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:1.2.1.13 (glyceraldehyde-3-phosphate dehydrogenase)
6,511 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A potential cytotoxic, self-destructive role of endogenously generated and exogenously supplied nitric oxide (NO) was studied in two mouse monocytic macrophage cell lines (RAW 264.7 and J774.1). Our attention centered on NO-mediated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) modification and inhibition of the Krebs cycle enzyme, aconitase, related to macrophage cell death. NO formed by an active inducible nitric oxide synthase significantly decreased cell viability in the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cytotoxicity assay. Similarly, cell viability was inversely and dose-dependently correlated to increasing concentrations of the NO-releasing compound, sodium nitroprusside. Biochemically, we noticed a correlation between endogenously derived or exogenously generated NO and inhibition of GAPDH as well as aconitase enzyme activity. The involvement of NO was further substantiated by the use of NG-monomethyl-L-arginine. Associated with decreased GAPDH enzyme activity, 32P-NAD(+)-dependent modification of the enzyme in the cytosol of pretreated cells was hindered. This reflects intracellular protein modification as a result of NO signalling. Using sodium nitroprusside we achieved GAPDH translocation from the cytosol to the plasma membrane or the nucleus of treated cells. However, despite GAPDH modification, lactate production was not rate limiting during NO intoxication. Furthermore, blocking the iron-sulfur-containing enzyme, aconitase, is insufficient to produce macrophage cell death. Although RAW 264.7 and J774.1 cells show substantial variation in their sensitivity towards NO it can be concluded that NO-mediated macrophage cell death is not linked to energy depletion. For GAPDH, NO-mediated protein modification may be related to functions of the enzyme, other than its glycolytic role.
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PMID:Modification of macrophage glyceraldehyde-3-phosphate dehydrogenase in response to nitric oxide. 879 Oct 5

We investigated the correlation between tumor sensitivity to 5-fluorouracil (5-FU) and enzymatic activities of thymidylate synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) in human gastric cancer specimens. Forty-one patients with advanced gastric cancer gave informed consent and were enrolled in the study. Biopsy specimens of gastric cancer were obtained preoperatively through gastrofiberscopy and used to determine TS and DPD messenger RNA (mRNA) levels. TS and DPD enzyme activity and mRNA levels were also measured in resected tumor tissue samples obtained after surgical resection. TS and DPD activity were measured using the TS-binding assay and a radioenzymatic assay, respectively, while mRNA levels were measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), with co-amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal standard. 5-FU sensitivity of resected tumor specimens was measured by the tetrazolium-based colorimetric assay (MTT assay). Both TS and DPD mRNA levels correlated well between biopsied and resected tumor specimens. A statistically significant correlation was also observed between mRNA levels in biopsied specimens and enzymatic activities in resected specimens. DPD levels significantly correlated with 5-FU sensitivity, such that high DPD activity and high DPD mRNA levels resulted in low sensitivity to 5-FU. In contrast, no correlation was observed between TS activity or TS mRNA levels and 5-FU sensitivity. We conclude that tumor DPD mRNA level, as assessed from biopsy specimens obtained by gastrofiberscopy, may be a useful indicator in predicting tumor sensitivity to 5-FU in patients with gastric cancer.
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PMID:Dihydropyrimidine dehydrogenase and messenger RNA levels in gastric cancer: possible predictor for sensitivity to 5-fluorouracil. 1074 51

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction is widely used to evaluate cell proliferation and viability. MTT reduction is interpreted to be indicative of cellular metabolic activity, and the site of reduction includes both mitochondrial and cytosolic redox reactions. Astrocytes are believed to rely mainly on glycolysis for ATP generation, whereas neurons are considered to depend more on oxidative metabolism. The present study, therefore, tested the substrate-preference of glucose and its metabolites for MTT reduction in cultures of rat type 1 astroglia and neurons.MTT specific activity of astroglia was much higher than that of neurons. Astroglial MTT reducing activity in glucose-free medium or 2mM glucose with iodoacetate (5mM) was completely blocked. In glucose-depleted medium, 2mM lactate, pyruvate, malate, or acetate elicited minimal increases in MTT reduction by astroglia. In contrast, MTT reducing activity in neurons was enhanced two-fold by pyruvate and the reducing activity of lactate was equivalent to that of glucose, while malate had a small and acetate had no effect on MTT reduction. These results indicate that these two cell types differ markedly in their substrate-preferences for MTT reduction. In astroglia, MTT reduction reflects mainly cytosolic redox activity and is dependent on glyceraldehyde-3-phosphate dehydrogenase. In neurons, pyruvate dehydrogenase supports MTT reduction more effectively than glucose or lactate, even though both of these substrates can produce NADH and pyruvate.
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PMID:Substrate-dependence of reduction of MTT: a tetrazolium dye differs in cultured astroglia and neurons. 1182 Nov 52

Resistance to glucocorticoids (GC) is an important adverse risk factor in the treatment of acute lymphoblastic leukemia (ALL). To induce apoptosis, GC bind to the GC receptor (GR), which is regulated by various (co)chaperone proteins such as heat-shock protein 70 (HSP-70), HSP-40, HIP (HSP-70-interacting protein), BAG-1 (BCL-2-associated gene product-1), HOP (HSP-70/HSP-90-Organizing protein), HSP-90, P-23, FKBP-51, FKBP-52 and CYP-40. In this study, we tested the hypothesis that mRNA expression levels of these molecules are determinants of GC resistance in childhood ALL. In all, 20 children with ALL cells in vitro sensitive to prednisolone (LC(50) < 0.1 microg/ml) were compared each with a resistant patient (LC(50) >150 mug/ml), matched for immunophenotype, age and white blood cell count. mRNA expression levels of the (co)chaperone molecules were measured by quantitative real-time RT-PCR and normalized to GAPDH and RNaseP levels. In vitro resistance to prednisolone was measured by MTT assay. HSP-90 mRNA expression levels were 2000-fold higher as compared to HSP-70. Using matched pair analysis, mRNA expression levels of the various (co)chaperone molecules were not significantly different between in vitro-sensitive and -resistant patients. GC resistance in childhood ALL cannot be attributed to different mRNA expression levels of the investigated (co)chaperone molecules involved in GC binding and transport to the nucleus.
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PMID:mRNA expression levels of (co)chaperone molecules of the glucocorticoid receptor are not involved in glucocorticoid resistance in pediatric ALL. 1575 36

To study the effects of tyrosine-kinase inhibitor STI571 on the adhesion of RPMI8226 cells to fibronectin (FN), cell adhesion mediated adriamycin-resistance and the Rac1 mRNA expression, the adhesion of RPMI8226 cells to fibronectin and drug resistance mediated by cell adhesion were determined by means of crystal violet staining and MTT assays respectively, Rac1 mRNA levels in RPMI8226 cells were examined by semi-quantitative RT-PCR. The results showed that STI571 could inhibit the adhesion of RPMI8226 cells to fibronectin. When RPMI8226 cells had been adhered to FN or BSA-coated wells for 1, 6 and 12 hours, the adhesion rates were (43.71 +/- 2.18)%, (55.63 +/- 1.56)%, and (63.42 +/- 2.46)% respectively. After treatment with STI571 20 micromol/L, the adhesion rates decreased to (15.12 +/- 1.04)%, (17.58 +/- 1.32)% and (17.24 +/- 1.59)% respectively (P < 0.05). The experiment revealed that growth of RPMI8226 cells adhered to FN-coated plates had a significant advantage over growth on BSA-coated plates when exposed to adriamycin (Adr) for 1 hour followed by a 24-hour culture period, and the mean IC(50) value for FN-adhered cells was (1.46 +/- 0.04) micromol/L while mean IC(50) value for BSA control was (0.78 +/- 0.03) micromol/L (P < 0.05). Following treatment with 20 micromol/L STI571, the mean IC50 values for FN and BSA adhered cells were (0.81 +/- 0.05) micromol/L, (0.74 +/- 0.02) micromol/L respectively, there was no significant difference between them (P > 0.05). RT-PCR demonstrated that the relative Rac1 mRNA level (Rac1/GAPDH) in RPMI8226 cells was downregulated following being treated with 20 micromol/L STI571. It is concluded that STI571 can inhibit the adhesion of RPMI8226 cells to fibronectin, reverse cell adhesion mediated adriamycin-resistance, and downregulate Rac1 mRNA level.
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PMID:[Tyrosine kinase inhibitor reverses adriamycin resistance mediated by cell adhesion in RPMI8226 cells]. 1663 94

We studied the ability of prolyl oligopeptidase (POP) inhibitors, Z-Pro-Prolinal and JTP-4819, to prevent translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and formation of reactive oxygen species (ROS), in 6-hydroxydopamine (6-OHDA) and cytosine arabinoside (Ara-C) treated monkey fibroblast (CV1-P) and human neuroblastoma (SH-SY5Y) cells. The cells were pretreated with POP inhibitors (30 min) before addition of toxicants. GAPDH was analyzed by Western hybridization, ROS by fluorescent 2'7'-dichlorodihydro-fluorescein diacetate, and viability by the MTT method. Both toxicants induced GAPDH translocation to the particulate fraction (mitochondria and nuclei). Z-Pro-Prolinal was able to inhibit the translocation in 6-OHDA-exposed CV1-P cells. In SH-SY5Y cells and in JTP-4819 pretreated cells, no prevention of translocation was seen. However, the intensity of GAPDH in cytosolic fraction increased. Both inhibitors blocked 6-OHDA-induced ROS-production to the control level in CV1-P but, not in SH-SY5Y cells, without affecting their viability. In conclusion, POP inhibitors are able to prevent certain cell stress related factors such as ROS production or GAPDH translocation.
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PMID:A prolyl oligopeptidase inhibitor, Z-Pro-Prolinal, inhibits glyceraldehyde-3-phosphate dehydrogenase translocation and production of reactive oxygen species in CV1-P cells exposed to 6-hydroxydopamine. 1694 54

We investigated the effects of celecoxib on the cell proliferation and the expression and activity of P-glycoprotein in the human gastric carcinoma multidrug resistance sublines SGC7901/adriamycin and SGC7901/vincristine. The cell proliferation was measured by [3H]thymidine incorporation assay and MTT test. The expression of the multidrug resistant gene (MDR1) was detected by real-time quantitative reverse transcription-polymerase chain reaction. P-glycoprotein was measured by Western blot analysis. The intracellular rhodamine 123 accumulation was analyzed by flow cytometry to evaluate the activity of P-glycoprotein. After treatment with celecoxib, the proliferation inhibitions of SGC7901 cell line and the SGC7901/adriamycin and SGC7901/vincristine sublines increased linearly in a positive dose-dependent pattern in both the [3H]thymidine incorporation assay and in the MTT test. The IC50 value of the MDR1/GAPDH ratio was 5.50 x 10(-6) mol/l in SGC7901/adriamycin and 3.89 x 10(-6) mol/l in SGC7901/vincristine. P-glycoprotein expression levels in the two multidrug resistance sublines treated with celecoxib were significantly lower than those in control groups, 0.28 vs. 0.71 in the SGC7901/adriamycin subline and 0.21 vs. 0.83 in the SGC7901/vincristine subline, respectively, P<0.05. After treatment with celecoxib, intracellular rhodamine 123 accumulation in the SGC7901/adriamycin and SGC7901/vincristine sublines increased positively in a dose-dependent pattern (P<0.05), and reached more than 50% of that in the SGC7901 cell line at the concentration of 1 x 10(-4) mol/l of celecoxib. In conclusion, celecoxib could inhibit proliferation of multidrug resistance in gastric carcinoma sublines. The reversal of multidrug resistance was caused by downregulation of the expression and activity of P-glycoprotein. The results may suggest a new way to reverse P-glycoprotein-dependent multidrug resistance in human gastric carcinoma.
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PMID:Effects of celecoxib on the reversal of multidrug resistance in human gastric carcinoma by downregulation of the expression and activity of P-glycoprotein. 1770 58

Ascertaining the time-dependent regulation of induced apoptosis and radioresistance is important to understand the relationship between the level of spontaneous apoptosis in cells and their radiosensitivity. Accordingly, we investigated the time-dependent expression of apoptosis related genes and radioresistance in neuroblastoma cells. Serum-starved human SK-N-MC cells were exposed to low linear energy transfer (LET) radiation (2 Gy) and incubated for 15, 30, 45 min, and 48 h. Radioresistance was investigated by examining the NF kappa B DNA-binding activity, cellular toxicity, DNA fragmentation, and expression of apoptotic signal transduction molecules. NF kappa B DNA binding activity was analyzed using electrophoretic mobility shift assay (EMSA). Cellular toxicity was measured using MTT assay. DNA fragmentation was quantified by labeling with fluorescein-conjugated deoxynucleotides. Microarray analysis was performed using cDNA microarray and relative gene expression was measured as % GAPDH and, subsequently validated using Q-PCR. Induction of NF kappa B analyzed using EMSA showed an increased DNA-binding activity at all time points investigated. Induced DNA fragmentation was observed after 15, 30, and 45 min post-radiation. Relatively, induced fragmentation was reduced after 48 h. Compared to the untreated controls cellular toxicity was induced with low LET radiation after 15, 30, and 45 min. Conversely, cytotoxicity was relatively less at 48 h after low LET radiation. Microarray analysis after low LET radiation revealed time-dependent modulation of apoptosis-related genes that are involved in radio-adaptation, spontaneous apoptosis-related early-responsive genes and late response genes. These results suggest that the time-dependent regulation of apoptotic response may determine the relationship between the level of spontaneous apoptosis in cells and their radiosensitivity.
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PMID:Alteration of apoptotic signaling molecules as a function of time after radiation in human neuroblastoma cells. 1806 12

We assessed changes in the apoptosis-related genes BCL2, BAX, BCL2L12, FAS and CASPASE-3 in OVCAR-3 human ovarian cancer cells and BT-20 human breast cancer cells to provide an insight into the molecular mechanisms involved in the response of these cells to treatment with anticancer drugs and to assess their value as potential biomarkers of chemotherapy response in breast and ovarian cancer. Cells were treated with different chemotherapeutic drugs (cisplatin, carboplatin, doxorubicin, etoposide and taxol) and assessed for changes in the expression of apoptosis-related genes at the mRNA level. Total RNA was extracted, reverse-transcribed into cDNA and amplified by PCR using gene-specific primers. GAPDH was used as a housekeeping gene. Cytotoxicity was assessed by MTT assay. Both cancer cell lines responded differentially at the molecular level to the drug treatments. OVCAR-3 cells showed more pronounced sensitivity and changes compared to BT-20 cells at the mRNA level for different apoptosis-related genes, leading to cell and cancer type dependence in conjunction with drug dependence.
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PMID:Molecular profile of breast versus ovarian cancer cells in response to treatment with the anticancer drugs cisplatin, carboplatin, doxorubicin, etoposide and taxol. 1878 38

The BCL2 (bcl-2)family of genes is highly involved in the apoptotic mechanisms. We have recently discovered and cloned a novel member of the same family, BCL2L12. The aim of this study was to investigate the modulations in the BAX, BCL2 and BCL2L12 mRNA levels in gastric cancer cells, after their treatment with the anticancer drugs 5-fluorouracil and irinotecan as well as the antioxidant substance leucovorin. AGS gastric cancer cells were examined for their sensitivity to antineoplastic drugs and/or antioxidants using the MTT assay. Total RNA was extracted and reverse transcribed into cDNA. A highly sensitive quantitative real-time PCR method for the mRNA quantification was developed using the SYBR Green chemistry. GAPDH was used as a housekeeping gene. Relative quantification analysis was performed using the comparative threshold cycle method. Treatment of AGS cells with 5-fluorouracil (5 microM), leucovorin (10 microM), a combination of the latter and, eventually, irinotecan (1 microM) for 3 time periods (24, 48 and 72 h), resulted in significant modulations of the BCL2, BAX and BCL2L12 mRNA levels compared with the untreated cells. Our experimental data demonstrate that the molecular profile mainly of BCL2 and BCL2L12 genes may serve as a new potential molecular biomarker predicting treatment response in gastric cancer cells.
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PMID:Treatment of gastric cancer cells with 5-fluorouracil/leucovorin and irinotecan induces distinct alterations in the mRNA expression of the apoptosis-related genes, including the novel gene BCL2L12. 1944 6


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