NAD(P)H:quinone oxidoreductase gene expression in human colon carcinoma cells: characterization of a mutation which modulates DT-diaphorase activity and mitomycin sensitivity.
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.
Traver
R D
RD
University of Southern California, School of Pharmacy, Los Angeles, California 90033.
Horikoshi
T
T
Danenberg
K D
KD
Stadlbauer
T H
TH
Danenberg
P V
PV
Ross
D
D
Gibson
N W
NW
D000199:Actins / Q000235:genetics; D001483:Base Sequence; D003110:Colonic Neoplasms / Q000201:enzymology / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007700:Kinetics; D016685:Mitomycin / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics* / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D000199:Actins; D004273:DNA, Neoplasm; D009838:Oligodeoxyribonucleotides; D012334:RNA, Neoplasm; D016685:Mitomycin; D016660:NAD(P)H Dehydrogenase (Quinone)