Mechanisms underlying the role of reactive oxygen species (ROS) generated by flavin-containing oxidases in regulating cell cycle progression were examined in human and rodent fibroblasts. Incubation of confluent cell cultures with nontoxic/nonclastogenic concentrations of the flavoprotein inhibitor, diphenyleneiodonium (DPI), reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase activity and basal ROS levels, but increased proteolysis of cyclin D1, p21Waf1 and phospho-p38MAPK. When these cells were allowed to proliferate by subculture in DPI-free medium, an extensive G1 delay was observed with concomitant activation of p53/p21Waf1 signaling and reduced phosphorylation of mitogen-activated kinases. Compensation for decreased oxidant generation by simultaneous exposure to DPI and nontoxic doses of the ROS generators, γ-radiation or t-butyl-hydroperoxide, attenuated the G1 delay. Whereas the DPI-induced G1 checkpoint was completely dependent on PHOX91, ATM and WAF1, it was only partially dependent on P53. Interestingly, G1 to S progression was not affected when another flavin-containing enzyme, nitric oxide synthase, was inhibited nor was it associated with changes in mitochondrial membrane potential. Proliferating cells treated with DPI also experienced a significant but attenuated delay in G2. We propose that ATM performs a critical function in mediating normal cellular proliferation that is regulated by nonphagocytic NAD(P)H oxidase enzymes activity, which may serve as a novel target for arresting cancer cells in G1.
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cumulative labeling index
mitogen-activated protein kinase
p53-binding protein 1
relative fluorescence units
relative luminescence units
reactive oxygen species
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We thank Debkumar Pain, Prasad Neti, Ling Li and Mitchell Coleman for suggestions and assistance, and Philip Leder and Francis Miller for providing p21Waf1 and gp91phox knockout cells. Research grants CA92262 from the National Institutes of Health (EIA), FG02-02ER63447 and FG02-05ER64050 (EIA and DRS) and FG02-98ER62685 (JBL) from the US Department of Energy, and a VA Merit Grant and a Career Investigator Award from the American Lung Association (ABC) supported this investigation.
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Venkatachalam, P., de Toledo, S., Pandey, B. et al. Regulation of normal cell cycle progression by flavin-containing oxidases. Oncogene 27, 20–31 (2008). https://doi.org/10.1038/sj.onc.1210634
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