Venkatachalam P, de Toledo S M, Pandey B N, Tephly L A, Carter A B, Little J B, Spitz D R, Azzam E I
Department of Radiology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ, USA.
Oncogene. 2008 Jan 3;27(1):20-31. doi: 10.1038/sj.onc.1210634. Epub 2007 Jul 16.
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).
我们在人和啮齿动物成纤维细胞中研究了含黄素氧化酶产生的活性氧（ROS）在调节细胞周期进程中所起作用的潜在机制。用无毒/无致断裂浓度的黄素蛋白抑制剂二亚苯基碘鎓（DPI）孵育汇合的细胞培养物，可降低烟酰胺腺嘌呤二核苷酸磷酸（NAD(P)H）氧化酶活性和基础ROS水平，但会增加细胞周期蛋白D1、p21（Waf1）和磷酸化p38（MAPK）的蛋白水解。当这些细胞在不含DPI的培养基中传代培养以使其增殖时，观察到广泛的G1期延迟，同时p53/p21（Waf1）信号激活，有丝分裂原激活激酶的磷酸化减少。同时暴露于DPI和无毒剂量的ROS生成剂γ射线或叔丁基过氧化氢以补偿氧化剂生成的减少，可减弱G1期延迟。虽然DPI诱导的G1期检查点完全依赖于PHOX91、ATM和WAF1，但仅部分依赖于P53。有趣的是，当另一种含黄素酶一氧化氮合酶被抑制时，G1期到S期的进程不受影响，也与线粒体膜电位的变化无关。用DPI处理的增殖细胞在G2期也经历了显著但减弱的延迟。我们提出，ATM在介导由非吞噬性NAD(P)H氧化酶活性调节的正常细胞增殖中发挥关键作用，这可能是将癌细胞阻滞在G1期的一个新靶点。
