DIFFERENTIAL GROWTH-FACTOR PRODUCTION, SECRETION, AND RESPONSE BY HIGH AND LOW METASTATIC VARIANTS OF B16BL6 MELANOMA

Low levels of tyrosine and phenylalanine alter the metastatic phenotyp e of B16BL6 murine melanoma. In this study, we investigated expression and secretion of fibroblast growth factor-like (FGF-like) and transfo rming growth factor beta-like (TGFbeta-like) molecules as well as the biological effect of basic FGF (bFGF) and TGFbeta1 on high (NDP) and l ow (LTP) metastatic variants of B16BL6 melanoma. Both NDP and LTP cell s expressed bFGF-like and TGFbeta-like polypeptides as detected by Wes tern blot analysis. An M(r) 29,000 bFGF-like form eluted from heparin- Sepharose by 0.6 M NaCl was found in extracts of both NDP and LTP cell s. Elution at 0.6 M NaCl suggested that this M(r) 29,000 form might be more closely related to FGF-5 than to bFGF. In addition, cell extract s of LTP, but not NDP cells, contained an M(r) 47,000 monomeric bFGF-l ike form that was not retained on heparin-Sepharose. Three major speci fic immunoreactive forms of M(r) 44,000, 36,000, and 29,000 were prese nt in conditioned medium from NDP cells. The M(r) 29,000 form present in the conditioned medium of NDP cells was retained on heparin-Sepharo se. Only the M(r) 44,000 and 36,000 FGF. like molecules were detected in conditioned medium from LTP cells, and they were also not retained on heparin-Sepharose. Anti-TGFbeta antibody that recognized both TGFbe ta1 and TGFbeta2 detected 3 different TGFbeta-like forms (M(r) 25,000, 23,000 and 22,000) in NDP and LTP cell extracts. Conditioned medium f rom NDP cells contained an M(r) 38,000 form of TGFbeta; however, no im munoreactive forms were found in conditioned medium from LTP cells. Th us, the NDP-LTP differences in this melanoma system were primarily in growth factor secretion, not expression. The effect of exogenous bFGF and TGFbeta1 on proliferation of LTP and NDP cells was determined by [ methyl-H-3]thymidine uptake. bFGF stimulated proliferation of NDP cell s; whereas, LTP cells exhibited no increase in proliferation. Both NDP and LTP cells responded to TGFbeta1. Proliferation of NDP cells was i nhibited more by this growth factor than was proliferation of LTP cell s. When NDP and LTP cells were incubated with 5 ng/ml TGFbeta1 and var ious amounts of bFGF, the effect of TGFbeta1 was masked. Antibody depl etion of bFGF-like molecules from NDP conditioned medium resulted in t he decreased proliferation of NDP cells but not LTP cells. Depletion o f TGFbeta-like molecules resulted in increased proliferation of LTP ce lls but did not affect NDP cells. These data suggest (a) that bFGF-lik e and/or TGFbeta-like molecules are important markers of the metastati c phenotype of murine B16BL6 melanoma and (b) that the inability of B1 6BL6 melanoma cells to secrete FGF-like and TGFbeta-like molecules may contribute to a decreased metastatic capability.