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.