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Research Article |
Center for Women's Medicine,3 Division of Reproductive Endocrinology and Infertility, Greenville Hospital System, Greenville, South Carolina 29605
Greenwood Genetic Center,4 Greenwood, South Carolina 29646
Departments of Medicine5
and Obstetrics and Gynecology,6 University of North Carolina, Chapel Hill, North Carolina 27599
ABSTRACT
Endometrial cancer cell lines have provided a valuable model to study endometrial epithelial cells in vitro. Since the first development of HEC1B over 35 yr ago, many different cell lines have been isolated and described. One valuable cell line that maintains hormone responsiveness and unique stability over time is the ECC-1 cell line, developed originally by the late P.G. Satyaswaroop. In this study, we investigated some of the properties of these cells and present their salient characteristics. Like Ishikawa cells, ECC-1 cells maintain both estrogen receptors (ESR1 [ER alpha] and ESR2 [ER beta]), progesterone receptors (PR A and B; PGRs), and androgen receptors (ARs), along with the p160 steroid receptor coactivators NCOA1 (formerly SRC1), NCOA2 (formerly TIF2), and NCOA3 (formerly AIB1). The karyotype of these cells is abnormal, with multiple structural rearrangements in all cells analyzed. Unlike Ishikawa cells that express glandular epithelial antigens, ECC-1 cells maintain a luminal phenotype, with expression of KRT13 (cytokeratin 13) and KRT18 (cytokeratin 18). Apparent differences in the regulation of ESR2 also were evident in ECC-1 cells compared to Ishikawa cells. Like other endometrial cell lines, ECC-1 cells express the steroid receptor coactivators and exhibit epidermal growth factor-stimulated expression of known luminal proteins thought to be involved in implantation, including the hyaluronate receptor CD44 and SPP1 (formerly osteopontin) and CD55 (decay-accelerating factor). These characteristics appear to be stable and persistent over multiple cell passages, making this well-differentiated cell line an excellent choice to study endocrine and paracrine regulation of endometrial epithelium in vitro.
adenocarcinoma, androgen receptor, coactivators, endometrial cell line, endometrium, estradiol receptor, female reproductive tract, mechanisms of hormone action, progesterone receptor, steroid receptors
INTRODUCTION
The endometrium is a unique uterine tissue that undergoes cyclic regeneration after each menstrual cycle in anticipation of pregnancy. Like the breast, the endometrium is a primary steroid hormone target tissue [1, 2]. The developmental changes in response to estrogen, progesterone, and androgens likely account for the establishment of receptivity toward embryo implantation, and steroid priming sets the stage for menstruation if conception does not occur. As a target tissue for estrogen, the endometrium is a frequent site of hyperplasia and cancer. Likewise, the sensitivity to the sex steroids accounts for many of the gynecologic ailments affecting women, including endometrial polyps, endometriosis, and adenomyosis. As a complex tissue composed of a large number of resident cell types, the isolated endometrial epithelium has been difficult to study.
The study of most human tissues has been significantly advanced by the availability of a wide variety of immortalized cell lines. Endometrial cell lines were established over 35 yr ago by Kuramoto, who derived the first endometrial cell line, HEC-1 cells, in 1968 [3]. Since that time, many different endometrial lines have been established and form the basis for numerous in vitro investigations into endometrial biology. One of the best-characterized endometrial cell lines is the Ishikawa cell line [4–9]. These cells maintain functional steroid receptors to estrogen, progesterone, and androgen, making them particularly useful for a wide range of studies of the endometrium. In 1987, Clarke et al. [10] established an endometrial adenocarcinoma cell line that responded appropriately to estradiol to produce progesterone receptors (PGRs) and could be cultured either in vitro or in nude mice. Although the ECC-1 cells have not yet been well characterized, they appear to be a stable and highly responsive cell line that maintain both estrogen receptors (ESR1 [ER alpha] and ESR2 [ER beta]), PGRs A and B, and androgen receptors (ARs), along with the steroid receptor coactivators NCOA1, NCOA2, and NCOA3. Unlike Ishikawa cells, the ECC-1 cells appear to exhibit a phenotype reminiscent of the luminal or surface epithelial cells, which may have advantages for the study of events related to endometrial-embryonic interactions or for the study of the expression and regulation of luminal proteins. The present study describes the characterization of this cell line and compares ECC-1 cells to another commonly used endometrial cell line, the Ishikawa cells.
MATERIALS AND METHODS
Cell Culture
The endometrial cancer cell lines, including the Ishikawa cells and ECC-1 cell line, were obtained from Nishida (Ibaraki, Japan) and P.G. Satyaswaroop (Hershey Medical Center, Hershey, PA), respectively. Cells were maintained in 50-cm2 flasks (Costar, Cambridge, MA), with minimal essential medium for Ishikawa cells and RPMI-1640 for ECC-1 cells at 37°C in the presence of 5% CO2. Typically, each cell line-specific medium is supplemented with 5% charcoal-stripped fetal calf serum, 1% (vol/vol) penicillin-streptomycin (Life Technologies, Inc., Grand Island, NY), and 2 mM L-glutamine (Life Technologies) before being used. For the specific experiments described, cells were treated with phenol red-free media containing 0.5% (vol/vol) charcoal-stripped fetal calf serum (Hi-Clone, Logan, UT), 1% penicillin-streptomycin (Life Technologies) and 2 mM L-glutamine (Life Technologies). Cells plated on 60-mm cell culture plates were exposed to medium alone (control) or medium containing either the synthetic estrogen diethylstilbestrol (DES) plus or minus medroxyprogesterone acetate, with a range of concentrations from 10–6 to 10–12 M. In some cases, 17ß-estradiol was used at a 10–8 M concentration, and epidermal growth factor (EGF; 10 ng/ml) was used to treat cells in certain experiments. All reagents were obtained from Sigma (St. Louis, MO) except when otherwise specified. The time of exposure in each experiment was between 24 and 72 h.
In Vitro-Binding Assays
To estimate the number of specific high-affinity binding sites in ECC-1 and Ishikawa cells, we used a single-concentration competition-binding assay for ESR1 and ESR2, PRGs, and ARs. Ishikawa and ECC-1 cells were cultured at a density of 100 000 cells per well (24-well plate) in their respective media supplemented with 20 mM Hepes, penicillin/streptomycin, and 200 mM L-glutamine (pH 7.2) with or without DES priming for 48 h (10–8 M). Media were aspirated and replaced with 600 µl of serum-free, phenol red-free Dulbecco modified Eagle medium F12 containing radiolabeled (total binding) or radiolabeled plus unlabeled (nonspecific binding) solution. This solution contained 3H-17ß-estradiol (10–8 M), 3H-R5020 (10–6 M), or 3H-R1881 (10–8 M; New England Nuclear, Boston, MA), which was incubated in the presence or absence of a 100-fold excess of unlabeled ligand (estradiol, R5020, or R1881) for 2 h at 37°C. Media were aspirated into a radioactive waste flask, and the wells were washed with 1x PBS to remove unbound ligand and aspirated to dry. The cells were then harvested in 200 µl of 1x SDS sample buffer (10 mM Tris [pH 6.8], 2% SDS, and 10% glycerol) and counted in a scintillation counter. Results were presented as the amount of total binding minus nonspecific binding of triplicate determinations. Cell binding for each radioactive ligand was compared before and after estrogen priming and normalized against cell protein on the basis of the concentration of cell homogenates by the Bio-Rad protein assay method (Bio-Rad Laboratories, Hercules, CA). Statistical differences in specific binding between conditions were calculated by a two-tailed t-test with Prism 4 Software (GraphPad Software, Inc., San Diego, CA).
Karyotype Analysis of ECC-1 Cells
Chromosomes for G-banding analysis and fluorescent in situ hybridization (FISH) analysis were prepared by standard methods, with the addition of ethidium bromide to produce prometaphase spreads [11, 12]. Cells were grown on coverslips in small Petri dishes in the appropriate media. To produce metaphase spreads, cells were incubated with standard medium containing ethidium bromide (10 ug/ml) for 20 min; then, colcemid at 60 ng/ml was added to prevent spindle fiber formation and arrest nuclear division at metaphase. After 20 min in colcemid, the cells were placed in hypotonic solution (75 mM KCl) and incubated for 30 min at room temperature to achieve expansion of the cells. Several drops of Carnoy fixative (methanol:acetic acid, 3:1) were added to each Petri dish to begin the fixation process. After 5 min, the cells were gently washed in fixative solution. This wash was repeated three times. The coverslips were then dried in a warm, humid chamber to allow optimal chromosome spreading. The dried coverslips were glued to standard microscope slides, and the slides were baked for 30 min at 90°C. The chromosomes were analyzed following GTG banding.
For FISH analysis, fresh metaphase spreads were prepared the day before use and baked for 4 h at 65°C. Slides were denatured for 2 min at 70°C, dehydrated, and allowed to air dry. Labeled probes (80 ng) were mixed with hybridization buffer and human CoT-1 DNA. The mixtures were denatured for 5 min at 70°C. The probes were immediately added to denatured slides and incubated overnight. The slides were washed in 50% formamide and 2x saline-sodium citrate (SSC), pH 7.0, at 42°C and then in 2x SSC, pH 7.0. The chromosomes were counterstained with 4',6'-diamidino-2-phenylindole (DAPI) and examined with a Zeiss Axioscop fluorescent microscope equipped with fluorescein isothiocyanate, DAPI, and triple band pass filter sets. Digital images were captured with a computer by SmartCapture imaging software (Applied Imaging, Pittsburgh, PA) and printed on a Techtronix Phaser 440 color printer.
RT-PCR Technique
Total cellular RNA from the human endometrial cancer cell lines was extracted with a TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The RT-PCR was performed with AccessQuick RT-PCR system on a GeneAmp PCR system 9700 (PE Applied Biosystems, Foster City, CA). The sequence of primers and the expected product size for RT-PCR are summarized in Table 1. The routine RT-PCR program was 2 Pre-PCR of 45 min at 48°C and 5 min at 94°C and 30 cycles of PCR amplification of target genes. All PCR products from a single experiment were run on a 2% agarose gel and stained with ethidium bromide.
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Western Blot Analysis
Steroid receptors and the steroid receptor coactivators NCOA1, NCOA2, and NCOA3 were evaluated by Western blot analysis. Cells were harvested, and total cell lysates made with a three-detergent lysis buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, and 50 mM Tris-HCl [pH 8.0]) containing protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN). After incubating on ice for 30 min, cell lysates were centrifuged at 12 000 x g for 10 min at 4°C. The supernatant was collected, and the protein concentration was measured by a DC protein assay kit (Bio-Rad). A total of 10 µg of protein was added per lane, resolved on 7.5% SDS-PAGE, and transferred to polyvinylidene fluoride (PVDF) membranes. Antibodies against ESR1 (Santa Cruz Biotechnologies, Santa Cruz, CA), ESR2 (Santa Cruz), PRG (Santa Cruz), AR (Santa Cruz), NCOA1 (Santa Cruz), NCOA2 (Santa Cruz), and NCOA3 (Santa Cruz) were used to detect these proteins. Antibodies to ACTB (beta actin) (Sigma) were used as a control for equal loading.
Endometrial cancer cell lines were also examined for known glandular and luminal markers. Cells cultured as described above were terminated with an ice-cold PBS rinse and addition of the appropriate amount of cell lysis buffer (RIPA extraction buffer: 10 mM Tris-HCl [pH 8.0], 10 mM EDTA [pH 8.0], 150 mM NaCl, 1% NP-40, and 0.5% SDS) containing protease-inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN). Cells were scraped from each well, mechanically disrupted by pipetting up and down several times, and transferred to chilled 1.5-ml Eppendorf tubes and incubated for 30 min on ice. Cell lysates thus obtained from the respective treatments were centrifuged at 16 000 x g for 20 min at 4°C; the total protein in each supernatant was quantified by the Bio-Rad protein assay kit (Bio-Rad), and lysates were stored at –80°C. Equal amounts (100 µg) of cell lysate total protein from each treatment were subjected to electrophoresis on 10% SDS-containing polyacrylamide gel and transferred to nitrocellulose membranes. Blots were rinsed once in Tris-buffered saline (TBS), blocked for 1 h in TBS-0.4% Tween (TBS/T)-10% nonfat dry milk, and rinsed three times in TBS/T before overnight incubation at 4°C in TBS/T-10% nonfat milk containing primary antibody diluted 1:1000 for the beta 3 integrin subunit (CD 51; SSA6, a monoclonal antibody previously characterized [13]), CD44 at 1 µg/ml (mouse monoclonal antibody; R&D Systems, Minneapolis, MN), and a 1:1000 dilution for CD55 (decay-accelerating factor; Serotec, MCA1614, Oxford, U.K.) while rocking at 4°C. The blots were then washed twice for 30 min each time with TBS/T and incubated for 1 h at room temperature with peroxidase-conjugated anti-mouse immunoglobulin G (IgG) secondary antibody while rocking. After washing twice for 30 min each time with TBS/T, the immunoreactive protein complexes were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech Inc., Piscataway, NJ). The results depicted are representative of at least two independent experiments, and the data shown are representative of those experiments. Equal loading was documented with ACTB (Sigma) as a control (data not shown in Fig. 5 or 6).
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Immunocytochemistry
Fluorescence immunocytochemistry was performed with formalin-fixed Ishikawa or ECC-1 cells that had been cultured on coverslips by a Vectastain kit (Vector Laboratories, Burlingame, CA). After the initial incubation with blocking antibody for 15 min at room temperature (1:100 dilution of nonimmune mouse serum), the primary antibody was applied for 1 h. The primary antibody consisted of monoclonal antibodies directed against two cytokeratins, KRT13 (cytokeratin 13) and KRT18 (cytokeratin 18) (AE8; ICN Biomedicals Inc., Aurora, OH; and CY-90; Sigma, respectively). After a 5-min wash in PBS (pH 7.2–7.4), biotinylated goat anti-mouse IgG conjugated to fluorescein isothiocyanate (1:100 dilution) was applied to the coverslips. After a 30-min incubation at room temperature, the coverslips were rinsed three times with PBS and mounted. Photomicrographs were prepared with Kodak (Rochester, NY) TMZ 3200 ASA film in a Nikon Optiphot fluorescence microscope (Melville, NY).
Full-thickness human endometrial samples were obtained from women at the time of hysterectomy by approved Institutional Review Board protocols at the University of North Carolina. Samples were staged according to histological dating criteria to designate the proliferative vs. the secretory phase of the cycle. For tissue staining, sections of endometrial tissues were placed in OCT embedding media (Tissue-Tek; Fisher Scientific, Pittsburgh, PA) and frozen in liquid nitrogen. Cryosections 4–6 µm in thickness were fixed with 4% formaldehyde in 0.1 M phosphate buffer, pH 7.2, at 4°C for 30 min. Sections were rinsed in TBS (20 mM Tris-HCl [pH 8.0], 150 mM NaCl, and 0.05% Tween 20) and then blocked in TBS containing 2.5% BSA (Fisher) and 5% normal donkey serum. Sections were next incubated with primary antibodies against KRT13 or KRT18. Sections were then incubated with secondary antibody, with intervening washes in TBS buffer. The secondary antibody was an affinity-purified donkey anti-mouse IgG from Jackson ImmunoResearch (West Grove, PA).
RESULTS
Karyotype results from ECC-1 cells were quite complicated, with multiple structural rearrangements noted. Twenty G-banded cells were completely analyzed, with each cell being slightly different from the next. Because of the complicated nature of the structural rearrangements seen on the G-banded metaphases, FISH analysis on metaphase spreads with whole-chromosome painting probes for chromosomes 1, 3, 6, 8, 9, 10, 12, 15, 16, and 19 was done. This FISH analysis identified the marker chromosomes and the more complicated chromosome rearrangements. The consensus karyotype, which includes rearrangements seen in the majority of cells, was determined to be the following: 45–47,X,-X,der(1)t(1;8)(p36.3;q13), +add(3)(p26), der(3)t(3;1;8)(p11.2;q11.2-q41;q23), t(10;17)(p11.2; p11.2),+del(12)(q15),der(15)t(10;15) (q22;p11.2), +del(16)(q22),del(16)(q22),der(16)t(8;16)(p21; q24),der(19)t(16;19)(q22;p13.3),-22[cp20]. The structurally rearranged chromosomes appeared to be fairly stable, with the total number of chromosomes being different between cells. Representative photomicrographs of the karyotype are shown in Figure 1.
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Like Ishikawa cells, ECC-1 cells maintain ESR1 and ESR2, PRGs, and ARs. By in vitro-binding assays with radioactive (3H-labeled) steroids, we show that ECC-1 cells and Ishikawa cells maintain specific binding sites for these receptors in excess of that found in Ishikawa cells (Fig. 2). While we cannot distinguish between ESR1 and ESR2 with this technique, we note that overall specific 3H-estradiiol binding goes down following estrogen pretreatment in ECC-1 cells (P < 0.05), while in Ishikawa cells, there is no change. The two cell lines are similar with respect to induction of the other steroid receptors, with both PRG and AR binding increasing following estrogen priming. However, it should be noted that ECC-1 maintained basal levels of both PGR and AR binding that were higher than those seen in Ishikawa cells.
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Steroid receptors and associated proteins were compared in ECC-1 cells with the Ishikawa cell line by RT-PCR and Western blot analysis. Expression of mRNA for the steroid receptor isoforms and coactivators is shown in Figure 3, before and after estrogen priming. Western blot analysis for the same proteins is shown in Figure 4. ARs may be slightly increased in both cell lines following DES treatment, though further studies would be required to document this. ESR1 was present in both ECC-1 and Ishikawa cells but did not appear regulated. While ESR2 appeared to be increased in ECC-1 cells in response to estrogen treatment, this could not be determined on the basis of these studies. Both Ishikawa and ECC-1 cells express three of the steroid coactivators: NCOA1, NCOA2, and NCOA3.
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Of the many known endometrial biomarkers that are regulated throughout the menstrual cycle, several display differential patterns of expression between luminal and glandular epithelium on the basis of the endocrine or paracrine milieu [14]. Three endometrial proteins that are expressed on endometrial epithelium were studied by Western blot analysis. CD55 (decay-accelerating factor) has a distribution similar to CD51 (beta 3 integrin subunit), expressed on both glandular and luminal epithelium [15], while SPP1 (formerly osteopontin) is thought to be primarily glandular in terms of its cell of origin and is also expressed during the mid- to late-secretory phase [16]. As shown in Figure 5, CD55 is expressed and present in both Ishikawa cells and ECC-1 cells. Its regulation is similar to that published for CD51, i.e., it is stimulated by EGF and inhibited by estrogen in both cell lines [17]. SPP1 is regulated by progesterone [16, 18] but not by estrogen or EGF; in Ishikawa cells, this lack of regulation is apparent, and little expression was noted in ECC-1 cells. The hyaluronate receptor, CD44, also appears to be specific to the glandular epithelium and is regulated during the menstrual cycle (Fig. 6, A–D). As shown in Figure 6, Ishikawa cells strongly express CD44 and exhibit regulated expression by EGF but not estrogen. Reduced expression of CD44 was observed in ECC-1 cells compared to Ishikawa cells.
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There are other differences in the pattern of protein expression between the luminal and glandular epithelium in human endometrium. Recent evidence pointed to a differential expression of cytokeratin types KRT13 and KRT18 in glandular and luminal epithelium in both rabbit and human endometrium [19]. The pattern of immunostaining in human endometrium is shown in Figure 7, demonstrating preferential immunolocalization of KRT13 in luminal epithelium, while KRT18 is present in both luminal and glandular epithelium. Comparing Ishikawa cells to ECC-1 cells, we also found a differential expression of KRT13 in ECC-1 cells, while both cells exhibited KRT18 expression (Fig. 8).
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DISCUSSION
Over the past 15 yr, we have investigated biomarkers of uterine receptivity, with a focus on the luminal and glandular epithelium. Integrin subunits were shown in the early 1990s to be useful markers of cellular subtypes and to characterize the changing hormonal milieu of the secretory endometrium [13, 14, 20]. Along with other researchers, we have extensively characterized an endometrial cell line, the Ishikawa cells, as a surrogate cell to study endometrial epithelium [4, 6, 17, 21–24]. We have shown that these cells express functional receptors to progesterone and androgens and have a pattern of integrins that is reminiscent of the glandular epithelium. Like normal endometrial glands, Ishikawa cells express both CD51 (beta 3 integrin) and its ligand SPP1 (osteopontin) [16].
Another well-differentiated cell line with similar characteristics was developed by P.G. Satyaswaroop [10, 25] and generously provided to us prior to his death. This cell line has now been submitted to the American Type Culture Collection posthumously and will be readily available. ECC-1 cells were originally cultured in nude mice and shown to be hormonally responsive with the induction of PGRs by estradiol, similar to normal endometrial epithelium. In the characterization, karyotype analysis of this cell line showed it to be abnormal and somewhat heterogeneous from a genetic perspective. Chromosome counts ranged from 45 to 47 chromosomes, with the loss of chromosomes 22 and X in every cell studied. Structural aberrations and marker chromosomes involving chromosomes 1, 3, 8, 10, 12, 16, and 19 were also present in all cells. Several complex rearrangements involving chromosomes 1, 3, and 8 were observed, which resulted in a partial trisomy of the long arms of chromosomes 1 and 8 and a partial monosomy of the short arm of chromosome 3. Trisomy of the short arm of chromosome 12 was observed as an additional marker chromosome. A deletion of the long arm of chromosome 16 was present in most every cell, and an extra copy of the deleted chromosome 16 was also seen in many cells, resulting in trisomy for most of chromosome 16. An apparently balanced translocation involving the short arms of chromosomes 10 and 17 was observed in the majority of cells.
We have previously demonstrated that both Ishikawa cells and ECC-1 cells activate ERE and PRE-CAT reporter gene activity when treated with estrogen, progesterone, or both [26]. In the present study, we characterized the steroid receptor and coactivator repertoire with Western blot and RT-PCR as well as with vitro-binding studies. The in vitro-binding assay is only an estimate of the number of specific binding sites for each receptor, but it suggests that ECC-1 cells maintain higher concentrations of each of the steroid receptor proteins than those seen in Ishikawa cells. Like Ishikawa cells, ECC-1 cells express ESR1 and ESR2, PGRs, ARs, and many of the steroid receptor coactivators on the basis of results obtained by Western blot and RT-PCR analysis. Unlike the expected increase in ESR binding in response to estrogen, neither Ishikawa cells nor ECC-1 cells increased the estrogen-binding capacity in response to estrogen priming. Indeed, ECC-1 cells appeared to downregulate estrogen binding following estrogen treatment. This phenomenon will be interesting to study and may involve the inducible ESR2 that is present in ECC-1 cells compared to Ishikawa cells. The ECC-1 cells also express many of the same surface biomarkers as luminal epithelium, including the differential expression of cytokeratin 13 previously reported for luminal epithelium [19].
Many of the proteins found in endometrial epithelium are found in ECC-1 cells. Like the CD51 (beta 3 integrin subunit), luminal and glandular endometrial cells express CD55 [15]. CD55 is positively regulated by EGF and heparin-binding EGF [15]. The regulated expression appears complex and, like CD51, is inhibited by estrogen while stimulated by EGF. This is demonstrated in both cell types and may explain their synchronous pattern of expression during the midsecretory phase corresponding to the downregulation of the estrogen receptor [27–29]. In contrast, CD44 has preferential expression on the glandular epithelium and stroma. Like CD55, this protein appears at the midsecretory phase on glandular epithelium and is stimulated by EGF. In articles included in the present study, the inhibition by estrogen is not apparent. ECC-1 cells do not express as much of this protein as do the Ishikawa cells, and regulation by EGF is minimal.
Endometrial cancer can arise from different cell types within the endometrium. It may not be unexpected, then, to find a cancer cell line with characteristics of luminal epithelium. The availability of an endometrial cell line that exhibits luminal characteristics and hormone responsiveness is notable; however, none has yet been described that convincingly exhibits this phenotype. In addition to the luminal characteristics reported in the present study, we have found that the ECC-1 cells are quite stable from passage to passage, a feature not typical of Ishikawa cells. In addition, the amount of estrogen, progestin, and androgen binding is both robust and hormone regulated.
To summarize, we have characterized the endometrial cell line ECC-1 derived by Dr. P.G. Satyaswaroop from a well-differentiated endometrial adenocarcinoma. This cell line provides a steroid-responsive endometrial cell line with luminal characteristics that maintain ESR1, ESR2, PGRs, and ARs and several steroid receptor coactivators. Many of the expected proteins made by luminal epithelium are expressed in this cell line. Regulation of others, including ESR2, appears to be different from that seen in Ishikawa cells, though further study is needed to explain this phenomenon. Studies to investigate the hormonal or growth factor regulation of key luminal proteins may now be more reliably investigated by researchers using this cell line. These cells provide a fresh opportunity to study the differences and similarities between luminal and glandular epithelium and have already been shown useful for DNA microarray studies [30]. Further experiments are contemplated, including the use of these cells to screen for new proteins that may be present on the luminal epithelium at the time of implantation. Coculture of these cells in combination with trophoblast or other embryonic-derived cells may also provide a novel means to investigate endometrial-embryonic interactions.
ACKNOWLEDGMENTS
We wish to dedicate this manuscript to the memory of Dr. P.G. Satyaswaroop, a generous colleague, collaborator, and friend. We acknowledge the skill and technical support of Melissa Hansen and Angela Houwing. We would also like to acknowledge Dr. Gary Olson, Vanderbilt University, for his technical assistance with the cytokeratin immunofluorescence studies.
FOOTNOTES
1 Supported by National Institute of Child Health and Human Development/National Institutes of Health through cooperative agreement U54 HD-30476 (B.A.L.) as part of the Specialized Cooperative Centers Program in Reproduction Research. 
2 Correspondence. FAX: 864 455 3095; blessey{at}ghs.org
Received: 19 February 2006.
First decision: 6 March 2006.
Accepted: 8 May 2006.
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ß3 integrin are coexpressed in the human endometrium during the menstrual cycle but regulated differentially. J Clin Endocrinol Metab 2001 86:4991-5000This article has been cited by other articles:
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