In most self-renewing tissues, one control point in the regulation of cell growth is availability of hormones and/or growth factors produced by other tissues. One method by which cancer cells may achieve autonomous growth is through production of autostimulatory growth factors. We have found that Hs0294 human melanoma cell line produces at least two growth stimulatory activities: a monolayer mitogen (MGSA) and a class I transforming growth factor (TGF-alpha). The monolayer mitogen is at least partially responsible for the growth of Hs0294 cells in serum-free medium. The melanoma growth stimulatory activity (MGSA) produced by this mitogen is autostimulatory in that it produces an increased incorporation of [?3?H]-thymidine into DNA and an increase in cell number in the Hs0294 cells which produce it. MGSA is stable to heat (100~C/10 min) and acid (IN acetic acid), is sensitive to dithiothretol and trypsin, and has no associated proteolytic activity. MGSA can be purified by molecular sieve chromatography, followed by RP-HPLC and preparative electrophoresis. In RP-HPLC the major peak of MGSA elutes from a mu-Bondapak C?18? column at 35+3% acetonitrile and is separated from the TGF-alpha activity eluting at 30+4% acetonitrile which does not compete with [?125?I]-EGF for binding to EGF receptors. When RP-HPLC purified MGSA is subjected to preparative electrophoresis under non-reducing conditions, the major bioactivity is recovered from a 16 kilodalton moiety. Monoclonal antibodies have been raised to a heterogenous preparation of MGSA and antibodies produced by five of the hybridoma clones will inhibit the growth of Hs0294 cells. An enzyme-linked immunoabsorbent assay developed with one of the antibodies, FB2AH7, shows peaks of immunoreactive protein coincident with the major MGSA peak. Immunoaffinity columns prepared with F82AH7 antibody bind the 16 kilodalton MGSA moiety. Our studies will now be directed toward: (1) characterizing the MGSA receptor; (2) determining the cellular specificity of production and response to MGSA; and (3) determination of effects of MGSA and antibodies to MGSA on the growth of human melanoma tumors growing in nude mice. (J)
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