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NAD(P)H:quinone oxidoreductase gene expression in human colon carcinoma cells: characterization of a mutation which modulates DT-diaphorase activity and mitomycin sensitivity.

Abstract
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.
AuthorsR D Traver, T Horikoshi, K D Danenberg, T H Stadlbauer, P V Danenberg, D Ross, N W Gibson
JournalCancer research (Cancer Res) Vol. 52 Issue 4 Pg. 797-802 (Feb 15 1992) ISSN: 0008-5472 [Print] United States
PMID1737339 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)
Chemical References
  • Actins
  • DNA, Neoplasm
  • Oligodeoxyribonucleotides
  • RNA, Neoplasm
  • Mitomycin
  • NAD(P)H Dehydrogenase (Quinone)
Topics
  • Actins (genetics)
  • Base Sequence
  • Colonic Neoplasms (enzymology, genetics)
  • DNA, Neoplasm (genetics, isolation & purification)
  • Gene Expression Regulation, Enzymologic
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Kinetics
  • Mitomycin (pharmacology)
  • Molecular Sequence Data
  • NAD(P)H Dehydrogenase (Quinone) (genetics, metabolism)
  • Oligodeoxyribonucleotides
  • Polymerase Chain Reaction
  • RNA, Neoplasm (genetics, isolation & purification)
  • Transcription, Genetic
  • Tumor Cells, Cultured

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