Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:A9QXG9 (bcl-2)
 7,497 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cholangiocytes are the target of a group of liver diseases termed the cholangiopathies that include conditions characterized by periductal inflammation and cholangiocyte apoptosis. Because inflammation is associated with oxidative stress, we developed the hypothesis that cholangiocytes exposed to oxidative stress will be depleted of endogenous cytoprotective molecules, leading to cholangiocyte apoptosis. To begin to test this hypothesis, we explored the relationships among glutathione (GSH) depletion, expression of Bcl-2 (a protooncogene that inhibits apoptosis), and apoptosis in a nonmalignant human cholangiocyte cell line. Monolayers of human bile duct epithelial cells, derived from normal liver and immortalized by SV40 transformation, were depleted of GSH using buthionine sulfoximine (BSO). Bcl-2 expression was assessed by quantitative immunoblot analysis, and apoptosis quantified by fluorescence microscopy using the DNA binding dye 4', 6'-diamidino-2-phenylindole. Bcl-2 message was assessed by RNase protection assay, and Bcl-2 protein synthesis and half-life by pulse-chase analysis. Exposure of human cholangiocytes in culture to BSO reduced GSH levels by 93 +/- 3% (P < 0.01). In addition, treatment of cholangiocytes with BSO reduced Bcl-2 levels by 87 +/- 2% (P < 0.01) and was associated with a time-dependent increase in the number of cells undergoing apoptosis; approximately 11 +/- 1% of cultured cells demonstrated morphological changes of apoptosis by 72 h compared with 1.5 +/- 0.1% in untreated cholangiocytes (P < 0. 01). Maintenance of GSH levels by addition of glutathione ethyl ester in the presence of BSO blocked the BSO-associated increase in apoptosis in BSO-treated cholangiocytes and also prevented the decrease in Bcl-2 protein. BSO treatment of cholangiocytes did not change steady-state levels of bcl-2 mRNA or Bcl-2 protein synthesis. However, Bcl-2 protein half-life decreased 57% in BSO-treated vs. untreated cells. Our results using a human cholangiocyte cell line demonstrate that reduction in the cellular levels of an antioxidant such as GSH results in increased degradation of Bcl-2 protein and an increase in apoptosis. These data provide a mechanistic link between the consequences of oxidative stress and cholangiocyte apoptosis, an observation that may be important in the pathogenesis of the inflammatory cholangiopathies.
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PMID:Glutathione depletion is associated with decreased Bcl-2 expression and increased apoptosis in cholangiocytes. 975 6

Incubation of mock-transfected PC12 rat pheochromocytoma cells (PC12) for 2 h with increasing concentrations of glutamate caused progressive loss of viability (e.g., 67% with 15 mM glutamate). In contrast, the viability of bcl-2-transfected cells (PC12/bcl-2) was unaffected by glutamate. Neither PC12 nor PC12/bcl-2 cells showed a significant incidence of apoptosis in response to glutamate. Conventional phospholipid analysis by high-performance TLC and phosphorous determination showed no significant changes in the phospholipid composition of either cell line incubated with </=15 mM glutamate. Phospholipid peroxidation was quantified in the cells using our newly developed method based on fluorescence-HPLC analysis of metabolically incorporated oxidation-sensitive and fluorescent fatty acid, cis-parinaric acid. Unlike previous studies that measured total phospholipid oxidation, this novel technology permitted quantitation of oxidative stress in different classes of labeled phospholipids (the amount of labeled phospholipids in the cells did not exceed 1% of total phospholipids). Significant peroxidation of phosphatidylcholine and phosphatidylethanolamine occurred in PC12 cells treated with >5 mM glutamate. The peroxyl radical initiator 2,2'-azobis(2,4-dimethylvaleronitrile) caused a pronounced loss of all major phospholipid classes in PC12 cells, but no loss of cell viability. No phospholipid peroxidation was detected in PC12/bcl-2 cells incubated with </=15 mM glutamate or with 2, 2'-azobis(2,4-dimethylvaleronitrile). These results directly demonstrate that peroxidation of membrane phospholipids is not responsible for the cytotoxicity of glutamate in PC12 cells. Total cellular thiol, protein thiol and GSH reserves were quantified by a previously described electron paramagnetic resonance spectrometric method. Total thiols were ca. 1.5-fold greater in PC12/bcl-2 than in PC12 cells. Glutamate (</=5 mM) caused a progressive and equally significant decrease in total thiols and GSH in both PC12 and PC12/bcl-2 cells. High glutamate concentrations caused oxidation of protein sulfhydryls in PC12 cells, but not in PC12/bcl-2 cells. The results suggest that the changes in cellular milieu caused by bcl-2 gene transfection protect PC12 cells from the toxic effects of glutamate in a manner consistent with prevention of protein sulfhydryl oxidation.
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PMID:Glutamate-induced cytotoxicity in PC12 pheochromocytoma cells: role of oxidation of phospholipids, glutathione and protein sulfhydryls revealed by bcl-2 transfection. 975 62

The glutathione (GSH) level of CC531 rat colorectal cancer cells is readily decreased by exposure to buthionine sulfoximine (BSO), an inhibitor of GSH synthesis; at 25 microM BSO, these cells died in a non-apoptotic fashion. By continuous exposure of CC531 cells to increasing concentrations of BSO, we obtained a BSO-resistant cell line (CCBR25) that was 50 times more resistant to BSO than the parental cell line. Whereas the GSH content of CCBR25 and CC531 cells was similar, the former contained a much higher level of the Bcl-2 protein. After stable transfection of CC531 cells with the human bcl-2 gene, the resulting Bcl-2-overexpressing cell line appeared to be 9 times more resistant to BSO than the parental cell line. These findings suggest that the Bcl-2 protein offers resistance against the cytotoxic effect of severe GSH depletion.
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PMID:Development of resistance to glutathione depletion-induced cell death in CC531 colon carcinoma cells: association with increased expression of bcl-2. 1079 52

Several organochlorinated pesticides including DDT, PCBs and dieldrin have been reported to cause immune suppression and increase susceptibility to infection in animals. Often this manifestation is accompanied by atrophy of major lymphoid organs. It has been suggested that increased apoptotic cell death leading to altered T-B cell ratios, and loss of regulatory cells in critical numbers leads to perturbations in immune function. The major objective of our study was to define the mechanism by which endosulfan, an organochlorinated pesticide, induces human T-cell death using Jurkat, a human T-cell leukemic cell line, as an in vitro model. We exposed Jurkat cells to varying concentrations of endosulfan for 0-48 h and analyzed biochemical and molecular features characteristic of T-cell apoptosis. Endosulfan lowered cell viability and inhibited cell growth in a dose- and time-dependent manner. DAPI staining was used to enumerate apoptotic cells and we observed that endosulfan at 10-200 microM induced a significant percentage of cells to undergo apoptotic cell death. At 48 h, more than 90% cells were apoptotic with 50 microM of endosulfan. We confirmed these observations using both DNA fragmentation and annexin-V binding assays. It is now widely being accepted that mitochondria undergo major changes early during the apoptotic process. We examined mitochondrial transmembrane potential (deltapsim) in endosulfan treated cells to understand the role of the mitochondria in T-cell apoptosis. Within 30 min of chemical exposure, a significant percentage of cells exhibited a decreased incorporation of DiOC6(3), a cationic lipophilic dye into mitochondria indicating the disruption of deltapsim. This drop in deltapsim was both dose- and time-dependent and correlated well with other parameters of apoptosis. We also examined whether this occurred by the down regulation of bcl-2 protein expression that is likely to increase the susceptibility of Jurkat cells to endosulfan toxicity. Paradoxically, the intracellular expression of bcl-2 protein was elevated in a dose dependent manner suggesting endosulfan-induced apoptosis occurred by a non-bcl-2 pathway. Based on these data, as well as those reported elsewhere, we propose the following sequence of events to account for T-cell apoptosis induced by endosulfan: uncoupling of oxidative phosphorylation --> excess ROS production --> GSH depletion --> oxidative stress --> disruption of deltapsim --> release of cytochrome C and other apoptosis related proteins to cytosol --> apoptosis. This study reports for the first time that endosulfan can induce apoptosis in a human T-cell leukemic cell line which may have direct relevance to loss of T cells and thymocytes in vivo. Furthermore, our data strongly support a role of mitochondrial dysfunction and oxidative stress in endosulfan toxicity.
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PMID:Evidence for the induction of apoptosis by endosulfan in a human T-cell leukemic line. 1082 22

Cellular production of reactive oxygen species (ROS) has been implicated as an important mechanism of chemical teratogenesis and developmental toxicity. Unfortunately, the lack of relevant model systems has precluded studies targeting the role of ROS in human teratogenesis and prenatal toxicity. In the current study, we have used cultured precision human prenatal liver slices to study the effects of the human teratogen phenytoin (diphenylhydantoin; Dilantin) on cell toxicity, glutathione redox status, and steady-state mRNA expression of a panel of oxidative stress-related biomarker genes. The biomarker genes analyzed were p53, bcl-2, alpha class glutathione S-transferases isozymes A1 and A4 (hGSTA1 and hGSTA4), and the catalytic subunit of gamma-glutamylcysteine synthetase (gammaGCS-HS). Liver slices (200 microm) were prepared from second trimester prenatal livers and cultured in the presence of 0, 250 microM, and 1000 microM phenytoin for 18 h. Exposure to 1000 microM phenytoin elicited 41% and 34% reductions in slice intracellular potassium and reduced glutathione (GSH) concentrations, respectively. The reduction in slice GSH concentrations at 1000 microM phenytoin was accompanied by a 2.2-fold increase in the percentage of total slice glutathione consisting of GSSG, and a 3.9-fold increase in hGSTA1 steady-state mRNA expression. Exposure to 250 microM or 1000 microM phenytoin also elicited a relatively minor (less than 2-fold) but significant increase in p53 steady-state mRNA expression. In contrast, the steady-state levels of gammaGCS-HS, hGSTA4, and bcl-2 mRNAs were not affected by phenytoin exposure. Our findings in a relevant human model system are supportive of a protective role of GSH and hGSTA1 against phenytoin toxicity and teratogenesis. These studies also demonstrate the utility of using cultured human prenatal liver slices as a relevant tool for developmental toxicology studies.
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PMID:Effects of phenytoin on glutathione status and oxidative stress biomarker gene mRNA levels in cultured precision human liver slices. 1113 51

Oxidative stress is one of the major causes of cellular injury. Various reactive oxygen (ROS) and nitrogen (RNS) species such as superoxide, hydroxyl radical, peroxynitrite, and nitric oxide are involved in the manifestations of different types of organ toxicity and the resultant syndromes, symptoms, or diseases. Hypothermic conditions have been reported to reduce the oxidative stress in various in vitro and in vivo studies. In the present study, we sought to determine the effect of lowered temperatures on oxidative stress-induced cell death in Chinese hamster ovary (CHO) cells. We also investigated the oxidative stress-induced alterations in the expression of anti-apoptotic protein, bcl-2, in CHO cells at lowered temperatures. CHO cells were incubated at four different temperatures of 30, 32, 35, and 37 degrees C (control temperature) from 1 to 4 d. In another set, the cells were incubated with 100 microM hydrogen peroxide (H(2)O(2)) for 30 min before harvesting at different time points. The cells were harvested at 1, 2, 3, and 4 d. Cell survival was significantly higher at 30 degrees C as compared to 37 degrees C over 4 d of incubation. In cells incubated with H(2)O(2), significantly higher cell viability was observed at lower temperatures as compared to the cells incubated at 37 degrees C. The activity of glutathione peroxidase (GSH-Px) also increased significantly at lower temperatures. Lowered temperature also provided a significant increase in the expression of anti-apoptotic protein, bcl-2 after 4 d of incubation. These data suggest that hypothermic conditions lowers the risk of oxidative stress-induced cellular damage and programmed cell death by increasing the activity of GSH-Px and by the induction in the expression of the anti-apoptotic protein, bcl-2.
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PMID:Hypothermia enhances bcl-2 expression and protects against oxidative stress-induced cell death in Chinese hamster ovary cells. 1146 79

L-glutamine (Gln) sensitizes tumor cells to tumor necrosis factor (TNF)-alpha-induced cytotoxicity. The type and mechanism of cell death induced by TNF-alpha was studied in Ehrlich ascites tumor (EAT)-bearing mice fed a Gln-enriched diet (GED; where 30% of the total dietary nitrogen was from Gln). A high rate of Gln oxidation promotes a selective depletion of mitochondrial glutathione (mtGSH) content to approximately 58% of the level found in tumor mitochondria of mice fed a nutritionally complete elemental diet (standard diet, SD). The mechanism of mtGSH depletion involves a glutamate-induced inhibition of GSH transport from the cytosol into mitochondria. The increase in reactive oxygen intermediates (ROIs) production induced by TNF-alpha further depletes mtGSH to approximately 35% of control values, which associates with a decrease in the mitochondrial transmembrane potential (MMP), and elicits mitochondrial membrane permeabilization and release of cytochrome c. Mitochondrial membrane permeabilization was also found in intact tumor cells cultured with a Gln-enriched medium under conditions of buthionine sulfoximine (BSO)-induced selective GSH synthesis inhibition. Enforced expression of the bcl-2 gene in tumor cells could not avoid the glutamine- and TNF-alpha-induced cell death under conditions of mtGSH depletion. However, addition of GSH ester, which delivers free intracellular GSH and increases mtGSH levels, preserved cell viability. These findings show that glutamine oxidation and TNF-alpha, by causing a change in the glutathione redox status within tumor mitochondria, activates the molecular mechanism of apoptotic cell death.
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PMID:Glutamine potentiates TNF-alpha-induced tumor cytotoxicity. 1152 49

Reduced cell proliferation and increased levels of cellular glutathione (GSH) are characteristic for cells that overexpress the anti-apoptotic Bcl-2 protein. We investigated the influence of various Bcl-2 domains on both these characteristics. Rat CC531 colorectal cancer cells were stably transfected with the human bcl-2 gene (CCbcl2 cells) or with bcl-2 gene constructs missing a coding sequence for a func-tional domain, BH1 (CCDeltaBH1 cells), BH3 (CCDeltaBH3 cells), BH4 (CCDeltaBH4 cells) or the transmembrane region (CCDeltaTM cells). We measured GSH levels in exponentially and confluent growing bcl-2-transfected cell populations. The fraction of S-phase cells during exponential growth was significantly reduced in CCbcl2, CCDeltaBH1, CCDeltaBH3, and CCDeltaTM cells compared with parental CC531, neo-transfected CC531 and CCDeltaBH4 cells. GSH levels in these bcl-2 transfectants were significantly higher than in the parental line measured at 50% confluence; at 100% confluence they reached a similar level as found in parental cells. Independently from the presence of BH1, BH3 or TM domains, overexpression of Bcl-2 reduces cellular proliferation under conditions of increased GSH levels. This apparent link is lost in CCDeltaBH4 cells; these cells are not reduced in cellular proliferation and harbour significantly higher GSH levels than found in the other transfectants. Studies on the subcellular localization revealed an extremely low expression of the Bcl-2 protein lacking the N-terminal BH4 domain in nuclear fractions. Nuclear translocation of Bcl-2 requires the presence of the BH4 domain and seems prominent in reducing cellular proliferation.
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PMID:The role of various Bcl-2 domains in the anti-proliferative effect and modulation of cellular glutathione levels: a prominent role for the BH4 domain. 1255 59

A substantial body of data from clinical and laboratory studies indicates that reactive oxygen intermediates are implicated in the pathogenesis of diverse human diseases, including cancer, diabetes, and neurodegenerative disorders. Oxidative stress induced by reactive oxygen intermediates often causes cell death via apoptosis that is regulated by a plenty of functional genes and their protein products. Bcl-2, which is an integral intermitochondrial membrane protein, blocks apoptosis induced by a wide array of death signals. In spite of extensive research, the molecular milieu that characterizes the antiapoptotic function of Bcl-2 is complex and not fully identified. Recently, there are several lines of evidence that Bcl-2 functions via antioxidant pathways to prevent apoptosis. Thus, bcl-2-overexpressing cells exhibit elevated expression of antioxidant enzymes and higher levels of cellular GSH compared with the control cells transfected with the vector alone. There has been increasing evidence supporting that the redox-sensitive transcription factor nuclear factor kappaB regulates the activity and/or expression of antioxidative and antiapoptotic target genes and promotes cell survival against oxidative cell death. This commentary focuses on the antioxidative functions of Bcl-2 and underlying molecular mechanisms in relation to its antiapoptotic property. The role of Bcl-2 in regulation of nuclear factor kappaB signaling pathways and possible cross-talk with mitogen-activated protein kinases are also discussed.
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PMID:Potentiation of cellular antioxidant capacity by Bcl-2: implications for its antiapoptotic function. 1455 11

To clarify the molecular basis of the cytoprotective properties of immunophilin ligands (IPLs), the anti-apoptotic effects of IPLs were determined in human glioma U251 cells. GPI1046 and V10367, non-immunosuppressive IPLs (NI-IPLs), as well as FK506, an immunosuppressive IPL (I-IPL), had cytoprotective effects against hydrogen peroxide (H20O)-induced apoptotic cell death in U251 cells. H2O2 increased both the ratio of bax/bcl-2 and the p53 mRNA expression. However, pre-treatment with FK506 and V10367 significantly prevented any increase in this ratio or p53 mRNA expression. GPI1046 also reduced the ratio of bax/bcl-2 to the normal level. In addition, H2O2 significantly increased activities of all three caspases, caspase-3, caspase-8, and caspase-9, in comparison with non-H2O2 controls. However, FK506 prevented the increase of these caspase activities. On the other hand, it is well-known that glutathione (GSH) and neurotrophic factor (NTF) is related to the induction of apoptosis in neuronal cells. In U251 cells, FK506, GPI1046 and V10367 had GSH-activating and NTF-activating effects. Thus, the immunosuppressive effect is not essential for the cytoprotective properties of IPLs, and IPLs have multiple beneficial properties such as the anti-apoptotic effect, GSH-activating effect, and NTF-activating effect, although the anti-apoptotic effect of NI-IPLs is independent of the regulation of apoptotic activators such as caspase-3.
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PMID:Molecular basis of anti-apoptotic effect of immunophilin ligands on hydrogen peroxide-induced apoptosis in human glioma cells. 1526 Jan 30


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