The investigations focus on the characterization of an endogenous growth factor produced by malignant melanoma cells and the description of the early expression of this growth factor in premalignant nevi. An autostimulatory growth factor for melanocytes has been isolated and partially characterized. This melanoma growth stimulatory activity (MGSA) resides in a family of antigenically related, acid and heat stable polypeptides which appear to be different from other previously described growth factors. MGSA is not detectable in chromosomally normal, inactive nevi, but nevi exhibiting chromosomal abnormalities are MGSA positive. MGSA can be purified from acetic acid extracts of lyophilized culture medium conditioned by the Hs0294 human melanoma cell line. When this extract is subjected to molecular sieve chromatography, RP-HPLC and preparative gel electrophoresis, low (greater than 14-16Kd) and high (24-26Kd) molecular weight forms of MGSA can be isolated. MGSA is separable from the 125I-EGF competing Class I TGF activity which is also produced by this cell line. MGSA can also be purified by immunoaffinity chromatography using a monoclonal antibody to MGSA, followed by gel filtration HPLS. When immunoprecipitates from 35S-methionine labeled extracts of Hs0294 cells were subjected to reducing SDS-PAGE followed by autoradiography, the major labeled bands had a Mr of greater than 20Kd. Efforts will now be directed toward: (1) determining the relationship between below the low and high molecular weight forms of MGSA and developing an invitro translation system to determine the nature of the precursor form of MGSA; (2) developing a radioreceptor assay for MGSA; (MGSA will be purified by the methods described above, iodinated using Bolton-Hunter reagent and binding assays will be developed using Hs0294 and NRK cells) (3) characterizing the MGSA receptor in isolated plasma membrane preparations using 125I-MGSA and the bifunctional cross-linking reagent, disuccinimidyl suberate; (4) comparing the cellular distribution of bioactive and immunoreactive MGSA and the distribution of MGSA receptors in normal nevi, malignant melanomas, and non-malignant and non-melanoma malignant controls. Nevus, melanoma and other tumor cells will be cultured in vitro, MGSA production will be determined by immunohistochemical and bioactivity assays, and MGSA receptor distribution will be evaluated using the MGSA-radioreceptor assay. Comparisons will be made regarding MGSA binding versus biological response to MGSA in these cultures.
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