Analysis of mechanism of estrogen action on estrogen receptor cDNA transfected endometrial cancer cells

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1. Purpose: This study was designed to establish estrogen responsive human endometrial cancer cell lines positive for estrogen receptor (ER) by trasfecting ER expression vector, and to investigate a mechanism of estrogen action.2. Methods: Ishikawa cell line was used for the study. It was originally was positive for ER but it became less responsive to estrogen, as the level of ER was gradually decreased. pSG5-HEGO was used as ER expression vector that contained human ER cDNA, wild type. Superfect method was carried out to transfect the vector into the cells. ER transfected cells were cloned by using Neomycin. The levels of ER expression was measured by immunoblotting or enxyme-linked immunoassay. Analysis of cell proliferation was performed by cell growth curve. Effects of estrogen on regulation of cell cycle regulators were examined by immunostaining and Western blotting.3. Results: ER level of null Ishikawa cells was very low. Thirty six clones were analyzed and three of them expressed high level of ER. Maximal effect of estrogen on cell proliferation was observed at a concentration of 10nM. An 20% increased of growth was detected on Day 6. Levels of Ki-67, Cyclin A, D1, E was also enhanced by estrogen.4. Conclusion: Stable endometrial cancer cell lines that express high ER and are responsive to estrogen were established. They could be useful to investigate the molecular mechanism of estrogen action.