Syncytiotrophoblastic giant cells in teratocarcinoma-like tumors derived from Parp-disrupted mouse embryonic stem cells.
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.
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ABSTRACT: An immune system capable of discriminating between self and nonself evolved in nature long before the appearance of the viviparous mode of pregnancy, which brings maternal cells into a direct physical contact with genetically disparate cells of fetal origin. In the hemochorial type of placentation, the former include cells of the maternal immune system. This article briefly reviews the possible mechanisms that may protect the semiallogeneic conceptus in nature, with special reference to the role of the cells at the fetomaternal interface. We also present some new data on the antigenicity of pre- and postimplantation trophoblast cells and the immunobiology of decidual cells. Systemic changes in the maternal immune system appear to represent homeostatic responses to the presence of a semiallogeneic conceptus, unrelated to its protection; mechanisms for this protection must reside locally at the fetomaternal interface. We find that the lack of immunogenicity of the outer (trophoblast) cells of the preimplantation blastocyst can be explained by a transient disappearance of the major histocompatibility (MHC) antigens on their cell surface. However, following implantation and the formation of the placenta, class 1 MHC antigens reappear on certain classes of trophoblast cells, i.e., labyrinthine and spongiotrophoblast cells of the murine placenta. Similarly, cytotrophoblast cells of the early human placenta exhibit the presence of class 1 MHC antigens. An absence of class 2 MHC antigens despite the presence of class 1 antigens cannot entirely explain the lack of trophoblast immunogenicity. A local immunosuppression mediated by trophoblast cells themselves as well as maternal cells of hemopoietic origin in the decidua remain as a strong possibility. Typical decidual cells appear to play a central role in the maintenance of pregnancy because of their numerous functions: nutritive, endocrine, and immunoregulatory. Our studies reveal that they are descendants of bone-marrow-derived precursors, have unique surface markers recognizable with monoclonal antibodies nonreactive with other hemopoietic cell lineages, and have the ability to abrogate mixed lymphocyte reactions in vitro in a genetically unrestricted manner. Further studies directed at the cells of the fetomaternal interface should provide a better insight into the mode of survival of the nature's most commonplace allograft.American Journal of Anatomy 08/1984; 170(3):501-17.
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ABSTRACT: Poly(ADP-ribosyl)ation is catalyzed by NAD+: protein(ADP-ribosyl) transferase (ADPRT), a chromatin-associated enzyme which, in the presence of DNA breaks, transfers ADP-ribose from NAD+ to nuclear proteins. This post-translational modification has been implicated in many fundamental processes, like DNA repair, chromatin stability, cell proliferation, and cell death. To elucidate the biological function of ADPRT and poly(ADP-ribosyl)ation in vivo the gene was inactivated in the mouse germ line. Mice homozygous for the ADPRT mutation are healthy and fertile. Analysis of mutant tissues and fibroblasts isolated from mutant fetuses revealed the absence of ADPRT enzymatic activity and poly(ADP-ribose), implying that no poly(ADP-ribosyl)ated proteins are present. Mutant embryonic fibroblasts were able to efficiently repair DNA damaged by UV and alkylating agents. However, proliferation of mutant primary fibroblasts as well as thymocytes following gamma-radiation in vivo was impaired. Moreover, mutant mice are susceptible to the spontaneous development of skin disease as approximately 30% of older mice develop epidermal hyperplasia. The generation of viable ADPRT-/-mice negates an essential role for this enzyme in normal chromatin function, but the impaired proliferation and the onset of skin lesions in older mice suggest a function for ADPRT in response to environmental stress.Genes & Development 04/1995; 9(5):509-20. · 12.44 Impact Factor
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ABSTRACT: Posttranscriptional modification of nuclear proteins by poly(ADP-ribosyl)ation in response to DNA strand breaks plays an important role in DNA repair, regulation of apoptosis, and maintenance of genomic stability. A 113-kDa human poly(ADP-ribose) polymerase (PARP) has previously been identified and cloned. However, there is evidence that additional enzymes with PARP activity exist in mammalian cells. I have identified and cloned the cDNAs of two novel approximately 60-kDa human proteins that are 40 and 31% identical to the catalytic C-terminal domain of PARP. These proteins, named PARP-2 and PARP-3, lack the DNA-binding and automodification domains. PARP-2 and PARP-3 mRNAs were detected in 16 different human tissues as major bands of 2.0 and 2.2 kb, respectively. Radiation hybrid analysis assigned the PARP-2 gene (HGMW-approved symbol ADPRTL2) to chromosome 14q11.2-q12 and the PARP-3 gene (HGMW-approved symbol ADPRTL3) to 3p21.1-p22.2. This report shows the existence of a human PARP gene family with at least three closely related members.Genomics 06/1999; 57(3):442-5. · 3.01 Impact Factor
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?, and Takashi Sugimura*
*Division of Biochemistry,§Section for Studies of Metastasis, and¶Common Laboratory, National Cancer Center Research Institute, 1–1, Tsukiji 5-chome,
Chuo-ku, Tokyo, 104-0045 Japan;‡Department of Pathology, School of Medicine, Mie University, 2–174, Edobashi, Tsu, Mie 514-8507 Japan;
and?Chugai Pharmaceutical Co., Ltd., 1–135, Komakado, Gotemba, Shizuoka, 412-0038 Japan
Contributed by Takashi Sugimura, September 17, 1999
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-ribo-
syl)ation of chromosome-associated proteins is suggested to be
implicated in the regulation of gene expression and cellular dif-
ferentiation, 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
neither STGC nor hemorrhage was observed in tumors of both
wild-type J1 cells and Parp?/?clones. Electron microscopic exam-
and contained secretory granules in the cytoplasm. Furthermore,
the cytoplasms of STGCs were strongly stained with antibody
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 pos-
sibly induced by the lack of Parp during the development of
teins by using NAD as a substrate after activation by single- or
double-strand breaks of DNA. Recent studies using Parp knock-
out 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 con-
formational changes on acceptor proteins, it is suggested that
poly(ADP-ribosyl)ation of proteins could affect the local chro-
mosome organization and consequently alter various gene ex-
pressions. In fact, studies have indicated that Parp is involved in
transcriptional regulation of genes (8–10) and cellular differen-
tiation processes (11–14). Poly(ADP-ribose) synthesis dramati-
cally 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
Parp inhibitor, 5-iodo-6-amino-1,2-benzopyron, also induces the
phenotypic reversions of tumorigenic endothelial cells trans-
formed with H-ras and of prostate carcinoma cells (14). This
evidence thus suggests that Parp could be involved in tumori-
genesis through affecting cellular differentiation. However, be-
ribosyl)ation reaction of Parp itself and other nuclear pro-
cause 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 inhibi-
tors. Therefore, Parp-disrupted cells and animals are useful as
relevant experimental tools to elucidate the Parp function
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 develop-
ment 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 immunohisto-
chemical features of trophoblast cell origin, and massive intra-
tumoral 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%
phoblastic giant cells.
†To whom reprint requests should be addressed. E-mail: firstname.lastname@example.org.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
November 9, 1999 ?
vol. 96 ?
no. 23 ?
CO2-95% air in DMEM (GIBCO?BRL) supplemented with
20% FBS, nonessential amino acids (GIBCO?BRL), 55 ?M
?-mercaptoethanol, 0.3 mM each of adenosine, guanosine, and
thymidine, 0.1 mM uridine, and 103units?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 ? 106ES 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 injec-
tion 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,
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 micro-
scopic observation. For electron microscopic examination, ul-
trathin 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 ?m) 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 meth-
anol for 30 min and blocking with PBS containing 2% normal
goat serum and 0.1% BSA for 30 min, sections were incubated
against mouse prolactin (Biogenesis, Bournemouth, U.K.) di-
luted 200-fold in PBS containing 2% goat serum and 0.1% BSA.
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, dupli-
cated sections were immunostained without exposure to the
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.
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
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 hemor-
rhage in tumors derived from Parp?/?clones, there was no
cells and Parp?/?and Parp?/?clones.
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). Micro-
scopic 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 ob-
served 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
10. Bar indicates the average size of the tumors.
Size of the tumors derived from wild-type J1 cells and Parp-deficient
www.pnas.org Nozaki et al.
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.
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
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).
Parp-deficient ES cells were able to develop into teratocarci-
noma 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 pro-
cesses, 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
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
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-aminobenz-
amide. This discrepancy could be explained either by the low
specificity of Parp inhibitors they used or the difference of the
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 ?m.
Photomicrograph of the intratumoral STGCs and hemorrhage in tumors derived from Parp?/?clones. (A) The loupe findings of paraffin sections.
Nozaki et al. PNAS ?
November 9, 1999 ?
vol. 96 ?
no. 23 ?
biological properties between teratcarcinoma EC-A1 cells and
The STGCs observed in tumors derived from Parp?/?clones
exhibited the similar features to placental syncytiotrophoblasts,
which represent the terminal differentiation stage of tropho-
blasts (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 kera-
tinocytes 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
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
appearance of STGCs and their invasion into the surrounding
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 poly-
tene 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
Magnifications: ?1,740. m, microvillus; g, granule; r, red blood cell; N, nucleus; n, nucleolus.
Electron microscopic findings around the STGCs. (A) Microvilli on the surface of STGCs and secretion granules observed in the cytoplasm of STGCs.
Table 1. Comparison of the components in the tumors derived
from Parp???, Parp???, and Parp???ES cells
Mature neural tissue
Hairs and follicles
www.pnas.org Nozaki et al.
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 differenti-
ation 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 in-
volved 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 tropho-
blast cell differentiation. Additional studies should be con-
ducted 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
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 undif-
ferentiated embryonal carcinoma cells in parenchymatous re-
gion showed no difference between tumors derived from J1 cells
The appearance of STGCs in human trophoblastic or chorio-
carcinomatous 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 be-
tween tumors derived from J1 cells and Parp?/?clones. How-
ever, 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
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 Pharma-
ceutical 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.
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mic fractions of STGCs in tumors derived from Parp?/?clone were stained.
Some granules in the STGCs are strongly stained. Bar indicates 100 ?m.
Magnification: ?160. The negative control sections, from which the primary
antibody was omitted, showed no positive staining (data not shown).
The STGCs stained with anti-mouse prolactin antibody. The cytoplas-
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