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 tumo r necrosis factor (TNF) kills transformed cells involves oxidative stress, NAD(P)H:(quinone acceptor) oxidoreductase (NQO1) is an antioxidant enzyme w ith particular relevance to cancer, The MCF-7 breast cancer cell line was s tably transfected with rat NQO1 cDNA to determine whether increased NQO1 ac tivity alters sensitivity to TNF-induced apoptosis, Five clones, with a ran ge 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 NQ O1 activity in the transfectants was attributable to expression from the tr ansfected rat sequence. Based on sulforhodamine B assays for the number of viable cells, the NQO1 clones showed increased sensitivity to EO9, an indol oquinone that undergoes bioactive reduction by NQO1. Viability studies also demonstrated that the NQO1 transfectants H ere significantly more sensitiv e to TNF than the control transfectants or MCF-7 parent. This increased sen sitivity could not be explained by changes in superoxide dismutase or catal ase activity or to increased sensitivity to oxidative stress in general, as assessed by response to hydrogen peroxide and paraquat treatment. Using di chlorodihydrofluorescein diacetate as a probe, we found that the NQO1 trans fectants had no difference in baseline level of oxidative stress compared t o the control cells but did exhibit greater intracellular oxidative stress after TNF treatment. We conclude that NQO1 can affect the TNF-mediated path way to apoptosis.