Endothelin expression and responsiveness in human ovarian carcinoma cell lines

To elucidate the potential role of endothelins (ETs) as growth regulators in ovarian carcinoma cells in culture, expression of endothelins and their receptors were measured in two ovarian cancer cell lines (PEO4 and PEO14), together with the effect of the exogenous addition of endothelins on the growth of these cell lines in vitro. RT-PCR analysis of mRNA prepared from PEO4 and PEO14 indicated the presence of ET-1 and ET-3 mRNA. Immunoreactive ET-1-like peptide was found in media from cultures of both PEO4 (1.7 +/- 0.4 fmol/10(6) cells/72 h) and PEO14 (20.2 +/- 6.8 fmol/10(6) cells/72 h) cell lines. Radioligand binding studies using 125I-ET-1 and membrane fractions were consistent with PEO4 cells having two receptor sites of either high affinity (Kd = 0.065 nM, Bmax = 0.047 pmol/mg protein) or lower affinity sites (Kd = 0.49 nM, Bmax = 0.23 pmol/mg protein). Studies using membrane fractions of PEO14 cells indicated that this cell line has only a single lower affinity binding site (Kd = 0.56 nM, Bmax = 0.31 pmol/mg protein). However, RT-PCR analysis indicated the presence of mRNA from both ETA and ETB receptors in PEO4 and PEO14 cell lines. Exogenous addition of ETs to PEO4 and PEO14 cells at concentrations of 10(-10)-10(-7)M resulted in specific dose-dependent increases in cell number for ET-1 (with maximum effects at 10(-10) and 10(-9)M for PEO4 and PEO14, respectively) and ET-2 (maximum effects at 10(-8) and 10(-9)M for PEO4 and PEO14, respectively) but not for ET-3. Experiments on the growth of PEO14 cells using BQ123 (ETA-R) antagonist and "antisense" oligonucleotide against the ETA-R, in the absence of exogenous ETs, suggested that immunoreactive ET-1-like material secreted by PEO14 cells can affect their growth in an autocrine manner. These results would be consistent with ET-1 acting as a possible autocrine growth regulator in human ovarian carcinoma cells.