Superoxide Signaling Mediates N-acetyl-l-cysteine–Induced G1 Arrest: Regulatory Role of Cyclin D1 and Manganese Superoxide Dismutase

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Abstract
Thiol antioxidants, including N-acetyl-l-cysteine (NAC), are widely used as modulators of the intracellular redox state. We investigated the hypothesis that NAC-induced reactive oxygen species (ROS) signaling perturbs cellular proliferation by regulating the cell cycle regulatory protein cyclin D1 and the ROS scavenging enzyme Mn–superoxide dismutase (MnSOD). When cultured in media containing NAC, mouse fibroblasts showed G1 arrest with decreased cyclin D1 protein levels. The absence of a NAC-induced G1 arrest in fibroblasts overexpressing cyclin D1 (or a nondegradable mutant of cyclin D1-T286A) indicates that cyclin D1 regulates this G1 arrest. A delayed response to NAC exposure was an increase in both MnSOD protein and activity. NAC-induced G1 arrest is exacerbated in MnSOD heterozygous fibroblasts. Results from electron spin resonance spectroscopy and flow cytometry measurements of dihydroethidine fluorescence showed an approximately 2-fold to 3-fold increase in the steady-state levels of superoxide (O2•−) in NAC-treated cells compared with control. Scavenging of O2•− with Tiron reversed the NAC-induced G1 arrest. These results show that an O2•− signaling pathway regulates NAC-induced G1 arrest by decreasing cyclin D1 protein levels and increasing MnSOD activity. [Cancer Res 2007;67(13):6392–9]