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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 G(1) 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, gamma-radiation or t-butyl-hydroperoxide, attenuated the G(1) delay. Whereas the DPI-induced G(1) checkpoint was completely dependent on PHOX91, ATM and WAF1, it was only partially dependent on P53. Interestingly, G(1) 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 G(2). 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 G(1).

Evidence accumulated over the past two decades has indicated that exposure of cell populations to ionizing radiation results in significant biological effects occurring in both the irradiated and nonirradiated cells in the population. This phenomenon, termed the 'bystander response', has been shown to occur both in vitro and in vivo and has been postulated to impact both the estimation of risks of exposure to low doses/low fluences of ionizing radiation and radiotherapy. Several mechanisms involving secreted soluble factors, oxidative metabolism and gap-junction intercellular communication have been proposed to regulate the radiation-induced bystander effect. Our current knowledge of the biochemical and molecular events involved in the latter two processes is reviewed in this article.

To further understand the mechanism(s) by which DNA damage activates p53, we analysed the expression levels of p53 and HDM2 (the human homolog of murine MDM2) in various human diploid fibroblast and tumor cell strains during the period that precedes activation of known downstream effectors of p53. In X-irradiated human cells, HDM2 protein was rapidly phosphorylated in serine/threonine residues in a p53, p14ARF and p73-independent manner. In p53 wild-type cells, HDM2 phosphorylation precedes a detectable increase in the levels of p53 and is not observed in ataxia telangiectasia (AT) fibroblasts. The transfection of AT cells with a vector expressing ATM restored the ability to rapidly phosphorylate HDM2 following X-irradiation, confirming a role for ATM in its phosphorylation. We also show that ATM complexes with HDM2. The DNA lesions signaling the early rapid phosphorylation of HDM2 are a result of X-ray and not UV-type damage. The ATM-promoted early covalent modification of HDM2 in X-irradiated human cells may provide a mechanism to activate p53.