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

Abstract

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.

Keywords: birth defects; developmental toxicity; embryonic stress; teratogen.

FIG. 1

Hydroxyurea significantly impacts gene expression profiles in GD 9 embryos and activates several cell cycle and cell death related pathways. A, Left: Volcano plot of all detected entities in the microarray. Blue dots indicate transcripts that were significantly changed (-Log P-value = 1.3; P < .05) by at least 1.5-fold. Right: Bar graph of detected probes that were significantly upregulated or downregulated by at least 1.5-fold in response to hydroxyurea treatment in GD 9 embryos. B, Data collected from GeneSpring were analyzed using the Ingenuity Pathway Analysis (IPA) software to predict and examine the molecular pathways that were most affected by hydroxyurea exposure. Vertical bars indicate the level of significance of each pathway indicated by –Log P-value; boxes (▪) indicate the ratio between detected genes in the microarray and total number of known genes in the database for that pathway. Statistical analysis was conducted with the IPA software. N = 4 for each treatment group.

FIG. 2

Pathway analysis predicts P53 as the most activated upstream regulator in response to hydroxyurea exposure in GD 9 embryos. A, Schematic representation of interactions between P53 and downstream targets detected in the microarray. Colors indicate the level of transcript expression as determined by the Log fold change extracted from the microarray data using GeneSpring; red denotes upregulation, blue denotes downregulation, whereas grey denotes no effect. B, Venn diagram of common and unique genes associated with the transcription factors P53, P63, and P73. Data were extracted from analysis of predicted activated upstream regulators as determined by the IPA software.

FIG. 3

P53 and Phospho-P53 protein levels increased in a dose-dependent fashion after hydroxyurea exposure. A, Top: Representative blots of P53 protein expression. Bottom: Quantification of P53 levels in embryos from the 400 mg/kg hydroxyurea treatment group (HU400) (Fold change [FC] = 2.83, SEM = ±0.36) and the 600 mg/kg group (HU600) (FC = 3.96, SEM = ±0.48). B, Top: Representative blots of phospho-P53 protein expression. Bottom: Quantification of phospho-P53 levels in HU400 (FC = 3.1, SEM = ±1.5) and HU600 treated embryos (FC = 5.55, SEM = ±1.2). P53 and phospho-P53 levels were significantly upregulated in both hydroxyurea treated groups compared with control. All protein levels were normalized to ACTIN protein expression. N = 4–5 for each treatment group. *P < .05, **P < .01, one-way ANOVA with Bonferroni post-hoc test.

FIG. 4

Hydroxyurea exposure induced a widespread increase in phospho-P53 immunoreactivity. A, Representative multiphoton and confocal microscopy images taken at × 20 of whole embryo sections showing increasing phospho-P53 immunoreactivity (red, top panel) and the DAPI nuclear counterstain (blue, bottom panel). B, Average mean intensity of phospho-P53 immunoreactivity in whole embryo sections. A significant increase in phospho-P53 intensity was detected in the HU400 and HU600 groups. N = 4–5 for each treatment group. ***P < .001, ****P < .0001, one-way ANOVA with Bonferroni post-hoc test.

FIG. 5

Nuclear translocation of phospho-P53 increased significantly with hydroxyurea treatment. A, Representative multiphoton and confocal images of phospho-P53 immunoreactivity taken at × 63 magnification. Three tissues were analyzed per embryo across all treatment groups (Heart, CNE: caudal neuroepithelium, RNE: rostral neuroepithelium). B, Quantification of phospho-P53 colocalization with DAPI. N = 4–5 for each tissue and treatment group. *P < .05, ***P < .001, ****P < .0001 compared with control, 2-way ANOVA with Bonferroni post-hoc test. There were no statistically significant differences in phospho-P53 nuclear translocation between tissues in the embryo, and no statistically significant interaction between the treatment and tissue variables.

FIG. 6

Transcription levels of P53 downstream targets in response to hydroxyurea exposure. A, Trp53 transcript levels after hydroxyurea treatment were not significantly different compared with control, N = 3. B–D, Hydroxyurea exposure significantly induced transcript levels of Cdkn1a (HU400 FC = 12.02, SEM = ±1.90; HU600 FC = 26.11, SEM = ±7.95, N = 5), Fas (HU400 FC = 2.83, SEM = ±0.54; HU600 FC = 5.6, SEM = ±1.59, N = 4–5), and Trp53inp1 (HU400 FC = 7.77, SEM = ±0.93; HU600 FC = 7.52, SEM = ±0.44, N = 5). Each bar represents the fold change of the mean quantity of the transcript relative to Hprt1. *P < .05, **P < .01, ***P < .001, ****P < .0001, one-way ANOVA followed by a Bonferroni post-hoc test.

FIG. 7

Upregulation of P53INP1 in response to hydroxyurea treatment. A, Representative blot of P53INP1 protein expression levels in embryos exposed to hydroxyurea compared with controls. B, Quantification of immunoblots showed that hydroxyurea treatment significantly upregulated P53INP1 in the HU400 (FC = 9.95, SEM = ±0.75) and HU600 (FC = 10.78, SEM = 2.29) groups. N = 4–5 for each treatment group. ****P < .0001, one-way ANOVA followed by a Bonferroni post-hoc test.