CHARACTERIZATION OF AN OVARIAN-CANCER ACTIVATING FACTOR IN ASCITES FROM OVARIAN-CANCER PATIENTS

Ascites from ovarian cancer patients contain potent growth-promoting a ctivity toward human ovarian cancer cells both in vitro and in vivo. T his activity is associated with rapid increases in cytosolic free calc ium ([Ca2+](i)) as a consequence of phosphoinositide hydrolysis. In th is study, we describe the purification, characterization, and identifi cation of an ovarian cancer activating factor (OCAF) from ascites of o varian cancer patients. We have isolated OCAF by a combination of solv ent extraction, silica gel chromatography, and TLC. Mass spectral anal ysis, phospholipase sensitivity, and gas chromatographic behavior of p urified OCAF indicate that OCAF is composed of various species of lyso phosphatidic acid (LPA), including LPAs with polyunsaturated fatty acy l chains (linoleic, arachidonic, and docosahexaenoic acids). However, OCAF is more potent than sn-l palmitoyl, oleoyl, or stearoyl LPA in in creasing [Ca2+](i) in ovarian cancer cells. The ability of OCAF to alt er [Ca2+](i) is sensitive to the effects of lipoxidase, whereas the ac tivity of sn-l oleoyl, stearoyl, or palmitoyl LPA is not, suggesting t hat polyunsaturated bonds in the fatty acyl chain of OCAF may account for its increased ability to activate ovarian cancer cells. Furthermor e, a sn-2 linoleoyl LPA generated by phospholipase A(1) treatment of s ynthetic phosphatidic acid is much more active than are sn-1 palmitoyl , stearoyl, or oleoyl LPA in increasing [Ca2+](i) in ovarian cancer ce lls. Taken together, these data suggest that the ability of OCAF to in crease cellular calcium may reside in the structure and/or location of the fatty acyl chain of LPA. Purified OCAF, at concentrations similar to those present in ascites from ovarian cancer patients, was suffici ent to induce proliferation of ovarian cancer cells, as indicated by t hymidine incorporation, reduction of (4,5-dimethylthiazol-2-yl)-2,5-di phenyltetrazolium bromide, or colony formation. However, even at optim al concentrations of OCAF, proliferation was lower than that induced b y FCS or ascites from ovarian cancer patients, indicating that, althou gh OCAF may be a major regulator of ovarian cancer cells in vivo, it i s not the sole mediator present in ascites, and it likely functions in concert with other growth factor activities.