Molecular and Cellular Endocrinology, 87 ( 1992)

Rl 1-R17 Q 1992 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/92/$05.00

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MOLCEL 02846

Rapid paper

Transfected

~nd~~etrial

cultured cells: a system to study gene regulation by estrogens

C. Vuillermoz, M, Jouvenot, I. Pellerin, C. Ordener, M. Royez and G.L. Adessi INSERM U 198, 25000 Besan, streptomycin (100 pg/mi), fungizone (2.5 pg/ml), Na,SO, (600 PM), insulin (10 pg/ml) and FCS (5% v/v>. The modified MPC was MPC in which 5% FCS were replaced by 5% of dextran-coated charcoal stripped FCS (DCC-FCS). Serum free chemically defined medium (CDM) consisted of phenol-red free Ham’s F-12, Hepes buffer (20 mM), penicillin (100 U/ml>, streptomycin (100 pg/ml), fungizone (2.5 pg./ml), transferrin (10 pg/ml), sodium selenite (10 pg/ml), Na,SO, (600 FM) and bovine serum albumin (1 mg,/mIf. Culture of endometrial cells. The isolation of glandular epithelial (GE) cells or stromal cells was performed using collagenase treatment as previously reported (Alkhalaf et al., 1987). Cells were then grown separately in MPC in tissue culture flasks. The MPC was changed every 2 days. Subconfluent GE or stromal cells were obtained within 6-8 days. Subconfluent cells were submitted to trypsinization as previously described (Mahfoudi et al., 1991). At the end of the procedure, GE or stromal cells were resuspended either in MPC for experiments of transfection with pCAT plasmid or in modified MPC for experiments studying E,-17/3 induction of a reporter gene, and plated in 35 mm wells at a density of 6.2 x lo4 GE cells per cm’ or 3.5 X lo4 stromal cells per cm2. Subcultures were incubated at 37°C for 48 h in a humidified atmosphere of 5% CO, in air. The medium was renewed 4 h before the end of this period. After 48 h subculture, the cells were either transfected or used to measure the estrogen receptors (Taylor et al., 1984). Just before transfection, about 5 X 10’ stromal cells or 4 x 10” GE cells per 35 mm well

were counted.

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Transient transfection and cell stimulation. Subconfluent epithelial or stromal cells (after 48 h subculture) were transfected using the calcium phosphate-DNA coprecipitation method (Graham et al., 1973). In order to define the suitability of the method and the transfection conditions, preliminary experiments were performed using the pCAT reporter plasmid, as reported in Results. After the transfection step, cells were washed 3 times with phosphate-buffered saline (PBS, 10 mM, pH 7.2) and kept in MPC at 37°C for 48 h. At the end of this period, they were harvested for CAT assay. In the case of the ERE-tk-CAT plasmids, the tranfection conditions were those defined after assays with pCAT plasmid and will be noted in Results. In these experiments, cells were cotransfected with an ERE-tk-CAT plasmid and a P-galactosidase plasmid used as internal control of transfection. Following three washes with PBS, cells were kept for 24 h in CDM supplemented with antiestrogen ICI 164,384 (10e7 Ml. The medium was then removed and the cells were washed with PBS and then incubated for a further 48 h with CDM with or without the appropriate stimulus. CAT enzyme assay was performed after normalization for P-galactosidase activity (Norton and Coffin, 1985). CAT assays. CAT assays were performed in whole cell extracts (Seed and Sheen, 1988). The acetylated and non-acetylated forms of [ “C]Chloramphenicol were separated by thin-layer chromatography (TLC). The TLC plates were exposed to X-ray films (Kodak X-Omat AR X-ray) for 24 h at -80°C. The radioactive spots were cut from the TLC plates and quantified by liquid scintillation counting. The ratio of radioactivity in acetylated chloramphenicol spots to that in acetylated plus non-acetyled chloramphenicol spots evaluated the relative CAT activity. For each stimulation. three dishes of transfected cells were treated separately from the transfection step to CAT assay. The experiments were always repeated 3 times with similar results. Statistical analysis was performed using the Student’s t-test. Results Characterization endometrial cells.

and transfection of cultured Transfection of subcultured

endometrial cells, i.e. GE or stromal cells has not been reported to our knowledge in the relevant literature. Thus, the first step of our study was to define the conditions of tranfection of these cells. At the end of the primary culture of GE cells, the percentage of anti-cytokeratin immunostained cells ranged from 69% to 78%. Selective trypsinization and subculture were used to enhance the percentage of epithelial cells as reported previously (Mahfoudi et al., 19911. More than 95% of the subcultured GE cells were positively stained. In the same way, primary cultures of stromal cells displayed about 80% of antivimentin immunostained cells and this percentage was increased to 98-99% after trypsinization and subculture. Consequently, transient transfection studies were done with enriched subcultures, i.e. containing a percentage of GE or stromal cells higher than that obtained in primary cultures. The content of total cellular estrogen receptors (ERs) was measured in subcultured cells just before transfection. A high level of ERs was observed. For a typical experiment, the number of ERs per cell was 16,800 binding sites with a K, value of 0.10 nM for subcultured stromal cells and 14,200 binding sites with a K, value of 0.09 nM for subcultured GE cells. In order to determine if the calcium phosphate coprecipitation method was suitable for transfection of endometrial cells, the pCAT control plasmid was used. It contains the SV40 promoter and enhancer sequences resulting in strong and constitutive expression of CAT activity in many types of eucaryotic cells. In the experiment shown in Fig. 1, subcultured stromal cells were exposed for 4 h or 8 h to a calcium phosphate-DNA precipitate containing a constant amount of pCAT plasmid (2.5 pg per 35 mm well) and varying amounts of carrier DNA (0 or 5 or 10 pg per well). The cells were then submitted to a glycerol shock (15% in MPC) at 37°C for 3 min. After three washes in PBS, they were kept in MPC for 48 h at 37°C and CAT assay was performed. Stromal cells transfected by the pCAT plasmid displayed a CAT activity whereas control cells treated with a precipitate without this plasmid did not display this activity (in Fig. 1, compare lanes l-6 with lane 7). The presence of carrier DNA increased transfection efficiency. When cells were exposed

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cipitate containing 5 pg of carrier DNA, 2.5 pg of P-galactosidase plasmid and 2.5 Kg of ERE-tkCAT plasmid for 4 h. After three washes with PBS, they were kept for 24 h in CDM plus ICI

A

Fig. 1. Assays of transfection of subcultured endometrial stromal cells using pCAT control plasmid. Cells were exposed for 4 or 8 h to a CaPO, precipitate containing a constant amount of control plasmid (2.5 Fg per 35 mm per well) and various amounts of carrier DNA (0 or 5 or 10 @g per well). After glycerol shock and washes, cells were incubated for 48 h in medium for primary culture (MPC). Then CAT assays were performed. The results are representative of three determinations performed in three separate wells of subcultured cells.

to the precipitate for 4 h, 5 pg or 10 pg of carrier DNA per well led to transfection efficiency higher than that obtained in the absence of carrier DNA (Fig. 1, compare lanes 2 and 3 with lane 1). When cells were exposed to the precipitate for 8 h, transfection with 10 pg of carrier DNA was slightly more efficient than with 5 pg (Fig. 1, lanes 6 and 5 respectively). However, after an exposure to the precipitate for 8 h, the microscopic morphological characteristics of the cells were altered and cell attachment was decreased. Thus, subsequent experiments were performed with a 4 h exposure to a precipitate containing 5 pg of carrier DNA. We also observed that the results, in terms of CAT assay, were the same whatever the amount of pCAT plasmid (2.5, 5 or 10 pg per well) and with or without glycerol shock (not shown). Thus, the following transfection experiments were performed with a precipitate containing 5 pg of carrier DNA and 5 Kg of reporter DNAs added to the cells for 4 h and without subsequent glycerol shock. Effect of E,-17p on expression of Llarious EREThe ERE-tk-CAT plasmids directed CAT genes. tested are described in Fig. 2A. At the end of subculture, GE cells were transfected with these plasmids as follows: they were exposed to a pre-

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Fig. 2. Stimulation with E,-17p of ERE-directed CAT genes transfected in subcultured glandular epithelial cells. (A) Structure of the ERE-tk-CAT plasmids: tk is the HSV thymidine kinase gene promoter ( - l&5/+52) and CAT is the bacterial chloramphenicol acetyltransferase gene coding region. Palindromic EREs were inserted upstream of the tk-CAT gene into a EarnHI site. Inverted arrows represent the arms of the palindromic elements. ERE wt and ERE 32 are the wild type and two base pair deleted vitellogenin A2 gene EREs respectively. BI ERE and 45 Bl ERE are the entire and 5’ deleted vitellogenin Bl gene EREs respectively. (B) CAT assays performed after P-galactosidase normalization on extracts of GE cells transfected with the different constructs and incubated in CDM (1) or in CDM plus E,-17P (2) or in CDM plus Ez-17P plus EGF (100 ng/ml) plus insulin (IO pg/ml) (3).

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164,384 (lo-’ MI. Then they were incubated in either CDM or CDM plus E,-17j3 (lo-’ MI or CDM plus E,-17/3 plus EGF (100 ng/ml) plus insulin (10 pg/ml). This last stimulation experiment was performed to determine whether the presence of growth factors could modify the response of the ERE-tk-CAT gene to E,-17P since it had previously been observed that E,-17P alone was ineffective in c-fos gene induction (Jouvenot et al., 1990). CAT activity measured after normalization with /?-galactosidase activity is presented in Fig. 2B. A low CAT activity was detected in cells transfected with plasmids containing a consensus ERE and kept in CDM without E2-17P (see ERE wt and Bl ERE, lane 1). An activity of the same order was observed in cells transfected with plasmids containing deleted EREs and incubated with either CDM or E,-17P or E,-17P plus EGF plus insulin (see ERE A2 and A5’ Bl ERE, lanes 1, 2 and 3). In contrast, treatment by E,-17P of cells transfected with ERE wt-tk-CAT (lane 2) or Bl ERE-tk-CAT (lane 2) increased CAT activity and there was respectively a 10.3- and 6.1-fold induction compared with control cells transfected with the same plasmids and kept in CDM (lanes 1). Stimulation by E,-17P plus EGF plus insulin did not induce a further increase in CAT activity, compared with the effect of E,-17P alone (see lanes 2 and 3 for ERE wt or Bl ERE). Transfection experiments were performed under the same conditions with stromal cells and led to similar results (not shown). After E,-17/I (lo-’ MI treatment, there was a 6.5-fold induction of CAT activity in cells transfected with ERE wt-tk-CAT and a 4.5-fold induction in cells transfected with Bl ERE-tk-CAT. Effect of carious estrogens on citellogenin A2 ERE-tk-CAT gene expression. ERE wt-tk-CAT plasmid was transfected in stromal cells under the above defined conditions. The effects of E,17a (a biologically weak estrogen) and of DES (an active chemical estrogen) were compared with the E,-17P effect, in terms of CAT activity induction. In the same way, inhibition of the E,-17p effect by antiestrogen ICI 164,384 was studied. For each stimulation, three wells of transfected cells were treated separately in order to evaluate intra-individual variations. One of the three CAT assays for the different stimulation conditions is

A

B

Fig 3. Effect of various estrogens on the induction of vitellogenin A2 ERE wt in subcultured stromal cells. Cells were transfected with ERE wt-tk-CAT plasmid. (A) CAT assays were performed after P-galactosidase normalization on extracts of transfected cells which had been incubated in CDM or CDM plus E,-17P (lO~x Ml or CDM plus DES (10-k Ml or CDM plus E,-170 (IO-’ Ml. Each treatment was carried out in triplicate. Only one spot is presented. (B) Effect of ICI 164,384 on the Ez-l7P-dependent induction. Transfected cells were stimulated with E,-17P (IO-’ M) and various concentrations of ICI 164,384 (0, lo-’ M, lo-’ M, IO-’ Ml. Each stimulation was carried out in triplicate. Only one spot is presented.

presented in Fig. 3 (A and B). A significant 5-fold induction of CAT activity (P < 0.05, n = 3) was observed with extracts from cells stimulated by either E,-17/3 or DES (lop8 MI. In contrast, CAT activity in the presence of E,-17a (lo-’ MI was not significantly different from that observed for control cells in CDM without hormone. The E,-17/3-dependent CAT induction was inhibited

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by ICI 164,384 in a dose-dependent manner (from 10p6 M to lo-” M). No induction of CAT activity was observed in the presence of E,-17P at It)-’ M and ICI 164.384 at 1O-6 M. Discussion The transfection of expression vectors containing a reporter gene into cultured cells facilitates the characterization of regulatory elements such as promoters and enhancers. Transformed or tumor cells are extensively used for such experiments. Information concerning the transfection of normal cells in culture is very limited. Transient transfection was developed in this study, using either subcultured glandular epithelial or stromal endometrial cells. Subculture was chosen rather than primary culture in order to control cell seeding in each dish and thus to obtain transfection efficiency which was not greatly dependent on the cell number. As reported by several authors, a too high cell density leads to a decrease of transfection efficiency. Thus, subcultures were performed to obtain subconfluent cells at the time of transfection, i.e. 5 x lo5 stromal cells or 4 x 10’ GE cells per 35 mm well. The epithelial nature of the cells was confirmed by immunocytochemical staining of cytokeratin, a marker for epithelial cells (Gentola et al., 1984; Benali et al., 1989). The stromal cells are of mesenchymal origin (Gentola et al., 1984) and were stained for vimentin intermediate filaments (Glasser and Julian, 1986). The specificity of immunostaining was confirmed in guinea-pigs uterus (Mahfoudi et al., 1991). The immunostaining was performed on both primary cultures and subcultures and revealed that the purity of the cell population was increased in subculture. The calcium phosphate-DNA transfection method was chosen because it is simple, sensitive and easy to perform. It was concluded that, for transfection of primary pituitary cell cultures, calcium phosphate precipitation technique was similar in efficiency to lipofection (Burrin and Jameson, 1989). To determine if our model was suitable for the study of gene regulation by estrogens, we transfected the cultured endometrial cells with EREtk-CAT plasmids. In previous studies, these plasmids had been transfected either in MCF-7 cells

(Martinez et al., 1987) or in Drosophila Schneider SL-3 cells (Martinez et al., 1991) and the effect of E,-17P tested. Their results have shown that ERE wt and Bl ERE were active EREs (6and 12-fold induction respectively in MCF-7 cells) and that ERE A2 and A.5’ Bl ERE were inactive or partially inactive EREs. The results of our experiments using GE or stromal endometrial cells, which had a high estrogen receptor content at the time of transfection, agreed with Martinez et al.‘s study: E2-17P stimulated ERE wt-tk-CAT (from 5- to 6.5-fold induction in stromal cells and 10.3-fold induction in GE cells) and Bl ERE-tkCAT gene expression (4.5- and 6.1-fold induction in stromal and GE cells respectively) and did not affect expression of CAT genes dependent on ERE A2 or A5’ Bl ERE. Thus it would appear that in subcultured endometrial cells, E2-17P is able to induce a CAT gene if this gene is under the control of a functional ERE. The Ez-17P effect on CAT gene expression would be due to the enhancer capacity of ERE wt and Bl ERE and not to a stimulation of the tk promotor since there was no increase in CAT activity when inactive EREs were linked to the CAT gene. The induction of the ERE directed CAT genes was dependent on estrogenic activity since it was abolished by ICI 164,384, DES was as effective as E,-17P and E,-17cu was ineffective. It is believed that subcultured GE or stromal cells can now be used as a model system for investigating the molecular mechanisms of E,-17P on different promoters, particularly those of early regulatory genes such as c-fos gene. Up until now, to investigate the regulatory region of the c-fos gene that mediates transcriptional activation by estrogens, recombinants containing segments of the 5’ region linked to a CAT reporter have been transfected into tumoral cell lines such as HeLa uterine cervix cancer cells (Weisz et al., 1990) and MCF-7 human breast cancer cells (Hyder et al., 1991). In guinea-pig endometrial cells in primary culture (Jouvenot et al., 1990; Pellerin et al., 1992) the regulation of cellular c-fos gene by E,-17j3 has been different from that observed in other in vitro models particularly human breast cancer cells (Van Der Burg et al., 1989). Comparative studies of the E,-17P effects on a defined fos-CAT recombinant in both endometrial cell

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cultures and tumoral cell lines could way of investigating these discrepancies.

thus be a

Acknowledgments The authors wish to thank W. Wahli for the kind gift of the ERE-tk-CAT plasmids and for helpful discussion, G. Panneton for photographic work. This research was supported by fellowships from ‘La Ligue Nationale Contre le Cancer’ and the ‘Minis&e de la Recherche et de la Technologie’, and funds from the ‘Institut National de la Sante et de la Recherche Medicale’ and the ‘Association pour la Recherche sur le Cancer’. References Alkhalaf, M., Chaminadas, G., Propper, A.Y. and Adessi, G.L. (1987) Exp. Clin. Endocrinol. 89, 201-210. Alkhalaf, M., Propper, A.Y. and Adessi, G.L. (1991) J. Steroid Biochem. Mol. Biol. 38, 345-350. Benali, R., Dupuit, F., Jaquot, J., Fuchey, C., Hinnrasky, J., Ploton, D. and Puchelle, E. (1989) Biol. Cell 66, 263-270. Burrin, J.M. and Jameson, J.L. (1989) Mol. Endocrinol. 3, 1643-1651. Chen, L., Lindner, H.R. and Lancet, M. (1973) J. Endocrinol. 59, 87-92. Di Augustine, R.P., Petrusz, P., Bell, G.I., Brown, CF., Korach, K.S., McLachlan, J.A. and Teng, C.T.I. (1988) Endocrinology 122, 2355-2363. Dickson, R.B. and Lippman, M.E. (1987) Endocr. Rev. 8, 29-43. Gentola, G.M., Cisar, M. and Knab, D.R. (1984) In Vitro Cell. Dev. Biol. 20, 451-461. Glasser, S.R. and Julian, J. (1986) Biol. Reprod. 35, 463-467.

Graham, F.L. and Van Der Eb, A.J. (1973) Virology 52, 456-467. Green, S. and Chambon, P. (1988) Trends Genet. 4,309-314. Hyder, S.M., Stance], G.M. and Loose-Mitchell, D.S. (1991) Steroids 56, 4988503. Inaba, T., Wiest, W.G.. Strickler, R.C. and Mori, J. (1988) Endocrinology 123, 1253-1258. Jouvenot. M., Pellerin. I., Alkhalaf, M., Marechal, G., Royez, M. and Adessi, G.L. (1990) Mol. Cell. Endocrinol. 72, 149-157. Lee, A.E. and Dukelow, W.R. (1972) J. Reprod. Fertil. 9, 473-476. Mahfoudi, A., Nicollier, M., Propper, A.Y., Coumes-Marquet. S. and Adessi, G.L. (1991) Biol. Cell 71, 255-265. Martinez, E., Givel. F. and Wahli, W. (1987) EMBO J. 6. 3719-3727. Martinez, E.. Givel, F. and Wahli. W. (1991) EMBO J. 10. 263-268. Murphy, L.J.. Murphy, L.C. and Friesen, H.G. (1987a) Mol. Endocrinol. 1, 445-450. Murphy, L.J., Murphy, L.C. and Friesen, H.G. (1987b) Endocrinology 120, 1882-1888. Norton, P.A. and Coffin, J.M. (1985) Mol. Cell Biol. 5. 281290. Pellerin, I., Vuillermoz, C., Jouvenot, M., Royez, M., Ordener, C., Marechal, G. and Adessi, G. (1992) Endocrinology 131 (in press). Seed, B. and Sheen, J.Y. (1988) Gene 67, 271-277. Taylor, C.M., Blanchard, B. and Zava, D.T. (1984) J. Steroid Biochem. 20, 1083-1088. Van Der Burg, B., Van Selm-Miltenburg, A.J.P.. De Laat, S.W. and Van Zoelen, E.J.J. (1989) Mol. Cell. Endocrinol. 64, 223-228. Weisz, A. and Bresciani, F. (1988) Mol. Endocrinol. 2. 816824. Weisz, A. and Rosales, R. (1990) Nucleic Acid Res. 18, 5097-5106. Weisz, A., Cicatiello, L., Persico, E.. Scalona, M. and Bresciani, F. (1990) Mol. Endocrinol. 4, 1041-1050.

Transfected endometrial cultured cells: a system to study gene-regulation by estrogens.

Glandular epithelial (GE) and stromal cells were isolated from guinea-pig endometrium, cultured and subcultured separately. At the end of subculture, ...
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