Onset of direct 17-β estradiol effects on proliferation and c-fos expression during oncogenesis of endometrial glandular epithelial cells

Experimental Cell Research 296 (2004) 109 – 122 www.elsevier.com/locate/yexcr

Onset of direct 17-h estradiol effects on proliferation and c-fos expression during oncogenesis of endometrial glandular epithelial cells Christophe Nemos, Re´gis Delage-Mourroux, Miche`le Jouvenot, * and Pascale Adami Laboratoire de Biochimie Biologie Mole´culaire, Universite´ de Franche-Comte´, U.F.R. Sciences et Techniques, Besancßon cedex, France Received 7 October 2003, revised version received 19 January 2004 Available online 14 March 2004

Abstract In normal endometrial glandular epithelial cells (GEC), 17h-estradiol (E2) enhances proliferation and c-fos expression only in the presence of growth factors. On the contrary, growth factors are not required for the E2 effects in cancerous cells. Thus, a repression of E2 action could exist in normal cells and be turned off in cancerous cells, allowing a direct estrogen-dependent proliferation. To verify this hypothesis, we established immortalized and transformed cell models, then investigated alterations of E2 effects during oncogenesis. SV40 large T-antigen was used to generate immortalized GEC model (IGEC). After observation of telomerase reactivation, IGEC model was transfected by activated c-Ha-ras to obtain transformed cell lines (TGEC1 and TGEC2). The phenotypic, morphological, and genetic characteristics of these models were determined before studying the E2 effects. In IGEC, the E2 action on proliferation and c-fos expression required the presence of growth factors, as observed in GECs. In TGECs, this action arose in the absence of growth factors. After IGEC transformation, the activation of ras pathway would substitute the priming events required for the release of repression in GEC and IGEC and thus permit direct E2 effects. Our cell models are particularly suitable to investigate alterations of gene regulation by E2 during oncogenesis. D 2004 Elsevier Inc. All rights reserved. Keywords: Endometrial glandular epithelial cell; Immortalization; Transformation; SV40 Large T-antigen; Ras oncogene; 17h-estradiol; Telomerase activity; Cell proliferation; c-fos expression

Introduction Estrogens induce DNA replication, cellular proliferation in the uterus of mammals [1 – 3], and have a role in promoting the growth and progression of uterus cancers [4,5]. Estrogen action on cell proliferation is mediated by receptors ERa and ERh. These receptors (ERs) are members of the nuclear receptor superfamily and function as ligandmodulated transcriptional regulators. In the classical mechanism of estrogen action, upon ligand activation, ER forms a dimer that binds with high affinity to specific DNA sequences, named estrogen-responsive element (ERE) and located within the regulatory regions of target genes. The DNAbound receptors then contact the general transcription apparatus. These interactions can involve cofactor proteins [6]. They allow the stabilization of transcription pre-initiation * Corresponding author. Laboratoire de Biochimie-Biologie Mole´culaire, Universite´ de Franche-Comte´, U.F.R. Sciences et Techniques, 16 route de Gray, 25030 Besancßon cedex, France. Fax: +33-381-666267. E-mail address: [email protected] (M. Jouvenot). 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.01.028

complex and the remodeling of chromatin at the target gene promoter. Subsequently, depending on the cell and promoter context, the receptor modifies the target gene expression. c-fos proto-oncogene is one of the estrogen-target genes and a functional ERE has been identified on the human and mouse c-fos genes [7,8]. Several in vivo studies have reported that acute administration of E2 to female rats elicited DNA synthesis and cell proliferation in the uterine luminal and glandular epithelia [9,10]. This cellular proliferation was linked with an early induction of the c-fos mRNA in both epithelia [10,11]. A link between proliferation and c-fos gene expression has also been observed at the time of estrogenic peak during the rat normal estrous cycle [12]. In human endometrium and breast cancer cell lines, E2 also induced early c-fos expression and cell proliferation [7,13,14]. However, results obtained in vitro with primary cultures of normal cells did not corroborate with those in vivo. Indeed, in cultured guinea pig endometrial glandular epithelial cells (GEC), E2 alone did affect neither growth nor c-fos gene expression [15,16]. An E2 effect on GEC proliferation linked to c-fos gene expression was observed

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in the presence of growth factors. Furthermore, c-fos gene was also induced by E2 in the presence of cycloheximide, an inhibitor of protein synthesis [17]. According to our findings, in normal cells, a labile repressor could prevent the E2 action on c-fos gene and consequently on cell proliferation. This repressor could be inactivated by growth factors in vivo or in cultured cells and its synthesis inhibited by Chx. In further studies, GEC were transfected with human c-fos gene recombinants known to be E2-responsive in cancerous HeLa cells. The lack of E2 effect on some of theses recombinants in GEC argued for a repression of E2 action which is dependent on the ERE environment [18]. These results observed in GEC and HeLa cells suggest that the molecular mechanism of estrogen regulation of c-fos transcription is different in normal and cancerous cells. We hypothesized that the repression of E2 effect would be turned off during oncogenesis allowing a direct estrogendependent proliferation. In the present study, we established cell models to verify this hypothesis. We chose a sequential approach of GEC transformation. First, we undertook GEC immortalization by SV40 large T-antigen [19 –22] and then immortalized GEC (IGEC) were transfected by activated c-Ha-ras oncogene [23 –25] to obtain transformed cell lines (TGEC1 and TGEC2). We investigated, in these three cell lines, estrogen effect on cell proliferation and c-fos gene expression.

Materials and methods Recombinant plasmids The pS701-ts-A58 plasmid used to immortalize GEC was a gift of Dr Feunten (Institut Gustave Roussy, Villejuif, France). This plasmid codes for a temperature-sensitive SV40 large T-antigen. The pSFF Vneo plasmid [26], expressing the neomycin resistance gene, was a gift of Dr Hoeveler (Poverty-related Diseases Unit, Brussels, Belgium). The plasmid pZeoSV2 (Invitrogen) contains a large T-antigen-dependent SV40 promoter and a zeocin resistance gene. The pEJ plasmid was generously provided by Dr. Maume (INRA, Dijon, France). It contains a mutated c-Haras gene cloned from human bladder carcinoma. This oncogene has a point mutation (conversion of GGC into GTC) in the first exon at codon 12 [24,25]. The pEJ plasmid was digested by BamHI, and the 6.6-kpb fragment containing c-Ha-ras gene was subcloned into the BamHI site of pZeoSV2. The recombinant plasmid pZeoSV2ras was used to transform IGEC. Cell transfection and isolation of stable transfected cell lines

a density of 28,500 cells/cm2 in complete medium (CM) consisting of Phenol Red Free Ham’s-F12 (HF-12) supplemented with 1 Ag/ml insulin, 100 Ag/ml penicillin, 250 ng/ ml amphotericin B, 100 Ag/ml streptomycin and 10% Fetal Bovine Serum (FBS). After 2 days of culture in CM, the cells in CM without FBS were co-transfected for 12 h with 2.5 Ag of pS701-ts-A58 and 2.5 Ag of pSFF Vneo in 15 Al of Lipofectink (Invitrogen). After 2 days of culture in CM at the permissive temperature for large T-antigen (33jC), the transfected cells were harvested, seeded at a density of 5700 cells/cm2, and selected for 2 weeks in 2 mg/ml neomycin analogue G-418 medium. Resistant clones were isolated using cloning rings, amplified and analyzed for large Tantigen expression. The positive identified clones were called IGECs. Once immortalized, one IGEC clone was characterized and, after 60 passages in CM at 33jC, this clone (IGEC p60) was transfected for 12 h with 5 Ag of pZeoSV2ras in 15 Al of Lipofectink. After selection in zeocin (400 Ag/ml) medium, resistant clones were amplified as described for IGEC and analyzed for mutated c-Ha-ras expression. Two positive clones, called TGEC1 and TGEC2, were further characterized. Reverse transcription-polymerase chain reaction (RT-PCR) RT-PCR was used to investigate expression of activated ras oncogene. One microgram of total RNA from TGECs, IGEC, and endometrium was reverse-transcribed with 100 U SuperScript II (Invitrogen) at 42jC for 50 min and 70jC for 10 min in the presence of 1 mM each of dATP, dCTP, dGTP, and dTTP, 5 mM MgCl2, 1 RT-PCR buffer (Invitrogen), and 10 mM DTT. From cDNAs, c-Ha-ras was amplified by PCR with sense primer (5V-TGGTGGTGGGCGCCGT-3V)-specific for mutated c-Ha-ras and reverse primer (5V-TCAGGAGAGCACACAGAGTTG-3V) located on mutated c-Ha-ras 4th exon. The PCR conditions were 94jC, 30 s; 58jC, 1 min; 72jC, 30 s for 33 cycles then 72jC, 7 min. The amplified DNA samples were electrophoresed on 1.2% agarose gel and stained with ethidium bromide. Karyotype analysis Exponentially growing IGEC was arrested by adding 0.03 Ag/ml colchicine for 2 – 4 h and treated with 10 Ag/ ml ethidium bromide to prevent chromosome condensation and to increase banding resolution [28]. Cells were then harvested, treated with hypotonic solution (75 mM KCl) at pH 8.0 for 20 min at 37jC, and fixed three times in cold Carny’s fixative (3 vol. of methanol/1 vol. of acetic acid) for 15 min. Chromosomes of 25 mitoses were counted after GTG banding (G-bands by trypsin using Giemsa) [29]. Proliferation assays

Isolation of glandular organoids from guinea pig endometrium and primary culture of GEC have been previously described [27]. GEC from a primary culture were seeded at

IGEC and TGECs were seeded at 102 cells/cm2 in six-well multiplates. IGEC were cultured in BM1 consisting of HF-12

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supplemented with antibiotic mixture and 0.2% steroid-free FBS (sfFBS) or in BM1 supplemented with either 100 ng/ml EGF plus 10 Ag/ml insulin or 100 ng/ml EGF plus 10 Ag/ml insulin plus 10 8 M E2. TGECs were cultured in CM or in BM2 consisting of HF-12 plus antibiotic mixture or in BM2 supplemented with either 10 8 M E2 or 100 ng/ml EGF plus 10 Ag/ml insulin or 100 ng/ml EGF plus 10 Ag/ml insulin plus 10 8 M E2. The culture medium was replaced 2 h after plating to discard non-adhering cells and every 48 h. Cells were harvested with trypsin/EDTA and counted in triplicate, first after the 2-h adhesion (t = 0) and then at different time intervals over 10 days. DNA content IGEC (106 cells) was cultured in CM or in BM1. TGECs (106 cells) were cultured in CM or in BM2. Cells were harvested by trypsinization, centrifuged, suspended in 0.1 ml of PBS, and then fixed by addition of 1.0 ml of 70% cold ethanol. After pelleting and removal of ethanol, RNA was removed by 1 unit of RNase A (Sigma), and the DNA was stained with propidium iodide (50 Ag/ml; Sigma) for 30 min at room temperature. The DNA content was then analyzed by cytofluorometry, using human lymphocytes as standard. Cell cycle analysis was performed using FACScan software (Becton Dickinson). Measurement of telomerase activity Telomerase enzymatic activity in GEC and in IGEC was determined using a TRAPezeR Telomerase Detection Kit (Intergen) connected with sensitive silver staining detection method [30]. Briefly, 2 Al of protein extract (140 to 0.4 ng for control cell extract, 46.6 ng for GEC and IGEC extracts and 1.5 Ag for tissue extract) was incubated in 48 Al of reaction mixture containing 1 TRAP buffer, 50 AM dNTPs, 0.1 Ag TS primer, 0.1 Ag RP primer, 0.1 Ag K1 primer, 0.1 Ag TSK, and 2 U Taq DNA polymerase at 30jC for 30 min. After telomerasemediated extension of the TS primer, reaction mixture was submitted to 33 cycles of PCR (Minicycler TM, MJ Research) using the following conditions: 94jC for 30 s, 60jC for 30 s, and 72jC for 30 s. Each analysis included a negative control (lysis buffer instead of protein extract) and a RNase heat-inactivated control (10 Al of protein extract was incubated for 10 min at 37jC in the presence of 0.1 Ag RNase, then heated at 95jC for 10 min). The quantification standard was obtained by performing the TRAP assay with 2 Al TSR8 (0.2 amoles) instead of protein extract. Samples were electrophoresed on 8– 15% polyacrylamide minigel. Detection by silver staining method, signal quantification, and TPG determination have been described previously [30]. One unit of total product generated (TPG) is defined as 0.001 amol, or 600 molecules, of primer TS extended for at least three telomeric repeats by telomerase present in the extract. Levels of

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telomerase activity were based on the results obtained from at least duplicate experiments. Determination of estrogen receptors Estrogen receptor content of GEC, IGEC, and TGECs was determined according to the method described by Taylor et al. [31]. Cells were incubated with increasing [3H]-E2 concentrations in 0.5 ml HF-12 –0.1% BSA with or without 100-fold molar excess of unlabeled diethylstilbestrol (DES). After incubation at 33jC for 1 h, the medium was removed, and the cells were washed twice with 1 ml cold phosphate buffer. Ethanol (1 ml) was added to each well to extract radioactivity. After 30 min at room temperature, ethanol solution was transferred to vials containing scintillation fluid (FluoranSafe, Scitran, Sigma) and [3H] radioactivity was counted (Minaxi TriCarb, Packard). Three experiments for each determination of estrogen receptors were performed. Differences between means F SD were compared using ANOVA test. Immunofluorescent analysis GEC, IGEC, and TGECs were grown on cover slips, rinsed two times in PBS, and fixed in 100% methanol for 20 min at 20jC. Cover slips were incubated with the first antibody diluted in 5% FBS-PBS for 1 h at room temperature. Mouse monoclonal antibody (1/40) (Oncogene Science) was used to detect SV40 Large T-antigen expression in cells. Cells were incubated with mouse monoclonal antibody of cytokeratin 18 (1/1) (Roche), or with mouse monoclonal antibody of vimentin (1/4) (Roche) or with rabbit polyclonal antibodies of sulfhydryl oxydase SOx-3 (1/500), specifically expressed in epithelial cells in endometrium [32]. Antigen antibody complexes were revealed by incubation for 1 h at room temperature with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse (1/40) or with pig anti-rabbit immunoglobulins (1/40). In each series of analysis, specimens incubated without the primary antibody were included as controls. Slices of endometrium were prepared as previously described [32]. They were subjected to the following incubations: 1 h in PBS-0.1% Tween 20, 2 h in primary antibody, and 30 min with FITC-secondary antibody. Samples were mounted in 50% PBS, 50% glycerol, 1 mg/ml DABCO and observed with Nikon Eclipse TE 300 microscope equipped for epifluorescence illumination. Noble agar assay The ability to grow in semi-solid medium was studied for Ishikawa and L87/4 cell lines as positive controls [33,34], IGEC, TGECs. The polystyrene flat bottom of wells had been precoated with 1% Noble agar in DMEM supplemented with 10% FBS for Ishikawa and L87/4 cells and with 1% Noble agar in CM for IGEC and TGEC cells. Ishikawa and L87/4 cells (103 cells/cm2) were suspended in

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0.3% Noble agar (Sigma) in DMEM supplemented with 10% FBS, plated on polystyrene flat bottom, and cultured at 37jC. IGEC and TGEC cells (103 cells/cm2) were suspended in 0.3% Noble agar in CM, plated and cultured at

33jC. The plates were incubated at 33jC, 5% CO2. The number of clones (> 100 Am) was counted by phase contrast microscopy after 21 days. Wells with cell aggregates were discarded 24 h after plating.

Fig. 1. Morphological and phenotypic characteristics of IGEC. (A) Polygonal epithelial-like morphology of IGEC at passage 20 (p20) (magnification: 100). (B) IGEC p30: phenomenon of cell crisis (arrows) (magnification: 100). (C) Nuclear expression of large T-antigen of SV40 in IGEC p50 (magnification: 200). (D) IGEC p15: complete appearance of vimentin antigen in cytoplasm (magnification: 200). (E) Slice of uterus: immunocytochemical expression of SOx-3 in glands (magnification: 100). (F) Perinuclear expression of SOx-3 in GEC (magnification: 200). (G) IGEC p60: perinuclear expression of SOx-3 (magnification: 200).

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Study of c-fos expression GEC, IGEC, and TGECs were grown in CM at 33jC. Subconfluent IGEC and TGEC were obtained within 3 –4 days (500,000 cells/cm2). IGEC were made quiescent in BM1 for 60 h as previously described [35]. For TGEC, cell quiescence was obtained by serum depletion for 60 h in BM2. After serum depletion (t = 0), the culture dishes were separated into several groups. The control groups were incubated in BM1 for IGEC and in BM2 for TGEC. The IGEC stimulated groups were incubated in BM1 supplemented with either 10 Ag/ml cycloheximide (Chx) or Chx plus E2 (10 8 M) with and without ICI 182,780 (10 6 M) (AstraZeneca, France) or 100 ng/ml EGF plus 10 Ag/ml insulin or with 100 ng/ml EGF plus 10 Ag/ml insulin plus E2 with and without ICI 182,780. For TGEC-stimulated group p34, stimulations were performed in BM2 supplemented with E2 (10 8 M) with and without ICI 182,780 (10 6 M). Total RNA was submitted to Northern blotting as previously described [17]. The v-fos and gapdh DNA probes were labeled with a [32P]dCTP by the nick translation

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system (Invitrogen). Labeled probes were purified by gel filtration and the specific radioactivity was estimated by liquid scintillation. Specific radioactivity was never less than 108 cpm/g of probe. The filters were prehybridized, hybridized, submitted to stringent washes, and were rehybridized [17]. Filters were exposed to Kodak X-Omat AR X-ray films (Kodak) with an intensifying screen for 24 h at 80jC and to Phosphoimager (Biorad) for signal quantification. Integration of peak areas was performed by Multianalyst software (Biorad). c-fos mRNA levels were normalized with the gapdh mRNA level.

Results Establishment and characterization of a SV40-immortalized endometrial glandular epithelial cell line To establish stable cell lines that might maintain some differentiated characteristics of endometrial glandular epithelial cell primary cultures, the SV40 large T-antigen was

Fig. 2. Genetic characteristics of IGEC. (A) Chromosome number distribution of IGEC p40. (B) Cell cycle distribution of IGEC p40. The cells were cultured in CM (on the left of C) or in BM1 (on the right of C) during 60 h then DNA content was analyzed.

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Fig. 3. Telomerase activity in GEC and IGEC p8, p18, p28, and p58. (A) TRAP products in 8 – 15% polyacrylamide minigel revealed by silver staining (representative experiment). Lanes 1 to 10: 46.6 ng of protein from GEC or IGEC at different passages (103 cell equivalents) were assayed. The reaction mixture was treated (+) or not treated ( ) with RNase and heat. Lane 11: quantification control with TSR8 instead of protein extract. Lane 12: negative control with lysis buffer instead of protein extract. Lane 13: 103 HeLa cell equivalents (46.6 ng). M: DNA molecular weight marker V, 8 – 587 bp (Boehringer Mannheim). (B) Quantification of telomerase activity; TPG units are mean values F SD of three independent experiments.

chosen as an immortalization promoter [19]. Subconfluent secondary culture of GEC was transfected with plasmids encoding SV40 large T-antigen and aminoglycoside phosphotransferase (for neomycin/G418 resistance). Resistant clones were obtained after 2 weeks on G418 selection medium. Subculture of these clones in CM corresponded to passage 1 (p1). These resistant clones were sub-cultured until the crisis (p27 – p32). This phenomenon of crisis (Fig. 1B) was distinguished by large dimensions of cells (>100 Am), spiral distribution, and abundance of cytoplasm in cells (arrows in Fig. 1B), a lot of cell death and a cell prolifer-

ation decrease. Most of the clones did not survive to crisis. One clone (IGEC), which bypassed this step, was selected for additional phenotypic and genetic characterization, from its clear polygonal epithelial-like morphology (Fig. 1A). Integration in IGEC genome of large T-DNA sequence was confirmed before crisis by Southern blotting analysis (data not shown). All nuclei of IGEC were shown to contain large T-antigen as revealed by indirect immunofluorescence staining (Fig. 1C). This large T-antigen expression persisted along the subcultures. Analysis of intermediate filament protein expression in IGEC was performed between p5 and p60. Expression of epithelium-specific cytokeratin 18 completely disappeared at p15, while vimentin expression completely appeared at this passage (Fig. 1D). Like epithelial glands of endometrium (Fig. 1E) and GEC in primary culture (Fig. 1F), IGEC, whatever the passage, were immunoreactive to anti-SOx-3 antibody (Fig. 1G) directed to a 68-kDa epithelium-specific sulfhydryl oxidase [19]. These results showed that some epithelial markers were expressed and maintained in IGEC. Guinea pig genome arrangement is 2n = 64 chromosomes [36]. Chromosome analysis of 25 metaphases at passage 40 (Fig. 2A) revealed that 96% of IGEC had a polyploid karyotype. The distribution of chromosome number showed a peak in the hypotetraploid region with 84% of metaphases containing 90 to 115 chromosomes. However, study of the cell cycle based on DNA content (Fig. 2B) showed a single population of IGEC p40. So, it appeared that in spite of chromosomal rearrangements, neither gain nor loss of DNA occurred in IGEC p40. As previously reported for GEC [16], a 60-h serum depletion in BM1 induced IGEC quiescence (Fig. 2B): in BM1, 72.5% of cells were in G0/G1 against 34.3% in CM. As telomerase activity can appear during immortalization [37], we have analyzed this activity in GEC and IGEC.

Fig. 4. RT-PCR analysis of ras oncogene expression in TGECs. Lanes 2 and 3: RT-PCR on RNAs (1 Ag) from guinea pig endometrium and IGEC p20, respectively. Lanes 4 – 9: RT-PCR on total RNAs (1 Ag) from zeocinresistant clones. Lanes 4, 6, 8, and 9 show the four clones that expressed ras oncogene (called TGEC1 to TGEC4, respectively). Lane 10: PCR on pZeoSV2ras-EJ DNA (100 ng). Lanes 1 and 11: 1 kb DNA ladder (Invitrogen).

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Fig. 5. Morphological and phenotypic characteristics of TGEC1 and TGEC2. (A and B) Phase-contrast microscopy of TGEC1 p34 and TGEC2 p34 grown in CM (magnification: 200). (C) Expression of cytokeratin-18 in TGEC2 p20: (magnification: 200). (D and G) Growth of L87/4 and Ishikawa cells (used as positive controls) in Noble agar DMEM medium. (E and F) Growth of TGEC1 p10 in 0.3% Noble agar and on 1% Noble agar precoating. (H) Growth of TGEC2 p10 in 0.3% Noble agar. Scale bars in D, E, F, G, H: 100 Am.

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TRAP assay was performed with 46.6 ng of protein (1000 cell equivalents) and a positive control was obtained using protein extract from 1000 HeLa cells. Telomerase activity was weak in GEC (Fig. 3A, lanes 1 – 2 and Fig. 3B) and increased in IGEC passage by passage (Fig. 3A lanes 3 to 10 and Fig. 3B). To quantify telomerase activity, TPG units were calculated from three experiments. Telomerase activity was evaluated at 22.1 TPG for telomerase-positive cells and at 0.74 TPG for GEC. For IGEC p8, p18, p28, and p58, telomerase activities were 2.3, 7.4, 17.6, and 22.2 TPG, respectively. It can be concluded that an abrupt increase of telomerase activity went together with immortalization of endometrial cells: between IGEC p8 and IGEC p58, there was a 9.6-fold increase. There was no further increase after passage 58 (results not shown). As telomerase activity for 1000 IGEC p58 cells was identical to that observed for 1000 control cells, apparently, 100% of IGEC at passage 58 were telomerase-positive. Establishment and characterization of a SV40-ras transformed endometrial glandular epithelial cell lines IGEC p60 was transfected with pZeoSV2ras expressing zeocin-resistance gene and mutated hyperactive ras. Discernible colony outgrowths were observed after 2 weeks in zeocin selection medium, and clones were derived from emerging foci. Clonal expansion of individual single variant cells was subcultured, and several individual resistant cell lines were retained. Among these resistant cell lines isolated, four clones (TGEC1, TGEC2, TGEC3, and TGEC4) expressed activated ras oncogene as shown by the 651 bp band corresponding to the amplification of ras exon 1/exon 4 mRNA (Fig. 4, lanes 4, 6, 8, and 9). Only two clones (TGEC1 and TGEC2) were then selected for additional characterization from their high proliferative capacity. The morphology of both clones was distinct: TGEC1 was a clone of epithelial-like cells but these cells were larger than IGEC (Fig. 5A), while TGEC2 was a clone of very small cells forming piles (Fig. 5B). In TGEC1, whatever the passage, antigenic characteristics were the same as those observed for IGEC p60: expression of large T-antigen, vimentin, and SOx-3 (not shown). In TGEC2, only large T-antigen and SOx-3 expressions were conserved. Indeed, vimentin was not detected in this clone, while cytokeratin18 was re-expressed (Fig. 5C). Ishikawa and L87/4 cell lines (used as positive controls), TGEC1 p10, TGEC2 p10, and IGEC p60 were evaluated for their ability to grow in Noble agar. After 21 days of culture in Noble agar, IGEC p60 did not form colonies. On the other hand, TGECs as well as positive controls showed anchorage-independent growth with different characteristics. As L87/4 (Fig. 5D), TGEC1 p10 formed diffuse colonies with spreading peripheral cells in Noble agar (Fig. 5E) and was able to form precoating anchoragedependent colonies (Fig. 5F). As Ishikawa cells (Fig. 5G), TGEC2 p10 formed compact colonies in Noble agar (Fig.

5H) but not on 1% Noble agar precoating. Moreover, differences between percentages of transformation levels were observed (Fig. 6): 6.2% for TGEC1 p10 and 10.7% for TGEC2. For Ishikawa and L87/4 controls, they were 7% and 11.5%, respectively. In other respects, telomerase activity was quantified in both TGEC1 and TGEC2 and was the same as activity determined in IGEC p58 (data not shown). Expression of estrogen receptors in IGEC and TGECs According to Scatchard analysis described in Materials and methods, the number of E2 receptors/cell (Fig. 7A), expressed as mean of triplicate determinations F SD, was 21,000 F 540 in GEC and 19,600 F 480 in IGEC p5. It decreased along passages of IGEC and was 6500 F 205 at p27. It stabilized at p52 (6990 F 230) with average KD = 5 F 1.7  10 9 M. In TGEC1 p24, the number of E2 receptors/cell was 7048 F 422 and thus similar to the stabilized one in late passages of IGEC. In TGEC2 p24, it strongly increased (17,399 F 1094) and the average KD was 6.3 F 2.1  10 9 M. So, comparatively to ER level in GEC, IGEC at passage 52 lost 66.7% of E2 receptors and TGEC2 p24 had almost similar levels. Estrogen and growth factor effects on IGEC and TGEC growth TGECs, maintained in CM, presented higher proliferation rates than IGEC cultured in the same conditions (not shown): doubling times were 19 and 16 h for TGEC1 p34 and TGEC2 p34, respectively whereas they were 37 and 48 h at early (15th) and late (50th) passages of IGEC. Then, to test the E2 effect on proliferation, cells were submitted to serum depletion in BM1 or BM2 and stimulated either by E2 alone or by EGF plus insulin or by E2 plus EGF plus insulin. In basal medium (control), the cell number slightly increased with time (Figs. 7B – E). When the basal medium was supplemented with E2,, the IGEC cell number (not

Fig. 6. Determination of transformation levels in TGECs. Noble agar plates (as shown in Fig. 5) were scored for colony formation. Colonies greater than 100 Am in diameter were counted 3 weeks after seeding 5000 cells/well.

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Fig. 7. Determination of estrogen receptor levels and proliferation of IGEC and TGECs. (A) Estrogen receptor levels in GEC, IGEC p5, p15, p27, p31, p51, p52, TGEC1 p24 and TGEC2 p24. ER number/cell are mean values F SD. (B and C) Proliferation of IGEC p15 and p50, respectively, in the presence of EGF + insulin F E2. The culture medium was changed on day 1 and then every 2 days. Unstimulated cells (in BM1) were used as control. Data are the mean values of three culture dishes (FSD). *P < 0.01 vs. control group; **P < 0.01 vs. EGF plus insulin group. (D and E) Proliferation of TGEC1 p34 and TGEC2 p34, respectively, in the presence of E2 and/or EGF + insulin. The culture medium was changed on day 1 and then every 2 days. Unstimulated cells (in BM2) were used as control. Data are the mean values of three culture dishes (FSD). *P < 0.01 vs. control group; **P<0.01 vs. EGF plus insulin group.

shown) was similar to that observed in control while there was a significant increase of TEC1 and TGEC2 cell numbers (Figs. 7D and E): compared with control, this increase on day 9 was 1.6- and 1.3-fold, respectively. An increase of cell number was achieved with EGF plus insulin for all clones (1.3-, 1.5-, 2.1-, and 2.4-fold increases for IGEC p15, IGEC p50, TGEC1 p34, and TGEC2 p34, respectively). Except for IGEC p50, the most effective action on cell number was obtained when E2 was added to EGF plus insulin (Figs. 7B, D and E): in the presence of E2 plus EGF plus insulin, the fold increases on day 9 (compared with EGF plus insulin stimulation) were 2, 1.3, and 1.3 for IGEC p15, TGEC1 p34, and TGEC2 p34, respectively. Our findings demonstrate that E2 effect on proliferation of IGEC at early passages requires the presence of growth factors as

previously demonstrated for GEC [15], whereas E2 direct proliferative effect occurs in TGECs. E2 and growth factor effects on c-fos expression in IGEC and TGECs To determine if c-fos gene regulation in immortalized and transformed cells was different or not from that observed in GEC [15], we analyzed the c-fos mRNA levels after treatment of IGEC and TGECS with various combinations of E2, EGF, insulin, Chx, and ICI 182,780 at concentrations described in Materials and methods. When IGEC p15 were maintained in basal medium for 120 min, c-fos mRNA was not detectable and E2 was not able to promote c-fos expression (not shown). As shown

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in Fig. 8A, in IGEC p15 treated with EGF plus insulin, the c-fos mRNA level increased rapidly within 45 min, peaked at 90 min, and decreased. The transient increase of c-fos mRNA level was enhanced by E2: in the presence of E2 plus EGF plus insulin, the c-fos mRNA levels were higher than those observed in the presence of EGF plus insulin (3.9-, 2.8-, 1.7-, and 1.6-fold higher at 45 min, 60,

75, and 90 min, respectively). This E2 effect was abolished by an E2 antagonist, ICI 182,780, whereas the EGF/ insulin effect persisted in the presence of this antagonist. Similar results were obtained with IGEC p20, p30, and p50 (not shown). Thus, in IGEC, as in GEC, an E2 effect on c-fos gene expression can occur in the presence of growth factors.

Fig. 8. E2 and growth factor effect on c-fos expression in IGEC and TGECs. Northern blot analysis of 20 Ag total RNA obtained from unstimulated (t0) and stimulated cells. (A) IGEC p15 stimulated by EGF + insulin F E2 F ICI 182,780. (B) IGEC p15 stimulated by Chx F E2 F ICI 182,780. (C) TGEC1 p34 cultured in BM2 F E2 F ICI 182,780. (D) TGEC2 p34 cultured in BM2 F E2 F ICI 182,780. (A, B, C and D): top, autoradiographic signals (representative experiment); bottom, c-fos mRNA levels corrected by gapdh mRNA levels.

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We then investigated if Chx could permit the E2 effect on c-fos gene expression as previously demonstrated for GEC. As shown in Fig. 8B, after simultaneous addition of E2 and Chx, the c-fos mRNA level increased in a time-dependent manner: compared with the level in cells treated with Chx alone, there were 2.6- and 3.7-fold increases at 30 and 210 min. The E2 effect observed in the presence of Chx was partially abolished by ICI 182,780. To determine if repression of estrogenic activity on cfos persisted in TGECs, we analyzed c-fos expression using E2 alone or in combination with ICI 182,780. As shown in Figs. 8C and D, a strong and transient increase of c-fos mRNA level was observed for both transformed cells lines treated with E2 alone. Compared with the level in BM2, a maximal 5-fold increase was observed for TGEC1 p34 at 120 min (Fig. 8C) and for TGEC2 p34 at 60 and 120 min (Fig. 8D). ICI 182,780 alone had no effect during the same time course (not shown), and it could partially abolish the E2 effect in TGEC1, but not in TGEC2 (Figs. 8C and D).

Discussion Here, one immortalized glandular epithelial cell line (IGEC) and two transformed glandular epithelial cell lines (TGEC1 and TGEC2) were established as models to gain new insights into changes of estrogenic effects during oncogenesis. GECs were transfected by SV40 large Tantigen gene known to be a promoter of immortalization [38 – 40]. As observed for other cell types, the expression of large T-antigen extended life span of GEC [19] reactivated the telomerase activity [37] and finally immortalized GEC [41]. During this immortalization process, no change of cell morphology was observed except during the crisis. Otherwise, phenotypic conversions occurred: cytokeratin 18 completely disappeared at p15 while vimentin appeared. Although IGECs did not express cytokeratin 18, they still displayed epithelial features like morphology and SOx-3 expression specific for epithelial cells in endometrium [32]. Previous studies have shown that SV40 large T-antigen induced changes in cell differentiation program [42 – 45]. The classic epithelial-to-mesenchymal transition that was essential to normal mammalian development was hallmarked by a change from cytokeratin expression to vimentin expression [46]. Moreover, it was shown that exogenous expression of vimentin in human breast cancer cells resulted in conversion from an epithelial to mesenchymal phenotype, reminiscent of the interconversion that occurs during development [47]. Thus, it is possible that IGECs express a transitional phenotype with both epithelial and stromal features. The main interest of the IGEC model is the relative abundance of E2 receptors (about 7000 ER/cell at late passages). Few studies have reported the ER level after endometrial cell immortalization in immortalized ovine

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[48] and porcine [49] epithelial cells, ERa was just detected by immunostaining and Western blotting, respectively; the ER levels were low or undetectable in almost all rat endometrial cell lines, the only exception being RENT4 cells (about 9000 ER/cell) [45]. Thus, as RENT4 cells, IGECs are a suitable alternative model to study estrogen effects. Mutations of ras cellular oncogenes are associated with tumorigenicity, invasiveness, and metastatic potential of estrogen-dependent type I endometrial cancers [50]. Several authors have used mutated c-Ha-ras [24,25] to transform human epithelial cells [51 – 54]. In this study, we showed that this oncogene can transform IGEC. The two transformed cell lines, TGEC1 and TGEC2, presented two cancer hallmarks: anchorage- and growth factor-independent growth. However, they had distinct morphological and phenotypic characteristics. In particular, unlike TGEC1, TGEC2 did not express anymore vimentin but re-expressed cytokeratin 18. A similar observation has been reported for transformed rat endometrial cells (RENTR01 and RENTR02) in which epithelial phenotype was enhanced by RAS protein [44]. The authors have concluded that RAS acts on the differentiation pathway at an early step before the distinction between decidual and glandular epithelial lineage. In TGEC1, the ras oncogene, because of its integration site, could be less expressed than in TGEC2 and the RAS level insufficient to induce stromal –epithelial interconversion. In fact, preliminary results obtained in immunoblotting experiments (not shown) suggest that RAS expression level is higher in TGEC2 than in TGEC1. The ER levels of both TGECs were also very different: about 7000 and 17,000 ER/cell in TGEC1 and TGEC2, respectively. An ER level increase has been previously reported after chemical cell transformation [55]. High levels have also been observed in ras oncogene-transfected cells [56,57] and in endometrial tumors with ras mutations [58,59]. In these studies, it appeared that ER expression was increased by RAS and that ER activated the RAS pathway. This positive feedback would increase ras oncogenic potency in human endometrial lesions, and thus high ER levels would be linked with tumorigenicity and tumor aggressiveness. Our study reinforced this suggestion since TGEC2 had the highest percentage of transformation. In IGEC at early passages, the positive E2 action on cell proliferation and c-fos expression required the presence of EGF plus insulin. The c-fos up-regulation by E2 was also observed when this hormone was associated with Chx. The E2 effect on c-fos expression implicated estrogen receptors since it was abolished by the antagonist ICI 182,780 [60]. These findings are identical in every respect to those previously reported for GEC [15] and argue for persistence of repression of E2 action in immortalized cells. At late passages, while E2 in the presence of appropriate factors still induced c-fos expression, it had no mitogenic effect likely because of the low ER level. Indeed, in the same conditions,

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diethylstilbestrol, which binds ER with higher affinity than does E2 [61], induced weak IGEC proliferation (data not shown). Interestingly, in TGECs, we showed that, in the absence of growth factors, E2 was able to induce c-fos expression and proliferation. In other words, this E2 action was direct and comparable to that observed in breast and endometrium cancer cells [7,13,14] and differed from the indirect action demonstrated for IGEC and previously reported for GEC [15]. Although the direct c-fos up-regulation by E2 was observed in both TGEC cell lines, it was abolished by antiestrogen ICI 182,780 in TGEC1 only. On the basis of the classical ICI 182,780 mechanism [60], this result would suggest that the E2 effect does not only involve ERs in TGEC2 despite their high ER expression level. It can also suggest that ICI 182,780 has an E2 agonist effect as recently reported [62]. However, in two separate experiments, no cfos mRNA increase was observed when TGEC2 was treated by ICI alone (not shown). Further studies are needed to elucidate the differential molecular mechanisms of E2 effect in both TGECs. Anyway, the direct E2 effect observed in TGECs on c-fos expression and cell proliferation clearly demonstrated that the hormone action in transformed cells is no more repressed. After the process of IGEC transformation, the constitutive activation of phosphorylation cascades in the ras pathway [63] would substitute the priming events required for the release of repression in normal or immortalized GEC. Various alterations of signal transduction could have similar effect to those induced by mutated RAS, explaining estrogen-dependent cancer cell growth. Thus, our different cell models turn out to be particularly suitable to investigate the alterations of gene regulation during oncogenesis.

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[7]

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Acknowledgments [16]

This research work was supported by a fellowship from the Re´gion de Franche-Comte´ and by grants from the Ministe`re de l’Enseignement Supe´rieur et de la Recherche (E.A. 3182) and the Ligue Nationale Contre le Cancer (Comite´ du Doubs and Comite´ du Jura). We are grateful to AstraZeneca (Reims, France) for providing ICI 182,780, to Dr Feunten (Villejuif, France) for generous gift of pS701-tsA58, to Dr Hoeveler (Bruxelles, Belgium) for generous gift of pSFF Vneo plasmid and to Dr Maume (Dijon, France) for generous gift of pEJ.

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