Increased tumor necrosis factor-alpha sensitivity of MCF-7 cells transfected with NAD(P)H:quinone reductase.
Evidence from a number of studies suggests that the mechanism by which tumor necrosis factor (TNF) kills transformed cells involves oxidative stress. NAD(P)H:(quinone acceptor) oxidoreductase (NQO1) is an antioxidant enzyme with particular relevance to cancer. The MCF-7 breast cancer cell line was stably transfected with rat NQO1 cDNA to determine whether increased NQO1 activity alters sensitivity to TNF-induced apoptosis. Five clones, with a range of NQO1 enzyme activities from 5- to 50-fold greater than the MCF-7 line, and two control transfectants were examined. Northern blot hybridization analyses and reverse transcription-PCR demonstrated that the increase in NQO1 activity in the transfectants was attributable to expression from the transfected rat sequence. Based on sulforhodamine B assays for the number of viable cells, the NQO1 clones showed increased sensitivity to EO9, an indoloquinone that undergoes bioactive reduction by NQO1. Viability studies also demonstrated that the NQO1 transfectants were significantly more sensitive to TNF than the control transfectants or MCF-7 parent. This increased sensitivity could not be explained by changes in superoxide dismutase or catalase activity or to increased sensitivity to oxidative stress in general, as assessed by response to hydrogen peroxide and paraquat treatment. Using dichlorodihydrofluorescein diacetate as a probe, we found that the NQO1 transfectants had no difference in baseline level of oxidative stress compared to the control cells but did exhibit greater intracellular oxidative stress after TNF treatment. We conclude that NQO1 can affect the TNF-mediated pathway to apoptosis.
Department of Pathology, University of Arizona, Tucson 85724-5043, USA.
L M
LM
Siemankowski
J
J
Morreale
B D
BD
Butts
M M
MM
Briehl
D000230:Adenocarcinoma; D000818:Animals; D001943:Breast Neoplasms; D002374:Catalase / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D005260:Female; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics / Q000378:metabolism*; D010269:Paraquat / Q000633:toxicity; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000633:toxicity*
D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D006861:Hydrogen Peroxide; D002374:Catalase; D016660:NAD(P)H Dehydrogenase (Quinone); D010269:Paraquat