Transfection of COS-1 cells with DT-diaphorase cDNA: role of a base change at position 609.
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.
Misra
V
V
Department of Medical Biophysics, University of Toronto, Ontario Cancer Institute, Canada.
Klamut
H J
HJ
Rauth
A M
AM
D000818:Animals; D001482:Base Composition; D015153:Blotting, Western; D019556:COS Cells / Q000201:enzymology*; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D006801:Humans; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D011110:Polymorphism, Genetic; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D016660:NAD(P)H Dehydrogenase (Quinone)