H₂O₂-induced AP-1 activation and its effect on p21^WAF1/CIP1-mediated G2/M arrest in a p53-deficient human lung cancer cell

Cellular response to oxidative stress is a complex process that is often connected to cell cycle regulation. The present study examines the effect of H₂O₂ on cell cycle regulation and involvement of reactive oxygen species (ROS) in these H₂O₂-induced responses in a p53-deficient human lung carcinoma cell line, H1299. Treatment of the cells with H₂O₂ caused a G2/M phase arrest. Among the redox-sensitive transcription factors, NF-κB and AP-1, we found that only AP-1 was activated by 200μM H₂O₂ in human lung cells. Furthermore, electrophoretic mobility shift assays revealed that H₂O₂ enhanced the DNA binding of AP-1 to a putative AP-1 binding element (TGAGGAA) in the p21^WAF1/CIP1 promoter region (between -2203 and -2197 nucleotides upstream of the transcription initiation site). An increase in c-Jun phosphorylation by ERK was also found to accompany the increased AP-1 activity as detected by Western blot. PD98059, a specific inhibitor of MEK, diminished H₂O₂-induced phosphorylation of c-Jun and DNA binding activity of AP-1, decreased expression of p21^WAF1/CIP1, and released the cells from G2/M arrest. Taken together, these results revealed a novel AP-1 binding site in the promoter region of p21^WAF1/CIP1 and a possible cell cycle regulation mechanism mediated by activation of a redox-dependent ERK signaling pathway.