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. 2012 Feb 21;106(6):1205–1213. doi: 10.1038/bjc.2012.26

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Copyright © 2012 Cancer Research UK

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Sex steroid-receptor expression in, and aromatase activity of, immortalised epithelial cells from ovarian endometrioma. (A) RT–PCR analysis of expression of the oestrogen receptor α (ERα) or the progesterone receptor B (PRB). EM-E6/E7/TERT/ER cells are immortalised endometrial epithelial cells in which ERα cDNAs were stably transfected and were used as a positive control for ERα. EM-E6/E7/TERT/PRB cells are immortalised endometrial epithelial cells in which PRB cDNAs were stably transfected. Because our primer sets for PRB were designed to amplify the sequences containing PRB gene promoter in order to distinguish from PRA transcript, they can detect only intrinsic PRB mRNA but not extrinsic, overexpressed PRB mRNA that lacks promoter sequences. The weak PRB band in EM-E6/E7/TERT/PRB cells is therefore derived from intrinsic PRB. BJ cells were used as a negative control for ERα and PRB expression. GAPDH was used as a loading control. (B) Western blot analysis of expression of the progesterone receptor. EM-E6/E7/TERT/PRA or EM-E6/E7/TERT/PRB cells are immortalised endometrial epithelial cells in which PRA or PRB cDNAs were stably transfected and were used as a positive control for PRA or PRB expressions, respectively. Although EM-E6/E7/TERT/PRA cells showed a clear PRA band by western blotting (94 kDa), EM-E6/E7/TERT/PRB cells displayed two bands; one band was of the expected size of intact PRB (114 kDa); the other band was located just below the PRA band (identified by the symbol: ★) and was not a PRA band but a degraded PRB band, which was confirmed by another western blot analysis using a PRB-specific antibody (data not shown). EMosis-CC/TERT1 cells exhibited a weak, but distinct, PRB band but not a PRA band. M: protein weight marker. (C) Western blot analysis of expression of the ER. There was no detectable protein expression of ERα in EMosis-CC/TERT1 or EMosis-CC/TERT2 cells. EMosis-CC/TERT1/ER cells, generated by the introduction of ERα cDNA into EMosis-CC/TERT1 cells, were confirmed to have significant ERα expression. MCF7 cells were used as a positive control of ERα expression. (D) Analysis of aromatase activity using a tritiated water assay. Primary endometriotic stromal cells isolated from the ovarian endometrioma of another patient were used as a positive control of aromatase activity. Both EMosis-CC/TERT1 and EMosis-CC/TERT2 cells lacked aromatase activity.