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BOR - Papers in Press, published online ahead of print February 6, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.026252
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BIOLOGY OF REPRODUCTION 70, 1762–1767 (2004)
DOI: 10.1095/biolreprod.103.026252
© 2004 by the Society for the Study of Reproduction, Inc.

Toxicology

Involvement of p53 in 1-ß-D-Arabinofuranosylcytosine-Induced Trophoblastic Cell Apoptosis and Impaired Proliferation in Rat Placenta

Hirofumi Yamauchi1, Kei-ichi Katayama, Masaki Ueno, Koji Uetsuka, Hiroyuki Nakayama, and Kunio Doi

Department of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo,Tokyo 113-8657, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1-ß-D-Arabinofuranosylcytosine (Ara-C), a DNA-damaging agent, severely inhibits fetal growth and has teratogenicity. Recently, we reported that Ara-C also causes placental growth retardation and increases placental apoptosis. The aim of the present study is to elucidate the mechanisms of placental injury induced by genotoxic stress and involvement of p53, which mediates apoptosis and cell-cycle arrest after DNA damage. We injected Ara-C into pregnant rats on Day 13 of gestation and examined the placentas from 1 to 48 h after the administration. Terminal deoxynucleotidyltransferase-mediated dUTP end-labeling (TUNEL) revealed that the apoptosis of trophoblastic cells in the placental labyrinth zone increased from 3 h after the treatment and peaked at 6 h before returning to control levels at 48 h. An increase in cleaved caspase-3 immunoreactivity was also detected at 6 h. Proliferative activity as measured by immunohistochemistry for topoisomerase II{alpha} and by mitotic index significantly decreased after the treatment in the labyrinth zone. Immunoreactivity for p53 protein in the placental labyrinth zone was remarkably enhanced and peaked at 3 h after treatment, although no increase in p53 mRNA expression was detected with a reverse transcription- polymerase chain reaction. Regarding p53 target genes, p21, cyclinG1, and fas mRNA levels increased significantly and peaked at around 9 h after the treatment. These results indicate that Ara-C would induce apoptosis and impair cell proliferation in the placental labyrinth zone, and p53 and its transcriptional target genes may play an important role in the pathogenesis of the Ara-C-induced placental toxicity.

apoptosis, conceptus, placenta, toxicology, trophoblast


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adequate placental growth and development are crucial to the development of the fetus, and dysfunctions of the placenta may be closely related to fetal developmental disabilities [1]. In the placenta during normal pregnancy in humans and experimental animals, apoptosis is observed in various kinds of component cells and is thought to play physiological roles in placental growth, turnover, and parturition [2–5]. In contrast, increased apoptosis is reported in human placenta complicated with intrauterine growth retardation or other abnormal pregnancies [6–10]. In experimental animals, placental apoptosis is induced concomitant with developmental disabilities such as fetal growth retardation, preterm delivery, and increased resorptions by the administration of NG-nitro-L arginine methyl ester [11], lipopolysaccharides [12], and glucocorticoids [5] to dams. Thus, the placenta is thought to be susceptible to endocrinological abnormality, inflammatory cytokines, and oxidative stress, and increased placental apoptosis may cause placental dysfunction, resulting in abnormal fetal development.

Embryos and fetuses are vulnerable to genotoxic stress such as DNA damaging agents or radiation, and congenital anomalies are easily induced [13–16]. Genotoxic stress induces apoptosis and cell-cycle arrest in fetal tissues in a p53-dependent way, and this is recognized as a cause of congenital anomalies [15, 17–19]. The p53, a tumor suppressor gene, is involved in the regulation of apoptosis and cell-cycle arrest after DNA damage mainly through the transcriptional activation of its target genes, thereby preventing the propagation of damaged cells in a number of different paradigms [20].

However, little attention has been given to the effect of genotoxic stress on the placenta. Recently, ethylnitrosourea, a teratogenic DNA alkylating agent, was reported to induce apoptosis and growth arrest in trophoblastic cells in the placental labyrinth zone with the up-regulation of p53 protein in vivo [21]. 1-ß-D-Arabinofuranosylcytosine (Ara-C), a cytidine analogue, is also known as a DNA-damaging agent. Ara-C is used in the clinical treatment for myelogenous leukemia and is associated with fetal growth restrictions and malformations such as microtis, auditory canal atresia, digit anomalies, and lower limb defects in humans [22–24]. It has a teratogenic effect and causes fetal growth retardation also in experimental animals [13, 25, 26]. Previously, we demonstrated that the administration of Ara-C to pregnant rats caused significant decreases of placental weight and thickness of the labyrinth zone with a marked increase in the number of apoptotic cells among trophoblastic cells of the placental labyrinth zone and confirmed that fetal weight was also restricted at 48 h after the administration [27].

The aim of the present study was to investigate the toxic effect of Ara-C in detail and to explore the mechanisms of increased apoptosis and growth inhibition in the placental labyrinth zone. For this purpose, we injected Ara-C into pregnant rats and investigated the sequential changes in the incidence of apoptotic cell death and kinetics of proliferative activity in the placental labyrinth zone histopathologically. In addition, to clarify the involvement of p53 in Ara- C-induced placental toxicity, we examined the sequential expression patterns of p53 protein and the mRNA of p53 and its transcriptional target genes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Chemicals

Pregnant Slc:Wistar rats (plug day: Day 0 of gestation) were obtained from Japan SLC Inc. (Shizuoka, Japan). They were kept under controlled conditions (temperature, 23 ± 2°C; relative humidity, 55% ± 5%) using an isolator caging system (Niki Shoji Co., Tokyo, Japan) and were fed commercial pellets (MF, Oriental Yeast Co., Tokyo, Japan) and water ad libitum. Ara-C (Sigma, St. Louis, MO) was dissolved in PBS and its concentration was adjusted to 50 mg/ml. The protocol of the present study was approved by the Animal Care and Use Committee of the Graduate School of Agricultural and Life Sciences, The University of Tokyo.

Treatments

Pregnant rats were injected intraperitoneally (i.p.) with 250 mg/kg of Ara-C on Day 13 of gestation (GD13). Under the conditions of this experiment, congenital anomalies and growth retardation were detected at a high rate in perinatal fetuses, although the incidence of fetal death was not markedly increased [25, 26]. At 1, 3, 6, 9, 12, 24, and 48 h after the treatment, six dams each were killed by heart puncture under ether anesthesia, and the placentas were collected. As controls, six pregnant rats were injected i.p. with an equivalent volume of PBS on GD13 and killed at the same time point as Ara-C-treated groups. Of the six dams obtained at each time point, three were used for histopathological analyses and three for reverse transcription-polymerase chain reaction (RT-PCR) analysis.

For the histopathological analysis, collected placentas were fixed in 10% neutral-buffered formalin, and 4-µm paraffin sections were stained with hematoxylin-eosin (HE). The sections were also subjected to the detection of fragmented DNA and immunohistochemical staining.

Detection of Fragmented DNA

Cells with fragmented DNA were detected by the terminal deoxynucleotidyltransferase-mediated dUTP end labeling (TUNEL) method, which was first proposed by Gavrieli [28] and is now widely used for the detection of apoptotic cells, using an apoptosis detection kit (Apop Tag; Intergen, Purchase, NY). In brief, multiple fragmented DNA 3'-OH ends on the section were labeled with digoxigenin-dUTP in the presence of terminal deoxynucleotidyl transferase (TdT). Peroxidase-conjugated anti-digoxigenin antibody was then reacted with the sections. The positive signals were visualized using a peroxidase-diaminobenzidine (DAB) reaction. The sections were then counterstained with methylgreen.

Immunohistochemical Staining

Immunohistochemical staining for cleaved caspase-3, topoisomerase II{alpha} (TII{alpha}), and p53 was carried out on paraffin sections. Cleaved caspase- 3 is one of the key executioners of apoptosis and is responsible for the proteolytic cleavage of many key proteins to yield the apoptotic phenotype [29]. TII{alpha} is a proliferation marker of rat and human tissues that is detected in the S to G2/M phase of the cell cycle [30]. Rabbit anticleaved caspase- 3 polyclonal antibody (Cell Signaling Technology, Beverly, MA), mouse anti-TII{alpha} monoclonal antibody (DAKO, Carpinteria, CA), and rabbit anti- p53 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) were used as primary antibodies. Sections were stained by the labeled streptavidin-biotin (LSAB) method with streptavidin (DAKO) for cleaved caspase-3 and TII{alpha}, and by EnVision+ polymer reagent (DAKO) for p53. The positive signals were visualized with a peroxidase-DAB reaction and then the sections were counterstained with methylgreen.

Morphometry

To examine the incidence of apoptotic cell death, TUNEL-positive trophoblastic cells in the placental labyrinth zone were counted in three randomly chosen placentas from a dam. Three hundred cells were counted in randomly chosen fields of labyrinth zone in each placenta under a light microscope. For the assessment of proliferative activity, TII{alpha}-positive trophoblastic cells were counted on the immunohistochemically stained sections and mitotic cells on the HE-stained sections in the same way. To examine p53 protein expression, p53-positive trophoblastic cells were also counted on the immunohistochemically stained sections. The apoptotic, mitotic, and TII{alpha}- or p53-labeling indices (%) were expressed as the mean ± standard deviation (SD) for 3 dams, and statistical analysis was carried out with Student t-test.

RNA Extraction and Semiquantitative RT-PCR

The mRNA expression of p53 and three of its well-known target genes, p21 [31], cyclinG1 [32], and fas [33], was examined. Protein p21 is an inhibitor of cyclin-dependent kinases and induces cell-cycle arrest at the G1 phase [34]. CyclinG1 dephosphorylates mdm2, a negative regulator of p53, and modulates its function [35]. Fas is a type I membrane protein that belongs to the tumor necrosis factor receptor/nerve growth factor receptor family, and it induces apoptosis when it binds to fas ligand [36].

Three or four randomly chosen placentas from a dam were dissected to remove decidua and pooled. Then total RNA was extracted using Isogen (Nippon Gene Co. Ltd., Toyama, Japan). The reverse transcriptase reaction for synthesis of the first strand cDNA was carried out with 15 µg of sample in 60 µl of reaction mixture using oligo(dT)12–18 primer and a SUPERSCRIPT Preamplification System (Invitrogen, Carlsbad, CA). PCR was performed with pairs of oligonucleotide primers corresponding to the cDNA sequences of the rat mRNA (Table 1). PCR was carried out with 1 µl of RT product in a 100-µl reaction mixture containing 50 pM of sense and antisense primer, 1.25 U of rTaq, 10x PCR buffer and dNTP mixture (Takara, Ohtsu, Japan). This was immediately followed by preheating at 94°C for 7 min, denaturation at 94°C for 1 min, annealing at 58.5°C for 1 min, and extension at 72°C for 1 min using a Takara PCR Thermal Cycler MP (Takara). Cycle numbers for different reactions are shown in Table 1. Optimal cycle numbers were determined in a preliminary experiment to ensure that the amplification of each gene was in the linear range and not during the plateau phase. PCR products were identified by electrophoresis on 2% agarose gels (Nippon Gene Co. Ltd.) followed by ethidium bromide (Invitrogen) staining. Fluorescent-gel imaging was carried out using an ultraviolet-CCD video system Fas-III (Toyobo, Tokyo, Japan). The results are shown relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. The relative band density is presented as the mean ± SD for three dams and statistical analysis was carried out using Student t-test.


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  		TABLE 1. Primer sequences, cycle numbers, and accession numbers


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Incidence of Apoptosis

From 3 h after the Ara-C treatment, the number of TUNEL-positive nuclei, which showed pyknosis on HE- stained sections, began to increase in trophoblastic cells of the placental labyrinth zone (Fig. 1). In our previous study, it was confirmed that the ultrastructural characteristics of these pyknotic cells were consistent with those of apoptotic cells [27]. The number peaked at 6 h after the treatment and returned to control levels at 48 h (Fig. 2). Only a few apoptotic cells were observed in the placental labyrinth zone in control dams throughout the experimental period (Figs. 1 and 2). In the placental basal zone of both the Ara- C-treated and control dams, only a few pyknotic cells were detected (data not shown).



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  		FIG. 1. Placenta of the Ara-C-treated (a) and control (b) rats at 6 h after the treatment. Many TUNEL-positive trophoblastic cells are seen in the labyrinth zone in a. TUNEL staining; bar = 55 µm



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  		FIG. 2. Apoptotic index in the labyrinth zone. The number of apoptotic trophoblastic cells peaked at 6 h and returned to control levels at 48 h after the treatment. Data represent the mean ± SD (n = 3). **, P < 0.01; significantly different from controls. Open squares, Ara-C-treated group; closed triangles, control group

At 6 h after the Ara-C-treatment, many positive signals for cleaved caspase-3 were detected in the trophoblastic cells of the placental labyrinth zone by immunohistochemistry (Fig. 3). On the other hand, only a few positive signals were observed in the placental labyrinth zone of control dams at 6 h after the treatment (Fig. 3).



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  		FIG. 3. Placenta of the Ara-C-treated (a) and control (b) rats at 6 h after the treatment. Many positive signals for cleaved caspase-3 are detected in the trophoblastic cells of the labyrinth zone in a. Immunostaining for cleaved caspase-3; bar = 40 µm.

Proliferative Activity

With the immunohistochemistry for TII{alpha}, many positive signals were observed in the trophoblastic cells of the placental labyrinth zone in both the Ara-C-treated and control groups. A significant decrease in the immunoreactivity was detected at 6 h after the treatment (Figs. 4 and 5). The number of mitotic figures in the placental labyrinth zone decreased rapidly and reached a minimum at 3 h after the Ara-C treatment. The mitotic index was suppressed significantly until 24 h after the treatment, and then returned to control levels at 48 h (Fig. 5).



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  		FIG. 4. Placenta of the Ara-C-treated (a) and control (b) rats at 6 h after the treatment. The number of TII{alpha}-positive nuclei of trophoblastic cells is decreased in the labyrinth zone in a. Immunostaining for TII{alpha}; bar = 55 µm



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  		FIG. 5. TII{alpha}-labeling (a) and mitotic (b) indices in the labyrinth zone. A significant decrease in the immunoreactivity for TII{alpha} was detected at 6 h after the Ara-C-treatment. The number of mitotic figures was significantly suppressed from 3 to 24 h after the treatment. Data represent the mean ± SD (n = 3). **, P < 0.01; significantly different from controls. Open squares, Ara-C-treated group; closed triangles, control group.

Expression of p53 Protein

In the placental labyrinth zone of the Ara-C-treated group, the number of p53-positive signals in the nuclei of trophoblastic cells began to increase from 1 h and peaked at 3 h after the treatment (Fig. 6). Then the number declined from 6 h and returned to control levels at 48 h after the treatment (Fig. 7). In the control group, only a few p53- positive signals were observed in the labyrinth zone throughout the experimental period (Figs. 6 and 7).



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  		FIG. 6. Placenta of the Ara-C-treated (a) and control (b) rats at 3 h after the treatment. Many positive signals for p53 are detected in the nuclei of trophoblastic cells in the labyrinth zone in a. Immunostaining for p53; bar = 55 µm



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  		FIG. 7. The p53-labeling index in the labyrinth zone. The number of p53-positive nuclei of trophoblastic cells peaked at 3 h and returned to control levels at 48 h after the treatment. Data represent the mean ± SD (n = 3). **, P < 0.01; significantly different from controls. Open squares, Ara-C-treated group; closed triangles, control group.

Expression of p53 and Its Transcriptional Target Genes mRNAs

The expression of p53 mRNA changed little throughout the experimental period. The expression of p21, cyclinG1, and fas mRNAs significantly increased in the Ara-C-treated group. The expression of p21, cyclinG1, and fas mRNAs gradually increased from 3 h and peaked at around 9 h, then returned to control levels at 24 or 48 h after the treatment (Figs. 8 and 9).



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  		FIG. 8. Sequential changes in the mRNA expression of p53 and its target genes. The expression of p53 target genes is enhanced after the Ara-C treatment. Agarose gel electrophoresis



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  		FIG. 9. Sequential changes in the expression of p53 (a), p21 (b), cyclinG1 (c), and fas (d) mRNAs in the placenta exposed to Ara-C. Data represent the mean ± SD (n = 3). *, P < 0.05, **, P < 0.01; significantly different from controls. Open squares, Ara-C-treated group; closed triangles, control group.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
As mentioned above, we previously demonstrated that the administration of Ara-C to pregnant rats caused significant decreases of placental weight and thickness of the labyrinth zone concomitant with increased apoptosis in the trophoblastic cells of the placental labyrinth zone [27]. In the present study, we detected a marked increase in the number of TUNEL-positive cells from 3 h to 24 h after the exposure to Ara-C. We also observed increased immunoreactivity for cleaved caspase-3. Caspase-3 is an effector of apoptosis thought to have a tissue- and stimulus-specific function [29]. We considered that caspase-3 would be an important executor of apoptosis also in placenta exposed to genotoxic stress. In addition, an analysis of TII{alpha}-labeling and mitotic indices showed that proliferative activity was substantially suppressed in trophoblastic cells of the placental labyrinth zone. These results suggest that both increased cell death and suppressed cell proliferation result in the growth inhibition of the placenta observed in our previous report.

Several investigators recently demonstrated that, strictly speaking, the TUNEL technique is not specific for apoptosis and it also detects a small population of necrotic cells [37]. However, in our studies, TUNEL-positive trophoblastic cells were considered to be apoptotic ones judging from their electron microscopical features [27] and immunoreactivity for cleaved caspase-3.

Increased apoptosis is reported in human placenta complicated with intrauterine growth retardation or other abnormal pregnancies, and it is believed to be associated with placental dysfunction [6–10]. Trophoblastic cells in the placental labyrinth zone are a barrier to transport between maternal and fetal blood that actively facilitate the feto-maternal exchange of nutrients such as glucose, amino acids, fatty acids, and nucleosides [38]. Thus, a functional placental labyrinth zone would be indispensable for the growth, development, and well being of the fetus. In the present study, we demonstrated that Ara-C caused increased apoptotic cell death and impaired cell proliferation in the placental labyrinth zone. These toxic effects on trophoblastic cells would disrupt the function of the placental labyrinth zone, resulting in abnormal fetal development.

In the placenta, the expression of various genes that regulate apoptosis and proliferation, including p53, has been reported [39–42], and the death as well as growth of placental component cells is believed to be strictly controlled by these genes. In the present study, immunohistochemical examination revealed that a substantial elevation of p53 protein expression preceded Ara-C-induced changes in the apoptosis and proliferation of trophoblastic cells. In response to DNA damage, p53 protein is modified via phosphorylation or acetylation and becomes stabilized and activated. In a lot of cases, the function of p53 is mediated by those posttranscriptional mechanisms increasing the half-life and transcriptional activity of the protein [43, 44]. In our case, overexpression of p53 mRNA was not detected in spite of the increase in the expression of p53 protein, and this suggests that p53 is regulated by such a posttranscriptional mechanism. On the other hand, the up-regulation of the p53 target genes mRNAs was observed following the increase in the expression of p53 protein, implying that p53 exerts its function by transactivation. Therefore, it is suggested that p53 and its transcriptional target genes are involved in the Ara-C-induced trophoblastic cell apoptosis and inhibition of cell proliferation in the placental labyrinth zone.

Ethylnitrosourea, a DNA alkylating teratogenic drug, was also reported to cause apoptosis and cell-cycle arrest with up-regulation of p53 protein in trophoblastic cells in the placental labyrinth zone [21]. In fetal central nervous system, targeted disruption of p53 critically suppressed apoptotic cell death induced by the administration of Ara-C [17] and ethylnitrosourea [45] to dams. The results of studies using Ara-C and ethylnitrosourea indicate that trophoblastic cells in the placental labyrinth zone are highly susceptible to genotoxic stimuli and p53 plays an important role in mediating the toxic effect of these stresses. Increased expression of p53 protein is also reported in human placenta complicated with fetal growth restriction [46]; thus, there is a possibility that stimuli such as hypoxia other than genotoxic stress cause placental disability in a p53-mediated way.

In addition to DNA damaging agents, endocrinological abnormality, inflammatory cytokines, and oxidative stress are demonstrated to induce placental apoptosis, and the distribution of the lesion is specific to the stimulus [5, 11, 12], suggesting that the pathways of increased placental apoptosis differ. Trophoblastic cells in the placental labyrinth zone actively proliferate and synthesize DNA [47], and this would have some relation to their sensitivity because Ara- C is cytotoxic to proliferating cells, especially in the DNA synthetic phase of the cell cycle [48]. However, the mechanism of the zone-dependent placental apoptosis under various pathological conditions is still obscure, and further study is needed to clarify this point.

In conclusion, the placenta is susceptible to genotoxic stress, including DNA damaging agents, which disrupt the regulation of trophoblastic cell death and proliferation. In addition, our findings suggest that p53 and its transcriptional target genes play an important role in the pathogenesis of Ara-C-induced placental toxicity. Probably, this induces placental growth inhibition and subsequent dysfunction of the placenta, which severely affects the development of the fetus, resulting in the induction of fetal growth restriction and other abnormal fetal development.


   FOOTNOTES
 
1 Correspondence: Hirofumi Yamauchi, Department of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-8657 Tokyo, Japan.FAX: 81 3 5841 8185; yamauchi-h{at}umin.ac.jp Back

Received: 5 December 2003.

First decision: 29 December 2003.

Accepted: 3 February 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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H. Yamauchi, K.-i. Katayama, M. Ueno, H. Kanemitsu, C. Nam, T. Mikami, A. Saito, Y. Ishida, K. Uetsuka, K. Doi, et al.
Etoposide Induces TRP53-Dependent Apoptosis and TRP53-Independent Cell Cycle Arrest in Trophoblasts of the Developing Mouse Placenta
Biol Reprod, April 1, 2009; 80(4): 813 - 822.
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