Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.5.2 (NQO1)
 6,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A polymerase chain reaction (PCR)-based method was used to quantitate the expression levels of low abundance genes relevant to cancer drug activity. RNA from tumor samples as small as 20 mg was isolated and converted to cDNA using random hexamers. The 5' primers for the PCR contained a T7 polymerase promoter sequence, allowing the PCR-amplified DNA to be transcribed to RNA fragments. In each sample, the linear ranges of amplification of each cDNA of interest were established. Relative gene expressions were calculated by extrapolating the amounts of PCR products generated within the linear amplification regions of each gene to equal volumes of the cDNA solution. The method was accurate to less than a 2-fold difference in expression levels. Using beta 2-microglobulin and beta-actin gene expressions as internal reference standards and cDNA from HT-29 cells as an external linearity standard, we measured the relative expressions of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase in a number of clinical tumor samples. The expressions of these genes varied from 50- to 100-fold among different tumors, although most of the values were grouped within about a 10-fold range. The amount of thymidylate synthase gene expression in tumor tissues was directly proportional to the content of thymidylate synthase protein. Those tumors with the lowest thymidylate synthase expression had the best response to both the 5-fluorouracil-leucovorin and 5-fluorouracil-cisplatin combinations.
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PMID:Quantitation of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase gene expression in human tumors using the polymerase chain reaction. 172 69

NAD(P)H:quinone oxidoreductase (DT-diaphorase; DTD) is an obligate two-electron reductase which may play a role in the bioactivation of antitumor quinones such as mitomycin C (MMC). We studied 10 colon carcinoma cell lines showing different levels of DTD activity (range, 0-3447 nmol/min/mg protein), as measured by the reduction of dichlorophenolindophenol. Expression of the NAD(P)H:quinone reductase gene (NQO1), which codes for the DTD enzyme, as measured by a polymerase chain reaction amplification technique was then correlated with enzymatic activity in all cell lines. HT-29 cells, which have intermediate DTD activity (769 +/- 144 nmol/min/mg protein, mean +/- SD) and are sensitive to MMC, showed high NQO1 expression relative to beta-actin (taken as 100% here for comparative purposes). BE cells which have no detectable DTD activity and are resistant to MMC showed moderate NQO1 expression (91% of HT-29). RNA single-strand conformational polymorphism analysis and subsequent sequencing of BE complementary DNA revealed a C to T mutation in the NQO1 complementary DNA. This confers a proline to serine substitution in the amino acid sequence of the protein. Additionally, HCT-116 cells showed both moderate DTD activity (390 +/- 41 nmol/min/mg protein) and NQO1 expression (41% of HT-29), while resistant subclones of these cells, exposed to MMC during 11 and 44 weeks, showed low gene expression (5 and 9% of HT-29 respectively) and enzymatic activity (11 +/- 6 and 36 +/- 16 nmol/min/mg protein). These results support the ideas that reductive activation of MMC by DTD may be important in the cytotoxicity of MMC and that polymerase chain reaction may be a useful technique for quantitating the relative expression of genes in human tumors.
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PMID:NAD(P)H:quinone oxidoreductase gene expression in human colon carcinoma cells: characterization of a mutation which modulates DT-diaphorase activity and mitomycin sensitivity. 173 39

The levels of NAD(P)H:(quinone-acceptor) oxidoreductase (EC.1.6.99.2) (DT-diaphorase) mRNA and enzyme activity have been studied in paired human normal lung and non-small cell lung tumor samples from patients with a history of cigarette smoking. There were significantly higher levels of DT-diaphorase mRNA (1.2 kilobases) in lung tumor compared to normal lung tissue of patients who had stopped smoking more than 6 months before surgery, with relative values (normalized to beta-actin mRNA) of 29.6 +/- 7.8 (SE) in the lung tumor compared to 11.7 +/- 2.2 in normal lung tissue (P < 0.05). There was no significant difference in DT-diaphorase mRNA between lung tumor and normal lung tissue of subjects who were smokers at the time of surgery, with values of 16.5 +/- 2.1 and 15.3 +/- 2.5 (P > 0.05), respectively. DT-diaphorase enzyme activity in normal and tumor lung tissue was positively correlated with DT-diaphorase mRNA (r = 0.908, P < 0.01). The results of the study suggest that DT-diaphorase does not function as an inducible protectant enzyme in human lung against oxidant species and carcinogens present in cigarette smoke. Metabolism of some anticancer drugs by DT-diaphorase can alter their activity. Differences in DT-diaphorase between lung tumors of smokers and past smokers might alter the response to these drugs.
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PMID:Cigarette smoking is a determinant of DT-diaphorase gene expression in human non-small cell lung carcinoma. 822 86

Mitomycin C (MMC) is activated by DT-diaphorase (DTD) and cytochrome P450 reductase (P450R). In cancer cell lines, MMC cytotoxicity is correlated with DTD and P450R expression levels. The present study investigated the relationship between enzyme expression/activity and MMC cytotoxicity in patient bladder tumors. DTD and P450R expression was detected by competitive reverse transcription-PCR and their activity was measured by bioreductive assays. The expression of DTD and P450R in patient tumors (n = 29), as ratios to beta-actin levels, varied from 0 to 90% and 0 to 29%, respectively. The DTD expression was significantly correlated with P450R expression (r(2), 0.32; P < 0.01), whereas the average DTD level was 2-fold higher than that of P450R (P < 0.01). Among the 29 tumors, 21 provided sufficient materials to evaluate tumor sensitivity to MMC. The concentration of MMC required to produce 50% inhibition (IC(50)) of DNA precursor incorporation for a 2-h treatment ranged from 0.17 to 18.1 microg/ml, indicating a 110-fold intertumor variation, with the high-grade and more invasive tumors being less chemosensitive compared with the low-grade and less invasive tumors. Tumor sensitivity to MMC, as indicated by the inverse of IC(50) values, was positively correlated with the expression of DTD (r(2), 0.28; P < 0.05) and P450R (r(2), 0.26; P < 0.05). Multivariate analysis indicates DTD expression and P450R expression as better determinants of MMC activity compared with other pathobiological factors (e.g., tumor grade, stage, and labeling index) that have been shown to significantly correlate with MMC activity. Eleven tumors were studied for the relationship between gene expression level and enzyme activity of DTD and P450R. The DTD activity was significantly correlated with the gene expression level (r(2), 0.84; P < 0.001). For P450R, there is a trend of a correlation between enzyme activity and its mRNA level, but the correlation was not statistically significant (r(2), 0.28; P = 0.09). These data indicate that the sensitivity of patient bladder tumors to MMC is correlated with the expression of DTD and P450R in tumors and suggest that the lower expression of these enzymes in the high-grade and more invasive tumors is a cause of the lower efficacy of intravesical MMC in these tumors.
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PMID:Expression of DT-diaphorase and cytochrome P450 reductase correlates with mitomycin C activity in human bladder tumors. 1135 Sep

NAD(P)H:quinone oxidoreductase (NQO1) and dihydronicotinamide riboside:quinone oxidoreductases (NQO2) are cytosolic flavoproteins that catalyze the two-electron reduction of quinones and quinoid compounds to hydroquinones, thereby promoting detoxification and preventing the formation of highly reactive oxygen species, which lead to DNA and cell damage. Two NQO isoforms, designated NQO1 and NQO2, have been cloned and sequenced. To elucidate their role in carcinogenesis, the gene expression of human NQO1 and NQO2 in paired normal and tumor tissue samples was examined. Quantitative triplex reverse transcriptase polymerase chain reaction was employed to analyze NQO1 and NQO2 mRNA expression in normal hepatic and biliary tissue as well as in cholangiocellular carcinomas (CCC), hepatocellular carcinomas (HCC), and focal nodular hyperplasias (FNH). Coexpression of beta-actin RNA was used as an internal reference standard and linear ranges of transcript amplification were established for each sample. In normal hepatocellular tissue, the two NQO isoforms were differentially regulated, with a higher expression of NQO2 than NQO1. Malignant hepatocellular tissue (HCC), however, displayed up-regulation of NQO1 and down-regulation of NQO2. Regulation of either transcript was not seen in benign hepatocellular tumor tissue (FNH), which indicates a reciprocal control of NQO genes in hepatocarcinogenesis. Normal biliary tissue expressed a significantly higher level of NQO1 transcripts compared with normal liver, whereas biliary NQO2 levels were significantly lower than in hepatocellular tissue. Comparing the levels of expression in normal and malignant biliary tissue (CCC), no significant differences were noted between the expression levels of either transcript. Thus, this study provides evidence for differential hepatic and biliary regulation of both NQO1 and NQO2.
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PMID:Differential gene expression of NAD(P)H:quinone oxidoreductase and NRH:quinone oxidoreductase in human hepatocellular and biliary tissue. 1180 56

The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) contains two transcription activation domains, Neh4 (Nrf2 ECH homology 4) and Neh5, which co-ordinately regulate transactivation of cytoprotective genes. In the present study we aimed to clarify the role of the Neh5 domain in Nrf2-mediated gene regulation. Deletion of the complete Neh5 domain reduces expression of endogenous Nrf2 target genes, such as HO-1 (haem oxygenase 1), NQO1 [NAD(P)H:quinone oxidoreductase 1] and GCLM (glutamate cysteine ligase modulatory subunit), in human kidney epithelial cells. Furthermore, the deletion of Neh5 markedly repressed CBP [CREB (cAMP-response-element-binding protein)-binding protein] and BRG1 (Brahma-related gene 1) from associating with Nrf2, diminishing their co-operative enhancement of HO-1 promoter activity. Mutational analysis of the Neh5 domain revealed a motif that shares significant homology with beta-actin and ARP1 (actin-related protein 1). Mutagenesis of this motif selectively decreased HO-1, but not NQO1 and GCLM, expression. Taken together, these results indicate that the Neh5 domain has the ability to regulate Nrf2 target gene transcription, yet the role of the Neh5 domain in transcription varies from gene to gene.
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PMID:Nrf2 Neh5 domain is differentially utilized in the transactivation of cytoprotective genes. 1731 70