Redox control of nitric oxide-mediated G0/G1 cell cycle arrest and cytotoxicity

The intracellular redox milieu is known to be an important determinant of NO's biological activity, but the relationship between the redox milieu and NO mediated cytostasis and cytotoxicity is less well characterized. In this study, we established an experimental model in which NIH3T3 fibroblasts were exposed to a long half life NO donor diethylenetriamine NONOate (DETA/NO). We investigated (1) the role of the intracellular antioxidant glutathione (GSH) in regulating specific signaling events that are associated with NO-mediated cell cycle arrest, and (2) the effects of GSH depletion on NO's cytotoxicity. We found that: (1) GSH depletion through pharmacological inhibition of glutamate-cysteine ligase (GCL) potentiated nitrosative stress, DNA damage, phosphorylation of the tumor suppressor p53 (Ser-18) and upregulation of p21 cip1/waf1 upon NO stimulation. (2) Neither overexpression of a dominant negative p53 nor pharmacological inhibition of p53 with cyclic pifithrin-alpha (cPFT-alpha) was sufficient to reverse NO-mediated cell cycle arrest or hypophosphorylation of retinoblastoma protein (pRb). (3) NO mediated cytostasis was associated with a redox sensitive downregulation of cyclin D1, which indicated that the redox regulation of NO-mediated cytostasis was a multifaceted process and that both p53/p21cip1/waf1 and p53 independent cyclin D1 pathways were involved in. (4) Pharmacological depletion of GSH by BSO alone was not sufficient to induce cytotoxicity upon NO stimulation. (5) NO induced the upregulation of some antioxidant genes including glutathione peroxidase 1 (GPX1) and glutathione reductase (GR) in addition to the catalytic subunit of GCL (GCLc). Taken together, these results implied that: (1) GSH serves as an important component of cellular protective mechanisms against NO-derived nitrosative stress to regulate DNA damage checkpoint. (2) Multifaceted cellular defense systems in addition to GSH may participate in the protective responses to mitigate damage induced by NO derived nitrosative stress. A better understanding of the functional redundancy and cooperative interactions between this collection of defense pathways during NO mediated cytostasis and cytotoxicity will provide new insights into chemoprevention and immuno-modulatory strategies associated with NO production.