Transfection of COS-1 cells with DT-diaphorase cDNA: role of a base change at position 609

Br J Cancer. 1998 Apr;77(8):1236-40. doi: 10.1038/bjc.1998.208.

Abstract

DT-diaphorase, a homodimeric flavoenzyme, can provide for a defence mechanism against carcinogenesis mediated by dietary or environmental quinones as well as bioactivate quinone-containing chemotherapeutic drugs. Human cell lines and strains have been identified with very low or undetectable enzymatic activity and a C to T transition at nucleotide 609 of the DT-diaphorase cDNA. This single base change is predicted to result in a proline to serine change in amino acid 187. Human cells homozygous for this base transition fail to exhibit Western blot reactivity for DT-diaphorase, suggesting that this substitution results in protein instability. To directly test whether this base change affects DT-diaphorase enzymatic activity and/or protein stability in vivo, mammalian expression vectors containing DT-diaphorase cDNA with or without the nucleotide 609 base transition were transiently transfected in COS-1 cells. Co-transfection with a human growth hormone expression vector allowed normalization for transfection efficiency. COS-1 transfectants expressing the C to T base change displayed at least a tenfold reduction in DT-diaphorase activity (P < 0.001) and a two- to threefold reduction in protein levels compared with wild-type transfectants. These results are the first to detect the presence of DT-diaphorase protein coded for by the 609 base transition in mammalian cells and confirm its predicted reduced enzymatic activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Composition
  • Blotting, Western
  • COS Cells / enzymology*
  • Cells, Cultured
  • Chlorocebus aethiops
  • DNA Primers / chemistry
  • DNA, Complementary / genetics*
  • Gene Expression Regulation, Enzymologic / genetics*
  • Humans
  • NAD(P)H Dehydrogenase (Quinone) / genetics*
  • NAD(P)H Dehydrogenase (Quinone) / metabolism
  • Point Mutation*
  • Polymorphism, Genetic
  • Transfection*

Substances

  • DNA Primers
  • DNA, Complementary
  • NAD(P)H Dehydrogenase (Quinone)