[CANCER RESEARCH 63, 4967– 4977, August 15, 2003]
Anomalous Expression of Epithelial Differentiation-determining GATA Factors in
Ovarian Tumorigenesis1
Callinice D. Capo-chichi, Isabelle H. Roland, Lisa Vanderveer, Rudi Bao,2 Tetsuya Yamagata, Hisamaru Hirai,
Cynthia Cohen, Thomas C. Hamilton, Andrew K. Godwin, and Xiang-Xi Xu3
Ovarian Cancer and Tumor Biology Programs, Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 [C. D. D., I. H. R., L. V., R. B.,
T. C. H., A. K. G., X-X. X.]; Department of Pathology, Emory University School of Medicine, Atlanta, Georgia 30322 [C. C.]; and Third Department of Internal Medicine,
University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan [T. Y., H. H.]
ABSTRACT
Tumor cells often appear in a deviant differentiated stage, and dedifferentiation is a hallmark of malignancy; however, the causative mechanism of
the global changes in dedifferentiation is not understood. The GATA transcription factors function in cell lineage specification during embryonic development and organ formation. The transcriptional targets of the GATA
factors in early embryonic development include Disabled-2 and collagen IV,
markers for epithelial lineages. GATA-4 and GATA-6 are expressed strongly
and are localized in the nucleus in ovarian surface epithelial cells in tissues or
primary cell cultures. By immunohistochemistry, we found that 82% of the
50 tumors analyzed had lost GATA-6 function, either by a complete absence
of expression or by cytoplasmic mislocalization. The frequent loss of GATA-6
was also confirmed in a panel of ovarian surface epithelial and tumor cell
lines. Although GATA-4 is absent only in a small percentage (14%) of ovarian
tumors, it is lost in the majority of established cell lines in culture. The loss of
GATA-6 correlates with the loss of Disabled-2, collagen IV, and laminin,
markers for epithelial cell types. Loss of GATA factors was also found in an
in vitro model for spontaneous transformation of rat ovarian epithelial cells.
Repression of GATA-6 by small interfering (si)RNA approach in cultured
cells leads to dedifferentiation as indicated by the loss of Disabled-2 and
laminin expression. Restoration of GATA factors expression by ectopic transfection suppresses cell growth and is incompatible with the maintenance of
the cells in culture. However, restoration of GATA-4 and GATA-6 expression
is not able to induce expression of endogenous Disabled-2 in tumor cells,
suggesting that the loss of GATA factors and dedifferentiation are irreversible processes. In conclusion, we observed the inappropriate expression and
cellular localization of the GATA transcription factors in ovarian tumor
tissues and cancer cell lines, and we have demonstrated that down-regulation
of GATA factor expression leads to dedifferentiation. We propose that alterations of GATA transcription factor expression and aberrant nucleocytoplasmic localization may contribute to the anomalous epithelial dedifferentiation
of the ovarian tumor cells.
INTRODUCTION
It is well recognized that tumor cells often appear in an inappropriately differentiated stage (1– 4); however, the causative mechanism
of the dedifferentiation is not well defined. The majority of solid
tumors are carcinomas, derived from cells of tissue surface epithelium. The epithelial-derived cancer cells often lose the epithelial
characterizations, one hallmark of which is the loss of ability to
produce a basement membrane. In ovarian cancer, tumor cells often
lose their ability to synthesize collagen IV and laminin (5), components of the basement membrane (6). Another important and consistReceived 12/30/02; revised 5/29/03; accepted 6/5/03.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported by NIH Grants R01 CA79716, R01 CA75389, and R01 CA095071 (to
X-X. X.) from National Cancer Institute, by funds from Ovarian Cancer Research Foundation (OCRF, New York, NY; to X-X. X.), and by an appropriation from the Commonwealth of Pennsylvania.. T. C. H., A. K. G., and X-X. X. are also supported by funding
from Ovarian Cancer SPORE P50 CA83638.
2
Current address: Novartis Oncology/Pharmacology, Summit, NJ 07901.
3
To whom requests for reprints should be addressed, at Ovarian Cancer and Tumor
Cell Biology Programs, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA
19111-2497; Phone: (215) 728-2188; Fax: (215) 728-2741; E-mail: [email protected].
ent characteristic of epithelial dedifferentiation is the loss of apicalbasolateral polarity, and the loss of Dab24 expression is thought to
account for the loss of epithelial polarity (7–9). Dab2, a cargoselective endocytic adaptor, has been shown to be a critical determinant of epithelial polarity and surface positioning (9).
The GATA transcription factors are conserved in insects and vertebrates from fly to humans and function in cell lineage specification during
embryonic development and organ formation (10). In mammals, there are
six GATA family members: GATA-1, -2, and -3 are involved mainly in
the development of the hematopoietic systems (11, 12); GATA-4, -5, and
-6 are expressed in a wide range of tissues and function in the formation
of most, if not all, organs during embryonic development (10). GATA-4
and GATA-6 are first expressed during the formation of extraembryonic
endoderm differentiated from the pluripotent embryonic stem cells of the
inner cell mass during early embryonic development (13–15). In vitro
analysis has shown GATA-4 and GATA-6 to be two of the most
upstream factors during the primitive endoderm (an epithelial cell type)
differentiation of pluripotent embryonic stem cells (16). GATA-4 and
GATA-6 are expressed in the heart (17), liver (18), lung (19), gastric
epithelium (20), intestine and colon (21), testis (22), and ovary (23, 24)
and play critical roles in the development of these organs. GATA factors
are not tissue specific but rather function in the specification and differentiation of cell lineages within an organ, such as the differentiation of an
epithelial cell lineage from stromal cells. In a study of the differentiation
of embryonic stem cells to endoderm cells, Dab2, GATA-4, and collagen
IV were among 10 of the genes identified to be regulated by GATA-6
(25). Dab2 is a candidate tumor suppressor of ovarian cancer expressed
mainly in the surface epithelial cells, and its expression is often lost in
ovarian tumors at an early stage of tumor development (7, 26, 27). The
loss of Dab2 closely correlates with the morphological transformation of
the ovarian surface epithelial cells (8) and the disorganization of the
primitive endoderm, the first polarized epithelial structure of the early
embryos (9).
In adult tissues, GATA factors likely function in maintaining the
differentiated states of cells (10). One possibility is that the loss of
GATA factors or their cognate regulatory pathways leads to dedifferentiation of epithelial cells and contributes to tumorigenicity. Previously, the expression of GATA factors has been investigated in tumor
cells. GATA-4 is expressed in sex cord-derived ovarian and gonad
tumors (24) and gastric cancer cell lines (28). It was found that
GATA-4 and GATA-6 are reciprocally altered in adrenal tumors (29).
In this report, we have investigated the expression of GATA-4 and
GATA-6 in ovarian tumors and cell lines, and examined the correlation with dedifferentiation of the tumor cells using the expression of
Dab2, collagen IV, and laminin as markers for epithelial differentiation. We observe the inappropriate expression and subcellular localization of the GATA transcription factors in ovarian tumors and
4
The abbreviations used are: Dab2, disabled-2; FBS, fetal bovine serum; GFP, green
fluorescence protein; HIO, immortalized HOSE (cell); HOSE, human ovarian surface
epithelial (cell); Nutu, nude mice tumorigenic; OVCAR, ovarian carcinoma; ROSE, rat
ovarian surface epithelial (cell); Dapi, 4⬘,6-diamidino-2-phenylindole; siRNA, small interfering RNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
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INACTIVATION OF GATA-6 IN DEDIFFERENTIATION OF OVARIAN TUMORS
cancer cells, and propose that alterations of GATA transcription factor
expression and nucleocytoplasmic trafficking account for the dedifferentiation in ovarian tumorigenicity. Additionally in vitro suppression of GATA-6 led to cell dedifferentiation.
MATERIALS AND METHODS
Ovarian Tumor Tissues. A representative set of 50 archived human
ovarian tumors from the tissue collection held by the Department of Pathology,
Emory University School of Medicine, was used for this study. This set of
ovarian tumors has been used previously for other investigations (5). All of the
tumors have been identified as of ovarian surface epithelial origin, and the
histological subtypes, and grades were verified in sections stained with H&E.
Neighboring sections were used for analysis. Of the examined cases, there are
2 endometrial carcinomas, 36 serous papillary and cystadenocarcinomas, 5
mucinous adenocarcinomas, and 7 poorly differentiated adenocarcinomas. The
age of the patients ranged from 43 to 79 years, with a mean age of 58.5 years.
HOSE and “Immortalized” Lines (HIO). HOSE cells were derived from
freshly dissected nontumor ovarian tissues obtained from women undergoing
prophylactic oophorectomies. Briefly, the surface epithelium was scraped from
the ovarian surface, and the cells were cultured in medium 199 and MCDB-105
(1:1) supplemented with 4% FBS and 0.2 units/ml of insulin (Novagen). These
early passage HOSE cells were obtained in small quantities and were used
mainly for immunofluorescence analysis, but were not sufficient in quantity for
various biochemical characterization.
After culturing of the primary cultures for 1–2 months but before the cells
entered replicative senescence, the HOSE cells were transfected with an SV40
large T antigen (SV40Tag) expression vector. These cells are referred to as
HIO cells and can undergo an additional 20 –30 population doublings before
ceasing proliferation. The cells have been characterized and verified to be of
epithelial cell type as described previously (5). RNA used in the Northern blots
was isolated from HIO cells at the following passages (p): HIO-103, p11;
HIO-105, p10; HIO-107, p7; HIO-114, p11; and HIO-118, p34.
ROSE and Tumor (Nutu) Cell Lines. ROSE cell lines were derived and
characterized as described previously (30, 31). Briefly, 20 ovaries were aseptically
removed from 10 mature female Fisher rats (12–16 weeks of age) and were
trypsinized to selectively release cells from the surface epithelial layer. Cell
suspensions washed out from treated ovaries were pooled together, filtered with
two sheets of cheesecloth, and transferred to tissue culture flasks. The cells were
incubated overnight at 37°C, 5% CO2, in DMEM with 10% serum, and antibiotic
supplement. The next day, the floating cells and debris were washed off with warm
PBS, and the attached cells were further subcultured at 1- or 2-week intervals at a
split ratio of 1:5 (for early passage, i.e., ⬍10 subcultures). The cells were characterized and shown to be epithelial in more than 95% of the cell populations. The
early passage of these cells is known as ROSE cells. ROSE 23 is an early culture
of the cells and was tested to be nontumorigenic (30, 31).
Continuous passaging and subculturing resulted in spontaneously transformed
cell populations raised as foci in monolayer cultures. The cells from individual foci
were collected, expanded, and examined for tumorigenicity by injecting these
individual cell populations into female athymic nude mice. Late passages of ROSE
cell subcultures (numbers 12, 14, 19, and 26), formed tumors by 3– 6 weeks after
inoculations. Histopathologically, tumors were as adenocarcinomas with different
degrees of morphological dedifferentiation (30, 31). Tumors from cell populations
19 and 26 were poorly differentiated adenocarcinomas. Tumors from cell population 12 appeared to be well differentiated, and another tumor from cell population 14 was moderately differentiated. The cell lines derived from the tumor were
named Nutu (number 12, 14, 19, and 26) lines (30, 31).
Immunohistochemistry. The immunostaining of ovarian tissues and tumors was performed and analyzed as reported previously (5, 7, 8, 32). Sections
were first deparaffinized and rehydrated. Antigen retrieval was performed for
5 min at 120°C in citrate buffer (pH 6) using an electric steamer cooker for 5
min, followed by cooling for 10 min before immunostaining. All of the tissues
were then exposed to 3% hydrogen peroxide for 5 min, primary antibodies for
25 min, biotinylated secondary linking antibodies for 20 min, streptavidin
enzyme complex for 20 min, diaminobenzidine as chromogen for 5 min, and
hematoxylin as counterstain for 1 min. These incubations were performed at
room temperature; between incubations, sections were washed with TrisBuffered Saline (TBS) buffer. An avidin-biotinylated enzyme complex kit
(DAKO LSAB2) was used in combination with the automated DAKO
AUTOSTAINER (DAKO Corp.). Coverslipping was performed using the
Tissue-Tek SCA (Sakura Finetek USA, Inc., Torrance, CA) automatic coverslip. The sources of the primary antibodies were: monoclonal mouse anti-Dab2
IgG (Transduction Lab); anti-GATA-4 and anti-GATA-6 rabbit antiserum
(Santa Cruz Biotechnology). The immunostaining of GATA-4 and GATA-6
was verified for specificity using blocking peptides on tissue sections containing GATA-4- and GATA-6-positive ovarian surface epithelia.
The slides were scored independently by three persons (C. C., X. X. X.,
I. H. R.) including a pathologist (C. C.). Staining in both cytoplasm and
nuclear areas were scored separately. Positive scoring was given when the
epithelial staining of collagen IV and laminin was higher than 10% on the
slide. If tumor cytosolic Dab2 staining was positive in more than 10% of the
cells, the tumor was scored as Dab2-positive. The results from three independent determinations were then compared, any differences in scoring results were
discussed, and the slides were further examined to reach a common conclusion.
Cell Culture, Western and Northern Blot Analysis. Ovarian epithelial
and tumor cell lines were previously established (the OVCAR lines; Ref. 34),
or obtained from American Type Culture Collection (A2780, ES2, SKOV-3,
and OV1016). The cells were cultured in DMEM with 10% FBS. Total cell
lysate was used for Western blotting using antibodies against GATA-4,
GATA-6, Dab2, and laminin.
Total RNA was isolated from 100-mm plates of 80% confluent monolayers
using the guanidinium isothiocyanate/phenol/chloroform extraction procedure as
described previously (5). Northern blot analysis was performed using 32P-labeled
cDNA fragments with a random prime labeling kit, Prime-It II (Stratagene). cDNA
for human GATA-6 was a generous gift from Dr. David Wilson (Washington
University, St. Louis, MO) and from Dr. Kenneth Walsh (St. Elizabeth’s Medical
Center, Boston, MA). Plasmids containing the partial cDNA of collagen IV␣1 and
-␣2, and laminin ␤-1 were EST clones obtained from American Type Culture
Collection. Human Dab2 cDNA was reported previously (35). The gel-purified
cDNA fragments from restriction digestion were used as probes. All of the cDNA
fragments were sequenced to verify their identity before use.
Cell Transfection. Human GATA-4 (GenBank accession no. D78260;
Ref. 36) and GATA-6 cDNAs were cloned either in pcDNA3 vectors or in an
expression vector (pMT-CB6⫹) under an inducible metallothionein promoter.
Transfection of plasmid DNA were performed using Mirus Trans1T-LT1
reagent (Mirus Co., Madison, WI) according to the manufacturer’s protocols.
GATA-4 was transfected into ES2 cells, which are positive for GATA-6 and
Dab2 but negative for GATA-4 expression. GATA-4 was transfected in
SKOV3 cells, which are negative for GATA-4 and Dab2, but are positive for
GATA-6 protein expression. A plasmid-construct containing the GFP under
the metallothionein promoter was used as a positive control for the cell
transfection. After 72 h of transfection, the cells were cultured for 21 days in
DMEM containing 10% FBS and 1 mg/ml G418. This selection medium was
changed every 48 h. The stable clones selected were used to analyze GATA-4,
GATA-6, and Dab2 expression before and after treatment with zinc sulfate
(200 ␮M) for 2 days to induce gene expression.
Suppression of Gene Expression by siRNA Approach. The expression of
GATA-6 in cultured epithelial and tumor cells was reduced/suppressed using
siRNA technique. Three 21-bp oligonucleotide sequences specific for human
GATA-6 were tested in pSuppressorNeo vector (IMG:800; Imgenex, San Diego,
CA) for suppression of GATA-6 expression. The most successful sequence found,
hG6-110, is a 21-bp sequence 110 base 3⬘ of ATG site-specific to human GATA-6
without significant similarity to other genes. A synthetic double-strand oligonucleotide was inserted (see below) into pSuppressorNeo plasmid according to the
manufacturer’s recommendation (Imgenex).
The vector or the siRNA expression plasmid were transfected into GATA6-positive ES2 or HIO-118 cells using Mirus transIT-LT1 reagent (Mirus), by
incubating for 24 h before regular media was added back. The suppression of
GATA-6 and Dab2 expression was evaluated by Western blotting and immunofluorescence 4 days after transfection.
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Immunofluorescence Microscopy. Cells were plated on 22 ⫻ 40 mm cover
slides in 6-well dishes and were fixed with 4% paraformaldehyde when they had
reached 60% confluence. Cells were permeabilized with 0.5% Triton X-100 in
PBS for 5 min, were washed with PBS, and were blocked with 3% BSA in PBS
containing 0.1% Tween 20 (at room temperature for 30 min). GATA-4, GATA-6,
Dab2, and laminin antibodies were used at 1:200 dilution in 1% FBS in PBS
containing 0.1% Tween 20 and were incubated at 37°C for 2 h. The cellular
localization of the antigens was revealed by fluorescein- or Texas Red-conjugated
secondary antibodies (Jackson Immuno-Research Laboratories, West Grove, PA)
at 1:200 dilution. The secondary antibodies were: donkey antimouse IgG conjugated with Texas Red and donkey antirabbit IgG conjugated with fluorescein.
Rabbit anti-GATA-4 or anti-GATA-6 antibodies were used with mouse anti-Dab2
antibodies for double labeling. Goat anti-GATA-4 and rabbit anti-GATA-6 antibodies were used for detection of GATA-4 and GATA-6 on the same slides.
Nuclei were marked by Dapi staining. The Nikon Eclipse E 800 epifluorescence
microscope with ⫻60 oil immersion objective linked to a Roper Quantix CCD
(charged coupled device) camera were used for observation and image acquisition.
A Nikon Eclipse E800 fluorescence microscope with ⫻60 water immersion
objective linked to a Bio-Rad Radiance 2000 LSCM (laser scanning confocal
microscope) camera was also used to examine the slides. Images were merged by
overlaying, using the Adobe-Photoshop program.
RESULTS
Nuclear Expression of GATA-4 and GATA-6 in Ovarian
Surface Epithelial Cells. The majority of ovarian malignancies are
derived from ovarian surface epithelial cells, which consist of a
single-cell layer of flat or cuboidal epithelial cells organized by a
sheet of basement membrane (37–39). Normal ovarian surface epithelial cells have been shown to be polarized and to express Dab2 (7,
8, 27), and to be organized by a layer of collagen IV- and lamininpositive basement membrane (5, 8, 32). Here, we demonstrate by
immunostaining that both GATA-4 and GATA-6 are strongly expressed in the nucleus of epithelial cells of morphologically normal
human ovarian surface epithelium (Fig. 1A). All of the surface epithelial cells are intensely positive for both GATA-4 and GATA-6
staining in the nucleus. Some cells scattered in the ovarian stromas are
also GATA-4 and/or GATA-6 positive.
We isolated and analyzed primary surface epithelial cells from
human and rat ovaries to confirm the expression of GATA-4 and
GATA-6. Because of the limitation in the quantity of the materials,
immunofluorescence microscopy was chosen to analyze the cultured
primary cells. GATA-4 and GATA-6 were found to be strongly
positive in the nucleus as indicated by their colocalization with Dapi
staining, and Dab2 was present mainly in the cytoplasm of the primary
cultures of HOSE cells (Fig. 1B). Dab2 staining exhibited a cytoplasmic speckled pattern, in agreement with its association with endocytic
vesicles reported previously (33). Similarly, primary ROSE cells were
found positive for GATA-4, GATA-6, and Dab2 expression by both
Western blotting and immunofluorescence microscopy (not shown).
Ovarian cancer cell lines of known GATA-4, GATA-6, and Dab2
expression status (detailed below), A2780, ES2, and SKOV-3, were
used as positive and negative controls, respectively (detailed below).
Frequent Loss of GATA-6 in HOSE Cells and Tumors. Previously, the loss of Dab2 was found to be an early event in ovarian
tumorigenicity, correlating closely with dysplastic morphological
transformation of ovarian surface epithelia (7, 8). Because Dab2 and
collagen IV are regulated by GATA factors during the differentiation
of embryonic stem cells to epithelial-like extraembryonic endoderm
cells (25), we hypothesized that GATA factors might also function in
the maintenance of ovarian surface epithelial differentiation by regulating expression of Dab2 and collagen IV. Thus, we investigated the
expression of GATA-4 and GATA-6 in ovarian tumors to determine
whether the loss of Dab2 might be caused by a dysfunction of these
factors in ovarian tumor cells. Immunostaining of archived ovarian
tumor tissues showed that GATA-6 is completely lost in cancer cells,
in 15 (30%) of the 50 tumors analyzed (Fig. 1, C and D; Tables 1 and
2). In an example shown in Fig. 1C, morphologically normal ovarian
surface epithelial cells were positive for both GATA-4 and GATA-6.
However, in malignant areas of the same tumor, cells were positive
for GATA-4, but GATA-6 staining is absent (Fig. 1C). Additional
examples are shown for five tumors that are negative for GATA-6 and
positive for GATA-4 in the nucleus (Fig. 1D, Tumors 1–5), two
tumors that were negative for both GATA-4 and GATA-6 in the
nucleus (Fig. 1D, Tumors 6 and 7), and one tumor that was positive
for both GATA-4 and GATA-6 (Fig. 1D, Tumor 8). Unlike GATA-6,
however, GATA-4 was present in most of the tumors, and only 7
(14%) of the 50 tumors analyzed were GATA-4 negative (Table 2).
To verify the loss of GATA transcription factors in established ovarian
cancer cell lines, a panel of ovarian cancer cells lines and five HIO cell
lines were analyzed for GATA-4 and GATA-6 expression by Northern
blot (Fig. 1E). The expression of Dab2, collagen IV, and laminin, which
are indicators of epithelial differentiation or dedifferentiation of the tumor
cells, was also determined. Most of the tumor lines, including A2780,
OVCAR-2, -3, -4, -8, and -10, and OV1016, have little or no expression
of either GATA-4 or GATA-6, consistent with the observations of the
loss of GATA-6 in tumor tissues. ES2, in which both GATA-6 and Dab2
are expressed strongly, is one of the few exceptions among tumor cell
lines. Even in two of the five nontumorigenic HIO lines, GATA-6 is
already absent, correlating with the loss of expression of Dab2, collagen
IV, and laminin (Fig. 1E). In all of these tumor and HIO cell lines, with
the exception of SKOV-3, the loss of GATA-6 expression correlates well
with the loss of Dab2 expression. The expression of collagen IV, however, is present in OVCAR-2 and -10 despite the absence of GATA-6.
Remarkably, GATA-4 expression is found to be absent in most of the
tumor and nontumorigenic cell lines, in contrast to the observation from
tumor tissues.
Loss of Nuclear Localization of GATA-6 in Ovarian Tumors.
In some of the ovarian tumors in which GATA-6 expression was classified as positive, the staining appears to be cytoplasmic rather than
nuclear (Table 1). Examples of the cytoplasmic staining of GATA-6 are
shown in four tumors (Fig. 2A). In these tumors, GATA-6 staining is
absent in the nucleus of the tumor cells, which are counterstained blue by
hematoxilyn (instead of brown or dark staining for the GATA-6 antigen).
Brown staining, indicating the presence of GATA-6 protein, is visible
around the nucleus, demonstrating the cytoplasmic localization of
GATA-6 in these tumors. In contrast, GATA-4 staining is present in the
nucleus of tumor cells from adjacent sections.
To confirm the cytoplasmic localization of GATA-6 in ovarian tumor
cells, we examined the cellular localization of GATA-6 in established
ovarian tumor cell lines by immunofluorescence microscopy using both
CCD (Fig. 2B) and confocal (Fig. 2C) imaging. GATA-6 positive (primary HOSE cells, ES2, and SKOV-3) and GATA-6 negative (A2780)
cell lines were analyzed (Fig. 2). In SKOV-3 cells, GATA-6 is not
nuclear localized (Fig. 2, B and C), whereas it is nuclear in both HOSE
(Fig. 2, B and C) and ES2 (Fig. 2B) cells. Thus, examples exist in cell
lines to confirm the observation of GATA-6 nuclear exclusion in tumor
tissues. Laminin staining was observed in HOSE and ES2 cells but not in
A2780 and SKOV-3 cells (Fig. 2B), consistent with Northern blot results
(Fig. 1E). Presumably, the expressed laminin protein detected by immunofluorescence signal is associated with the outer cell membranes.
Both GATA-4 and GATA-6 are transcription factors, and their
function is believed to reside in the nucleus. Furthermore, GATA-4
and GATA-6 are known to work in collaboration in regulating myocardial gene expression (40) and embryonic cell differentiation (16).
Thus, by combining the number of tumors that have lost or have
nuclear exclusion of one of the GATA factors, the majority (41 of 50,
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INACTIVATION OF GATA-6 IN DEDIFFERENTIATION OF OVARIAN TUMORS
Fig. 1. Nuclear expression of GATA-4 and GATA-6 in HOSE cells and frequent loss of GATA-6 in ovarian tumors. A, archived nontumor human ovarian tissues were analyzed for GATA-4
and GATA-6 by immunostaining (brown or dark) of neighboring sections. A representative staining of five tissue samples is shown. Both GATA-4 and GATA-6 staining are positive in the
surface epithelium. B, primary HOSE cells were prepared freshly from oophorectomy surgery. The primary HOSE cells were used to analyze the expression of GATA-4 (green, fluorescein),
GATA-6 (green, fluorescein), and Dab2 (red, Texas Red) using fluorescence microscopy, with Dapi staining (blue) as a nuclear marker. The cells were doubled stained for Dab2 and GATA-4
or GATA-6. Dab2 signal was overlaid with either GATA-4, or GATA-6, and/or Dapi. C, 50 OVCARs were analyzed by immunostaining for GATA-6 and GATA-4. Sections of a tumor with
both morphologically normal epithelium and invasive tumor areas are shown. D, representative areas of two adjacent sections of each tumor stained with GATA-4 or GATA-6 are shown.
Tumors 1–5, five examples of tumors that are GATA-4 positive and GATA-6 negative in the nucleus. Tumors 6 and 7, two examples of tumor cells that are negative for both GATA-4 and
GATA-6 in the nucleus. Tumor 8, one example in which both GATA-4 and GATA-6 are positive in the nucleus of the tumor cells. E, northern blot analysis of GATA-6 (3.6 kb), GATA-4
(3.4 kb), Dab2 (4.4 and 2.8 kb), collagen IV ␣1 (6.0 kb), and laminin (5.2 kb) expression in ovarian surface epithelial and cancer cell lines. Right panel, total RNA was isolated from five
independently derived HIO and seven ovarian tumor cell lines and was analyzed by Northern blotting for collagen IV ␣1 and 2, laminin, and Dab2. Left panel, RNA isolated from duplicate
cultures of four additional ovarian tumor cell lines was analyzed.
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INACTIVATION OF GATA-6 IN DEDIFFERENTIATION OF OVARIAN TUMORS
Table 1 Immunostaining of GATA-4 and GATA-6 in 50 ovarian carcinomasa
The histological subtypes included 2 endometrial carcinomas (endometrioid),
36 serous papillary and cystadenocarcinomas (serous), 5 mucinous adenocarcinomas
(mucinous), and 7 poorly differentiated adenocarcinomas (undifferentiated).
No.
Subtypes
GATA-4
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
Serous
Serous
Serous
Serous
Serous
Serous
Endometrioid
Undifferentiated
Undifferentiated
Endometrioid
Mucinous
Serous
Serous
Serous
Serous
Undifferentiated
Serous
Serous
Serous
Serous
Mucinous
Serous
Mucinous
Mucinous
Serous
Undifferentiated
Undifferentiated
Serous
Serous
Serous
Serous
Serous
Mucinous
Serous
Serous
Serous
Undifferentiated
Serous
Serous
Serous
Serous
Serous
Undifferentiated
Serous
Serous
Serous
Serous
Serous
Serous
Serous
3⫹ nc
2⫹ nc
⫺ nc
⫺ nc
2⫹ nc
2⫹ nc
3⫹ nc
⫺ nc
3⫹ nc
2⫹ nc
3⫹ nc
3⫹ nc
3⫹ nc
3⫹ nc
3⫹ nc
3⫹ nc
3⫹ nc
3⫹ nc
3⫹ nc
3⫹ nc
3⫹ n
2⫹ nc
3⫹ n
3⫹ n
⫺ nc
2⫹ nc
3⫹ nc
3⫹ nc
3⫹ nc
3⫹ n
⫺ nc
⫺ nc
3⫹ nc
3⫹ nc
⫺ nc
3⫹ nc
3⫹ nc
3⫹ nc
3⫹ nc
3⫹ n
3⫹ nc
3⫹ nc
3⫹ nc
3⫹ nc
3⫹ nc
3⫹ nc
3⫹ n
3⫹ nc
3⫹ nc
3⫹ n
GATA-6
⫺ nc
⫺ nc
⫺ n, 2⫹
⫺ nc
⫺ n, 2⫹
⫺ nc
⫺ n, 2⫹
⫺ n, 2⫹
2⫹ nc
⫺ n, 2⫹
⫺ n, 2⫹
⫺ n, 2⫹
⫺ n, 2⫹
⫺ n, 3⫹
⫺ n, 2⫹
⫺ n, 2⫹
⫺ n, 2⫹
⫺ n, 3⫹
⫺ n, 2⫹
⫺ n, 3⫹
3⫹ nc
⫺ n, 2⫹
⫺ nc
3⫹ n, ⫺
⫺ n, 2⫹
⫺ nc
⫺ n, 2⫹
⫺ nc
⫺ n, 3⫹
⫺ nc
⫺ n, 3⫹
⫺ n, 3⫹
3⫹ nc
3⫹ nc
⫺ n, 2⫹
⫺ n, 3⫹
3⫹ nc
⫺ n, 2⫹
⫺ nc
3⫹ nc
⫺ nc
3⫹ nc
⫺ nc
⫺ nc
⫺ nc
⫺ nc
⫺ n, 1⫹
⫺ nc
3⫹ nc
⫺ n, 2⫹
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
⫺, no staining; 1⫹, weak staining; 2⫹, positive staining; 3⫹, strong staining; c,
cytoplasm; n, nucleus; nc, both nucleus and cytoplasm.
a
or 82%) of the tumors analyzed have lost the function of GATA
factors (Table 2).
Heterogeneity in GATA-6 Expression among Tumor Cells. In
the tumors classified as GATA-6-positive, heterogeneity in the expression of GATA factors among cancer cells of the same tumors is a general
feature, as shown by examples of GATA-4 and GATA-6 staining in three
ovarian tumors (Fig. 3A). In tumor 1 (Fig. 3A), ⬃20% of the tumor cells
are positive for GATA-6 staining, interspersed with the rest of GATA6-negative tumor cells. All of the tumor cells appear to have GATA-4
staining, both nuclear and cytoplasmic. In this tumor, there is no detectable morphological difference between the GATA-6 positive and negative cells. In the second example (Fig. 3A, tumor 2), the tumor cells are
stained heterogeneously for GATA-4, about 30% positive and 70%
negative. Uniquely, the GATA-6 staining in this tumor appears to locate
in the nucleolus, whereas the nuclei are free of GATA-6 staining. Such a
staining feature is deviant from the general observation for GATA-6
expression in ovarian surface epithelial cells. In the last example (Fig. 3A,
tumor 3), most tumor cells are positive for GATA-4 but negative for
GATA-6. Interestingly, about 10% of the tumor cells that are GATA-6
positive in the nucleus are scattered among GATA-6-negative tumor
cells. Thus, we observe variable GATA factor expression in morphologically indistinguishable tumors cells. This heterogeneity in GATA factor
expression may contribute to the heterogeneity among cancer cells within
a tumor mass (1, 41).
Improper Function of GATA-6 as an Early Event in Ovarian
Surface Epithelial Cell Transformation. In previous studies (5, 8),
we considered the ovarian surface epithelium immediately adjacent to
tumor areas to be preneoplastic. These preneoplastic lesions often lack
an intact basement membrane as indicated by the absence of collagen
IV and laminin staining, may have lost Dab2 expression, and may
exhibit altered and atypical morphology (5, 8). In examining morphologically normal epithelia immediately adjacent to tumor areas, we
observed the loss of GATA-6 and/or GATA-4 expression in these
preneoplastic epithelial cells (Fig. 3B). Examples are shown for the
loss of GATA-6 but not of GATA-4 nuclear expression (Fig. 3B,
tumor 1), cytoplasmic instead of nuclear staining of GATA-6 in the
epithelial cells of the morphologically nonneoplastic epithelium of
another tumor (Fig. 3B, tumor 2), and loss of both GATA-4 and
GATA-6 expression in the nuclei of epithelial cells of ovarian surface
epithelia (Fig. 3B, tumor 3). Such results indicate that the loss of
GATA-6 function, either by the absence of expression or by its
nuclear exclusion, is an early event in ovarian epithelial transformation. This conclusion is consistent also with the loss of GATA-6 in
some of the nontumorigenic HIO cells (Fig. 1E).
Frequent Loss of GATA Factors during Tumorigenicity of
Rodent Ovarian Epithelial Cells. We next determined whether the
loss of nuclear GATA factors in ovarian tumors could be recapitulated in
a rodent model for ovarian epithelial transformation. Previously, ROSE
cells that were isolated and cultured in vitro were shown to transform to
tumorigenic cells by repeated passage in culture (30, 31). Thus, we also
examined a panel of ovarian surface epithelial cells and derived tumorigenic cell lines (Nutu lines) for the expression of GATA transcription
factors. Primary ovarian surface epithelial cells were able to grow continuously in passage as monolayer cultures for up to 2–3 months. Rose
23, a nontumorigenic line derived from expansion of the primary ROSE
cells over an extensive period of culture (⬃2 months), exhibits wellorganized cell-cell adhesive morphology in a monolayer culture (Fig.
4A). The four tumorigenic lines, Nutu 12, -14, -19, and -26, appear to
have lost the organized cell-cell contacts (Fig. 4A). ROSE 23, the nontumorigenic control line, exhibits low GATA-6 expression (Fig. 4C) but
has lost GATA-4 expression (Fig. 4B). The continuous culturing and
passaging of the ROSE cells led to formation of foci on the monolayers,
and eventually tumorigenic sublines were produced, as determined by the
Nutu assay (30, 31). The four Nutu lines, numbers 12, 14, 19, and 26,
were derived independently from four ROSE cell preparations. Three of
the Nutu lines, numbers 12, 14, and 19, have lost their GATA-4 expres-
Table 2 Immunostaining of GATA-4 and GATA-6 in 50 ovarian carcinomas
Human ovarian carcinomas (50 samples) were formalin fixed and paraffin embedded,
and sections (5 ␮m) were cut and immunostained with anti-GATA-4 and anti-GATA-6
antibodies. The staining in the nucleus or cytoplasm of the tumor cells was scored as (⫺)
negative, lightly positive (⫹1), positive (⫹2), and strongly positive (⫹3) by three authors
including a pathologist (Cohen). The total percentage of loss of GATA-6 function in
ovarian tumors is 82%.
Cellular staining of GATA-4 and GATA-6 in ovarian carcinomas
GATA-4
GATA-6
⫺ N, ⫹ Ca
⫺ N, ⫺ C
Sum, ⫺ N
0
26 (52%)
7 (14%)
15 (30%)
7 (14%)
41 (82%)
a
⫺ N, ⫹ C, staining is negative in nucleus but is positive in cytoplasm; ⫺ N, ⫺ C,
staining is negative for both the nucleus and cytoplasm; ⫺ N, staining is negative for
nucleus, disregarding cytoplasmic staining; ⫹ N, staining is positive for nucleus, irrespective of cytoplasmic staining.
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immunofluorescence microscopy, the vector had no effect on either
GATA-6 or Dab2 expression in ES2 cells; however, the hG6-110 construct suppressed both GATA-6 and Dab2 expression. Laminin expression was also down-regulated, but ␤ actin as control, determined by using
the same blot, was not affected (Fig. 5A). The transfection efficiency was
estimated to be ⬎85%. In some cells in which GATA-6 is positive,
presumably untransfected by the GATA-6 siRNA suppression construct,
Dab2 is also positive (Fig. 5B, arrow).
Similarly, in HIO-118 immortalized ovarian surface epithelial
cells, vector-transfected cells exhibited no effect on GATA-6 and
Dab2 expression, but both GATA-6 and Dab2 expression were
greatly diminished after transfection of the GATA-6 suppression
construct hG6-110 (Fig. 5C). The nuclear morphology of HIO-118
but not of ES2 cells was affected by the GATA-6 suppression
construct, indicating that the loss of GATA-6 expression and
subsequent change in gene expression drastically alters the biology
of the cells. Perhaps, additional mutations in ES2 tumor cells allow
better tolerance of the changes in gene expression profile caused
by the abrupt loss of GATA-6 than do additional mutations in the
nontumorigenic HIO-118 cells.
Restoration of GATA Factor Expression in Tumor Cells. In
tumor cell lines with varied expression status of GATA-4 and
Fig. 2. Cytoplasmic staining of GATA-6 in ovarian tumors and cell lines. A, four
examples of ovarian tumors with cytoplasmic staining of GATA-6. Cellular brown
staining, the cytoplasmic localization of the GATA-6 protein. Hematoxylin counterstaining (blue, instead of brown to dark color), the absence of GATA-6 in the nucleus.
GATA-4 staining of a neighboring section is positive in the nucleus (brown to dark) of the
tumor cells. B, a panel of ovarian epithelial and tumor cell lines plated on coverslips were
analyzed for the expression and cellular localization of GATA-6, Dab2, and laminin using
immunofluorescence microscopy. Dapi was used to stain nuclei. The same slides were
used for determination of GATA-6 (green), Dab2 (red), and Dapi (blue) stainings. Dab2
staining was merged with Dapi staining. A set of similar slides was used for laminin
staining (green). In A2780 and SKOV-3 cells, which are laminin negative, the images
were merged with Dapi staining to indicate the location of cells. C, confocal microscopy
was used to determine the cellular localization of GATA-6 in SKOV-3 and HOSE cells.
sion (Fig. 4B) but retain a low level of GATA-6 expression (Fig. 4C). In
both ROSE 23 and Nutu lines, GATA-6 is not exclusively nuclear (Fig.
4, E and F). However, one of the lines (Nutu 26) expresses GATA-4,
whereas GATA-6 is absent or greatly reduced (Fig. 4, B and C). All of the
Nutu lines have lost the expression of Dab2 and laminin, and ROSE 23
expresses Dab2 and laminin only at low levels (Fig. 4, D–F). Thus, it
seems that GATA-4 or GATA-6 is lost during the tumorigenic transformation of ROSE cells and that the GATA transcription factors are often
lost early, before the tumorigenic phenotype is presented.
Down-regulation of GATA-6 Expression in Cultured Cells
Leads to Dedifferentiation. We tested whether down-regulation of
GATA-6 in cultured epithelial cells would lead to dedifferentiation using
the siRNA approach. Two of the cell lines we have, ES2 ovarian tumor
cells and ovarian surface epithelial cells HIO-118, are positive for
GATA-6 and Dab2, but negative for GATA-4. These two cell lines were
used to examine the consequences of down-regulation of GATA-6. After
testing several oligonucleotides targeting human GATA-6, an optimal
sequence (hG6-110) was found that was able to down-regulate GATA-6
in cells. As shown in Fig. 5A by Western blotting and in Fig. 5B by
Fig. 3. A, heterogeneity of tumor cells for staining of GATA factors in ovarian tumors.
GATA-4 and GATA-6 stainings of three ovarian tumors (tumors 1, 2, and 3) are shown
as examples of heterogeneous cellular expression of GATA factors in tumors. B, staining
of GATA-6 and GATA-4 in morphologically preneoplastic ovarian surface epithelia.
Three examples of GATA-4 and GATA-6 staining of ovarian tumor tissues in areas
containing morphologically preneoplastic ovarian surface epithelium adjacent to tumor
areas are shown. The GATA-4- or GATA-6-stained ovarian surface epithelia shown in a
higher magnification (⫻400) are indicated by arrows in an image of a lower magnification (⫻40). Tumor 1, GATA-4 is positive and GATA-6 is negative in the morphologically
normal ovarian surface epithelial cells. Tumor 2, GATA-4 is positive and GATA-6 is
negative in the nucleus but positive in the cytoplasm of the morphologically normal
ovarian surface epithelial cells. Tumor 3, both GATA-4 and GATA-6 are negative in the
nucleus of the morphologically nonneoplastic ovarian surface epithelial cells.
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Fig. 4. Expression of GATA factors in rodent ovarian surface epithelial and tumor
cells. ROSE cells of nontumorigenic (ROSE 23) line and tumorigenic (Nutu 12, 14, 19,
and 26) lines were analyzed for Dab2, GATA-4, and GATA-6 expression. A, morphology
of the cells in both low and high cell density. B, total lysates from these cell lines were
prepared and subjected to Western blotting analysis for the expression of GATA-4; kd,
molecular weight (Mr) in thousands. C, GATA-6 (3.8 kb) expression in these cells was
analyzed by Northern blotting using mouse GATA-6 cDNA as a probe. The anti-GATA-6
antibodies that we purchased were not sufficiently specific nor sensitive to detect GATA-6
by Western blotting in these rat cells. D, total lysates from these cell lines were prepared
and subjected to Western blotting analysis for the expression of Dab2. ␤ actin was used
as a loading control in Western blotting. The cell lines were also analyzed for expression
and cellular localization of GATA-4, GATA-6, Dab2, and laminin by immunofluorescence microscopy; examples are shown for ROSE 23 (E) and Nutu 26 (F).
inducible expression vector (pMT-CB6⫹). The expression of
GATA-4 is under the control of the metallothionein promoter that
is inducible by ZnSO4. The induction of GATA-4 expression in
SKOV-3 cells did not result in the induction of Dab2 (Fig. 6A), a
transcriptional target of GATA-4 and GATA-6 (35). Induction of
GATA-4 in all clones of SKOV-3 cells, however, dramatically
altered cell morphology to small and spindle-like, greatly inhibited
cell growth, and induced cell death by day 4 of zinc addition, as
illustrated by representative images of the cells on dishes (Fig. 6B).
All three clones of GATA-4-expressing cells, clone 4, -5, and -6,
behaved similarly, and control cells of a GFP-inducible clone were
not affected by the addition of zinc.
Several clones, clones 4 – 6, of GATA-4-transfected ES2 cells were
selected and expanded in culture for further analysis (Fig. 6C). In
these GATA-4-transfected ES2 cells, GATA-4 was detected in the
nucleus by immunofluorescence microscopy (Fig. 6D). In two clones
(clones 4 and 5) tested, the induction of GATA-4 resulted in an
increased expression of Dab2, with a smaller increase in the expression of the endogenous GATA-6 (Fig. 6E). Expression of GATA-4
also dramatically decreased the growth of ES2 cells, as determined by
both MTT assay and cell counting (Fig. 6F). Thus, we conclude that
reexpression of GATA-4 in either GATA-6 cytoplasmic-localized
(SKOV-3) or nuclear-localized (ES2) cells greatly inhibits cell growth
and/or survival under cell culture conditions.
In three attempts to introduce GATA-6 expression into the
ovarian tumor cell line A2780, which is negative for both GATA-4
and GATA-6, no GATA-6-expressing cell lines were obtained
after the analysis of more than 50 clones of G418-selected cells in
each transfection. The efficiency of transfection/expression of
GATA-6 in these cells was monitored by immunofluorescence
microscopy of the newly transfected cells. In all of these transfections, 3–10% of the cells in cultures were positive for GATA-6 4
days after transfection (not shown). However, after selection and
expansion over a 4-week period, the G418-resistent cells were no
longer positive for GATA-6 expression, as determined by either
immunofluorescence microscopy or Western blotting. Thus the
GATA-6-positive cells were presumably lost or the GATA-6 expression in the cells was suppressed during the selection and
expansion of the transfected clones. We speculate that the reexpression of GATA-6 is incompatible for maintenance of transformed ovarian surface epithelial cells in culture. Further study of
the effect of GATA-6 reexpression in tumor cells will be needed.
We also transfected A2780 cells with a mixture of both GATA-4
and GATA-6 expression constructs simultaneously (Fig. 7). Four days
after transfection, cells expressing both GATA-4 and GATA-6 could
be detected by immunofluorescence microscopy (Fig. 7A), and
GATA-4 and GATA-6 were found to express in the same cells (Fig.
7B). Dab2 expression was not recovered after GATA-4 and GATA-6
expression, because no Dab2 staining (red) was detectable in cells
expressing GATA-4 and/or GATA-6 (Fig. 7A). Thus, ectopic expression of GATA-4 and GATA-6 in transformed epithelial cells could
not revert cells to the differentiated state based on the absence of Dab2
expression. Again, the clones of GATA-4 and GATA-6 positive cells
could not be expanded and maintained in cultures.
DISCUSSION
GATA-6, we examined the effect of restoration of GATA factor
expression. We were able to establish several lines of inducible
GATA-4-expressing clones from OVCAR cells, SKOV-3 (GATA4-negative, GATA-6-positive but localized in cytoplasm; Fig. 6A),
and ES2 (GATA-4-negative, GATA-6 positive; Fig. 6C) using an
In the present investigation, we found that the function, expression,
and/or nuclear localization of GATA-4 or GATA-6 are lost during
tumorigenicity of both ROSE and HOSE cells. We conclude that the
loss is an early and general event and accounts for the dedifferentiation of epithelial characteristics in tumors.
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Fig. 5. Suppression of GATA-6 expression by siRNA in ES2 and HIO-118 cells.
GATA-4-negative, GATA-6-positive, and Dab2-positive ES2 ovarian tumor or HIO-118
ovarian surface epithelial cells were transfected with pSuppressorNeo vector alone or
were inserted with siRNA suppression sequence targeting human GATA-6. After transfection for 7 days, the cells were analyzed by Western blotting for the expression of
GATA-6, Dab2, and laminin; and ␤ actin was used as loading control (A). ES2 (B) and
HIO-118 (C) cells were also analyzed by immunofluorescence microscope for GATA-6
(green) and Dab2 (red) expression, and the nuclei were stained by Dapi.
GATA Factors Act in Collaboration to Maintain Epithelial
Differentiation. The GATA factors are known to function in epithelial
cell lineage determination during organ formation in embryonic development (17, 18, 20, 21, 42, 43). In extraembryonic endoderm differentiation, both GATA-4 and GATA-6 are expressed and participate in gene
induction (13, 44, 45), and ectopic expression of one induces the other in
embryonic stem cells (16). Presumably, a proper makeup of GATA
factors leads to differentiation toward a particular cell lineage. Both
GATA-4 and GATA-6 are expressed in many epithelial cell types (10).
However, in adult cells, the functions of these transcription factors have
not been as extensively investigated. One can speculate that the expression of a proper ratio of GATA-4 and GATA-6 functions to maintain the
differentiated states of cells in postdevelopment tissues.
Consistent with this notion, ovarian surface epithelial-derived tumor cells lose the expression of either GATA-4 (often in rat cells) or
GATA-6 (often in human cells). The loss of one of the GATA factors
often correlates with the loss of collagen IV expression, an indicator
of epithelial differentiation. Thus, the loss of GATA factors and
dedifferentiation would result in basement membrane-independence
of epithelial tumor cells. In several tumor cell lines examined, either
GATA-4 or GATA-6 is absent, although collagen IV and laminin are
expressed. It is possible that tumor cells lose the expression of a
GATA factor and collagen IV initially, and collagen IV expression is
a gain-of-function alteration in later stages of tumor development.
This would be consistent with the observation that ovarian cancer
cells often lose extracellular collagen IV and laminin initially, and the
restoration of collagen IV and laminin expression correlates with
tumor cell spreading in later stages (5).
The loss of Dab2 expression, however, closely correlates with the loss
of GATA-6 function. Dab2 is another known transcriptional regulatory
target of GATA factors (25). Dab2 is specifically expressed in ovarian
surface epithelial cells (7), and its loss in tumor cells closely correlates
with morphological transformation and disruption of the epithelia (8). It
was thus suggested that Dab2 functions in the maintenance of epithelial
organization (32). Dab2 deficiency in mice results in early embryonic
lethality (9). The Dab2 (⫺/⫺) phenotype was characterized as disorganization of the visceral endoderm layer, an epithelial structure in early
embryos (9). Thus, the phenotype of Dab2 deficiency in mouse embryos
supports the idea that Dab2 functions in epithelial cell positioning organization and Dab2 acts in establishing epithelial polarity.5 Dab2 expression is lost in most of the ovarian tumor cell lines, correlating with the
loss of GATA-6 (see Fig. 4), and Dab2 expression is a more consistent
marker of epithelial differentiation. Therefore, loss of Dab2 is a hallmark
of epithelial dedifferentiation because of the deviant expression and
function of GATA factors.
Mechanism of GATA Factors in Regulating Cell Lineage Specification and Differentiation. Chromosome remodeling and chromatin structure are thought to be responsible for establishing and maintaining states of differential gene expression and thus cell functional
differentiation during embryonic development (46). A role for GATA
factors and other zinc finger transcription factors in interaction with
chromatin and in gene regulation during cell lineage determination
and development has been speculated (47). One intriguing idea developed by Zaret and colleagues [Bossard and Zaret (18), Zaret (47,
and Cirillo et al. (48) in their investigation of hepatocyte cell fate
determination from the precursor cells of the gut endoderm is the
concept of the genetic potentiation by GATA-4. It was shown by in
vivo footprinting experiments that in precursor cells of the gut
endoderm, GATA-4 and HNF3 can alter chromatin conformation and
occupy the binding sites in the regulated genes (such as albumin)
without initiating their transcription (18, 48). The observation was
interpreted as the binding of the GATA-4 transcription factors to
chromatin as a gain of competence to differentiate while maintaining
pluripotency. A secondary event leads to the activation of gene
transcription and, thus, to the commitment of the cells to a GATA5
D-H. Yang and X-X. Xu, unpublished observations.
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INACTIVATION OF GATA-6 IN DEDIFFERENTIATION OF OVARIAN TUMORS
Fig. 6. Transfection and expression of GATA-4 in ovarian tumor cell lines. A and B,
transfection and expression of GATA-4 in SKOV-3 ovarian tumor cells. A, GATA-4negative and GATA-6-positive SKOV-3 ovarian tumor cells were transfected with GATA-4
under the regulation of metallothionien promoter in the pMTCB6⫹ vector. Clones were
selected with G418. The expression of GATA-4 in individual clones was determined by
Western blotting after induction with ZnSO4 (200 ␮M) for 4 days. Dab2 expression was also
determined in parallel by Western blot. ␤ actin was used as a loading control. The HOSE cell
lysate was used as a positive control for the level of GATA-6. B, cells were treated with or
without zinc (200 ␮M) for 4 or 6 days. The cell morphology of clones 4, 5, and 6, and the
GFP control clone are shown. (C–F) Transfection and expression of GATA-4 in ES2 ovarian
tumor cells. C, GATA-4-negative, GATA-6-positive ES2 ovarian tumor cells were transfected with GATA-4 under the regulation of metallothionien promoter in the pMT-CB6⫹
vector. Clones were selected with G418, and 42 clones were characterized. The expression
of GATA-4 in individual clones was determined by Western blotting after induction with
ZnSO4 (200 ␮M) for 2 days. D, the induction of GATA-4 or GFP control expression by zinc
in ES2 cells was monitored by immunofluorescence microscopy by detecting GATA-4 with
antibodies or by direct fluorescence to detect the expression of GFP. GATA-4 expression in
clone 4 is shown. E, in the two selected clones (clones 4 and 5), GATA-4, GATA-6, and
Dab2 expression were determined by Western blot 4 days after induction with zinc. F, cell
growth and survival were determined by MTT assay and cell counting.
4-positive differentiated cell fate, whereas the uncommitted cells can
adopt an alternative cell fate.
In postdevelopment adult cells, GATA-4 and GATA-6 may be
required to maintain chromatin structure of the differentiated cells
(49). In tumor cells, the expression of either GATA-4 or GATA-6 is
often lost, or they are mislocated in cellular compartments and are
unable to perform their nuclear function as transcription factors in
maintaining chromatin conformation. The absence of GATA factors
may allow the chromatin to drift from a “differentiated” conformation,
and the tumor cells may thus lose their epithelial differentiation. The
link between chromatin structure and cancer has been recognized (50),
and the functional losses of GATA factors may be the underlying
mechanism for the abnormalities in chromatin structure associated
with malignancy.
Another obvious question is how the expression of GATA factors
are regulated during development and how their expression is lost in
neoplastic transformation. The differentiation of embryonic stem cells
to visceral endoderm cells in vitro has provided some insights, that
GATA-4 and GATA-6 are upstream genes in the regulatory cascade
(16), and that environmental cues such as aggregation or morphogens
such as retinoic acid (44) can induce the expression of GATA factors.
However, the transcriptional regulatory mechanisms during embryonic development for GATA factors are complex and are yet to be
explored (51).
Implication of GATA Factors in the Mechanism of Tumorigenicity. In normal tissues, differentiation of epithelial cells renders the
growth and survival of the cells to regulation by tissue architecture
organization and endocrine signaling. The proliferating tumor cells no
longer obey the rules imposed on differentiated epithelial cells; these
cells no longer depend on a basement membrane for growth and
survival, and they often invade and colonize in adjacent (tumor
spreading) or distal (metastasis) tissues (5, 32). Such a lack of epithelial properties in morphology, behavior, and gene expression is
known as dedifferentiation (1). Presumably a large number of epithelial-determining genes are deregulated in the tumor cells. The present
thinking holds that only a few genetic mutations are required for the
development of neoplasia (52), and, thus, epigenetic mechanisms may
account for the vast deregulation of gene expression in cancers. We
propose that the inappropriate expression of GATA factors in epithelial cells of postdevelopmental tissues occurs as an error in the
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execution of epigenetic program for the maintenance of differentiated
cell lineages. The loss of GATA transcription factors may be a
fundamental mechanism for the tumor cells to abandon the differentiated states and thus escape the regulation imposed on epithelial cells
in normal tissues.
The loss of GATA factors is an early event in epithelial cell
transformation because the changes have occurred in preneoplastic
epithelium. Even in several lines of nontumorigenic, immortalized
ovarian surface epithelial cells, either GATA-4 or GATA-6 is
already lost. Therefore, the loss of GATA factors, and thus the
dedifferentiation of epithelial cells, may be an earlier event in
tumorigenicity that correlates with the morphological disruption of
epithelial layer structure. Additional genetic and epigenetic
changes may further drive the development of the epithelial cells
into a carcinoma.
In most established epithelial and tumor cell lines, GATA-4 is
absent, although it is present in most of the tumors. Additionally, in
three of four rat tumor cell lines, GATA-4 rather than GATA-6 is lost.
Even in the cultured nontumorigenic ROSE cells, ROSE 23, GATA-4
expression is absent. It is noted that, different from the development
of human ovarian tumors, the rat ovarian epithelial cells first undergo
tissue culture adaptation before tumorigenic transformation in vitro, in
tissue culture condition. Thus, the correlation exists between the loss
of GATA-4 and adaptation of the epithelial cells to in vitro tissue
culture condition. Nevertheless, the loss of GATA-6 appears to be
required for tumorigenic transformation in both cell lines and tumor
tissues, whereas the loss of GATA-4 is often associated with the
adaptation of both tumor and nontumorigenic ovarian surface epithelial cells to culture conditions (Fig. 8A).
We were able to restore the expression of GATA-4 and GATA-6
expression after either stable or transient transfection in ovarian tumor
cells. Expression of the GATA factors, however, was incompatible
with cell maintenance and growth in culture and did not revert the
differentiated state of the epithelial cells as judged by the lack of Dab2
expression. This is perhaps not surprising, because it is known that
fully transformed cells are not easily reverted to the original differ-
Fig. 7. Detection by immunofluorescence of Dab2, GATA-4, and GATA-6 expression
in transfected cells. A2780 OVCAR cells were transfected with a mix of GATA-4 and
GATA-6 expression constructs in the pcDNA3 vector. A, the slides were doubly labeled
with antibodies to Dab2 and GATA-4, or antibodies to Dab2 and GATA-6. Expression of
Dab2 (red), GATA-4 (green), and GATA-6 (green) were detected by immunofluorescence microscopy, and the nuclei were stained with Dapi (blue). Images of GATA-4
(green) or GATA-6 (green) staining were merged with either Dapi (blue) or Dab2 staining
(red). No Dab2 staining is detectable in GATA-4 and GATA-6 positive cells. B, the
transfected cells were labeled with antibodies to GATA-4 (goat) and GATA-6 (rabbit).
Expression of GATA-4 (red) and GATA-6 (green) were detected by immunofluorescence
microscopy, and the nuclei were stained with Dapi (blue). GATA-4 and GATA-6 were
expressed in the same cells.
Fig. 8. Models for the loss of GATA factors in cell transformation and chromatin
conformation. A, model for the loss of GATA-4 function in the adaptation of epithelial
cells to culture condition and loss of GATA-6 in tumorigenicity. It is proposed that loss
of GATA-4 and, thus, the expression of its regulated genes enable epithelial cells to grow
under culture conditions. Loss of GATA-6 and its regulated genes leads to dedifferentiation of epithelial cells, which then allows the cells to escape architectural restrain and
enables disorganized proliferation in tumorigenicity. B, model for the alteration of gene
expression pattern/chromatin conformations as a result of change in expression of GATA
factors. The differences of gene expression profile/chromatin structure in various differentiated and dedifferentiated states are abstractly symbolized by an assortment of geometric shapes of the cells. Differentiation to epithelial lineage associated with chromatin
alteration is a result of the expression of GATA factors. GATA factors function in
maintaining the chromatin conformation of the differentiated cells. Loss of GATA factors
leads to alteration of chromatin conformation/gene expression profile to undefined states.
Nevertheless, restoration of GATA factor expression cannot revert to the differentiated
chromatin conformation/gene expression profile. However, the reexpression of GATA
factors leads to a gene expression profile incompatible with cell survival.
entiated state. The chromatin structure of transformed and dedifferentiated cells is likely to be drastically altered. GATA factors function
in chromatin remodeling but are not tissue restricted and, thus, likely
contain no memory of the previous chromatin structure of the differentiated state. Thus, once the expression of GATA factors is lost
during epithelial cell transformation and dedifferentiation, chromatin
conformation is irreversibly changed. The reexpression of GATA
factors may further alter the chromatin conformation but may not be
able to restore to the original differentiated state of the parental cells
(Fig. 8B).
The present study of ovarian surface epithelial tumors presents a
scheme that the loss of expression or function of GATA-6 and/or
GATA-4 are general events associated with and account for dedifferentiation in the processes of adaptation of epithelial cells to cultures
and/or neoplastic transformation. This study concludes that GATA-4
and GATA-6 determine epithelial lineage and reveals a possible
mechanism underlying the phenomenon of epithelial dedifferentiation
in carcinomas.
4976
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Research.
INACTIVATION OF GATA-6 IN DEDIFFERENTIATION OF OVARIAN TUMORS
ACKNOWLEDGMENTS
We greatly appreciated the generous gift of human GATA-6 cDNA from
Dr. David Wilson (Washington University, St. Louis, MO) and from
Dr. Kenneth Walsh (St. Elizabeth’s Medical Center, Boston, MA). We
thank Drs. Elizabeth Smith, Cathy Bingham, Wan-Lin Yang, Dong-Hua
Yang, and Andrey Frolov for reading and commenting during the process
of preparing the manuscript. We acknowledge and thank the excellent
assistance of Jonathan Boyd in the studies using the immunofluorescence
microscope. We thank Carolyn Slater, Malgorzata Rula, and Jennifer
Smedberg for their excellent technical assistance, Diane Lawson for her
technical assistance in immunostaining, and Patricia Bateman for her
secretarial assistance.
25.
26.
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28.
29.
REFERENCES
1. Fusenig, N. E., Breitkreutz, D., Boukamp, P., Tomakidi, P., and Stark, H. J. Differentiation and tumor progression. Recent Results Cancer Res., 139: 1–19, 1995.
2. Lynch, R. G. Differentiation and cancer: the conditional autonomy of phenotype.
Proc. Natl. Acad. Sci. USA, 92: 647– 648, 1995.
3. Rheinwald, J. G., and Beckett, M. A. Defective terminal differentiation in culture as
a consistent and selectable character of malignant human keratinocytes. Cell, 22:
629 – 632, 1980.
4. Richardson, G. S., Scully, R. E., Nikrui, N., and Nelson, J. H., Jr. Common epithelial
cancer of the ovary (2). N. Engl. J. Med., 312: 474 – 483, 1985.
5. Capo-chichi, C. D., Smith, E. R., Yang, D-H., Roland, I. H., Vanderveer, L., Cohen,
C., Hamilton, T., Godwin, A. K., and Xu, X. X. Dynamic alterations of extracellular
environment of ovarian surface epithelial cells in pre-malignant transformation,
tumorigenicity, and metastasis. Cancer (Phila.), 95: 1802–1815, 2002.
6. Stenbeck, F., and Wasenius, V-M. Basement membrane structures in tumors of the
ovary. Eur. J. Obstet. Gynecol. Reprod. Biol., 20: 357–371, 1985.
7. Fazili, Z., Sun, W., Mittelstaedt, S., Cohen, C., and Xu, X. X. Disabled-2 inactivation
is an early step in ovarian tumorigenicity. Oncogene, 18: 3104 –3113, 1999.
8. Yang, D. H., Smith, E. R., Cohen, C., Patriotis, C., Godwin, A. K., Hamilton, T. C., and
Xu, X. X. Molecular events associated with dysplastic morphological transformation and
initiation of ovarian tumorigenicity. Cancer (Phila.), 94: 2380 –2392, 2002.
9. Yang, D. H., Smith, E. R., Roland, I. H., Sheng, Z., He, J., Martin, W. D., Lambeth,
J. D., and Xu, X. X. Disabled-2 is essential for endodermal cell positioning and
structure formation during early extraembryonic development. Dev. Biol., 251: 27–
44, 2002.
10. Molkentin, J. D. The zinc finger-containing transcription factors GATA-4, -5, and -6.
Ubiquitously expressed regulators of tissue-specific gene expression. J. Biol. Chem.,
275: 38949 –38952, 2000.
11. Orkin, S. H. GATA-binding transcription factors in hematopoietic cells. Blood, 80:
575–581, 1992.
12. Simon, M. C. Gotta have GATA. Nat. Genet., 11: 9 –11, 1995.
13. Koutsourakis, M., Langeveld, A., Patient, R., Beddington, R., and Grosveld, F. The
transcription factor GATA-6 is essential for early extraembryonic development.
Development, 126: 723–732, 1999.
14. Morrisey, E. E., Tang, Z., Sigrist, K., Lu, M. M., Jiang, F., Ip, H. S., and Parmacek,
M. S. GATA6 regulates HNF4 and is required for differentiation of visceral endoderm
in the mouse embryo. Genes Dev., 12: 3579 –3590, 1998.
15. Soudais, C., Bielinska, M., Heikinheimo, M., MacArthur, C. A., Narita, N., Saffitz,
J. E., Simon, M. C., Leiden, J. M., and Wilson, D. B. Targeted mutagenesis of the
transcription factor GATA-4 gene in mouse embryonic stem cells disrupts visceral
endoderm differentiation in vitro. Development, 121: 3877–3888, 1995.
16. Fujikura, J., Yamato, E., Yonemura, S., Hosoda, K., Masui, S., Nakao, K., Miyazaki
Ji, J., and Niwa, H. Differentiation of embryonic stem cells is induced by GATA
factors. Genes Dev., 16: 784 –789, 2002.
17. Charron, F., and Nemer, M. GATA transcription factors and cardiac development.
Semin. Cell Dev. Biol., 10: 85–91, 1999.
18. Bossard, P., and Zaret, K. S. GATA transcription factors as potentiators of gut
endoderm differentiation. Development, 125: 4909 – 4917, 1998.
19. Yang, H., Lu, M. M., Zhang, L., Whitsett, J. A., and Morrisey, E. E. GATA6 regulates
differentiation of distal lung epithelium. Development, 129: 2233–2246, 2002.
20. Jacobsen, C. M., Narita, N., Bielinska, M., Syder, A. J., Gordon, J. I., and Wilson,
D. B. Genetic mosaic analysis reveals that GATA-4 is required for proper differentiation of mouse gastric epithelium. Dev. Biol., 241: 34 – 46, 2002.
21. Gao, X., Sedgwick, T., Shi, Y. B., Evans, T. Distinct functions are implicated for the
GATA-4, -5, and -6 transcription factors in the regulation of intestine epithelial cell
differentiation. Mol. Cell. Biol., 18: 2901–2911, 1998.
22. Ketola, I., Rahman, N., Toppari, J., Bielinska, M., Porter-Tinge, S. B., Tapanainen,
J. S., Huhtaniemi, I. T., Wilson, D. B., and Heikinheimo, M. Expression and
regulation of transcription factors GATA-4 and GATA-6 in developing mouse testis.
Endocrinology, 140: 1470 –1480, 1999.
23. Heikinheimo, M., Ermolaeva, M., Bielinska, M., Rahman, N. A., Narita, N.,
Huhtaniemi, I. T., Tapanainen, J. S., and Wilson, D. B. Expression and hormonal
regulation of transcription factors GATA-4 and GATA-6 in the mouse ovary. Endocrinology, 138: 3505–3514, 1997.
24. Laitinen, M. P., Anttonen, M., Ketola, I., Wilson, D. B., Ritvos, O., Butzow, R., and
Heikinheimo, M. Transcription factors GATA-4 and GATA-6 and a GATA family
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
cofactor, FOG-2, are expressed in human ovary and sex cord-derived ovarian tumors.
J. Clin. Endocrinol. Metab., 85: 3476 –3483, 2000.
Morrisey, E. E., Musco, S., Chen, M. Y., Lu, M. M., Leiden, J. M., and Parmacek,
M. S. The gene encoding the mitogen-responsive phosphoprotein Dab2 is differentially regulated by GATA-6 and GATA-4 in the visceral endoderm. J. Biol. Chem.,
275: 19949 –19954, 2000.
Mok, S. C., Wong, K. K., Chan, R. K., Lau, C. C., Tsao, S. W., Knapp, R. C., and
Berkowitz, R. S. Molecular cloning of differentially expressed genes in human
epithelial ovarian cancer. Gynecol. Oncol., 52: 247–252, 1994.
Mok, S. C., Chan, W. Y., Wong, K. K., Cheung, K. K., Lau, C. C., Ng, S. W., Baldini,
A., Colitti, C. V., Rock, C. O., and Berkowitz, R. S. DOC-2, a candidate tumor
suppressor gene in human epithelial ovarian cancer. Oncogene, 16: 2381–2387, 1998.
Bai, Y., Akiyama, Y., Nagasaki, H., Yagi, O. K., Kikuchi, Y., Saito, N., Takeshita, K.,
Iwai, T., and Yuasa, Y. Distinct expression of CDX2 and GATA4/5, development-related
genes, in human gastric cancer cell lines. Mol. Carcinog., 28: 184 –188, 2000.
Kiiveri, S., Siltanen, S., Rahman, N., Bielinska, M., Lehto, V. P., Huhtaniemi, I. T.,
Muglia, L. J., Wilson, D. B., and Heikinheimo, M. Reciprocal changes in the
expression of transcription factors GATA-4 and GATA-6 accompany adrenocortical
tumorigenesis in mice and humans. Mol. Med., 5: 490 –501, 1999.
Godwin, A. K., Testa, J. R., Handel, L. M., Liu, Z., Vanderveer, L. A., Tracey, P. A.,
and Hamilton, T. C. Spontaneous transformation of rat ovarian surface epithelial
cells: association with cytogenetic changes and implications of repeated ovulation in
the etiology of ovarian cancer. J. Natl. Cancer Inst. (Bethesda), 84: 592– 601, 1992.
Testa, J. R., Getts, L. A., Salazar, H., Liu, Z., Handel, L. M., Godwin, A. K., and
Hamilton, T. C. Spontaneous transformation of rat ovarian surface epithelial cells
results in well to poorly differentiated tumors with a parallel range of cytogenetic
complexity. Cancer Res., 54: 2778 –2784, 1994.
Sheng, Z., Sun, W., Smith, E., Cohen, C., Sheng, Z., and Xu, X. X. Restoration of
positioning control following Disabled-2 expression in ovarian and breast tumor cells.
Oncogene, 19: 4847– 4854, 2000.
Morris, S. M., and Cooper, J. A. Disabled-2 colocalizes with the LDLR in clathrincoated pits and interacts with AP-2. Traffic, 2: 111–123, 2001.
Hamilton, T. C., Young, R. C., Louie, K. G., Behrens, B. C., McKoy, W. M.,
Grotzinger, K. R., and Ozols, R. F. Characterization of a xenograft model of human
ovarian carcinoma which produces ascites and intraabdominal carcinomatosis in
mice. Cancer Res., 44: 5286 –5290, 1984.
Sheng, Z., He, J., Tuppen, J. A., Sun, W., Fazili, Z., Smith, E. R., Dong, F. B., and
Xu, X. X. Structure, sequence, and promoter analysis of human disabled-2 gene
(DAB2). Genomics, 70: 381–386, 2000.
Yamagata, T., Nishida, J., Sakai, R., Tanaka, T., Honda, H., Hirano, N., Mano, H.,
Yazaki, Y., and Hirai, H. Of the GATA-binding proteins, only GATA-4 selectively
regulates the human interleukin-5 gene promoter in interleukin-5-producing cells which
express multiple GATA-binding proteins. Mol. Cell. Biol., 15: 3830 –3839, 1995.
Bast, R. C. Jr., Boyer, C. M., Jacobs, I., Xu, F. J., Wu, S., Wiener, J., Kohler, M., and
Berchuck, A. Cell growth regulation in epithelial ovarian cancer. Cancer (Phila.), 71:
1597–1601, 1993.
Godwin, A. K., Testa, J. R., and Hamilton, T. C. The biology of ovarian cancer
development. Cancer (Phila.), 71: 530 –536, 1993.
Bell, D. A., and Scully, R. E. Early de novo ovarian carcinoma. A study of fourteen
cases. Cancer (Phila.), 73: 1859 –1864, 1994.
Charron, F., Paradis, P., Bronchain, O., Nemer, G., and Nemer, M. Cooperative
interaction between GATA-4 and GATA-6 regulates myocardial gene expression.
Mol. Cell. Biol., 19: 4355– 4365, 1999.
Ali, I. U., Lidereau, R., and Callahan, R. Heterogeneity of genetic alterations in
primary human breast tumors. Cancer Treat. Res., 40: 25– 48, 1988.
Kuo, C. T., Morrisey, E. E., Anandappa, R., Sigrist, K., Lu, M. M., Parmacek, M. S.,
Soudais, C., and Leiden, J. M. GATA4 transcription factor is required for ventral
morphogenesis and heart tube formation. Genes Dev., 11: 1048 –1060, 1997.
Morrisey, E. E., Ip, H. S., Lu, M. M., and Parmacek, M. S. GATA-6: a zinc finger
transcription factor that is expressed in multiple cell lineages derived from lateral
mesoderm. Dev. Biol., 177: 309 –322, 1996.
Arceci, R. J., King, A. A., Simon, M. C., Orkin, S. H., and Wilson, D. B. Mouse
GATA-4: a retinoic acid-inducible GATA-binding transcription factor expressed in
endodermally derived tissues and heart. Mol. Cell. Biol., 13: 2235–2246, 1993.
Morrisey, E. E., Ip, H. S., Tang, Z., Lu, M. M., and Parmacek, M. S. GATA-5: a
transcriptional activator expressed in a novel temporally and spatially-restricted
pattern during embryonic development. Dev. Biol., 183: 21–36, 1997.
Wolffe, A. P. Chromatin and gene regulation at the onset of embryonic development.
Reprod. Nutr. Dev., 36: 581– 606, 1996.
Zaret, K. Developmental competence of the gut endoderm: genetic potentiation by
GATA and HNF3/fork head proteins. Dev. Biol., 209: 1–10, 1999.
Cirillo, L. A., Lin, F. R., Cuesta, I., Friedman, D., Jarnik, M., and Zaret, K. S.
Opening of compacted chromatin by early developmental transcription factors HNF3
(FoxA) and GATA-4. Mol. Cell, 9: 279 –289, 2002.
Urnov, F. D. A feel for the template: zinc finger protein transcription factors and
chromatin. Biochem. Cell Biol., 80: 321–333, 2002.
Klochendler-Yeivin, A., Muchardt, C., and Yaniv, M. SWI/SNF chromatin remodeling and cancer. Curr. Opin. Genet. Dev., 12: 73–79, 2002.
He, C. Z., and Burch, J. B. The chicken GATA-6 locus contains multiple control
regions that confer distinct patterns of heart region-specific expression in transgenic
mouse embryos. J. Biol. Chem., 272: 28550 –28556, 1997.
Knudson, A. G. Two genetic hits (more or less) to cancer. Nature Rev. Cancer
(Phila.), 1: 157–162, 2001.
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Anomalous Expression of Epithelial
Differentiation-determining GATA Factors in Ovarian
Tumorigenesis
Callinice D. Capo-chichi, Isabelle H. Roland, Lisa Vanderveer, et al.
Cancer Res 2003;63:4967-4977.
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