Mullerian Inhibiting Substance, a product of the fetal testic, is a large molecular weight glycoprotein which initiates regression of the Mullerian duct, the anlagan of the uterus, fallopian tube, and upper vagina. The underlying hypothesis of this proposal is that a fetal regressor which initiated morphogenic dissociation of an organ system in the embryo may produce the same effects against tumors derived from the cell types which respond to the fetal regressor in embryonic life. Mullerian Inhibiting Substance is detected by an embryonic organ culture assay and has been purified from the newborn calf testis. Using sequential ion exchange, carbohydrate affinity and dye affinity chromatography steps, we have achieved a 30,000-fold purification from starting testes and 2,000-fold from incubation media with 65% yield. Using hybridoma technology, we have developed monoclonal antibodies which both absorb out and block biological activity and from which we have eluted almost completely purified biologically active Mullerian Inhibiting Substance. Initial attempts at scale-up production (10 x) from 1,000 newborn calf testes were successful and will be continued from both bovine and avian sources. The availability of more purified material with MIS bioactivity makes broader oncological studies feasible. We propose to use purified MIS to study its effect against post-transplant, human ovarian tumor, as well as endometrial and cervical carcinomas, in nude mice and in the subrenal capsule assay. We continue to harvest fresh tumor from patients with ovarian and endometrial carcinoma to test their sensitivity to MIS. We have perfected PAP techniques and have localized MIS to the Sertoli cell endoplasmic reticulum and golgi apparatus. We will now attempt to localize MIS receptors with either monoclonal antibodies, antiidiotypic antibodies, polyclonal antibodies, or receptor antibodies. Phosphorylation events, which appear to be involved in Mullerian duct regression, will be studied both in the embryo and in the human ovarian cell line to investigate the mechanisms of MIS action and to propose newer chemotherapeutic strategies. We plan to study the ontogeny of steroid receptors both autoradiographically and biochemically during Mullerian duct regression and to correlate these events with the steroid modulation of MIS that we have observed. These findings will be applied to modulation of animal tumor hosts prior to treatment of Mullerian duct tumors with Mullerian Inhibiting Substance.
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