Hydrogen peroxide increases a 55-kDa tyrosinase concomitantly with induction of p53-dependent p21 waf1 expression and a greater Bax/Bcl-2 ratio in pigmented melanoma

Abstract

Differentiated melanocytic cells produce melanin, through several redox reactions including tyrosinase-catalyzed DOPA oxidation to DOPA quinone. We now developed a method based on DOPA oxidase in-gel detection and Sypro Ruby fluorometric normalization to investigate induction of specific DOPA oxidase isoforms in response to hydrogen peroxide-mediated stress, and to ask whether this is associated with p53-dependent adaptive responses. This report shows that hydrogen peroxide leads to comparable induction of 60 and 55 kDa DOPA oxidases in poorly pigmented B16 melanoma, in contrast to sole induction of a major 55 kDa DOPA oxidase in their highly pigmented counterparts. In the latter cells, this response also increases p53 concomitant with joint induction of p53-activated proteins like the cell-cycle inhibitor p21WAF1 and pro-apoptotic bax, with no comparable effect on expression of anti-apoptotic bcl-2. Together, these data suggest that response to hydrogen peroxide involves p53-mediated growth-restrictive signaling and unequal induction of specific DOPA oxidases in melanocytic cells with unequal basal pigmentation.