ToxSci Advance Access originally published online on October 26, 2006
Toxicological Sciences 2007 95(1):257-269; doi:10.1093/toxsci/kfl143
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Teratogen-Induced Activation of p53 in Early Postimplantation Mouse Embryos
* Department of Veterinary Physiology and Pharmacology, MS4466 435 VMR Building, Texas A&M University, College Station, Texas 77843
Birth Defects Research Laboratory, Division of Genetics and Developmental Medicine, Department of Pediatrics, University of Washington, Seattle, Washington 98195
1 To whom correspondence should be addressed. Fax: (979) 862-4929. E-mail: pmirkes{at}cvm.tamu.edu.
Received September 20, 2006; accepted October 23, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hyperthermia (HS) and 4-hydroperoxycyclophosphamide (4CP) activate the mitochondrial apoptotic pathway in day 9 mouse embryos. Previous microarray analyses Microarray analyses revealed that several p53 target genes are upregulated after exposure to HS or 4CP, suggesting a role for p53 in teratogen-induced apoptosis. To explore the role of p53, we assessed the activation of p53 in day 9 mouse embryos exposed to HS or 4CP in vitro. Both teratogens induced the accumulation of p53 and phosphorylation of p53 at ser-15, two hallmarks of p53 activation. HS and 4CP also induced an increase in Noxa and Puma mRNAs, transcripts of two known proapoptotic p53 target genes; however, these two teratogens did not induce significant increases in NOXA and PUMA proteins, suggesting that p53 does not activate the mitochondrial apoptotic pathway by transcriptionally upregulating the expression of NOXA and PUMA proteins. HS and 4CP also induced the expression of p21 mRNA and protein, suggesting a role for p53 in teratogen-induced cell cycle arrest. Previously, we also showed that HS and 4CP activate the apoptotic pathway in the embryo proper (head and trunk) but not in the heart. We now show that HS and 4CP induce a robust activation of p53 in the embryo proper but an attenuated induction in the heart. HS and 4CP induce the expression of p21 protein in majority of the cells in the embryo; however, expression of NOXA and PUMA proteins were not significantly induced in heads, hearts, or trunks of day 9 embryos. Overall, our results suggest that p53 may play a transcription-dependent role in teratogen-induced cell cycle arrest but a transcription-independent role in teratogen-induced apoptosis in day 9 mouse embryos exposed to HS or 4CP.
Key Words: day 9 mouse embryo; hyperthermia; 4-hydroperoxycyclophosphamide; embryotoxicity; p53; apoptosis; Noxa; Puma; cell cycle arrest; p21.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Over 1200 chemical and physical agents are known to cause structural and/or functional congenital anomalies in experimental animals (Shepard, 2001
). Although the mechanisms by which these agents disrupt normal development are often not well understood, it is known that many teratogens induce cell death in tissues that subsequently develop abnormally and give rise to structural malformations (Knudsen, 1997; Scott, 1977). In addition, teratogens often induce cell death in areas of normal programmed cell death (PCD) suggesting a mechanistic link between PCD and teratogen-induced cell death (Menkes et al., 1970; Milaire and Rooze, 1983; Sulik et al., 1988). Finally, mouse mutants, in which genes known to play a role in apoptosis have been deleted, exhibit abnormal levels of apoptosis associated with abnormal development often culminating in structural birth defects (Boya and de la Rosa, 2005). Taken together, available data indicate that the dysregulation of apoptosis is consistently correlated with embryo/fetal lethality and/or birth defects.
Previously, we showed that hyperthermia (HS), 4-hydroperoxycyclophosphamide (4CP), and staurosporine, known animal teratogens, induce apoptosis in early postimplantation rodent embryos by activating the mitochondrial apoptotic pathway. Activation of this pathway is characterized by the release of cytochrome c and the subsequent activation of caspases, cleavage of poly ADP-ribose polymerase, and DNA fragmentation (Little and Mirkes, 2002; Little et al., 2003; Mirkes and Little, 1998, 2000). Thus, at least for this small sampling of teratogens, teratogen-induced apoptosis in early postimplantaion mouse embryos involves activation of the mitochondrial apoptotic pathway.
Using vital dyes and TUNEL staining, we have also shown that teratogen-induced cell death is cell specific, that is, some cells in the mouse embryo die, particularly in areas of normal PCD, while other cells, often neighboring cells, survive (Mirkes and Little, 1998; Umpierre et al., 2001). For example, cells of the embryonic nervous system (neuroepithelial cells) are particularly sensitive to teratogen-induced cell death, whereas mesenchymal cells surrounding the neuroepithelium are less sensitive (Umpierre et al., 2001). In contrast, cells of the embryonic heart are resistant to cell death induced by a variety of teratogens (Gao et al., 1994; Umpierre et al., 2001). We have also shown that although teratogens activate the apoptotic pathway in sensitive cells, these hallmarks of apoptosis are not activated in cells of the heart (Mirkes and Little, 1998, 2000; Umpierre et al., 2001). These results indicate that the mitochondrial apoptotic pathway is blocked in heart cells at the level of the cytochrome c release from mitochondria or at some point upstream of cytochrome c release.
The rapid induction of the mitochondrial apoptotic pathway in teratogen-sensitive neuroepithelial cells and the failure to activate this pathway in teratogen-resistant heart cells suggest that the embryo must possess factors that regulate the efflux of cytochrome c and thereby the activation of the mitochondrial apoptotic pathway. To begin to identify proteins and signaling pathways that regulate cytochrome c release, we compared gene expression patterns in HS- or 4CP-treated and -untreated mouse embryos before and during the activation of the mitochondrial apoptotic pathway, using DNA microarray gene expression profiling. Our studies identified five candidate "apoptosis-related" genes (Mikheeva et al., 2004). Three of these genes, Mdm2, Gtse1, and Cyclin G, are coordinately upregulated by both HS and 4CP during the first 5 h after embryos are exposed to these teratogens. Because these three genes are all p53-regulated genes, our microarray data suggested that HS and 4CP both activate p53.
p53 is essential for preventing inappropriate cell proliferation and maintaining genomic integrity following a variety of stresses (Harris and Levine, 2005). The level of p53 is maintained at a low level because of its rapid turnover through proteolysis. Following DNA damage, hypoxia, oncogene expression, and nucleotide depletion, p53 undergoes extensive posttranslational modifications. These modifications result in the accumulation of p53, its translocation into the nucleus, enhanced binding to DNA, and transcriptional activation of its target genes. The protein products of these target genes subsequently regulate a number of cellular processes, the most well studied being cell cycle arrest and apoptosis.
The objectives of the current studies were (1) to assess the kinetics of p53 activation in day 9 embryos exposed to HS or 4CP, (2) to determine whether key p53 proapoptotic or cell cycle arrest genes/proteins are induced by HS and/or 4CP, (3) to assess whether p53 is activated in cells sensitive and resistant to teratogen-induced apoptosis, and (4) to determine whether key p53 proapoptotic or cell cycle arrest genes/proteins are induced by HS and/or 4CP in cells sensitive and resistant to teratogen-induced apoptosis. Our data show that p53 is rapidly activated in day 9 mouse embryos exposed to HS or 4CP and suggest that once activated, p53 plays an important role in teratogen-induced apoptosis and cell cycle arrest.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In vitro whole-embryo culture.
Primigravida Swiss-Webster mice were obtained from a local supplier. The morning following copulation was designated day 0 of gestation. On the morning of day 9, conceptuses from multiple litters were explanted using the whole rodent embryo culture system established by New (1978)
with the following modifications. Embryos from all litters were equally distributed among the different treatment groups. For each treatment group, 10–12 embryos were cultured in 12 ml of media containing 80% heat-inactivated rat serum, 20% Hanks' balanced salt solution (HBSS), 50 U/ml penicillin, 50 µg/ml streptomycin; gassed with a mixture of 20% O2, 5% CO2, 75% N2; and cultured for 1 h prior to treatment.
Embryo exposure conditions.
Treatment of the embryos with 4CP, a preactivated analogue of cyclophosphamide (a gift of Michael Colvin, Johns Hopkins University), was initiated by direct addition of freshly prepared 250x solution of 4CP (in HBSS) to the culture medium resulting in a final concentration of 40µM 4CP. HS-treated embryos were exposed to 43°C for 15 min and then returned to 37°C (Mirkes, 1985b). Embryos were continued in culture with drug or following heat shock for up to 5 h.
Embryo collections.
At indicated times, treated and control embryos were removed from culture, dissected free of associated membranes and rinsed in cold HBSS. Embryos from each treatment group were pooled prior to the preparation of embryo lysates. For those experiments requiring embryo parts, groups of embryo were further dissected into heads, hearts, and trunks using a fine lancet and forceps. The dissected head consisted of the prosencephalon and mesencephalon and the trunk consisted of the remainder of the embryo minus the heart. Dissected heads, hearts, and trunks were separately pooled prior to the preparation of head, heart, and trunk lysates.
Western blot.
Following treatment, washed embryos or embryo parts were sonicated in RIPA lysis buffer (10mM HEPES, pH 7.4, 42mM KCl, 5mM MgCl2, 0.1mM EDTA, 0.1mM EGTA, 1% Triton-X100 plus 1mM PMSF, 1mM DTT, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, and 5 µg/ml aprotinin). Aliquots were taken for protein quantification Bicinchoninic Acid (BCA) assay by BCA assay and equal amounts of protein of each sample in 2x laemmli buffer were applied to 12.5% PAGE (Laemmli, 1970) and transferred to PVDF membranes. Immunoblot analysis was carried out as previously described (Mirkes and Little, 1998) using 3% nonfat dry milk in tris-buffered saline (TBS)/0.5% Tween-20 (TW) for blocking and antibody dilutions. The primary antibodies used were mouse monoclonal anti-Pan p53 at 1:500, rabbit polyclonal anti-human phosphoserine-6, -9, -15, -20, -37, -46, -392 specific p53 (Cell Signaling, Beverly, MA) at 1:1000–1:2000 (note: human ser-15 is equivalent to mouse serine-18), mouse monoclonal anti-p21 at 1:625 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-NOXA at 1:1000 (Ingenex, San Diego, CA), rabbit polyclonal anti-
PUMA at 1:5000 (Oncogene Science, San Diego, CA), mouse monoclonal anti-GAPDH at 1:1,200,000 (Chemicon, Temecula, CA), and mouse monoclonal anti-actin at 1:300,000 (Sigma, St Louis, MO). Membranes were incubated overnight with primary antibodies and then washed four times with TBS/TW. HRP-linked anti-mouse or anti-rabbit secondary antibodies (Amersham Life Sciences, Arlington Heights, IL) were used at 1:3000 for 2 h, and membranes were washed twice with TBS/TW and three times with TBS. Antigen-antibody complexes were visualized by development with ECL Plus (Amershan Life Sciences) and autoradiography. For image analysis, antigen-antibody complexes were detected using the KODAK Image Station 440, and quantification of protein band densities was determined using ImageQuant (Molecular Dynamics, Sunnyvale, CA). Differences in levels of particular polypeptides were assessed using t-test (Statview 512).
Real-time PCR.
Embryos for real-time PCR were quick-frozen and stored at – 80°C. Total RNA was isolated from embryos using the RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. After treatment with 1 U DNase (Promega, Madison, WI) at 37°C for 30 min, followed by incubating with stop solution at 65°C for 10 min, 2 µg total RNA was reverse transcribed using 0.025 µg/µl oligo-d(T)15 primer and 0.5mM dNTP mix (Roche Applied Science, Indianapolis, IN) by heating at 70°C for 5 min, followed by cooling on ice. The mRNA was then copied into cDNA by diluting 10-fold with first-strand buffer containing 100 U SuperScript II (Gibco, Rockville, MD), 10mM DTT and 1 U/µl RNasin (Promega) and incubated at 45°C for 1 h. The reaction was terminated by placing at 70°C for 15 min and then brought to 4°C and stored at – 80°C until analyzed by real-time PCR. Fluorogenic 5' nuclease assays (TaqMan) were carried out in Functional Genomics Laboratory in the Center for Ecogenetics and Environmental Health at the University of Washington, Seattle, WA, using the ABI PRISM 7500 Sequence Detection System (Applied Biosystems Inc., Foster City, CA). Statistical differences of control to HS or 4CP samples were determined by ANOVA analysis followed by Dunnett post hoc test.
Immunohistochemistry.
Following treatment and embryo collections, embryos were immediately fixed in 4% paraformaldehyde for 2 h at 4°C. The embryos were then dehydrated in several changes in ethanol, embedded in paraffin, sectioned at 5 µm, and mounted on glass slides. Tissue sections were heated at 58°C for 20 min, deparaffinized in histoclear, and rehydrated. Sections were then boiled for 20 min with sodium citrate buffer (pH 6.0) for antigen unmasking. Endogenous peroxidase activity was blocked by placing slides in 3% H2O2 in dH2O for 10 min. Slides were briefly rinsed in distilled water and in TBS/TW. Sections were blocked with 1% BSA in TBS for 1 h. Primary antibody solutions, diluted in blocking solution, were placed on the sections overnight. The ser-15 p53 antibody (Cell Signaling) was prepared against a synthetic phosphopeptide corresponding to residues surrounding ser-15 of human p53, and the dilution used was 1:50. To confirm the specificity of ser-15 p53, the primary antibody was incubated with the blocking peptide (Cell Signaling) for 30 min at room temperature and then the adsorbed primary antibody was placed on tissue sections and incubated overnight at room temperature. The p21 antibody (Santa Cruz Biotechnology) was prepared against amino acids 1–159 representing full-length p21 of mouse, and the dilution used was 1:50. Slides were washed three times in TBST for 5 min each. Biotinylated donkey anti-rabbit or sheep anti-mouse IgG secondary antibodies (Amersham) were placed at 1:200 for 30 min. The slides were washed as before and incubated in Vectastain Universal Elite ABC Reagent (Vector Laboratories, Burlingame, CA) for 30 min. Antigen-antibody complexes are visualized as brown staining. Slides were then rinsed in water, dehydrated, counterstained with hematoxylin, and coverslipped.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To investigate the activation of p53, we measured two different but related aspects of teratogen-induced activation of p53, that is, p53 accumulation and site-specific phosphorylation, in day 9 embryos exposed to HS or 4CP. To study p53 accumulation, we used commercially available antibodies that recognize both unphosphorylated (Pan p53) and phosphorylated p53. To determine the kinetics of p53 activation, we assessed total p53 accumulation at 1, 2.5, and 5 h after initiation of exposure to HS or 4CP (Fig. 1). Figure 1A is a representative western blot showing that HS induces an apparent increase in total p53 as early as 2.5 h after exposure. Densitometric analysis of western blots from three independent experiments shows that HS induces a statistically significant increase in total p53 at the 2.5- and 5-h time points (Fig. 1B). We next determined whether another known teratogen, 4CP, also induced the activation of p53 in day 9 mouse embryos. Figure 1C shows that 4CP, like HS, induces an apparent increase in total p53 as early as 2.5 h after exposure to 4CP; however, densitometric analysis of western blots from three independent experiments indicates that a statistically significant increase is only observed at the 5-h time point as shown in Figure 1D.
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To study site-specific phosphorylation of p53, we initially used commercially available antibodies that recognize p53 phosphorylated at specific serine residues (ser-6, -9, -15, -20, -37, -46, -392) and western blot analysis to determine which, if any, sites are phosphorylated in response to HS. Although phospho-specific antibodies to ser-6 (Fig. 2, lanes 3 and 4), ser-9 (Fig. 2, lanes 5 and 6), ser-20 (Fig. 2, lanes 9 and 10), ser-37 (Fig. 2, lanes 11 and 12), and ser-46 (Fig. 2, lanes 13 and 14) p53 failed to detect an increase in phosphorylated p53, the phospho-specific ser-15 p53 antibody (Fig. 2, lanes 7 and 8) detected a HS-induced increase in ser-15 p53. In addition, there may be a slight increase in ser-392 p53 (Fig. 2, lanes 15 and 16) induced by HS.
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On the basis of these preliminary results, we next assessed the kinetics of HS- and 4CP-induced increases in ser-15 p53. Figure 3A is a representative western blot showing that HS induces an apparent increase in ser-15 p53 as early as 2.5 h after exposure. Densitometric analysis of western blots from three independent experiments shows that HS induces a statistically significant increase in ser-15 p53 (Fig. 3B) at the 2.5- and 5-h time points. Figure 3C shows that 4CP, like HS, also induces an apparent increase in ser-15 p53; however, densitometric analysis of western blots from three independent experiments indicates that a statistically significant increase is only observed at the 5-h time point (Fig. 3D).
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Having shown that HS and 4CP both activate p53, we next sought to determine whether these two teratogens activated both arms of the p53 pathway, that is, one leading to cell cycle arrest and the other to apoptosis. To do this, we used real-time PCR and western blot analysis to quantitate the levels of p21, a major p53 target gene involved in cell cycle arrest, and Noxa and Puma, two "proapoptotic" p53 target genes. With respect to the cell cycle arrest arm of the p53 pathway, both HS and 4CP induce a significant increase in p21 mRNA but only at the 5-h time point (Figs. 4A and B). Using western blot analysis, we show that p21 protein levels mirror p21 mRNA levels, with a significant increase in HS- and 4CP-induced p21 levels at the 5-h time point (Figs. 4C and D, respectively).
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With respect to the apoptosis arm of the p53 pathway, HS and 4CP induce apparent increases in Noxa mRNA at the 2.5- and 5-h time points; however, only the HS- and 4CP-induced increases at the 5-h time point are statistically significant (Fig. 5A). Similarly, HS and 4CP also induce apparent increases in Puma mRNA at the 2.5- and 5-h time points; however, only the HS-induced increase at 5 h and 4CP-induced increase at 2.5 and 5 h are statistically significant (Fig. 5B). Although HS and 4CP induced increases in Noxa and Puma mRNAs at the 5-h time point, western blot analysis did not show significant increases in NOXA or PUMA protein at any of the time points studied (Figs. 6A–H). It should be noted that although other studies have identified several isoforms of PUMA (
, ß, 
, and 
), only PUMA is expressed in the day 9 mouse embryo.
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Published research from our laboratory has shown that although HS and 4CP activate the mitochondrial apoptotic pathway leading to apoptosis in day 9 mouse embryos, activation of this pathway does not occur in cells of the embryonic heart, which are resistant to teratogen-induced apoptosis. On the basis of this information, we next compared the activation of p53 in day 9 mouse embryo hearts (resistant tissue) and the day 9 mouse embryo head and trunk (sensitive tissues). Figure 7A presents a representative western blot showing HS-induced increases in total p53 in heads and trunks but not hearts. Densitometric analysis of western blots from three independent experiments shows that HS induces a statistically significant increase in total p53 in heads and trunks (Fig. 7B). In the heart, HS appears to induce a modest increase in total p53; however, this increase is not statistically significant. The results for total p53 are mirrored by those for ser-15, that is, significant increases in ser-15 p53 in heads and trunks and a modest but not a significant increase in ser-15 p53 in hearts (Figs. 7C and D).
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Although the western blot data clearly show that HS induces a robust activation of p53 in the day 9 mouse embryo head and trunk but only a modest increase in the heart, this approach does not provide any information concerning HS-induced activation of p53 in specific cells/tissues of the head and trunk. To obtain such data, we used an anti–ser-15 p53 antibody and immunohistochemistry (Fig. 8). Figure 8A is a parasagittal section from a day 9 control embryo. Occasional p53-positive cells (brown staining) are observed in the neuroepithelium (arrows in Fig. 8B) but not in the heart (Fig. 8C) or trunk (Fig. 8D). In contrast, many more p53-positive cells are observed in a comparable parasagittal section from a day 9 embryo exposed to HS and harvested 5 h after exposure (Fig. 8E). Although p53-positive cells are present in all tissues of the embryonic head and trunk, for example, outer epithelium, neuroepithelium, loose mesenchyme, and somites, the majority of cells in any one tissue are unstained. Higher magnification photomicrographs of the prosencephalon (Fig. 8F) and the trunk region (Fig. 8H) show robust staining in cells and different tissues, for example, epithelial, mesenchymal, and neural cells in the head. Even higher magnification photomicrographs show that this staining is nuclear (data not shown). In contrast, heart cells exhibit either no apparent staining or much reduced staining intensity (Fig. 8G). The lack of staining in Figures 8I–L, in which an adjacent section was stained with the anti–ser-15 p53 antibody that had been adsorbed with the peptide used to make the primary antibody, confirms the specificity of the primary antibody. Overall, these immunohistochemical data confirm the western blot data showing that HS-induced activation is robust in some cells of the embryo proper but attenuated in cells of the heart.
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A western blot analysis of the effects of 4CP exposure on p53 activation in day 9 mouse embryo head, heart, and trunk shows that 4CP induces a statistically significant increase in total p53 (Figs. 9A and B) and ser-15 p53 (Figs. 9C and D) in heads and trunks. Unlike the situation in HS-treated embryos, 4CP induced an attenuated but statistically significant increase in total and ser-15 p53 levels in the heart (Figs. 9B and D).
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Having shown that HS and 4CP induce a robust activation of p53 in the head and trunk and a more attenuated activation in the heart, we next sought to determine the extent to which these two teratogens activated both arms of the p53 pathway, that is, one leading to cell cycle arrest and the other to apoptosis. The induction of p21 in heads, hearts, and trunks is presented in Figure 10. HS-induced activation of p53 in the heads and trunks (Fig. 7) is correlated with a statistically significant induction of p21 (Figs. 10A and B). Similarly, 4CP-induced activation of p53 in the heads and trunks (Fig. 9) is correlated with a statistically significant induction of p21 (Figs. 10C and D). Although there is no apparent activation of p53 in the heart (Fig. 7) and an attenuated but significant activation of p53 in heart cells from 4CP-treated embryos (Fig. 9), HS and 4CP both induced a statistically significant induction of p21 in the heart (Figs. 10B and D).
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To confirm the increased mRNA and protein expression levels of p21 in day 9 mouse embryos after the exposure to HS, and to provide information concerning HS-induced activation of p21 in specific cells/tissues of the head and trunk, immunohistochemistry was used to stain p21 protein in embryos (Fig. 11). Figure 11E is a parasagittal section from a day 9 embryo taken 5 h after exposure to HS and probed with an anti-p21 antibody. Many cells throughout all regions including the heart cells are uniformly stained. Higher magnification photomicropraphs of the prosencepahalon (Fig. 11F), heart (Fig. 11G), and the trunk region (Fig. 11H) show distinct staining in many cells and different tissues. Control embryos (Fig. 11A) stained with p21 showed an absence of staining in the head (Fig. 11B), heart, (Fig. 11C), and trunk (Fig. 11D), except for the specific staining of a subset of cells in somites. Overall, these immunohistochemical data show that HS-induced activation is robust in cells of the embryo, including heart cells, and confirm the western blot data.
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We next determined whether HS- and 4CP-induced activation of p53 was accompanied by an increased expression of NOXA and PUMA proteins, two proapoptotic proteins known to be transcriptional target genes of activated p53, in heads, hearts, and trunks of day 9 mouse embryos (Fig. 12). Western blot analysis (Figs. 12A and E) showed that HS does not induce a significant increase in NOXA (Fig. 12B) or PUMA (Fig. 12F) protein expression in either heads, hearts, or trunks; however, our results do show that NOXA and PUMA proteins are constitutively expressed in heads, hearts, and trunks of unexposed embryos. Similar results were obtained when embryos were exposed to 4CP (Figs. 12C, D, G, and H).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previously published gene expression profiling studies revealed that three known p53 target genes, Mdm2, Gtse1, and Cyclin G, were significantly upregulated in day 9 mouse embryos after exposure to HS or 4CP, thus implicating p53 as an important regulator of teratogen-induced apoptosis (Mikheeva et al., 2004
). Called the "guardian of the genome," p53 is known to play a key role in regulating whether a cell will arrest, undergo apoptosis, senesce, or differentiate in response to various stresses. To achieve this regulatory role, p53 must be activated by a variety of posttranslational modifications, for example, phosphorylation, acetylation, and ubiquitination (Brooks and Gu, 2003). Using a panel of phospho-specific p53 antibodies directed against human ser-6, -9, -15, -20, -37, -46, and -392 p53, we have now shown that p53 is phosphorylated at ser-15 after exposure to HS or 4CP. Phosphorylation at this serine is known to regulate apoptosis because p53-mediated apoptosis is significantly impaired when ser-15 is mutated to alanine (Chao et al., 2003; Sluss et al., 2004). Using an antibody that recognizes phosphorylated and nonphosphorylated p53 (Pan p53), we also showed that the increase in ser-15 p53 is correlated with an increase in total p53. Finally, using ser-15 p53 antibodies and immunohistochemistry, we have shown that activated p53 localizes to the nucleus of stained cells. Together, these results show that HS and 4CP, two teratogens that induce apoptosis in day 9 mouse embryos, also activate p53, a known regulator of apoptosis.
Our data also show that p53 is rapidly activated by both HS and 4CP, with activation occurring between 1 and 2.5 h after exposure. Thus, p53 is activated before HS- and 4CP-induced release of mitochondrial cytochrome c and activation of the caspase cascade, which occur between 2.5 and 5 h after exposure to these two teratogens (Little and Mirkes, 2002; Mirkes and Little, 1998, 2000). Although not definitive, the kinetics of p53 activation are consistent with a regulatory role for p53 in teratogen-induced apoptosis. We are currently using p53 mutant mice to determine whether p53 is required for teratogen-induced apoptosis.
Although our data are consistent with a regulatory role for p53 in teratogen-induced apoptosis, the mechanisms by which p53 activates the mitochondrial apoptotic pathway in the day 9 mouse embryo are unclear. One known mechanism, elucidated from cell culture studies, is the transcription-dependent expression of proapoptotic genes. Two of the major downstream targets of p53-mediated apoptosis are Noxa and Puma, transcripts of proapoptotic genes belonging to the Bcl-2 family. At least in some settings, NOXA protein is an essential mediator of p53-dependent apoptosis (Yakovlev et al., 2004) and activates the mitochondrial apoptotic pathway by interacting with Bcl-2 family members resulting in the release of cytochrome c and the activation of caspase-9 (Oda et al., 2000; Seo et al., 2003). Other studies suggest that PUMA protein interacts with BCL-2 and BCL-XL and thereby induces mitochondrial membrane potential change, cytochrome c release, and caspase activation (Chipuk et al., 2005; Yu et al., 2001, 2003).
Our results indicate that although HS and 4CP both induce increased expression of Noxa and Puma mRNAs, the increased expression of these mRNAs is not coupled with an increased expression of NOXA and PUMA proteins. Although we cannot explain the apparent disconnect between mRNA and protein levels, the explanation may involve a timing issue. Our data show that the increases in Noxa and Puma mRNA levels are significant at the 2.5- and 5-h time points, latter being the latest time point studied in terms of NOXA and PUMA protein levels. Thus, there may not have been sufficient time for the increases in Noxa and Puma mRNAs to be translated into increases in their respective proteins that could be detected by western blot analysis. Had we measured NOXA and PUMA protein levels at some later time point, we may have observed the expected increases. Nonetheless, even if this were observed, increases in NOXA and PUMA proteins at times later than 5 h would not be relevant to the initial activation of the mitochondrial apoptotic pathway, which is activated at some time between 2.5 and 5 h after exposure to these teratogens. Thus, our data suggest that p53-mediated upregulation of NOXA and PUMA proteins is not involved in HS- and 4CP-induced activation of the mitochondrial apoptotic pathway.
Although upregulation of NOXA and PUMA proteins may not be required, these proteins may still play a role in the activation of the mitochondrial apoptotic pathway. Of interest, our data show that NOXA and PUMA proteins are constitutively expressed in the day 9 embryos in the absence of any teratogenic exposure. The function of these proteins in mouse development is unknown but do not appear to be required for normal development because Noxa and Puma null mice are born at the expected frequency and exhibit a normal phenotype (Villunger et al., 2003). How the proapoptotic activity of NOXA and PUMA is blocked is also unknown; however, it may be that these proteins are sequestered in an inactive form that is then activated in response to appropriate apoptotic stimuli. The constitutive expression of proapoptotic Bcl-2 family members that are sequestered and then activated in response to various apoptotic stimuli is well documented. Examples include binding to other proteins (BIM and BMF binding to dynein motor complex), cleavage (inactive BID cleaved to active tBID by caspase-8), and phosphorylation-induced binding of BAD to 14-3-3 (Gross et al., 1999; Li et al., 1998; Puthalakath et al., 1999, 2001; Zha et al., 1996). However, we are not aware of any published data showing that NOXA or PUMA proteins are sequestered in the absence of an apoptotic stimulus and then activated after an appropriate cell death signal.
Even if p53-mediated upregulation of NOXA and PUMA proteins does not play a role in activating the mitochondrial apoptotic pathway in teratogen-exposed mouse embryos, p53 is known to upregulate other proapoptotic proteins, for example, BAX, p53AIP, and PIGs. Whether any of these or other p53 target genes play a role in HS- and 4CP-induced activation of the mitochondrial pathway is unknown; however, our studies have shown that there is no significant increase in BAX protein levels in mouse embryos exposed to HS or 4CP (unpublished data). Although p53 may regulate teratogen-induced apoptosis in the mouse embryo by transcriptionally upregulating proapoptotic target genes, we presently do not have any data to support this possibility.
Alternatively, recent evidence has uncovered a transcription-independent role for p53 in the regulation of apoptosis. Early studies, using cancer cell lines, reported that apoptosis occurred in the presence of inhibitors of transcription and translation or in cells expressing p53 mutants with abrogated transactivation activity (Caelles et al., 1994; Jimenez et al., 2000). Subsequent studies have shown that after apoptotic stimuli activated p53 rapidly translocates to mitochondria where it physically interacts with BCL-2 and BCL-XL to antagonize their antiapoptotic activities or BAX and BAK to promote their proapoptotic activities (Chipuk et al., 2004; Leu et al., 2004; Marchenko et al., 2000; Mihara et al., 2003; Park et al., 2005; Schuler et al., 2000). Current studies are underway to determine whether p53 may have a transcription-independent role in inducing apoptosis in mouse embryos exposed to HS or 4CP.
Our data also show that HS and 4CP induce the upregulation of cyclin-dependent kinase p21 mRNA and protein. Moreover, results from our immunohistochemical analysis indicate that p21 protein is upregulated in most, if not all, cells of the day 9 mouse embryo after exposure to HS. Because p21 is a known p53 target that plays a central role in arresting the cell cycle after various genotoxic stresses (Harris and Levine, 2005; Taylor and Stark, 2001), our results suggest that cells of the day 9 mouse embryo have activated the cell cycle arrest arm of the p53 pathway in response to teratogenic exposures. Although we have not shown that HS induces cell cycle arrest in early postimplantation rodent embryos, we have shown that phosphoramide mustard, the major teratogenic metabolite of 4CP, induces alterations in the cell cycle in postimplantation rat embryos (Little and Mirkes, 1992; Mirkes et al., 1989). In addition, Chernoff et al. (1989) and Francis et al. (1990) have shown that cyclophosphamide induced a dose-dependent increase in the percentage of limb bud cells in the S phase of the cell cycle. Together, these results demonstrate that cyclophosphamide/4CP induce alterations in the cell cycle in early postimplantation mouse embryos exposed in vitro or in vivo.
Published data consistently show that teratogens induce apoptosis in some cells of the embryos and not others (Gao et al., 1994; Mirkes, 1985a,b; Mirkes et al., 1991; Thayer and Mirkes, 1995). With respect to HS- and 4CP-induced apoptosis, cells within the neuroepithelium and neural crest cells that have migrated from the neuroepithelium are the most sensitive to teratogen-induced cell death, whereas surrounding mesenchymal cells as well as epithelial cells are less sensitive. In contrast, cells of the heart are completely resistant to teratogen-induced apoptosis (Umpierre et al., 2001). In addition, activation of the mitochondrial apoptotic pathway, characterized by cytochrome c release, activation of caspases, and the induction of DNA fragmentation, is completely blocked in heart cells from day 9 mouse embryos (Little and Mirkes, 2002; Mirkes and Little, 1998, 2000). In the present studies, we now show that heart cell resistance is associated with significant attenuation of the activation of p53 in heart cells. Despite the attenuated activation of p53 in heart cells in response to teratogenic exposures, our data indicate that both HS and 4CP induce increased levels of p21 in heart cells. These results suggest that p53 is activated in the heart and when activated subsequently upregulates the expression of p21, thereby arresting heart cells. One caveat, however, is that p21 expression can also be induced via p53-independent mechanisms (O'Reilly, 2005). Using p53 null mice, we are currently determining whether HS- and 4CP-induced upregulation of p21 is p53-dependent.
Our results showing robust activation of p53 in cells sensitive to teratogen-induced apoptosis and attenuated activation of p53 in cells resistant to teratogen-induced apoptosis, leads to the hypothesis that high levels of activated p53 induce apoptosis, whereas low levels of activation lead to cell cycle arrest. This hypothesis is supported by studies showing that high amounts of ectopic p53 induce apoptosis, whereas lower amounts result in cell cycle arrest (Chen et al., 1998; Lokshin et al., 2005; Ronen et al., 1996). More recently, Speidel et al. (2006) showed that low levels of UV-irradiation, which led to a relatively low-level activation of p53, induced temporary cell cycle arrest, whereas high levels of UV-irradiation, which induced a more robust activation of p53, led to apoptosis. Although our results are consistent with the cell culture data suggesting that low levels of p53 activation culminate in cell cycle arrest whereas more robust activation of p53 results in apoptosis, additional research will be required to determine whether the sensitivity/resistance of specific cells to teratogen-induced apoptosis in the day 9 mouse embryo is determined by the extent to which p53 is activated.
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ACKNOWLEDGMENTS |
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This research was supported by National Institutes of Health grants R01ES07026, R01ES08744, P30ES07033, and P30ES09106. We express sincere appreciation to Dr Michael Colvin for providing 4CP, Sean Quigley (University of Washington) for his assistance in real-time PCR, and Murat Russell (Texas A&M University) for his assistance in immunohistochemistry.
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