Nuclear overexpression of NAD(P)H:quinone oxidoreductase 1 in Chinese hamster ovary cells increases the cytotoxicity of mitomycin C under aerobic and hypoxic conditions.
The effects of the subcellular localization of overexpressed bioreductive enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) on the activity of the antineoplastic agent mitomycin C (MC) under aerobic and hypoxic conditions were examined. Chinese hamster ovary (CHO-K1/dhfr(-)) cells were transfected with NQO1 cDNA to produce cells that overexpressed NQO1 activity in the nucleus (148-fold) or the cytosol (163-fold) over the constitutive level of the enzyme in parental cells. Subcellular localization of the enzyme was confirmed using antibody-assisted immunofluorescence. Nuclear localization of transfected NQO1 activity increased the cytotoxicity of MC over that produced by overexpression in the cytosol under both aerobic and hypoxic conditions, with greater cytotoxicity being produced under hypoxia. The greater cytotoxicity of nuclear localized NQO1 was not attributable to greater metabolic activation of MC but instead was the result of activation of the drug in close proximity to its target, nuclear DNA. A positive relationship existed between the degree of MC-induced cytotoxicity and the number of MC-DNA adducts produced. The findings indicate that activation of MC proximal to nuclear DNA by the nuclear localization of transfected NQO1 increases the cytotoxic effects of MC regardless of the degree of oxygenation and support the concept that the mechanism of action of MC involves alkylation of DNA.
Seow
Helen A
HA
Department of Pharmacology and the Developmental Therapeutics Program, Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Penketh
Philip G
PG
Belcourt
Michael F
MF
Tomasz
Maria
M
Rockwell
Sara
S
Sartorelli
Alan C
AC
D000332:Aerobiosis; D000818:Animals; D001483:Base Sequence; D001711:Biotransformation; D016466:CHO Cells; D015687:Cell Hypoxia; D002467:Cell Nucleus / Q000201:enzymology; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology; D018736:DNA Adducts / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D016685:Mitomycin / Q000493:pharmacokinetics / Q000633:toxicity*; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D018736:DNA Adducts; D018076:DNA, Complementary; D011994:Recombinant Proteins; D016685:Mitomycin; D016660:NAD(P)H Dehydrogenase (Quinone)