Ms. Kurian et al., CLEAVAGE OF MULLERIAN-INHIBITING SUBSTANCE ACTIVATES ANTIPROLIFERATIVE EFFECTS IN-VIVO, Clinical cancer research, 1(3), 1995, pp. 343-349
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.