The Roles of P53 and Its Family Proteins, P63 and P73, in the DNA Damage Stress Response in Organogenesis-Stage Mouse Embryos.

Abstract

Members of the P53 transcription factor family, P53, P63, and P73, play important roles in normal development and in regulating the expression of genes that control apoptosis and cell cycle progression in response to genotoxic stress. P53 is involved in the DNA damage response pathway that is activated by hydroxyurea in organogenesis-stage murine embryos. The extent to which P63 and P73 contribute to this stress response is not known. To address this question, we examined the roles of P53, P63, and P73 in mediating the response of Trp53-positive and Trp53-deficient murine embryos to a single dose of hydroxyurea (400 mg/kg) on gestational day 9. Hydroxyurea treatment downregulated the expression of Trp63 and upregulated Trp73 in the absence of effects on the levels of Trp53 transcripts; Trp73 upregulation was P53-dependent. At the protein level, hydroxyurea treatment increased the levels and phosphorylation of P53 in the absence of effects on P63 and P73. Upregulation of the expression of genes that regulate cell cycle arrest and apoptosis, Cdkn1a, Rb1, Fas, Trp53inp1, and Pmaip1, was P53-dependent in hydroxyurea-treated embryos. The increase in cleaved caspase-3 and cleaved mammalian sterile-20-like-1 kinase levels induced by hydroxyurea was also P53-dependent; in contrast, the increase in phosphorylated H2AX, a marker of DNA double-strand breaks, in response to hydroxyurea treatment was only partially P53-dependent. Together, our data show that P53 is the principal P53 family member that is activated in the embryonic DNA damage response.