1. ¹Department of Radiology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ, USA
2. ²Department of Medicine, University of Iowa, Roy J and Lucille A Carver College of Medicine, Iowa City, IA, USA
3. ³Center for Radiation Sciences and Environmental Health, Harvard School of Public Health, Boston, MA, USA
4. ⁴Free Radical and Radiation Biology Program, Department of Radiation Oncology, Holden Comprehensive Cancer Center, University of Iowa, Iowa City, IA, USA

Correspondence: Dr E Azzam, Department of Radiology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 South Orange Avenue, MSB – F451, Newark, NJ 07101-1709, USA. E-mail: azzamei@umdnj.edu

Received 6 February 2007; Revised 30 April 2007; Accepted 25 May 2007; Published online 16 July 2007.

Abstract

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, p21^Waf1 and phospho-p38^MAPK. When these cells were allowed to proliferate by subculture in DPI-free medium, an extensive G₁ delay was observed with concomitant activation of p53/p21^Waf1 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, -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.