Endometrial cancer: experimental models useful for studies on molecular aspects of

Transcription

Endocrine-Related Cancer (2003) 10 23–42
REVIEW
Endometrial cancer: experimental models
useful for studies on molecular aspects of
endometrial cancer and carcinogenesis
G Vollmer
Molecular Cell Physiology and Endocrinology, Institute of Zoology, Dresden University of Technology,
Mommsenstr. 13, 01062 Dresden, Germany
(Requests for offprints should be addressed to G Vollmer; Email: Guenter.Vollmer@mailbox.tu-dresden.de)
Abstract
There is definitely a need for the development of new drugs for the treatment and cure of endometrial
cancer. In addition there are various new drugs or phyto-remedies under development which are
intended for use in the treatment and prevention of breast cancer, for the treatment of menopausal
symptoms and for hormone replacement therapy. The efficacy of novel drugs targeting steroid
receptors in endometrial cancers has to be evaluated and the safety of other endocrine measures on
endometrial cancers or on endometrial carcinogenesis has to be assessed. For these experimental
purposes five main classes of experimental models are available: spontaneous endometrial
tumorigenesis models in inbred animals (Donryu rats, DA/Han rats, BDII/Han rats), inoculation tumors
from chunks of tumors (rat EnDA-tumor, human EnCa 101 tumor) or from inoculated tumor cell lines
(rat RUCA-I cells, human Ishikawa and ECC-1 cells), developmental estrogenic exposure or chemical
carcinogen exposure of CD-1 and ICR mice, transgenic approaches such as mice heterozygous
regarding the tumor suppressor gene PTEN (pten + / − -mice) and endometrial tumor cell lines cultured
under conditions promoting in vivo-like morphology and functions e.g. cell culture on reconstituted
basement membrane. Although the number of models is comparatively small, most aspects related
to functions of estrogenic or gestagenic substances are assessable, particularly if various
experimental models are combined. Whereas models based on human endometrial adenocarcinoma
cells are widely used, the properties and advantages of animal-derived models have mainly been
ignored so far.
Introduction
Endometrial cancer is the most frequently diagnosed malignancy of the female genital tract. Incidence rates of endometrial cancer are described as 10 to 25 women per
100 000 with a clear geographic variation between e.g.
European (Spain, UK, France) and North American (USA
and Canada) countries, with a slightly higher incidence in
North America (Parazzini et al. 1991). In 1998, an estimate
of 36 000 new cases and 6300 deaths attributable to endometrial cancer were reported in the USA (Podratz et al.
1998) resulting in an overall frequency which is only
exceeded by breast, lung and colon cancer (Li et al. 1999).
However, the mortality rate of endometrial cancer if compared with other cancers is low and the prognosis is excellent if it is detected in the early stages (Creasman 1997).
The present knowledge of the role of endocrine factors in
the etiology of endometrial adenocarcinomas, their role in
the regulation of tumor growth, invasion and metastasis of
endometrial adenocarcinoma cells, as well as the potential
value of established and novel endocrine manipulations for
prevention and treatment of endometrial adenocarcinomas
have been exhaustively reviewed in a recent review article
(Emons et al. 2000).
In the concluding remarks of the same paper it was stated
that neither progestin treatment, the major hormonal therapy
of endometrial carcinoma, nor cytotoxic chemotherapy
showed substantial benefits in the adjuvant setting. Therefore, future research activities must evaluate new compounds
and new treatment strategies.
The endometrium is a classical hormone-dependent
tissue and most of the endometrial adenocarcinomas are
hormone-dependent tumors. A high percentage of these
tumors express the estrogen receptor(s) and/or progesterone
receptors. Investigators have, therefore, aimed at targeting
steroid hormone receptors by the development of novel sub-
1351-0088/03/010–023  2003 Society for Endocrinology Printed in Great Britain
Online version via http://www.endocrinology.org
Vollmer: Endometrial cancer
stances e.g. new antiestrogens, selective estrogen receptor
modulators (SERMs) and aromatase inhibitors. For testing
and characterization of these new substances appropriate
models have to be available.
A further need for endometrial tumor models stems from
safety considerations. Estrogens not only act as tumor promoters but also as carcinogens. This has consequences for
the approval of endocrine active substances intended for use
in tumor therapy e.g. breast cancer, tumor prevention or hormone replacement therapy. The concerns are that substances
which are beneficial in one organ might harm another, e.g.
tamoxifen, a common therapy in breast cancer, might
increase the risk for endometrial cancer (Emons et al. 2000).
Finally, plenty of phytoestrogens are recommended either as
semipure substances or, as in the case of soy or red clover
extracts, for use as supportive measures in hormone replacement therapy as well as for use in lifestyle medicine. None
of these substances has been exhaustively investigated
regarding its safety (Fugh-Berman & Kronenberg 2001, Balk
et al. 2002, Burton & Wells 2002).
These examples clearly demonstrate the need for experimental endometrial tumor models. The aim of this paper
is to review the knowledge of endometrial tumor models.
Particular emphasis is given to their applicability in vivo and
to their potential responsiveness to natural as well as synthetic estrogens and progestins. The latter feature is a prerequisite for the process of development and evaluation of
endocrine active substances.
Spontaneous endometrial
adenocarcinoma of rats
Longevity studies revealed that female animals of four rat
strains die with an uncommonly high incidence rate for endometrial adenocarcinoma if kept to their natural life end. Due
to their clinical, pathophysiological, histological and biochemical features they all represent excellent experimental
models for endometrial carcinogenesis. Their potential as
experimental tumor models has so far not been appreciated
by the scientific community.
Han:Wistar rats
Already in 1981 Deerberg et al. published a report showing
that virgin Han:Wistar rats die with an incidence rate of 39%
from tumors of the uterus if kept to their natural life end. A
total of 35% of these tumors were endometrial adenocarcinomas. This rat strain and the pathogenesis of its endometrial
adenocarcinomas have not been investigated any further.
Donryu rats
Female Donryu rats have been presented as a hormonedependent endometrial cancer model, an issue discussed in
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detail below. This model is not well characterized regarding
histopathological properties and metastatic potential. Genetically, in spontaneous endometrial cancers point mutations of
the K-ras locus have been found recurrently (Tanoguchi et
al. 1999). The latter mutation also has a rather frequent
occurrence in human endometrial adenocarcinoma (Boyd &
Risinger 1991, Semczuk et al. 1998).
Female animals of the Donryu strain are characterized
by a similar mortality rate for endometrial adenocarcinoma
as Han:Wistar rats and exhibit an incidence rate of 35.1%
for endometrial adenocarcinoma and a total of proliferative
lesions of approximately 60% (Nagaoka et al. 1990). The
pathogenesis of endometrial adenocarcinoma in this rat strain
is characterized by some features which are discussed as risk
factors contributing to endometrial carcinogenesis in humans.
Donryu rats are characterized by an imbalanced (increased)
estradiol/progesterone rate which at the age of 12 months is
increased almost fivefold if compared, for example, with
aged Fischer-344 rats (Nagaoka et al. 1990). In humans anovulatory cycles reflecting the estrogen/progesterone imbalance are regarded as a typical risk factor for endometrial
adenocarcinoma (Barakat et al. 1997).
It has been proposed that endometrial carcinogenesis in
Donryu rats is hormone dependent (Nagaoka et al. 1994).
However, this conclusion has been drawn from experimental
observations which should not necessarily be interpreted as
hormone dependency. The first piece of evidence derives
from the above mentioned estrogen/progesterone imbalance
in the serum of these animals. The other piece of evidence
has been deduced from comparative studies with Fischer-344
rats which have a normal cyclicity and almost no advanced
histological changes such as hyperplastic or neoplastic
lesions (Nagaoka et al. 1994) or alterations in the proliferative capacity of the uterine epithelium (Ando-Lu et al. 1998).
Further, reproductive experience reduced the incidence of
endometrial and mammary carcinoma. This finding is analogous to the situation discussed for humans (Terry et al.
1999), although it only becomes apparent in Donryu rats after
three periods of gestation. One- or twofold experiences were
ineffective if compared with nulliparous animals (Nagaoka
et al. 2000). However, the conclusive experiments proving
hormone dependency, namely comparative studies in normal
cyclic and ovariectomized animals are missing.
The overall yield of endometrial adenocarcinoma could
be increased and the onset of endometrial adenocarcinoma
could be considerably accelerated if animals were intraperitoneally treated four times with a combination of estradiol
diproprionate and N-methyl-N-nitrosourea (Nagaoka et al.
1993) or if they received a single intra-uterine administration
of N-ethyl-N′-nitro-N-nitrosoguanidine via the vagina
(Ando-Lu et al. 1994). High fat diets also led to an earlier
onset of endometrial carcinogenensis which was paralleled
by an earlier onset of imbalance in estrogen/progesterone
levels (Nagaoka et al. 1995). Transplacental administration
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of diethylstilbestrol on days 17 and 19 at a dose of 0.1 mg/
kg led to an increase in lesions of the female reproductive
tract of Donryu female offspring, including an increase in
endometrial adenocarcinoma (Kitamura et al. 1999).
Although the overall process of carcinogenesis is accelerated
by these chemical carcinogens or the tumor promoting estrogen, it is questionable whether results from studies applying
this approach can be interpreted more accurately than studies
using the spontaneous protocol. Treatment with chemical
carcinogens induces mutations which unpredictably add to
genetic alterations already present in Donryu rats, like the
frequent mutation of the Ki-ras locus (Tanoguchi et al.
1999).
So far two therapeutic studies have been described using
this model in the situation of spontaneous endometrial carcinogenesis. Treatment with dietary indole-3-carbinol
reduced the incidence rate of endometrial carcinogenesis
considerably. The study showed a clear correlation between
the induction of estradiol 2-hydroxylation and a more normalized estrogen/progesterone level (Kojima et al. 1994). On
a mechanistic level this means chemoprevention is due to
induction of estradiol 2-hydroxylase. The other chemoprevention study was performed on N-ethyl-N′-nitro-Nnitrosoguanidine-treated Donryu rats. Tamoxifen treatment
significantly reduced the number of proliferative lesions in
the uteri which also was limited to a few hyperplastic lesions
(Yoshida et al. 1998). The authors claimed that tamoxifen
acted as an antiestrogen, because of decreased serum estradiol levels. However, in none of the papers were any comments on estrogen receptor involvement made. Receptor
involvement is crucial for any antiestrogenic function. In
addition, the finding mentioned above does not reflect the
situation in humans. For the human situation it is well
accepted that tamoxifen increases the relative risk for endometrial carcinogenesis (ACOG Committee Opinion 1996,
Love et al. 1999), a calculation from epidemiological findings substantiated by the stimulation of proliferation in cultured endometrial adenocarcinoma cells by tamoxifen
(Gusberg 1994, Creasman 1997).
DA/Han rats
DA/Han rats are characterized by an inbred background.
Specific gene alterations are not yet documented. The overwhelming majority of tumors are moderately to well differentiated which makes the tumor phenotypically similar to most
of the human endometrial adenocarcinomas. This model
exhibits a highly metastatic phenotype (Fig. 1), but has not
so far been used for intervention studies, because the tumor
can be transplanted into syngenic animals. There it gives rise
to metastasizing endometrial adenocarcinomas which by
histopathological and biochemical means (expression of
estrogen receptor α (ER-α)) are undistinguishable from the
parental tumor (Horn et al. 1993, 1994).
Female DA/Han animals die from endometrial adenocarcinoma with an incidence rate of > 60% if kept to their natural life end at 24–27 months of age. Ovariectomy prior to
onset of cyclicity prevents endometrial carcinogenesis
(Deerberg et al. 1985). This has to be interpreted as a clear
indication of hormone dependency of the carcinogenic process. Sixty-three percent of all of the developing endometrial
adenocarcinomas of the DA/Han rats metastasize into the
lung, thereby using a lymphogenic pathway (Fig. 1; F Deerberg, unpublished observations). Lymphatic invasion is a
characteristic of type II endometrial carcinoma (Emons et al.
2000). Distant metastases are rare in cases of human endometrial cancer (Cook et al. 1999); however, among those few
cases the lung has been described as a frequent metastases
site (Bouros et al. 1996).
So far, these spontaneously occurring tumors have not
been used for experimental purposes. However, from these
tumors a serially transplantable tumor called EnDA (Horn et
al. 1993) and a cell line called RUCA-I (Schu¨tze et al. 1992)
which also gives rise to hormone-sensitive endometrial cancers if inoculated into syngenic rats or athymic nude mice
(see below) have been established (Fig. 2).
BDII/Han rats
In terms of an endometrial cancer model the female rats of
this strain are unique worldwide. Since being first described
in 1987 (Deerberg & Kaspareit 1987) it took almost a decade
before they were used for cancer treatment experiments, and
an additional five more years before investigators started to
unravel their genetic peculiarities which contribute to the
high incidence of endometrial cancer. This tumor model is
genetically well characterized (discussed in detail below),
although some important genetic information is still missing
e.g. mutation frequency of some tumor suppressor genes (e.g.
APC, PTEN). Most histopathological properties known for
human endometrial adenocarcinoma, e.g. tumor grading, are
also found in this spontaneous animal model for endometrial
adenocarcinoma (Deerberg & Kaspareit 1987). Furthermore,
this model has been investigated for the occurrence of precancerous lesions and their immunohistochemical properties.
In rat endometrial tissue the same precancerous lesions have
been found which are also detectable in human specimens.
In addition, as in hyperplastic and displastic human endometrium, tenascin-C, a stromal marker for proliferative epithelial disease, is strongly up-regulated in similar lesions of
rat endometrial tissue (Vollmer et al. 1990, 1991, Sasano et
al. 1993). These findings illustrate the large similarities in
cell biological aspects in the pathogenesis of endometrial
cancers in humans and rats. In experimental setups for the
understanding of the molecular mechanisms of endometrial
carcinogenesis this model is of high value and should be considered as one of the most important in vivo models. There
are several experimental pieces of evidence that endometrial
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Figure 1 Endometrial carcinogenesis and lung metastasis of DA/Han rats. In the upper panel the overall incidence of
endometrial carcinogenesis of DA/Han rats, dependent on age, is given. The lower panel shows the percentage of those tumors
developing lung metastases. ECA, endometrial carcinoma.
carcinogenesis of female BDII/Han animals is hormone
dependent and therefore represents an endometrial tumor
model for spontaneous hormonal carcinogenesis.
If female animals of this strain are kept to their natural
life end (around 27 months of age) they die from endometrial
carcinoma or metastases with an incidence rate of > 90%.
The overwhelming majority (87%–97%) of all carcinomas in
the various experimental groups were endometrial adenocarcinomas. The small remaining group consisted of anaplastic
carcinomas, adenosquamous carcinomas and squamous cell
carcinomas (Deerberg & Kaspareit 1987). This powerful
study was set up with 50 animals per experimental group
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initially. In parallel experimental groups it was demonstrated
that neither maintenance of animals in a germ-free environment nor feeding of a purified diet reduced the incidence rate
of tumors. The group of retired breeders also exhibited an
incidence rate of > 90% but was characterized by a significantly longer life span. This finding is a clear indication of
hormonal involvement in the carcinogenic process. Numbers
of pregnancies are regarded as a life style factor inversely
correlated to endometrial carcinogenesis in humans (Emons
et al. 2000 and references therein). But even more importantly, if BDII/Han rats are ovariectomized prior to estrous
cyclicity, they die from causes other than endometrial
Figure 2 The EnDA/RUCA model. This figure shows the experimental possibilities of the syngenic EnDA/RUCA model
consisting of spontaneous endometrial carcinogenesis of DA/Han rats, of the EnDA inoculation tumor and of cultured and/or
inoculated RUCA-I cells.
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carcinoma, the incidence rate for this tumor decreasing to
0%. This is the strongest piece of evidence in favor of hormone-dependent endometrial carcinogenesis in BDII/Han
rats (Deerberg & Kaspareit 1987). This latter finding was
further substantiated in a more recent study in which life long
treatment with the progestin, melengestrol acetate, in three
different doses (0.1, 0.2 and 0.4 mg/kg/day) completely suppressed endometrial carcinogenesis (Deerberg et al. 1995).
At the highest dose glucocorticoid-like side effects were
observed.
Only recently has the hormonal carcinogenesis of inbred
BDII/Han rats been recognized as a model system to study
genes involved in endometrial carcinogenesis. Using them as
models for the understanding of genetic elements involved
in the ontogeny of endometrial adenocarcinoma in general,
genetic changes occurring in the course of endometrial carcinogenesis could be demonstrated in crossbreeding experiments applying comparative genomic hybridization (Helou et
al. 2001). The most common aberration was amplification of
the proximal region of rat chromosome 4. The genes Cdk6
(cyclin dependent kinase 6) and Met (hepatocyte growth
factor receptor) were found to be located in the core of each
amplified region and amplified most recurrently and at the
highest level among the genes tested (Walentinsson et al.
2001). These data suggested that up-regulation of Cdk6 and/
or Met contributes to the development of endometrial cancers
in BDII/Han rats. The human homolog of Cdk6 has been
postulated to be an important player in cell cycle control.
Evidence exists that Cdk6 provides the link between growth
factor stimulation and onset of cell cycle progression
(Meyerson & Harlow 1994). However, the only documentation of Cdk6 overexpression in association with Cdk6
amplification comes from human gliomas (Costello et al.
1997). The Met protoocogene encodes a transmembrane
growth factor which binds the cytokine hepatocyte growth
factor/scatter factor (HGF/SF). The highest levels of this
tyrosine kinase receptor are found in epithelial tissues. The
ligand, in contrast, is predominantly expressed in mesenchymal tissues suggesting a paracrine mode of action
(Gherardi & Stoker 1991). The major effect of HGF/SF function mediated by Met is thought to be epithelial cell proliferation and motility (Sugawara et al. 1997), including endometrial epithelium as well (Wagatsuma et al. 1998). Further,
it has been shown that HGF/SF can stimulate invasion of
endometrial adenocarcinoma cell lines if they express MET
(Bae-Jump et al. 1999).
Extra copies of chromosomal regions were found for
chromosome 6 and eight cancer-related genes were predicted
to be located in this chromosomal region of BDII/Han rats.
These genes comprised the N-myc protooncogene, apolipoprotein B, the DEAD box gene, ornithine decarboxylase, proopiomelanocortin, ribonucleotide reductase, M2 polypeptide
and syndecan. Amplification of N-myc was by far the highest
suggesting that Mycn amplification and overexpression con-
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tributes to the development of this hormone-dependent tumor
(Karlsson et al. 2001). In addition, three major chromosomal
regions representing multiple susceptibility genes involved in
the development of endometrial adenocarcinoma in rat could
be identified (Roshani et al. 2001). Loss of heterozygosity
analysis revealed three major losses of heterozygosity
regions on chromosome 10 (Behboudi et al. 2001). By radiation hybrid mapping and single and dual color fluorescence
in situ hybridization techniques a detailed chromosomal map
of the proximal part of chromosome 10 could be established.
With this approach the regional localization of 14 genes,
most of them cancer related, and of 5 microsatellite markers
could be determined (Behboudi et al. 2002).
Finally, in crossbreeding experiments of female BDII/
Han rats with males of two strains with a low incidence of
endometrial adenocarcinoma, three chromosomal regions,
which give rise to susceptibility for endometrial adenocarcinoma, could be identified (Roshani et al. 2001). This is a clear
indication that endometrial carcinogenesis of BDII/Han rats
exhibits heritable features. Interestingly, the genes affecting
susceptibility to endometrial adenocarcinoma were different
in the two crosses, suggesting that genes behind the susceptibility in BDII/Han animals may interact with various genes
in different genetic backgrounds. Data in the literature suggest that this feature of heritability of endometrial carcinogenesis in BDII/Han rats is shared by at least two forms of
human endometrial cancer (Sandles et al. 1992, Gruber &
Thompson 1996, Lynch et al. 1996). Whether or not this
heritability of endometrial carcinogenesis resembles the heritable phenotype in human endometrial adenocarcinoma of
patients with hereditary non-polyposis colorectal cancer
(HNPCC) remains open. The latter phenotype would, at least,
require microsatellite instability, mutation or alteration of
promotor methylation of mismatch repair genes and
mutations of the pten locus (Kuismanen et al. 2002, Whelan
et al. 2002, Zhou et al. 2002a,b). A corresponding genetic
analysis of endometrial adenocarcinoma of BDII/Han rats is
still missing.
Mouse models
Mouse models historically have proved to be particularly
useful in studying the effects of developmental exposure to
hormones, particularly estrogens. This topic will be discussed
in more detail below. In addition, the ICR mouse model
which responds to chemical carcinogens by induction of
endometrial cancer is described.
Transgenic mouse models are emerging tools for carcinogenesis in general. In the context of endometrial carcinogenesis pten + / − transgenic mice have to be discussed. These
animals exhibit a phenotype strongly resembling human
Cowden syndrome, a syndrome associated with high incidence of breast and endometrial neoplasia (Stambolic et al.
2000). This particular mouse strain, therefore, has to be discussed as a potential endometrial cancer model.
Developmental hormone (estrogen) exposure
In various mouse and rat strains estrogens increase the incidence of tumorigenesis in several organs (Liehr 2001 and
references therein), including the uterus, particularly during
developmental exposure. In humans, epidemiologists have
recognized prolonged estrogen action or estrogen use unopposed by gestagens as a risk factor for endometrial carcinogenesis (Key & Pike 1988, Weiderpass et al. 1999). From all
these data the question has been raised whether estrogens act
as carcinogens (Boyd 1996). From mechanistic and molecular studies more and more pieces of evidence have accumulated to strengthen this hypothesis (reviewed by Liehr 2000,
2001).
The neonatal mouse has been proposed as a model for
hormonal carcinogenesis of the endometrium (Newbold et al.
1990). If outbred mice are treated neonatally (days 1–5) they
will develop endometrial adenocarcinoma in a dosedependent manner and dependent on the strength of the estrogen. In the initial paper, Newbold et al. (1990) showed that
diethylstilbestrol and derivatives thereof are more potent than
estradiol. This model has been further characterized in terms
of cellular differentiation and gene expression in the epithelium (Yoshida et al. 2000). In the meantime, this model
has been successfully applied to test for the developmental
toxicity of tamoxifen (Newbold et al. 1997), catecholestrogens (Newbold & Liehr 2000) and genistein (Newbold et al.
2001). All these substances induce endometrial adenocarcinoma if injected in neonatal mice during postnatal days 1–
5. Whether this model can be replaced by transgenic mice
overexpressing ER and which exhibit an accelerated onset of
endometrial carcinogenesis (Couse et al. 1997) remains to be
elucidated, because these tumors are characterized by a more
aggressive phenotype and may therefore be less representative of the human situation than endometrial carcinogenesis
in wild-type animals.
In summary, CD-1 mice appear to be an excellent model
to test for developmental toxicity of estrogens, thereby evaluating the capacity of test substances to induce hormonedependent endometrial cancers.
Chemical carcinogen-induced
endometrial cancers in ICR mice
ICR mice respond to treatment with N-methyl-N-nitrosourea
(NMU) or estradiol with the development of endometrial
cancers within 30 weeks. This process is significantly accelerated if ICR mice are treated with both agents simultaneously
(Niwa et al. 1991). The enhancing effects on endometrial
carcinogenesis initiated by NMU are not only detectable for
estradiol but also for estrone and estriol (Niwa et al. 1993).
In comparison to human endometrial carcinogenesis, histopathological examinations revealed that these tumors
develop from various preneoplastic, hyperplastic lesions,
resembling the human situation (Niwa et al. 1991). Interestingly, the carcinogen and estradiol induce different preneoplastic lesions. Estradiol induces glandular hyperplasia, while
adenomatous hyperplasia is induced by NMU. For the development of atypical hyperplasia the cooperative action of
estradiol and NMU is favorable (Niwa et al. 1996).
Whereas the histopathology of NMU-induced endometrial tumors in ICR mice resembles the situation described
for endometrial carcinogenesis, the limited genetic analysis
described for the mouse model is different to human endometrial carcinogenesis. In specimens of human endometrial
cancer the mutation of various Ras loci (Boyd & Risinger
1991, Ignar-Trowbridge et al. 1992) and the p53 gene
(Risinger et al. 1992) are found quite often. These kinds of
mutations are very rare in NMU-initiated endometrial tumors
in ICR mice (Murase et al. 1995).
With regard to steroid receptor expression and protein
levels this model is poorly characterized. In a short-time protocol in ovariectomized ICR mice, animals respond to estrogen treatment by up-regulation of steady state mRNA levels
of the proto-oncogenes fos and jun (Niwa et al. 1998).
The NMU-induced and estradiol-enhanced endometrial
carcinogenesis proved to be particularly useful for treatment
studies. Several hormonal and traditional treatment procedures were tested leading with one exception to inhibition
or prevention of all tumors in the ICR model. Carcinogenesis
induced by a combination of estradiol and NMU was
inhibited by gestagen treatment (medroxyprogesterone acetate; Niwa et al. 1995) or by treatment with the antiestrogen,
toremifen (Niwa et al. 2002). In contrast, treatment with the
selective estrogen receptor modulator, tamoxifen, stimulated
endometrial carcinogenesis in NMU-treated animals with or
without the combined treatment with estradiol (Niwa et al.
1998).
Prevention of endometrial carcinogenesis in the above
described mouse model including a reduction of hyperplastic
foci was detected following treatment of animals with danazol (Niwa et al. 2000) or following treatment with traditional
Japanese or Chinese remedies such as extracts from Glycyrrhiza radix (Niwa et al. 1999) and Juzen-taiho-to (Niwa
et al. 2001). Shimotsu-to was identified as the active principle in Juzen-taiho-to (Lian et al. 2002). Finally, prevention
studies with isoflavones revealed that both genistein and
daidzein exhibited an inhibitory effect on endometrial carcinogensis in the estradiol/NMU ICR mouse model, particularly on the indices of atypical endometrial hyperplasia (Lian
et al. 2001).
In summary, NMU-induced endometrial adenocarcinomas in ICR mice are particularly sensitive to various hormonal treatment procedures. The limitation of this model is
its poor characterization of hormone responsiveness at a
29
molecular level. Neither the levels nor the qualities of
expressed steroid hormone receptors are known nor is there
any information on hormonally triggered signal transduction
cascades or responsive genes except for fos and jun.
Heterozygous mouse models with pten +/−
-mice as an example
PTEN/MMAC1/TEP1 has been detected as one of the most
commonly mutated tumor suppressor genes in human cancer
(Li et al. 1997, Steck et al. 1997). A significant rate of PTEN
mutations has also been reported for human endometrial adenocarcinoma (Risinger et al. 1997, 1998, Tashiro et al. 1997).
This mutation has been found in up to 80% of cases of human
endometrial adenocarcinoma, and is already detectable in 33–
55% of the precancerous lesions (Latta & Chapman 2002,
Konopka et al. 2002). In addition to a mutation promoter,
hypermethylation is an alternative way to inactivate tumor
suppressor genes. There is one report in the literature showing
that the frequency of promoter hypermethylation of the PTEN
tumor suppressor gene in endometrial adenocarcinoma is
around 19% (Salvesen et al. 2001).
Although the importance of a functional PTEN protein
has been known for several years, the association of PTEN
expression with a prognosis is not yet clear. It appears as if
loss of PTEN expression is associated with metastatic disease
(Salvesen et al. 2002) and may serve as an independent prognostic marker for patients who undergo postoperative chemotherapy (Kanamori et al. 2002).
In order to understand the biological role of this dominant tumor suppressor gene, transgenic PTEN knock-out
mice have been created. A PTEN − / − mutation is lethal presumably due to defective chorio-allantoic development
(Suzuki et al. 1998).
Surprisingly, the mutation of one allele is sufficient to
cause neoplasia in multiple organ systems in these pten + / −
-mice (Podsypanina et al. 1999). A particularly high incidence has also been detected in breast as well as in endometrial neoplasia and preneoplasia, strongly resembling human
Cowden syndrome (Stambolic et al. 2000). Indeed, in the
human situation and in addition to the occurrence of PTEN
mutations in sporadic tumor, germ-line mutations are
believed to cause related autosomal dominant hamartoma
syndromes, among them Cowden syndrome (Liaw et al.
1997, Eng 1998). Although the phenotype detectable in
pten + / − -mice in terms of the development of Cowden syndrome and the association with the high incidence of development of precancerous and cancerous lesions of breast and
endometrium is strikingly similar to the human situation,
some differences do exist. Unlike the human situation, in
pten + / − -mice neoplasia of the skin and brain were notably
absent, whereas the observed changes in the endometrium
were very consistent (Podsypanina et al. 1999).
30
Endometrial carcinoma, except colon carcinoma itself, is
the most common malignancy in patients with hereditary
non-polyposis colon cancer (HNPCC). This disease is characterized by germ-line mutations in mismatch repair genes
and by microsatellite instability (Fishel et al. 1993, Leach et
al. 1993, Peltoma¨ki et al. 1993, Bronner et al. 1994, Nicolaides et al. 1994, Papadopoulos et al. 1994). Surprisingly,
patterns of microsatellite instability differ between endometrial and colorectal tumors from patients with HNPCC
(Kuismanen et al. 2002).
To learn more about the involvement of these genes in
the pathogenesis of endometrial carcinogenesis, tumor specimens from patients with HNPCC and an established mutation
record of either the MLH1 or MSH2 missmatch repair gene
locus were subjected to analysis of the PTEN locus. Mutation
of the mismatch repair loci and PTEN gene were very
common in HNPCC, with PTEN mutation presumably preceding mutations of the mismatch repair loci (Zhou et al.
2002b). Interestingly, crosses of pten + / − - with mlh1 − /
−
-mice were characterized by an accelerated endometrial
tumorigenesis (Wang et al. 2002).
In summary, endometrial cancers from pten + / − -mice
with or without mlh1 − / − may serve as an excellent model
for hereditary endometrial cancer, because of the strong
resemblance with human Cowden disease and human
HNPCC. In addition, Stambolic et al. (2000) proposed
pten + / − -mice as a model for endometrial adenocarcinomas
which develop in women with unopposed estrogen stimulation. These patients rather commonly suffer from loss of heterozygosity at the PTEN locus and/or mutations in all stages
of endometrial hyperplasia (Risinger et al. 1997, 1998,
Tashiro et al. 1997).
Whether pten + / − -mice can serve as a hormonedependent cancer model or if they are suitable as models for
hormonal treatment protocols remains open, because no data
are available on these issues. However, despite the lack of
the latter information it has to be stated that pten + / − -mice
represent a promising new endometrial cancer model,
because of the similarity to hereditary human endometrial
cancer.
Inoculation tumors
These tumors are derived by inoculating chunks of tumors or
defined numbers of cultured cells e.g. subcutaneously or into
the fat pad of syngenic animals, athymic nude mice or rats.
It is the scope of this paper to focus primarily on steroid
hormone dependent or at least steroid hormone sensitive in
vivo models. For this reason in the following sections evidence for hormone responsiveness from in vitro data is
briefly summarized prior to the review of in vivo application
data of either model.
The RUCA-I/EnDA model
From a spontaneous endometrial adenocarcinoma a transplantable tumor called EnDA-tumor (Horn et al. 1993) and
a cell line called RUCA-I (Schu¨tze et al. 1992) have been
derived. These models have primarily been used to test novel
antiestrogens, potential agonistic activities of phytoestrogens
and o,p′-DDT as an example of an endocrine-disrupting
chemical.
To test for antiestrogenic activities of antiestrogens
chunks of EnDA-tumors or RUCA-I cells have been inoculated into syngenic DA/Han rats or athymic nude rats (Fig.
2; Horn et al. 1993, Vollmer & Schneider 1996). Tumor
growth from chunks of EnDA-tumors at the ectopic site as
well as formation of lymphogenic and pulmonary metastasis
are estrogen-sensitive processes, because the tumor growth
rate at the ectopic site, as well as the weight of ipsilateral
axillary lymph nodes and the number of lung metastases is
reduced in ovariectomized animals if compared with intact
animals. These effects most likely are mediated by the ERα,
which unlike the progesterone receptor is expressed in these
tumors. This experimental model has been successfully
applied to study, for example, the antiestrogenic activity and
function of ZK 119010 in an endometrial-derived experimental tumor model (Horn et al. 1993, 1994). As indicated above
an endometrial adenocarcinoma cell line, the RUCA-I cell
line, has been established from this transplantable EnDAtumor. This cell line is characterized by the expression of the
ERα and by its estrogen responsiveness in vitro and in vivo
(Fig. 2). If RUCA-I cells are cultured on reconstituted basement membrane (matrigel) they respond to treatment with
estradiol by stimulation of proliferation and alteration of gene
expression e.g. by the upregulation of complement C3
expression (Vollmer et al. 1995a) which is the major estrogen responsive gene in the rat uterus (Sundstro¨m et al. 1989),
as well as by down-regulation of fibronectin expression
(Vollmer et al. 1995b) and up-regulation of clusterin gene
expression (Wu¨nsche et al. 1998). In vivo, if inoculated subcutaneously into the hind limb of syngenic DA/Han rats
RUCA-I cells form estrogen-sensitive metastasizing adenocarcinomas which by histological and biochemical means are
indistinguishable from tumors arising from chunks of EnDAtumors. Tumor growth and metastasis in both experimental
setups are very fast. After approximately 30–35 days animals
are clinically ill from the metastatic disease if 0.5–1 × 106
cells are inoculated at the ectopic site (Horn et al. 1994,
Vollmer & Schneider 1996).
In this transplantation approach RUCA-I cells at the inoculation site as well as metastases of these cells at lymphogenic
sites and in the lung react very sensitively to antiestrogen treatment. The weight of the primary tumor at the ectopic site, the
weight of the axillary ipsilateral lymph nodes as well as the
number of lung metastases were found to be reduced compared
with control animals in response to antiestrogen treatment of
inoculated animals (Vollmer & Schneider 1996). In
ectopically grown tumors, in lymph node metastases and in the
normal uterus (the latter organ was analyzed for control
purposes) expression of estrogen-dependent genes was downregulated significantly following treatment of animals with the
pure antiestrogen ICI 182,780 (Wu¨nsche et al. 1998), amongst
these genes were matrix metalloproteinases (Tu¨shaus L,
Hopert AC & Vollmer G, unpublished observations).
Inoculation of RUCA-I cells into ovariectomized DA/
Han rats for the purpose of screening for estrogenic properties of phytoestrogens and endocrine-disrupting chemicals in
an endometrial-derived tumor model led to unexpected
results. Although tumor weight increased following oral
application of ethinyl estradiol in a 28-day treatment protocol, the weight of the tumor tissue remained almost unaffected following treatment of animals with a selected phytoestrogen, genistein (Diel et al. 2001) or a representative
industrial chemical o,p′-DDT (Diel et al., in preparation).
Unlike ethinyl estradiol-treated animals, there was no significant alteration of gene expression detectable in tumor tissues of animals treated with genistein or o,p′-DDT. Control
investigations in the normal rat uterus showed that this organ
responded very sensitively to treatment with ethinyl estradiol, genistein and o,p′-DDT, both with an increase in tissue
weight as well as up-regulation of estrogen-dependent gene
expression (Diel et al. 2001).
In conclusion, RUCA-I cells if inoculated into syngenic
DA/Han rats represent a very sensitive estrogen-responsive
tumor model for the purpose of antiestrogen screening and
testing in an endometrial-derived tumor model. The RUCA-I
cell-derived model is of limited value for testing of agonistic
estrogenic properties of e.g. suspected phytoestrogens or
endocrine-disrupting chemicals. Experimentally, the EnDA/
RUCA-I model shows a unique feature: it is possible to work
in syngenic animals which means it is not necessary to use
athymic nude mice or rats having an imperfect immune
system which have to be kept in germ-free surroundings.
Ishikawa cells
Hormonal responsiveness of Ishikawa cells
in vitro
Since their first description (Nishida et al. 1985) Ishikawa
cells, a human endometrial adenocarcinoma cell line expressing the estrogen and the progesterone receptors, are the most
widespread human endometrial-derived cell culture model.
Later on, applying the limited dilution method eighteen different clones of Ishikawa cells were established (Nishida et
al. 1996), fifteen of them being estrogen receptor positive.
However, in terms of population doubling time, plating efficiency or saturation density there was no significant
31
difference among the clones (Nishida et al. 1996). In addition
to the expression of functional estrogen receptors (Holinka
et al. 1986a, b) and progesterone receptors (Lessey et al.
1996), Ishikawa cells express the androgen receptor (Lovely
et al. 2000) and the receptor for aryl hydrocarbons (Wormke
et al. 2000). There exists a large body of evidence for the
hormonal responsiveness of Ishikawa cells in vitro. In summary, the evidence for hormonal responsiveness is based
mainly on hormonal modification of proliferation, cellular
functions or gene expression (for summary see Table 1 and
references therein).
In vitro the cells are in use for the elucidation of
molecular mechanisms of hormone action e.g. in drug
development, in the drug discovery process, for testing of
potential agonistic functions of antiestrogens or selective
estrogen receptor modulators in an endometrial-derived
model (Labrie et al. 2001), in studies of ligand independent
activation of the estrogen receptor, in anchorage independent tumor growth (Holinka et al. 1989), in studies on
factors controlling hormonal receptivity (Appa Rao et al.
2001), in environmental toxicology studies on the function
of phytoestrogens (Markiewicz et al. 1993, Liu et al. 2001,
Frigo et al. 2002) and endocrine-disrupting chemicals
(Bergeron et al. 1999) in an endometrial model, in paracrine cell/cell-interaction studies (Yang et al. 2001, Arnold
et al. 2002), in studies on signaling cross-talk
(Ignar-Trowbridge et al. 1995, Wormke et al. 2000), and
others (Kanishi et al. 2000).
Involvement of a switch in the angiogenic pathways
during tumorigenesis has become a recent focus of interest.
The participation of the ERα in this process was studied in
Ishikawa cells following overexpression of the ERα protein.
Overexpression of ERα not only significantly inhibited the
growth of xenografted cells, but also down-regulated vascular endothelial growth factor expression in tumor xenografts,
resulting in a decreased vascularization of the tumors and the
inhibition of the angiogenic agent integrin αvβ3 (Ali et al.
2000). These experimental findings provide evidence in favor
of the assumption that high levels of ERα may be beneficial
in the control of endometrial cancer due to its inhibitory
effects on angiogenic pathways.
Finally, endometrial carcinoma tumorgenicity could be
suppressed following introduction of chromosome 18 into
Ishikawa cells (Yamada et al. 1995). This chromosome is
known to harbor the presumptive tumor suppressor gene
DCC and therefore experimental evidence is provided for the
hypothesis of DCC being a candidate for an endometrial carcinoma tumor suppressor gene.
In summary, Ishikawa cells represent a combined in
vitro/in vivo human endometrial tumor model which is particularly suitable for the study of hormonal growth control.
In addition, it may be useful in characterizing organ specificity of natural and synthetic estrogens, like phytoestrogens
or endocrine-disrupting chemicals.
Enca101 tumor and ECC-1 cells
Ishikawa cells as an in vivo tumor model
Because of their hormone responsiveness in vitro Ishikawa
endometrial adenocarcinoma cells have also been developed
as an estrogen sensitive in vivo tumor model (Nishida et al.
1986). As mentioned above, eighteen subclones were isolated, all of these subclones were found being transplantable
into athymic nude mice (Nishida et al. 1996). This model has
mainly been used for two purposes: (1) to elucidate general
mechanisms in tumor biology, particularly towards the
understanding of estrogen and progesterone function in endometrial cancer and (2) as an endometrial model for hormonal
treatment of endometrial cancer.
The Ishikawa endometrial adenocarcinoma model proved
to be useful for studies on the regulation of growth control
by steroid hormones in endometrial adenocarcinoma in vivo
(Gong et al. 1994). The most important result of this study
was that 4-hydroxytamoxifen, like estradiol, stimulates endometrial tumor growth in vivo. This effect most likely
coincides with alteration of levels of expression of transforming growth factor (TGF)-α and TGF-β. In a similar
approach following inoculation of Ishikawa cells into the fat
pad of nude mice it could be demonstrated that raloxifen
exhibits a growth stimulatory potential in this assay
(Barsalou et al. 2002).
32
ECC-1 cells as an in vitro model
ECC-1 endometrial adenocarcinoma cells (Satyaswaroop &
Tabibzadeh 1991) have been derived from a transplantable
endometrial adenocarcinoma called EnCa101 which was
established immediately after the technique of tumor growth
in athymic nude mice became available (Satyaswaroop et al.
1981). Most importantly, ECC-1 cells form tumors with
glandular structures if inoculated into athymic nude mice
(Satyaswaroop & Tabibzadeh 1991).
The EnCA101 tumor and the ECC-1 cell line respond
to estrogen treatment (Satyaswaroop et al. 1983) and are
particularly rich in progesterone receptors (Clarke &
Satyaswaroop 1985). In addition, tumors respond to
tamoxifen treatment by stimulation of growth (Jordan et al.
1991, Tonetti et al. 1998), a feature which from epidemiological studies is expected to increase the risk for sporadic
human endometrial cancer during breast cancer therapy with
tamoxifen (Emons et al. 2000 and references therein). There
are many pieces of experimental evidence proving the
responsiveness of this model to treatment with sex steroid
hormones (for summary see Table 2). For these reasons the
EnCA101/ECC-1 tumor is the most widely used in vivo
human endometrial adenocarcinoma model.
Table 1 Hormonal responsiveness of Ishikawa cells
Receptor
Ligand(s)
Functional parameter
Regulation
Reference
ER
ER
ER
ER
ER
ER
ER
E2
E2
4-OHT
4-OHT
E2
EM652, EM800
E2
Stimulation
Stimulation
Stimulation
Stimulation
Stimulation
No regulation
Stimulation
Holinka et al. (1986a,b)
Holinka et al. (1989)
Anzai et al. (1989)
Croxtall et al. (1990)
Holinka et al. (1986c)
Labrie et al. (2001)
Fujimoto et al. (1996b)
ER
Resveratrol
E2, Tam
Down-regulation
Down-regulation
No regulation
Up-regulation
Bhat & Pezzuto (2001)
ER
ER
ER
ER
E2
Tam
E2
ER
AR
PR
PR
E2
DHT, T
P
MPA
PR
PR
PR
P; E2 + P
P
P
Proliferation
Proliferation, colony formation
Proliferation
Proliferation
Alkaline phosphatase
Migration through basement
membrane
E2 induced alkaline phosphatase
ERα
ERβ
Proliferation, plasminogen
activator
Glycogen metabolism, gelatinase
Glycogen metabolism, gelatinase
Growth, cyclin D1, invasiveness,
MMP-1, -7, -9
AF2
Growth
Growth
P27Kip
PAI-1
Estrogen sulfotransferase
α1ß1 integrin
PR
PR
MPA
MPA
PR
P
Sex hormone binding globulin
8 products from differential
display RT-PCR
VEGFs
PR
P
Osteopontin
Up-regulation
Down-regulation
Up-regulation
Stimulation
Up-regulation
Stimulation
Suppression
Up-regulation
Up-regulation
Up-regulation
Up-regulation
Upregulation
Up-/down-regulation
(gene dependent)
Down-regulation of E2
induced induction
Up-regulation
Hochner-Celnikier et al.
(1997)
Mizumoto et al. (2002)
Sakamoto et al. (2002)
Markiewicz & Gurpide (1997)
Holinka & Gurpide (1992)
Shiozawa et al. (2001)
Fujimoto et al. (1996a)
Falany & Falany (1996)
Lessey et al. (1996),
Castelbaum et al. (1997)
Misao et al. (1998)
Sakata et al. (1998)
Fujimoto et al. (1999)
Appa Rao et al. (2001)
Abbreviations: ER, estrogen receptor; E2, estradiol; 4-OHT, 4-hydroxytamoxifen; Tam, tamoxifen; DHT, dihydrotestosterone; T,
testosterone; P, progesterone; MPA, medroxyprogesterone acetate; MMP, matrix metalloproteinase; PAI, plasminogen activator
inhibitor; VEGF, vascular endothelial growth factor.
EnCa101 and ECC-1 cells-derived tumors
In terms of hormonal regulation of tumor growth, inoculation
of EnCa101 tumors or ECC-1 endometrial adenocarcinoma
cells into nude mice appears to be the most complete model.
It is responsive to estrogens and progestins and it grows following tamoxifen stimulation. In addition, the EnCA101
tumors exhibit another feature which is highly advantageous
in experimental cancer research: it can be used as a multi-site
transplantation model thereby saving animals (Heitjan et al.
2002). In these tumors the most interesting features of hormonal influences on endometrial tumor growth can be
studied, e.g. testing of novel estrogen receptor antagonists,
treatment of tamoxifen-stimulated tumor growth – a major
concern as indicated above (ACOG Committee Opinion
1996, Love et al. 1999) – and characterizing the effects of
progestins, these being the adjuvant therapy for endometrial
carcinoma for decades (Emons et al. 2000 and references
therein).
Once the estrogen dependency of tumor growth of
EnCa101 tumors (Satyaswaroop et al. 1983, Jordan et al.
1991) and their estrogen-responsiveness as measured by gene
expression, particularly of progesterone receptor (Clarke et
al. 1987) and of c-fos, (Sakakibara et al. 1992) had been
established, in a first series of larger experiments the effects
of antiestrogens and progestins on the growth of these tumors
were investigated. In fact, for progestins schedules for progestin administration were designed in this model (Mortel et al.
1990).
For antiestrogens it could be shown that almost all antiestrogens tested stimulated tumor growth although to a lesser
degree than tamoxifen (Gottardis et al. 1990) with the exception of the pure antagonist ICI 164,384 which was not only
void of tumor growth stimulatory activity but also blocked
tamoxifen-induced tumor growth.
During combination treatments with tamoxifen and progestins, resistance of EnCA101 tumors to treatment became
apparent. This resistance was attributed to a desensitization
33
Table 2 Hormonal responsiveness of EnCa101 tumors and ECC-1 cells
Cell line/
tumor
Receptor
Ligand
Functional parameter
Regulation
Reference
EnCa101
EnCa101
EnCa101
ECC-1
ER
ER
ER
ER
ER
ECC-1
ER
Growth in vivo
Growth in vivo
Growth in vivo
Growth in vivo
Growth in vivo
Growth in vivo
Growth in vivo
Proliferation in vitro
Increase
Increase
Increase
Increase
No effect
Stimulation
No effect
Stimulation
Satyaswaroop et al. (1993)
Jordan et al. (1991)
Jordan et al. (1989)
Dardes et al. (2002a)
ECC-1
E2
Tam
E2/Tam
E2
GW5638
E2
Tam, Ral
E2
EnCa101
ECC-1
EnCa101
ECC-1
ECC-1
ECC-1
ER
ER
ER
ER
ER
ER
E2
E2, BPA
E2, Tam
E2
E2
E2, Ral
ICI182,780
E2
E2
Progesterone receptor
Progesterone receptor
c-fos
Cathepsin D
GREB1
ER-α
ER-α
pS2, VEGF
HER2/neu
Stimulation
Stimulation
Induction
Induction
Induction
Down-regulation
Degradation
Induction
Down-regulation
Dardes et al. (2002b)
Bergeron et al. (1999), Castro-Rivera
et al. (1999), Dardes et al. (2002b)
Clarke et al. (1987)
Bergeron et al. (1999)
Sakakibara et al. (1992)
Castro-Rivera et al. 1999
Ghosh et al. (2000)
Dardes et al. (2002b)
Abbreviations: ER, estrogen receptor; E2, estradiol; Ral, raloxifen; Tam, tamoxifen; BPA, bisphenol A; VEGF, vascular
endothelial growth factor; pS2, presenelin-2.
following down-regulation of the progesterone receptor
(Satyaswaroop et al. 1992). Based on the results of another
study in which progesterone levels, progesterone receptor
levels and rates of tumor growth were assessed, it was predicted that intermittent progestin administration may result
in better control of endometrial cancer growth in the nude
mouse model (Mortel et al. 1990).
The EnCa101 tumor model is characterized by another
feature – the estrogen-inducible expression of aromatase.
Aromatase expression has been reported to be up-regulated
in endometrial adenocarcinoma, particularly in the stromal
tissue compartment (Watanabe et al. 1995, Sasano et al.
1996). Analyses of aromatase (Cyp19) gene polymorphism
revealed that these polymorphisms might represent one genetic risk factor for endometrial cancer development (Berstein
et al. 2001). Despite the clear up-regulation of aromatase
activity in endometrial cancer, the use of aromatase inhibitors
in a clinical setting has not been exhaustively investigated
and the clinical outcome is questionable (Rose et al. 2000,
Berstein et al. 2002).
Estrogen-inducible aromatase activity, however, is
interesting for general tumor biology because the role of
androgens in the growth of endometrial carcinoma is poorly
understood. The question of androgen sensitive endometrial
tumor growth was addressed in this particular tumor model.
There was little detectable growth promoting activity of
androgens which was lower by far than that of estrogens.
Surprisingly, there was no difference in the effects of
androgens on the growth of EnCa101 tumors if the effects of
aromatizable and non-aromatizable androgens were compared (Legro et al. 2001).
34
In summary, the EnCa101/ECC-1 tumor model is the
most complete endometrial tumor model because the effects
of estrogens, progestins and androgens can be assessed due
to the stable and, in part, estrogen inducible expression of the
estrogen receptor, the progesterone receptor and aromatase.
These concluding remarks have recently been substantiated
by studies on the characterization of some novel antiestrogens: the tamoxifen analog GW5638 and the piperidin
derivative ERA-923. These substances, like tamoxifen, block
growth of breast cancer cells and, in addition, more effectively block EnCa101 endometrial tumor growth
(Greenberger et al. 2001, Dardes et al. 2002a).
Differentiation of endometrial
adenocarcinoma cells in vitro
Endometrial adenocarcinomas differ in their ability to form
glandular structures; however most of the sporadic occurring
endometrial adenocarcinomas are well to moderately well
differentiated. In conventional cell culture on plastic in the
presence of charcoal stripped fetal calf serum or in the presence of a serum-free defined medium, endometrial adenocarcinoma cells acquire a polygonal cell shape with a low
degree of differentiation, and in a more narrow view cannot
be regarded as correlated to the relatively high differentiated
status of endometrial adenocarcinoma cells in vivo.
For cells derived from a variety of organs, particularly
for normal and malignant mammary cells, a huge body of
evidence has accumulated (for review see Hansen & Bissell
2000) that culturing of glandular cells on reconstituted
Alteration of
gene expression
Adenocarcinoma cell
(dedifferentiated)
basement
membrane
TN-C rich
Stromal
matrix
Adenocarcinoma cell on
basement membrane
(differentiated)
Processes regulated by basement membrane
Reference
Cellular morphology and ultrastructure
Satyaswaroop & Tabibzadeh. (1991), Hopfer et al. (1996),
Behrens et al.(1996), Pinelli et al. (1998)
Proliferation
Tan et al. (1999)
Hormone responsiveness
Vollmer et al. (1995a,b), Wünsche et al. (1998)
Gene expression in general
Strunck et al. (1996)
Expression of integrins
Strunck and Vollmer (1996)
Expression of cytokeratins
Pastor U, Strunck E & Vollmer G, unpublished observations
Mitochondrial gene expression
Strunck E & Vollmer G, unpublished observations
icb-1 expression (differentiation marker)
Treeck et al. (1998)
Expression of phosphatases (L3-PSP)
Strunck et al. (2001)
Proteases (MMP2, MMP13)
Tüshaus L, Holpert A C & Vollmer G, in preparation
Figure 3 Responses of endometrial adenocarcinoma cells to contact with reconstituted basement membrane. The upper part is
a schematic drawing of the situation of an endometrial adenocarcinoma cell grown in conventional cell culture on plastic
(dedifferentiated) and on reconstituted basement membrane (differentiated). This figure also indicates the presence of the
stromal matrix underneath the basement membrane, which in cases of endometrial adenocarcinoma is particularly rich in
tenascin-C (TN-C). The lower part shows the processes regulated by the basement membrane. icb-1, induced by contact to
basement membrane; L3-PSP, L3-phosphoserine phosphatase; MMP, matrix metalloproteinase.
basement membrane induces differentiation processes,
thereby increasing physiological responsiveness considerably
if compared with cells kept under conventional cell culture
conditions. The evidence for morphological and functional
differentiation of human endometrial adenocarcinoma cells
of Ishikawa (Pinelli et al. 1998), of the ECC-1 cell line
(Satyaswaroop et al. 1991) and of the RUCA-I rat endometrial adenocarcinoma cells (Vollmer et al. 1995a) is summarized in Fig. 3. The most important observation is that the
morphological differentiation strongly correlates with functional differentiation, the latter being most importantly evidenced by the induction of hormone responsiveness.
With the culturing of endometrial adenocarcinoma cells
on reconstituted basement membrane a more pronounced
physiological phenotype of these cells is achieved, allowing
the assessment of physiological in vivo functions in an in
vitro model. This assay can be modified into an in vitro
invasion assay for the assessment of hormonal influences on
tumor cell invasion through basement membranes (Fujimoto
et al. 1996b). Finally, a modification of this assay has been
described by reconstituting the natural situation even further
and adding conditioned medium of endometrial stromal cells
or cultured endometrial stromal cells to the system. In this
way two important parameters in endometrial tumor growth
can be assessed: the contribution of paracrine cellular interactions to tumor growth and the hormonal regulation of cells
of the two tissue compartments in a single cell culture dish
(Arnold et al. 2002).
35
Conclusions
There is definitely a need for the development of substances
for the treatment and cure of endometrial cancer. In addition
there are various new drugs or phyto-remedies under development which are intended to be used in the treatment and
prevention of breast cancer, for the treatment of menopausal
symptoms and for hormone replacement therapy. The efficacy of novel drugs targeting steroid receptors in endometrial
cancers has to be evaluated and the safety of other endocrine
measures on endometrial cancers or on endometrial carcinogenesis has to be assessed. For these purposes a relatively
small number of animal models or combined in vivo/in vitro
tumor models, based on human endometrial adenocarcinoma
cells, are available. However, these models alone or in combination cover all needs for drug development or testing of
drug safety in relation to endometrial cancer. While models
consisting of human endometrial adenocarcinoma cells transplanted to athymic nude mice are commonly used, the potential of animal models consisting of spontaneous endometrial
carcinogenesis or transplantation of rat endometrial adenocarcinoma cells to syngenic animals is largely ignored. First
of all, with these models treatment protocols for chronic
treatment of both primary tumors and metastases can be
established. And even if the inoculation approach is used
there is still a considerable advantage because syngenic animals can be used. Thus the use of the cost intensive athymic
nude animal, a more artificial model which has to be kept
in germ-free surroundings because of its imperfect immune
system, can be avoided.
In the future, the number of studies using transgenic animals and therapy approaches in these animals will definitely
increase. The advantage of these tools is that the genetic
modification leading to the carcinogenic process is more precisely defined. In this way single genes or corresponding
signal transduction pathways can be targeted in quite a selective way.
Acknowledgements
The author is grateful to Dr Deerberg for giving authorization
for the use of unpublished data on lung metastasis of endometrial adenocarcinoma in DA/Han rats.
The author wishes to thank Drs Susanne Starcke, Jannette Wober and Oliver Zierau for critical reading of the
manuscript and for their helpful suggestions. In addition,
special thanks are due to Dr Oliver Zierau for his assistance
with the art work.
This paper was supported by the Deutsche Forschungsgemeinschaft Vo410/5-4, Vo410/6-1 and Vo410/6-3.
Dedication
This paper is dedicated to honour the work of the recently
deceased Dr Friedrich Deerberg, formerly of the German
36
Institute for Laboratory Animals. Dr Friedrich Deerberg systematically investigated spontaneous carcinogenesis in inbred
laboratory animals and developed experimental animal
models useful for studying cancer treatment and prevention.
Amongst these models are two spontaneous hormonedependent endometrial cancer models which are exhaustively
discussed in this paper. Personally, the author is indebted to
Dr Deerberg for supplying him with the DA/Han and BD/
Han rat endometrial cancer models and the cell lines developed thereof.
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