Mullerian Inhibiting Substance (MIS) is a gonadal factor with diverse differentiative and antiproliferative effects. The most effective MIS congener will be identified and employed as an anticancer agent and taken to clinical trials. We propose to clone the MIS receptor gene and to characterize the MIS receptor. The factors which regulate MIS expression in vivo and their role in gonadal development and hence sexual differentiation will be deciphered. Progress made during the past grant period will allow these goals to be accomplished. Among these advances are the ability to produce sufficient recombinant human MIS (rhMIS) for analytical experimental use, the discovery of and ability to label a biologically active domain of the rhMIS molecule, the ability to produce rhMIS preparations that give reproducible antiproliferative effects, the elucidation of cis regulatory elements and trans regulatory factors of the MIS gene and their role in gonadal differentiation, and the construction of several cDNA libraries from MIS responsive cell lines and tissues, for receptor cloning. We will produce holo rhMIS and its amino and carboxy-terminal domains, then determine and generate quantities of the most biologically active form of rhMIS sufficient for clinical trials against gynecological cancers with poor survival. Mutants that simplify proteolytic processing of nascent rhMIS to release the biologically active carboxy-terminal region of MIS, will be designed. To this same end, techniques will be devised to reactivate monomer MIS derived from E. coli. Using exogenous MIS and MIS transfected cell lines, we will explore the intracellular signals and MIS regulated genes which lead to its antiproliferative and differentiative effects. Radiolabelled carboxy-terminal rhMIS will be used to screen the several expression libraries produced from MIS responsive sources, to clone the MIS receptor. An alternative strategy is being employed which uses nucleotide primer sequences designed from the conserved regions of other recently cloned receptors, e.g., Activin and TGF-beta, members of the same gene family as MIS. The receptor gene will be stably transfected and the protein expressed for subsequent purification and characterization. Tumor response to MIS will be correlated with the presence of MIS receptor. MIS plays an important role in assuring sexual dimorphism during mammalian differentiation, hence we are studying regulation of MIS gene expression, with particular emphasis on the role of the testis determining factor, SRY, and of novel MIS transacting factors. Understanding MIS gene regulation may provide a means of activating the expression of MIS in tumors as a therapeutic strategy. The proposed studies are designed to give new insights into the role of MIS in normal development and to delineate its therapeutic applications.
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