Differentiation and growth potential of human ovarian surface epithelial cells expressing temperature-sensitive SV40 T antigen

The epithelial ovarian carcinomas arise in the ovarian surface epithelium ( OSE) which is the mesothelial covering of the ovary. Studies of human OSE h ave been hampered by the small amounts and limited lifespan of this epithel ium in culture. OSE cells expressing SV40 large T antigen (Tag) or the HPV genes E6 and E7 have increased growth potentials but lack some of the norma l characteristics of OSE. In this study, we used conditional SV40 Tag expre ssion to produce OSE cells with increased proliferative potentials but rela tively normal phenotypes. Primary OSE cultures from three women, one of who m had a BRCA1 mutation, were infected with a temperature-sensitive Tag cons truct (tsTag), and from these, 28 monoclonal and four polyclonal lines were isolated. The effects of temperature changes were examined in two monoclon al and two polyclonal lines. At the permissive temperature (34 degreesC), t hese cell lines underwent 52-71 population doublings (PD) compared to 15-20 PD for normal OSE. Nuclear SV40-Tag and p53 expression., demonstrated by i mmunofluorescence, showed that tsTag was uniformly present and biologically active in all lines. At 34 degreesC, culture morphologies ranged from epit helial to mesenchymal. The mean percentage of cells expressing the epitheli al differentiation marker, keratin, varied between lines from 20 to 97%. Co llagen type III, a mesenchymal marker expressed by OSE in response to expla ntation into culture, was present in 24-43% of cells. At 39 degreesC, tsTag was inactivated by 2 d while nuclear p53 staining diminished to control le vels over 2 wk. Over 3 d, the cells assumed more epithelial morphologies, k eratin expression reached 85-100% in all lines and collagen expression incr eased significantly in two lines. The cultures with the BRCA1 mutation expr essed the most keratin and the least collagen III at both temperatures. As indicated by beta -galactosidase staining at pH 6.0, changes leading to sen escence were initiated at 39 degreesC by 6 h and were present in all cells after 24 h. However, the cells underwent 1-3 population doublings over up t o 1 wk before growth arrest and widespread cell death, thus providing an ex perimental system where large numbers of OSE cells with different genetic b ackgrounds and growth potentials can be studied without the concurrent infl uence of Tag.