As₂O₃-induced c-Src/EGFR/ERK signaling is via Sp1 binding sites to stimulate p21^WAF1/CIP1 expression in human epidermoid carcinoma A431 cells

Arsenic has been effectively used to treat acute promyelocytic leukemia, and can induce cell cycle arrest or apoptosis in human solid tumors. Previously, we have demonstrated that As₂O₃ can induce p21 ^WAF1/CIP1 (p21) expression in A431 cells and then due to cellular cytotoxicity. Presently, we have clarified these signaling events and compared them with EGF. Using reporter assay, RT-PCR and Western blotting, we show that c-Src activation might be a prerequisite for As₂O₃-induced EGFR/Ras/Raf/ERK signaling. Furthermore, with the aids of 5′-deletion and site-directed mutagenesis, we demonstrate that Sp1 binding sites, ranging from - 64 to - 84 bp, are essential for As₂O₃- or EGF-regulated p21 expression. Finally, our experiments utilizing cycloheximide prompt the suggestion that the stability of mRNA or protein also contributes to As ₂O₃- or EGF-induced p21 expression. Taken together, we conclude that the Sp1 binding sites are required for As₂O ₃-induced p21 gene transcription through c-Src/EGFR/Ras/Raf/ERK pathway. Furthermore, post-transcriptional or post-translational stabilization mechanism is also essential for As₂O₃-induced p21 expression. EGF-induced p21 expression may involve similar mechanisms as those that operate in the As₂O₃-mediated reactions in A431 cells.