Increased tumor necrosis factor-α sensitivity of MCF-7 cells transfected with NAD(P)H: Quinone reductase

Linda M. Siemankowski, Jeanne Morreale, Brent D. Butts, Margaret M Briehl

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

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 sulforhodaminc 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.

Original languageEnglish (US)
Pages (from-to)3638-3644
Number of pages7
JournalCancer Research
Volume60
Issue number13
StatePublished - Jul 1 2000

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NAD(P)H Dehydrogenase (Quinone)
MCF-7 Cells
NAD
Tumor Necrosis Factor-alpha
Oxidative Stress
apaziquone
Indolequinones
Clone Cells
Apoptosis
Paraquat
Enzymes
Northern Blotting
Catalase
Hydrogen Peroxide
Reverse Transcription
Superoxide Dismutase
Complementary DNA
Cell Count
Antioxidants
Breast Neoplasms

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Increased tumor necrosis factor-α sensitivity of MCF-7 cells transfected with NAD(P)H : Quinone reductase. / Siemankowski, Linda M.; Morreale, Jeanne; Butts, Brent D.; Briehl, Margaret M.

In: Cancer Research, Vol. 60, No. 13, 01.07.2000, p. 3638-3644.

Research output: Contribution to journalArticle

Siemankowski, Linda M. ; Morreale, Jeanne ; Butts, Brent D. ; Briehl, Margaret M. / Increased tumor necrosis factor-α sensitivity of MCF-7 cells transfected with NAD(P)H : Quinone reductase. In: Cancer Research. 2000 ; Vol. 60, No. 13. pp. 3638-3644.
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