Characterization of a polymorphism in NAD(P)H: quinone oxidoreductase (DT-diaphorase)

Abstract

Summary NAD(P)H:quinone oxidoreductase (NQO1, EC 1.6.99.2) is an obligate two-electron reductase that can either bioactivate or detoxify quinones and has been proposed to play an important role in chemoprevention. We have previously characterized a homozygous point mutation in the BE human colon carcinoma cell line that leads to a loss of NOO1 activity. Sequence analysis showed that this mutation was at position 609 of the NQO1 cDNA, conferring a proline to serine substitution at position 187 of the NQO1 enzyme. Using polymerase chain reaction (PCR) analysis, we have found that the H596 human non-small-cell lung cancer (NSCLC) cell line has elevated NQO1 mRNA, but no detectable enzyme activity. Sequencing of the coding region of NQO1 from the H596 cells showed the presence of the identical homozygous point mutation present in the BE cell line. Expression and purification of recombinant wild-type and mutant protein from E. coli showed that mutant protein could be detected using immunoblot analysis and had 2% of the enzymatic activity of the wild-type protein. PCR and Northern blot analysis showed moderate to low levels of expression of the correctly sized transcript in the mutant cells. Immunoblot analysis also revealed that recombinant mutant protein was immunoreactive; however, the mutant protein was not detected in the cytosol of either BE or H596 cells, suggesting that the mutant proteins were either not translated or were rapidly degraded. The absence of any detectable, active protein, therefore, appears to be responsible for the lack of NQO1 activity in cells homozygous for the mutation. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis for the mutation at position 609 conducted on 90 human lung tissue samples (45 matched sets of tumour and uninvolved tissue) revealed a 7% incidence of individuals homozygous for the mutation, and 42% heterozygous for the mutation. These data suggest that the mutation at position 609 represents a polymorphism in an important xenobiotic metabolizing enzyme, which has implications for cancer therapy, chemoprevention and chemoprotection.

Induction of DT-diaphorase by 1,2-dithiole-3-thiones in human tumour and normal cells and effect on anti-tumour activity of bioreductive agents

“…The bars represent the mean DT-diaphorase activity ± standard error of four determinations. The means were compared using a t-test evaluating the significance of the difference of the DT-diaphorase activity in the control and D3T-treated cells the NQO1cDNA (Traver et al, 1992;Kuehl et al, 1995) that results in a protein with very low enzyme activity (Traver et al, 1997). Whether any of these mechanisms are responsible for the different effects of the D3T analogues on DT-diaphorase activity in the different human cell lines studied remains to be determined.…”

NAD(P)H:Quinone Oxidoreductase 1 Expression in Kidney Podocytes

“…This "fragile" podocyte will be particularly sensitive to the toxic effect of DTD-directed anticancer compounds. A polymorphism of the gene encoding DT-diaphorase has been described (Traver et al 1997). This mutation (a C-to-T transition at base pair 609 coding for a proline to serine substitution) causes a loss of enzyme activity Siegel et al 1999) and resistance to anticancer agents that require reductive activation (Lambert et al 1998).…”

Ubiquitin-independent p53 proteasomal degradation

“…68,69 The product of this NQO1 genotype is less active in stabilizing p53. 18 Several studies have found that the C609T allele is associated with increased risk of developing different types of tumors such as urothelial tumors, basal cell carcinoma, pediatric leukemia, colorectal cancer, esophageal squamous cell carcinoma, and gastric carcinoma.…”