Creation of immortalised epithelial cells from ovarian endometrioma

Endometriosis is a common gynaecological disorder associated with dysmenorrhoea, pelvic pain and subfertility and is a leading cause of disability and loss of productivity in women of reproductive age (Olive and Schwartz, 1993). Numerous studies have attempted to dissect the biology of endometriosis. These studies mainly use in vitro culture of stromal cells rather than culture of epithelial cells from endometriotic tissues, because the former cells are more easily and stably cultured for much longer periods than the latter cells (Noble et al, 1997; Gurates et al, 2002). In fact, it is difficult to culture endometriotic epithelial cells in vitro, because these cells lose their proliferative capacity during ongoing cultivation of primary cultures over several days. The inability of endometriotic epithelial cells to survive in vitro is an obstacle to gaining a better understanding of the biology of this disease. In particular, malignant change of endometriosis, especially of ovarian endometrioma, for which epithelial cells are exclusively responsible, has lately attracted considerable clinical attention (Kurman and Craig, 1972; McMeekin et al, 1995; Ness, 2003; Oral et al, 2003). There is therefore an urgent need to establish a stable system for the culture of endometriotic epithelial cells that can be used for research not only into the biology of endometriosis but also into its carcinogenesis. There are two major barriers within epithelial cells that inhibit cell division under usual culture conditions: premature senescence and telomere-dependent senescence (Kiyono et al, 1998). The former is observed during early passage in primary culture and is caused by the activation of Rb that leads to cell cycle arrest, whereas the latter is found at a later stage of culture and is caused by telomere shortening after a considerable number of cell divisions. Inhibition of both Rb function and telomere shortening is therefore required for long-term culture of epithelial cells. In previous studies, we successfully established a stable system for the culture of primary endometrial epithelial cells, in which the human papillomavirus type 16 E6/E7 genes and the human telomerase reverse transcriptase (hTERT) were introduced to inhibit Rb functions and to activate telomerase, respectively (Kyo et al, 2003). These immortal cells were not transformed but retained the original characteristics of endometrial epithelial cells, such as steroid responsiveness. Subsequent studies have demonstrated that overexpression of cyclin D1 or cdk4, instead of HPV E6/E7, effectively inhibited Rb activity and might be an alternative method of overcoming premature senescence in primary epithelial cells of other origins (Ramirez et al, 2004; Sasaki et al, 2009). In the present study, we sought to generate a stable culture of epithelial cells isolated from the ovarian endometriomas by the introduction of various genetic elements. These cells were successfully immortalised without generation of transformed phenotypes and were responsive to progestin and oestrogen. These cells are thus potentially useful as an experimental model for analysis of the mechanisms of steroid hormone functions as well as of carcinogenesis arising from ovarian endometrioma.