The p53-activated gene, PAG608, requires a zinc finger domain for nuclear localization and oxidative stress-induced apoptosis

Abstract

The p53-activated gene PAG608, which encodes a nuclear zinc finger protein, is a p53-inducible gene that contributes to p53-mediated apoptosis. However, the mechanisms by which PAG608 is involved in the apoptosis of neuronal cells are still obscure. In this study, we demonstrated that expression of p53 was induced by 100 μM 6-hydroxydopamine (6-OHDA), accompanied by increased PAG608 expression in PC12 cells. On the other hand, transient or permanent transfection of antisense PAG608 cDNA into PC12 cells significantly prevented apoptotic cell death induced by 100 μM 6-OHDA or 200 μM hydrogen peroxide but not by 250 μM 1-methyl-4-phenylpyridinium ion. The 6-OHDA-induced activation of caspase-3, DNA fragmentation, loss of mitochondrial membrane potential, and induction of p53 and Bax were also prevented in PC12 cells that stably expressed antisense PAG608 cDNA. These results suggest that PAG608 is associated with the apoptotic pathway induced by these oxidative stress-generating reagents, upstream of the collapse in the mitochondrial membrane potential in PC12 cells. Interestingly, transient transfection with PAG608 cDNA increased p53 expression in both PC12 cells and B65 cells, indicating that PAG608 induced by p53 is able to induce p53 expression in these cells inversely. Furthermore, transient transfection of a truncated mutant PAG608 cDNA, lacking the first zinc finger domain, inhibited 6-OHDA-induced cell death and altered the nuclear and nucleolar localization of wild-type PAG608 in PC12 cells. These results suggest that PAG608 may induce or regulate p53 expression and translocate to the nucleus and nucleolus using its first zinc finger domain during oxidative stress-induced apoptosis of catecholamine-containing cells.