Hydroxyurea embryotoxicity is enhanced in P53-deficient mice.

Hydroxyurea, a ribonucleotide reductase inhibitor, is a potent teratogen in mice, causing severe limb and skeletal defects. The exposure of gestation day nine murine embryos to hydroxyurea elicits an early embryonic stress response that involves activation of the P53 transcription factor. The impact of this P53 activation on the embryotoxicity of hydroxyurea- is not known. The goal of this study was to test the hypothesis that P53 acts to suppress hydroxyurea embryotoxicity. Trp53+/- timed pregnant mice were treated with saline or hydroxyurea (200 or 400 mg/kg) on gestation day nine; fetuses were examined for viability and external and skeletal malformations on gestation day eighteen. Neither the deletion of Trp53 nor hydroxyurea treatment significantly affected fetal growth although a trend towards a decrease in fetal weights was observed in Trp53-/- fetuses. However, hydroxyurea induced a significantly higher incidence of malformations and resorptions in Trp53-/- fetuses compared to their wildtype littermates. Thus, fetal P53 genotype is an important determinant of the effects of hydroxyurea on organogenesis-stage embryos.

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

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.

Editor's Highlight: Hydroxyurea Exposure Activates the P53 Signaling Pathway in Murine Organogenesis-Stage Embryos.

Hydroxyurea, an anticancer agent and potent teratogen, induces oxidative stress and activates a DNA damage response pathway in the gestation day (GD) 9 mouse embryo. To delineate the stress response pathways activated by this drug, we investigated the effect of hydroxyurea exposure on the transcriptome of GD 9 embryos. Timed pregnant CD-1 mice were treated with saline or hydroxyurea (400 mg/kg or 600 mg/kg) on GD 9; embryonic gene and protein expression were examined 3 h later. Microarray analysis revealed that the expression of 1346 probe sets changed significantly in embryos exposed to hydroxyurea compared with controls; the P53 signaling pathway was highly affected. In addition, P53 related family members, P63 and P73, were predicted to be activated and had common and unique downstream targets. Western blot analysis revealed that active phospho-P53 was significantly increased in drug-exposed embryos; confocal microscopy showed that the translocation of phospho-P53 to the nucleus was widespread in the embryo. Furthermore, qRT-PCR showed that the expression of P53-regulated genes (Cdkn1A, Fas, and Trp53inp1) was significantly upregulated in hydroxyurea-exposed embryos; the concentration of the redox sensitive P53INP1 protein was also increased in a hydroxyurea dose-dependent fashion. Thus, hydroxyurea elicits a significant effect on the transcriptome of the organogenesis stage murine embryo, activating several key developmental signaling pathways related to DNA damage and oxidative stress. We propose that the P53 pathway plays a central role in the embryonic stress response and the developmental outcome after teratogen exposure.