytochrome c from mitochondria. In turn, the low levels of cytochrome c released from mitochondria correlated with a lack of detectable translocation of proapoptotic Bax from the cytosol onto mitochondria in response to onconase. This suggests the possibility of involvement of a different, potentially Bax- and cytochrome c-independent mechanism of caspase-9 activation in onconase-treated cells. As one possible mechanism, we demonstrate that procaspase-9 is released from mitochondria in onconase-treated cells. A detailed understanding of the molecular determinants of the cytotoxic action of onconase could provide means of positive or negative therapeutic modulation of the activity of this potent anticancer agent.
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PMID:Molecular determinants of apoptosis induced by the cytotoxic ribonuclease onconase: evidence for cytotoxic mechanisms different from inhibition of protein synthesis. 1076 89

The present study investigates the molecular apoptotic pathway in germ cells following acute ischemia of the rat testis. Rats were subjected to ischemia-inducing torsion and testes were harvested after reperfusion. Apoptotic cells were identified with an antibody to single-stranded DNA. Seminiferous tubule RNA was examined by RNase protection assay or by reverse transcriptase-polymerase chain reaction (RT-PCR) for the presence and regulation of apoptotic molecules. Proteins from seminiferous tubules were used for Western blot analysis of cytochrome c. Germ cell apoptosis was maximal at 24 h after repair of torsion. Germ cells in stages II-III of the seminiferous epithelium cycle were the predominant early responders. The RNase protection assays revealed that Bcl-X(L) was the prominent mRNA species. Caspases 1, 2, 3, and Bax mRNA were consistently upregulated; however, the time of upregulation after torsion was variable. The Bcl-X(L) and Bcl-X(S) mRNAs were less consistently upregulated and there was no evidence for upregulation of Fas or Bcl-2. Fas ligand (FasL) was not detected by RNase protection assay, but RT-PCR revealed a significant increase in FasL expression 4 h after the repair of torsion. Western blot analysis for cytochrome c release demonstrated a significant increase 4 h after the repair of torsion. Results suggest that germ cell apoptosis following ischemia/reperfusion of the rat testis is initiated through the mitochondria-associated molecule Bax as well as Fas-FasL interactions.
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PMID:Molecular pathway of germ cell apoptosis following ischemia/reperfusion of the rat testis. 1105 53

Retinal pigment epithelial (RPE) cells have been proposed to play a part in maintaining the eye as an immune privileged organ. However, our knowledge of the implicated mechanism is still sparse. Fas ligand (FasL) expression of RPE cells is generally recognized to be essential for the immune privilege of the eye, but due to contradictory published results, it is unclear whether RPE cells express this molecule. The purpose of this study was to investigate the expression of FasL in RPE cells in vitro and in vivo. Cultured human fetal and adult RPE cells were examined by flow cytometry, Western blotting, RT-PCR and RNase Protection assay for FasL expression. Additionally, sections of ocular tissue were stained for FasL by immunohistochemistry. None of the used methods indicated FasL expression in cultured fetal or adult RPE cells of various passages. However, RPE cells in vivo, as judged from tissue sections, were positive for FasL, indicating a discrepancy between RPE cells in vitro and in vivo with regard to this molecule.
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PMID:Lack of FasL expression in cultured human retinal pigment epithelial cells. 1115 Aug 51

Fumonisin B(1) (FB(1)), a carcinogenic mycotoxin produced primarily by fungus Fusarium verticillioides in corn, causes several fatal animal diseases. In mice, liver is the primary site of its toxicity. Our previous study showed that maximum induction of interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) was observed at 4 and 8 h, respectively, after an acute po FB(1) treatment. To further investigate the time-related induction of other cytokines and genes involved in apoptosis signaling, male BALB/c mice were administered orally with either saline or 25 mg/kg of FB(1) and sampled 4 or 8 h after treatment. Expression of various genes was analyzed by ribonuclease protection assay. FB(1) treatment caused increased expression of TNFalpha and interleukin (IL)-1beta in both liver and kidney, whereas IL-1alpha and IL-1 receptor antagonist (IL-1Ra) expression was induced only in the liver. Expression of TNFalpha signaling molecules, TNF receptor 55 and receptor interacting protein, was increased in liver and kidney after FB(1) treatment. Caspase 8 expression was increased only in liver with no changes in kidney with FB(1). FB(1) treatment induced expression of Fas in liver and kidney with no alterations in Fas signaling molecules, Fas ligand, Fas-associated death domain and Fas-associated protein factor. Treatment of mice with FB(1) increased the expression of B-Myc, c-Myc and Max, oncogenic transcription factors in the kidney. FB(1) toxicity caused induction of cytokine network in liver with involvement of TNFalpha signaling pathway. Increased expression of caspase 8 involved in the TNFalpha signaling pathway may contribute to the apoptosis, whereas IL-1Ra induction could contribute to the proliferating effects observed in FB(1) toxicity.
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PMID:Fumonisin B(1)-induced alterations in cytokine expression and apoptosis signaling genes in mouse liver and kidney after an acute exposure. 1188 48

NO is a potent cellular mediator which has been shown to modulate several immune mechanisms. Using human T lymphocytes as responder cells in a primary mixed lymphocyte reaction, we demonstrated that, at the initiation of the culture, exogenously provided NO via sodium nitroprusside, in non-toxic concentrations, inhibited both allogeneic proliferative and primary cytotoxic responses in a dose-dependent manner. In contrast, it had no effect on the cytotoxic activity of established human TCR (alpha)beta and TCR (gamma)delta cytotoxic T lymphocyte (CTL) clones. The NO inhibitory effect on primary cytotoxic T cell response correlates with inhibition of T cell blastogenesis. Furthermore, under our stimulation conditions, NO induced an inhibition of IL-2 production, an alteration of IL-2R(alpha) expression, and a down-regulation of NF-AT translocation in CD4(+) and CD8(+)allostimulated T cells. Furthermore, we demonstrate that the inhibition of allospecific CTL activity by the NO donor was at least in part related to an inhibition of granzyme B and Fas ligand transcription as revealed respectively by RNase protection and RT-PCR analysis. These results suggest that NO may function to fine tune human CD3(+) T cell activation and subsequent CTL generation.
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PMID:Analysis of the mechanisms of human cytotoxic T lymphocyte response inhibition by NO. 1235 82

Large granular lymphocyte (LGL) leukemia is a lymphoproliferative disease often associated with autoimmune disorders such as rheumatoid arthritis. High levels of soluble Fas ligand have been implicated in development of chronic neutropenia. However, a comprehensive analysis of constitutive chemokine and lymphokine production in LGL leukemia has not previously been reported. Here, we utilized RNase protection assays and enzyme-linked immunosorbent assays (ELISAs) to address this question. RANTES, IL-8, MIP-1alpha, MIP-1beta, IL-1beta, IL-10, IL-12 p35, IL-18, IFN-gamma and IL-1Ra were the cytokine transcripts expressed in elevated levels from RNA of peripheral blood mononuclear cells of LGL leukemia patients. Confirmatory ELISAs indicated that sera from LGL leukemia patients have elevated levels of RANTES, MIP-1beta, IL-18, and to a lesser extent IL-8 and IL-1Ra. This pattern of cytokine upregulation is similar to that seen in some chronic infections or in autoimmune diseases, thus characterizing LGL leukemia as a proinflammatory disorder.
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PMID:Constitutive production of proinflammatory cytokines RANTES, MIP-1beta and IL-18 characterizes LGL leukemia. 1564 40


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