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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1999 Nov 9;96(23):13345–13350. doi: 10.1073/pnas.96.23.13345

Syncytiotrophoblastic giant cells in teratocarcinoma-like tumors derived from Parp-disrupted mouse embryonic stem cells

Tadashige Nozaki *, Mitsuko Masutani *,†, Masatoshi Watanabe ‡, Takahiro Ochiya §, Fumio Hasegawa , Hitoshi Nakagama *, Hiroshi Suzuki ‖, Takashi Sugimura *
PMCID: PMC23950  PMID: 10557323

Abstract

The enzyme poly(ADP-ribose) polymerase (Parp) catalyzes poly(ADP-ribosyl)ation reaction and is involved in DNA repair and cell death induction upon DNA damages. Meanwhile, poly(ADP-ribosyl)ation of chromosome-associated proteins is suggested to be implicated in the regulation of gene expression and cellular differentiation, both of which are important in tumorigenesis. To investigate directly the role of Parp deficiency in tumorigenicity and differentiation of embryonic stem (ES) cells during tumor formation, studies were conducted by using wild-type J1 (Parp+/+) ES cells and Parp+/− and Parp−/− ES clones generated by disrupting Parp exon 1. These ES cells, irrespective of the Parp genotype, produced tumors phenotypically similar to teratocarcinoma when injected s.c. into nude mice. Remarkably, all tumors derived from Parp−/− clones contained syncytiotrophoblastic giant cells (STGCs), which possess single or multiple megalo-nuclei. The STGCs were present within large areas of intratumoral hemorrhage. In contrast, neither STGC nor hemorrhage was observed in tumors of both wild-type J1 cells and Parp+/− clones. Electron microscopic examination showed that the STGCs possess microvilli on the cell surface and contained secretory granules in the cytoplasm. Furthermore, the cytoplasms of STGCs were strongly stained with antibody against mouse prolactin, which could similarly stain trophoblasts in placenta. These morphological and histochemical features indicate that the STGCs in teratocarcinoma-like tumors derived from Parp−/− clones belong to the trophoblast cell lineage. Our findings thus suggest that differentiation of ES cells into STGCs was possibly induced by the lack of Parp during the development of teratocarcinoma.

Poly(ADP-ribose) polymerase (Parp) catalyzes the poly(ADP- ribosyl)ation reaction of Parp itself and other nuclear proteins by using NAD as a substrate after activation by single- or double-strand breaks of DNA. Recent studies using Parp knockout mice and cells showed that Parp is involved in recovery from DNA damages and maintenance of genomic integrity (1–7). On the other hand, because poly(ADP-ribosyl)ation of nuclear proteins causes the accumulation of negative charges and conformational changes on acceptor proteins, it is suggested that poly(ADP-ribosyl)ation of proteins could affect the local chromosome organization and consequently alter various gene expressions. In fact, studies have indicated that Parp is involved in transcriptional regulation of genes (8–10) and cellular differentiation processes (11–14). Poly(ADP-ribose) synthesis dramatically decreases in teratocarcinoma EC-A1 cells during in vitro differentiation induced by retinoic acid (11). Furthermore, the teratocarcinoma cells undergo differentiation in vitro in the presence of the Parp inhibitor, 3-aminobenzamide (11). A potent Parp inhibitor, 5-iodo-6-amino-1,2-benzopyron, also induces the phenotypic reversions of tumorigenic endothelial cells transformed with H-ras and of prostate carcinoma cells (14). This evidence thus suggests that Parp could be involved in tumorigenesis through affecting cellular differentiation. However, because Parp inhibitors have various side effects on cells (15), it is not known whether Parp alone is involved in these phenomena. In addition, other Parp-related proteins, including Parp-2, Parp-3, and tankyrase, recently were found and reported to have poly(ADP-ribosyl)ation activity (16–20). Tankyrase was shown to be inhibited by the classical Parp inhibitors (17). Parp-2 and Parp-3 possibly could be inhibited by the classical Parp inhibitors. Therefore, Parp-disrupted cells and animals are useful as relevant experimental tools to elucidate the Parp function specifically.

In the present study, to clarify the effect of Parp disruption on tumorigenesis and cellular differentiation in vivo, Parp-deficient embryonic stem (ES) cell clones established by disrupting both alleles of Parp exon 1 by inserting neomycin-resistance gene and puromycin-resistance gene, respectively (21), in wild-type J1 ES cells (22) were used. Mouse ES cells are potentially tumorigenic and develop into teratocarcinoma when injected into extra-uterine sites in syngenic or nude mice (23). Mouse ES cells also are known to participate in normal mouse embryonic development when injected into blastocyst and generally are understood to have no serious genetic changes (23). Tumors derived from ES cells also might have no additional substantial genetic changes but could be associated with epigenetic changes as previously claimed by Mintz and Illmensee (24). During teratocarcinoma formation in vivo, the differentiation potential of ES cells also could be analyzed.

Parp-deficient ES clones derived from J1 ES cells were injected s.c. into nude mice, and the growth and histological characteristics of the tumors were analyzed and compared with those of the wild-type J1 cells. Tumorigenicity was not lost in Parp+/− and Parp−/− ES clones. However, tumors derived from Parp−/− clones unexpectedly showed two characteristic features: the frequent appearance of syncytiotrophoblastic giant cells (STGCs), which exhibited morphological and immunohistochemical features of trophoblast cell origin, and massive intratumoral hemorrhage around the STGCs. Both features were not observed in tumors derived from either J1 cells or Parp+/− ES clones. The present study suggests that the loss of Parp activity possibly triggers differentiation process of ES cells into STGCs during tumor formation.

Materials and Methods

Cells and Culture Condition.

The wild-type J1 as well as Parp-deficient ES cells were cultured as described (21). Briefly, cells were maintained in a humidified incubator at 37°C under 5% CO2-95% air in DMEM (GIBCO/BRL) supplemented with 20% FBS, nonessential amino acids (GIBCO/BRL), 55 μM β-mercaptoethanol, 0.3 mM each of adenosine, guanosine, and thymidine, 0.1 mM uridine, and 103 units/ml of mouse leukemia inhibitory factor (Amrad, Melbourne, Australia) on gelatin-coated dishes (Iwaki, Chiba, Japan). Parp+/− heterozygous ES cells analyzed in this study were clones 210 and 226. These ES clones were established by inserting neomycin-resistance gene in Parp exon 1. Parp−/− homozygous ES cells analyzed were clones 210–58 and 226–47, which were derived from Parp+/− clones 210 and 226 by inserting puromycin-resistance gene in Parp exon 1, respectively, as described (4, 5, 21).

Subcutaneous Injection of ES Cells into Nude Mice.

ES cells were grown in the absence of a STO cell feeder layer on 100-mm culture plates to near 50% confluence, harvested with a cell scraper, then resuspended in PBS. Aliquots of 2 × 106 ES cells of each Parp genotype were injected s.c. into both flanks of six 8-week-old female BALB/c nu/nu mice (CLEA Japan, Tokyo), and the animals were examined continuously over 3 weeks for the appearance and growth of tumors. Three weeks after injection of ES cells, mice were euthanized, and the weight of each tumor was determined immediately after resection. Differences in tumor weights were evaluated statistically by the Mann–Whitney U tests using the spss software (Macintosh version, SPSS, Chicago).

Morphological Analysis of Tumors.

After resection of the tumors, they were fixed about 12 hr in neutralized 10% formalin solution and embedded in paraffin blocks by using standard procedures. Paraffin sections were stained with hematoxylin/eosin, and histopathological analysis was performed under a light microscopic observation. For electron microscopic examination, ultrathin sections were prepared from tissues embedded in epon after fixation with 2% glutaraldehyde-phosphate buffer and 1% osmic acid (Merck), and the sections were stained with uranium acetate-lead. Electron microscopic examination was performed by using an H7000 electron microscope (Hitachi, Tokyo).

Immunohistochemical Staining.

Tissue sections (5 μm) were mounted on poly-l-lysine-coated slides, deparaffinized with xylene, and rehydrated with graded alcohol. After inactivating endogenous peroxidase with 0.3% hydrogen peroxide in methanol for 30 min and blocking with PBS containing 2% normal goat serum and 0.1% BSA for 30 min, sections were incubated for 12 hr at 4°C in a humidified chamber with polyclonal antibody against mouse prolactin (Biogenesis, Bournemouth, U.K.) diluted 200-fold in PBS containing 2% goat serum and 0.1% BSA. Biotinylated anti-rabbit IgG raised in goat (Vector Laboratories) was diluted 200-fold in PBS containing 2% goat serum and used as the secondary antibody. Staining was performed by using a Vectastain ABC kit (Vector Laboratories). The sections were counterstained with hematoxyline. As a negative control, duplicated sections were immunostained without exposure to the primary antibody.

Results

Tumorigenicity of Parp-Deficient ES Cells.

In vitro growth rate and survival of wild-type J1 cells, Parp+/−, and Parp−/− clones in the presence of leukemia inhibitory factor are similar, and doubling times are about 9 hr, as described (21). Three weeks after s.c. injection of J1 cells at 12 sites of six mice in total, tumors developed at 10 sites. Parp+/− clones 210 and 226 and Parp−/− clones 210–58 and 226–47 also gave rise to the similar number of tumors as shown in Fig. 1. The weight of the tumors derived from Parp−/− clones tends to be relatively small, although the difference between tumors derived from J1 cells and Parp−/− clones was not statistically significant.

Figure 1.

Figure 1

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Size of the tumors derived from wild-type J1 cells and Parp-deficient ES clones in nude mice. Three weeks after s.c. injection of ES cells at each flank, resected tumors were weighed. The observed number of tumors were: J1, n = 10; clone 210, n = 9; clone 226, n = 11; clone 210–58, n = 9; clone 226–47, n = 10. Bar indicates the average size of the tumors.

The microscopic findings of tumors derived from Parp−/− clones 210–58 and 226–47 were different from those of tumors derived from the cells with other Parp genotypes. As shown in Fig. 2A, the regions densely stained with eosin occupied large areas of tumors derived from Parp−/− clones. Using a higher magnification, these regions were found to contain mainly red blood cells, together with characteristic giant cells (see below). In contrast to the tumors derived from Parp−/− clones, none of the tumors derived from wild-type J1 cells or Parp+/− clones showed such hemorrhagic areas and the giant cells within the tumor.

Figure 2.

Figure 2

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Photomicrograph of the intratumoral STGCs and hemorrhage in tumors derived from Parp−/− clones. (A) The loupe findings of paraffin sections. Magnification: ×3.2. The hemorrhagic areas, seen as blood lakes, were present only in tumors derived from Parp−/− clones. (B) High-power magnification of the STGCs containing single or multiple megalo-nuclei and eosinophilic cytoplasm. The STGCs are present within the hemorrhagic area. Bars indicate 100 μm. Magnification: ×120.

A detailed comparison of tissues and cell types present in the tumors are summarized in Table 1. Irrespective of the Parp genotype, all tumors were composed of both undifferentiated and differentiated germinal components. Each tumor contained ectodermal, mesodermal, and endodermal tissue derivatives in various grades of differentiation, including cellular components of cartilage, smooth muscle, mucous glands, neuroectodermal tissue, and primitive gut. All tumors derived from J1 cells and Parp+/− and Parp−/− clones were phenotypically very similar to teratocarcinomas, containing elements of embryonal carcinoma and teratoma. Except for the giant cells and extensive hemorrhage in tumors derived from Parp−/− clones, there was no significant difference in the differentiated components among J1 cells and Parp+/− and Parp−/− clones.

Table 1.

Comparison of the components in the tumors derived from Parp+/+, Parp+/−, and Parp−/− ES cells

Tissue type Parp+/+ Parp+/− Parp−/−
Undifferentiated embryonal
  carcinoma cells + + +
 Hemorrhage − − +
 STGCs − − +
Ectodermal derivatives
 Primitive neuroepithelium + + +
 Mature neural tissue + + +
 Keratinized epithelium + + +
 Hairs and follicles − − −
Mesodermal derivatives
 Connective tissue + + +
 Cartilage + + +
 Bone + + +
 Blood vessel + + +
 Muscle + + +
 Adipose tissue + + +
Endodermal derivatives
 Ciliated epithelium + + +
 Gut epithelium + + +
 Mucus-secreting epithelium + + +
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Presence of STGCs and Extensive Hemorrhage in Tumors Derived from Parp−/− Clones.

As described in the previous section, the giant cells with single or multiple megalo-nuclei were present in tumors derived from Parp−/− clones 210–58 and 226–47, and these cells contained eosinophilic cytoplasm (Fig. 2B). Microscopic examination showed that these giant cells were present in extensive intratumoral hemorrhage in all tumors derived from Parp−/− clones, and these characteristic features were not observed in tumors derived from J1 cells or Parp+/− clones. There was no hematopoiesis or vascular endothelial cell growth at the boundary of the hemorrhagic region. To get further information on the fine structure of these giant cells, electron microscopic examination was performed as shown in Fig. 3. It revealed the presence of microvilli on the surface and secretion granules with high electron density in the cytoplasm, both of which are characteristically seen in trophoblasts in the normal placenta. These cells therefore were diagnosed as STGCs based on their characteristic features, namely the single or multiple megalo-nuclei, eosinophilic cytoplasm, microvilli on the cell surface, and the presence of secretion granules.

Figure 3.

Figure 3

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Electron microscopic findings around the STGCs. (A) Microvilli on the surface of STGCs and secretion granules observed in the cytoplasm of STGCs. Magnifications: ×1,740. m, microvillus; g, granule; r, red blood cell; N, nucleus; n, nucleolus.

Because rodent trophoblastic cells are known to produce prolactin, prolactin-related protein, or placental lactogen (25–27), the immunoreactivity of these STGCs to an antibody against mouse prolactin also was examined. As shown in Fig. 4, strong positive staining was clearly observed in the cytoplasms of almost all STGCs. Some of the stained spots were detected in the granules of cytoplasm in STGCs. No other cells present in the tumors derived from Parp−/− clones were stained. The tumor sections of J1 cells or Parp+/− clones did not show positive staining of prolactin (data not shown).

Figure 4.

Figure 4

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The STGCs stained with anti-mouse prolactin antibody. The cytoplasmic fractions of STGCs in tumors derived from Parp−/− clone were stained. Some granules in the STGCs are strongly stained. Bar indicates 100 μm. Magnification: ×160. The negative control sections, from which the primary antibody was omitted, showed no positive staining (data not shown).

Discussion

Parp-deficient ES cells were able to develop into teratocarcinoma in nude mice as wild-type J1 ES cells. We demonstrated that the tumors derived from two Parp-deficient ES clones, which were independently isolated, characteristically contain STGCs and show extensive intratumoral hemorrhage. It is suggested that the lack of Parp activity in ES cells possibly triggers differentiation of STGCs during tumor formation processes, although we could not negate whether other genetic or epigenetic changes associated with the establishment of the ES clones are involved in STGC induction or not. Further studies are required to clarify the Parp involvement in STGC induction. Previously, c-jun−/−(28), PTEN−/−(29), HIF-1−/− (30), and FGF-4−/− (31) ES clones were established, but none of these ES clones produced STGCs during tumor formation after s.c. injection into mice. Parp−/− clones possess inserted neomycin- and puromycin-resistance genes, which are not present in wild-type J1 cells. FGF-4−/− ES clones were similarly established by inserting these two antibiotic resistance genes in R1 ES cells (31), which are derived from the same 129/Sv mouse strain as J1 cells. Therefore, it is unlikely that expressions of neomycin- and puromycin-resistance genes in ES cells resulted in the formation of STGCs in tumors. Taken together, the induction of STGCs observed in this study is likely to be related to the Parp disruption.

Except for the presence of STGCs, the differentiation profile of ES cells in tumor was not different among Parp genotypes. This finding is an apparent discrepancy with the result of Ohashi et al. (11), who observed differentiation of teratocarcinoma EC-A1 cells by treatment with Parp inhibitor, 3-aminobenzamide. This discrepancy could be explained either by the low specificity of Parp inhibitors they used or the difference of the biological properties between teratcarcinoma EC-A1 cells and ES cells.

The STGCs observed in tumors derived from Parp−/− clones exhibited the similar features to placental syncytiotrophoblasts, which represent the terminal differentiation stage of trophoblasts (32). The STGCs contained single or multiple megalo-nuclei and possessed secretion granules and microvillous cell surface. Previously, Wang et al. (2) reported the presence of multinucleated kerationocytes in acanthosis observed in the exon 2-disrupted Parp−/− mice. The STGCs observed in this study are histologically different from the multinucleated keratinocytes because keratinocytes lack such secretion granules and microvillous cell surface. For the same reason, these cells could be distinguished from other kinds of giant cells such as megakaryoblasts or megakaryocytes. The further evidence of positive staining with mouse antiprolactin antibody, which also could stain trophoblasts in normal placenta, supports the idea that the STGCs belong to the trophoblast cell lineage.

It is noteworthy that the STGCs were detected inside of the hemorrhagic regions. Microscopic examination suggested that the hemorrhage could not have occurred at early stages of tumor development by, for example, damage of the vascular endothelial cell layer, a process known to occur along with tissue necrosis, because hemosiderin deposits or blood clots in the hemorrhagic areas was not observed. In addition, there was no hematopoiesis or vascular endothelial cell growth around the hemorrhagic region. Taken together, these observations suggest that the hemorrhage most likely represents a blood lake with continuous blood flow or recent bleeding. Because STGCs possess invasive characteristics like placental trophoblasts, which invade the uterine wall through the process of implantation and placental formation, the intratumoral hemorrhage observed in tumors derived from Parp−/− clones could be a secondary event after the appearance of STGCs and their invasion into the surrounding tissues.

The majority of STGCs contained single nuclei but some contained multinuclei. Syncytiotrophoblasts of rodent placenta give rise exclusively by continued rounds of DNA synthesis without intervening mitosis (endoreduplication) and have polytene chromosomes, although some cells are polyploid (33). It is not elucidated whether the STGCs in teratocarcinoma are formed by similar process to syncytiotrophoblast formation in rodent placenta, including endoreduplication and karyokinesis, as described above or entirely different processes, including cell fusion and subsequent nuclear fusions. Although the precursor cells of the STGCs in teratocacinoma are not known yet, the cytotrophoblasts or spongiotrophoblasts (34) are the possible precursors of the STGCs. These smaller trophoblasts were observed only occasionally in the tumor 3 weeks after injection of Parp−/− clones. These trophoblasts were not positively stained by antiprolaction antibody.

Under the in vitro culture condition in the presence of leukemia inhibitory factor, the differentiation of ES cells was not observed frequently for wild-type J1 cells or Parp+/− or Parp−/− clones (data not shown). Trophoblast cell differentiation is regulated by several transcription factors including Mash-2 (35), Hxt (36), and a zinc finger transcription factor, Snail family protein (37). A transcription factor AP2 is involved in teratocarcinoma formation (38). Interestingly, Parp recently was found to possess coactivator function of AP2 and cooperate in transcriptional regulation (39). Various studies also suggest that Parp is involved in transcription control of the genes (8–10). It is thus possible that loss of Parp affects the transcription of a certain subset of genes that control trophoblast cell differentiation. Additional studies should be conducted to elucidate the precise mechanisms of how the loss of Parp activity drives differentiation into the STGCs. This study further opens a question on the effect of Parp deficiency on placental formation and function in uterus during mouse development.

Tumorigenicity was not lost in Parp-deficient ES cells. There was no significant difference in the mean weight of the tumors derived from wild-type J1 cells and Parp−/− clones. However, because the tumors derived from Parp−/− clones contained large hemorragic areas, the tumor weight does not directly reflect tumor cell growth. The ratio of differentiated cells and undifferentiated embryonal carcinoma cells in parenchymatous region showed no difference between tumors derived from J1 cells and Parp−/− clones.

The appearance of STGCs in human trophoblastic or choriocarcinomatous germ cell tumor is known to be associated with metastasis and poor prognosis (40). Therefore, Parp deficiency could confer more malignant phenotype in germ cell tumor as a consequence. We tried to compare metastasis frequency between tumors derived from J1 cells and Parp−/− clones. However, even 3 months after transplantation of ES cells, no metastasis was observed. Because s.c. tumors seem to have low tendency of metastasis in general, experiments should be further conducted by changing injection site of ES cells. The present model could provide us with a good tool to investigate the biological role and induction mechanism of STGCs in germ cell tumors.

Acknowledgments

We are grateful to Dr. E. Winterharger of Essen University, Germany for helpful comments. We thank Dr. M. Furusato of the Jikei University School of Medicine, Drs. M. Sakamoto, M. Suzui, and K. Fukuda of the National Cancer Center Research Institute, Japan for the diagnosis of tissue sections, and Drs. N. Kamada and S. Uchida of Chugai Pharmaceutical Co. for technical assistance. We thank Dr. S. Hirohashi of the National Cancer Center Research Institute, Japan and Dr. S. Nishimura of Banyu Tsukuba Research Institute for critical discussions. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture (10877333) and a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan.

Abbreviations

Parp

poly(ADP-ribose) polymerase

ES

embryonic stem

STGCs

syncytiotrophoblastic giant cells

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