CLEAVAGE OF MULLERIAN-INHIBITING SUBSTANCE ACTIVATES ANTIPROLIFERATIVE EFFECTS IN-VIVO

Mullerian inhibiting substance (MIS), an inhibitor of growth and devel opment of the female reproductive ducts in male fetuses, requires prec ise proteolytic cleavage to yield its biologically active species. Hum an plasmin is now used to cleave and, thereby, activate immunoaffinity purified recombinant human MIS at its monobasic arginine-serine site at residues 427-428. To avoid the need for exogenous enzymatic cleavag e and to simplify purification, we created an arginine-arginine dibasi c cleavage site (MIS RR) using site-directed mutagenesis to change the serine at position 428 (AGC) to an arginine (cGC). The mutant cDNA wa s then stably transfected into a MIS-responsive ocular melanoma cell l ine, OM431, followed by cloning for amplified expression to test its b iological activity in vitro and in vivo. Media from each clone were as sayed for production of MIS RR by a sensitive ELISA for hole-MIS, and high- and low-producing clones were selected for further study. Media from the highest MIS RR producer caused Mullerian duct regression in a n organ culture bioassay. Other transfections were done with an empty vector (pcDNAI Neo) or a construct lacking the leader sequence and thu s failing to secrete MIS, to serve as controls. The OM431 clones conta ining the MIS RR mutant were growth inhibited in monolayer culture. Th e high- and low-producing MIS RR OM431 clones, along with transfected OM431 controls, were injected into the tail veins of immunosuppressed severe combined immunodeficiency mice for in vivo analyses. Four to 6 weeks later, pulmonary metastases were counted in uniformly inflated l ungs, OM431 clones containing the more easily cleaved MIS RR displayed a significant dose-dependent reduction in pulmonary metastases when c ompared to the lungs of animals given injections of OM431 clones conta ining empty vector, leaderless MIS, or wild-type MIS that requires act ivation by plasmin cleavage. Since the purification protocol of MIS RR is less complicated than that for wild-type MIS, which requires subse quent enzymatic activation, MIS RR can be used for scale-up production with increased yields for further therapeutic trials against MIS-sens itive tumors.