Malignant melanoma is a very serious disease with a relatively poor prognosis. Melanoma growth stimulatory activity (MGSA) is a growth regulatory protein which is over-expressed in melanoma and several other human tumors. Characterization of the mechanism by which MGSA acts may lead to new methods of therapeutic intervention for those tumors which overexpress identical with the human gro gene and closely related, if not identical, to the human PDGF inducible KC gene. The mature 73 amino acid protein is apparently derived form proteolytic cleavage of a 107 amino acid precursor. Antagonism of MGSA bioactivity with antibodies to MGSA slows the growth of Hs294T melanoma cells. When the MGSA/KC/gro DNA is transfected into Chinese hamster ovary cells, conditioned medium from these cultures has MGSA bioactivity which can be blocked with antibody to MGSA. 125I-MGSA binds to Hs294T melanoma cells and a number of other cell types which exhibit a bioresponse to MGSA, including PC3 prostatic carcinoma, NRK fibroblast, Calu 6, and A427 lung carcinoma, Balbc/3T3 fibroblasts, MeWo melanoma and nervus 106 cultures. 125I-MGSA disuccinimidyl suberate crosslinking studies reveal that MGSA binds to membrane associated proteins of 45,000, 64,000 and 184,000 Mr in Hs294T cells. For some cells this binding event results in phosphorylation of an 80,000 Mr protein on ser/thr. Experiments described here aim to (1) determine the effects of over-expression of MGSA on the growth of nontransformed cells [MGSA DNA will be transfected into nevus and fibroblast cultures. Transfectants will be studied for changes in double times, growth in serum-free medium, tumor formation in nude mice, and induction of collagen, laminin, and fibronectin synthesis]. (2) examine the effects of MGSA antagonist [including antisense MGSA oligonucleotides, antisense MGSA DNA transfection, and liposome mediated transfection with antibodies to MGSA] on growth of cells overexpressing MGSA. (3) utilize ligand affinity chromatography to purify the MGSA receptor [Recombinant MGSA will be purified in sufficient quantity to utilize ligand affinity purification and to obtain enriched MGSA- receptor preparations. These enriched preparations will be further purified by RP-HPLC and/or gel filtration and ion exchange HPLC and antibodies to the receptor will be raised in the rabbit.] (4) determine the signal transduction mechanism for MGSA [specifically, tyrosine kinase, protein kinase C, Ca++, inositol lipids cAMP dependent protein kinase, and G-proteins will be studied].
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