Regulation of normal cell cycle progression by flavin-containing oxidases

Oncogene (2008) 27, 20–31 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE Regulation of normal cell cycle progression by flavin-containing oxidases P Venkatachalam1, SM de Toledo1, BN Pandey1, LA Tephly2, AB Carter2, JB Little3, DR Spitz4 and EI Azzam1 1 Department of Radiology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ, USA; Department of Medicine, University of Iowa, Roy J and Lucille A Carver College of Medicine, Iowa City, IA, USA; 3Center for Radiation Sciences and Environmental Health, Harvard School of Public Health, Boston, MA, USA and 4Free Radical and Radiation Biology Program, Department of Radiation Oncology, Holden Comprehensive Cancer Center, University of Iowa, Iowa City, IA, USA 2 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, c-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. Oncogene (2008) 27, 20–31; doi:10.1038/sj.onc.1210634; published online 16 July 2007 Keywords: flavin-containing oxidases; NAD(P)H oxidase/ nitric oxide synthase; reactive oxygen species; cellular proliferation/G1 checkpoint/G2 checkpoint; ATM/p53/ p21Waf1/p38MAPK/cyclin D1 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: Received 6 February 2007; revised 30 April 2007; accepted 25 May 2007; published online 16 July 2007 Introduction Cellular exposure to high levels of reactive oxygen species (ROS) produced by endogenous enzymatic reactions or induced by external agents damages DNA, proteins and lipids (Halliwell, 1996), and contributes to numerous human disorders (Droge, 2002). In contrast, low levels of ROS participate in signaling pathways that control essential cellular functions including proliferation (Burdon, 1995). ROS regulate expression of specific genes, modulate ion channel activities and mimic or affect intermediates (for example, second messengers) in signal transduction (Schulze-Osthoff et al., 1997). Hence, homeostatic cellular functions require tight control of the cellular redox environment (Spitz et al., 2004). Disruption of the balance between oxidant production and antioxidant capacity alters this environment, resulting in detrimental consequences to the cell (Schafer and Buettner, 2001; Spitz et al., 2004). Mitochondrial oxidative metabolism, nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidases and lipoxygenases are the major cellular sources of ROS (Droge, 2002). Most of the oxidants they generate and their byproducts are metabolized by antioxidants. However, ROS that escape antioxidant defense are believed to contribute to homeostatic regulation of redox signaling (Droge, 2002). Superoxide anions produced by the multicomponent NAD(P)H oxidase (a flavin-containing oxidase) were initially detected in phagocytes, and were found to be essential to their antimicrobial activity (Ushio-Fukai et al., 1996). Recently, similar ROS-generating oxidases have been shown to exist also in nonphagocytic cells, including fibroblasts (Babior, 1999). NAD(P)H oxidase affects signal transduction by growth factor receptors (Ammendola et al., 2002) and promotes proliferation in a variety of cell types (Irani and Goldschmidt-Clermont, 1998). It is thought that mitogenic signaling elicited by cytokines or growth factors induces intracellular ROS production via activation of the PI3K pathway resulting in stimulation of Rac1, which upregulates NAD(P)H oxidase activity (Bae et al., 1997). However, the overall mechanism(s) by which NAD(P)H oxidase (and/or Flavin oxidases regulate G1 to S transition P Venkatachalam et al 21 a control 1 +0.5 µM (7 days) 0.5 Treatment b 80 Labeled cells (%) Exposure of MEFs to DPI results in reversible decrease in clonogenic survival While examining the role of flavin oxidases in the proliferative response of irradiated C3H-10T1/2 mouse embryo fibroblasts (MEFs), an apparent cell killing effect of the flavin-oxidase inhibitor, diphenyleneiodonium (DPI), was observed following prolonged exposure (Figure 1a). Cells treated with 0.5 mM DPI for 7 days were completely inhibited from proliferating. Removal of DPI-containing medium and feeding with fresh medium resulted in about 50% of the cells recovering to form colonies consisting of over 200 cells/colony 10 days later. These data suggest that inhibition of flavincontaining oxidases by chronic exposure to low concentrations of DPI induces reversible cell cycle arrests. They support the concept that ROS have a major role in regulating cellular proliferation (Murrell et al., 1990; Menon et al., 2003). Surviving fraction Results - 0.5 µM (10 days) on progression of normal human fibroblasts from G1 to S phase. AG1522 cells synchronized in G0/G1 by density inhibition of growth were incubated with 0.05 or 0.15 mM DPI for 20 h. After treatment, control and DPI-treated cells were subcultured in DPI-free medium containing [3H]-thymidine (1 mCi/ml) and allowed to progress through the cell cycle. Movement into S phase was monitored autoradiographically by measuring the cumulative labeling indices (CLI) at multiple time points up to 50 h after subculture. Compared with shamtreated cells, significant delays in progression into + 0.5 µM (7 days) other flavin-containing oxidases or lipoxygenases) mediates cellular proliferation under normal endogenous oxidative conditions is not clear. Using genetic and biochemical ap (...truncated)