Mullerian duct regression and antiproliferative bioactivities of mullerian inhibiting substance reside in its carboxy-terminal domain.
A 25-kilodalton dimeric carboxy-terminal fragment of the recombinant human Mullerian inhibiting substance protein (rhMIS) was produced by proteolytic cleavage with plasmin and purified by size-exclusion chromatography. The identity of the isolated dimer as the carboxy-terminal fragment was confirmed by gel electrophoresis and Western analysis. As was true of every sample of the holo molecule, all preparations of the carboxy-terminal domain of rhMIS (n = 10), when added in the 0.5-5.0 micrograms/ml range, exhibited a dose-dependent partial to complete regression of the 14.5-day fetal rat Mullerian duct in an organ culture assay. The carboxy-terminal dimer also inhibited, in a dose-dependent manner, the growth of A431 cells in monolayer cultures. Daily addition of 5, 10, or 20 micrograms carboxy-terminus for 3 days resulted in 0%, 25%, and 100% inhibition of cell proliferation, respectively. Similar and higher doses of holo rhMIS had no or inconsistent antiproliferative activity (0-34% inhibition), even though the preparations caused Mullerian duct regression. All amino-terminal fragments prepared using this separation protocol were found to be inactive in these assays. These findings suggest that the bioactivity of rhMIS as a regressor of fetal Mullerian ducts and an inhibitor of A431 cell growth resides in its carboxy-terminal domain. These results indicate that the urogenital ridge tissue, but not A431 cells in culture, may be capable of cleaving intact MIS to a biologically active conformation.
MacLaughlin
D T
DT
Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Boston 02114.
Hudson
P L
PL
Graciano
A L
AL
Kenneally
M K
MK
Ragin
R C
RC
Manganaro
T F
TF
Donahoe
P K
PK
D000595:Amino Acid Sequence; D000818:Animals; D054304:Anti-Mullerian Hormone; D015153:Blotting, Western; D016466:CHO Cells; D002455:Cell Division / Q000187:drug effects; D006224:Cricetinae; D005260:Female; D006023:Glycoproteins; D006131:Growth Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D009095:Mullerian Ducts / Q000187:drug effects / Q000502:physiology*; D009924:Organ Culture Techniques; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D013735:Testicular Hormones / Q000737:chemistry / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D014846:Vulvar Neoplasms / Q000473:pathology
D006023:Glycoproteins; D006131:Growth Inhibitors; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013735:Testicular Hormones; D054304:Anti-Mullerian Hormone