Differential Growth Factor Production, Secretion, and Response by High and Low Metastatic Variants of B16BL6 Melanoma¹

Low levels of tyrosine and phenylalanine alter the metastatic phenotype of B16BL6 murine melanoma. In this study, we investigated expression and secretion of fibroblast growth factor-like (FGF-like) and transforming growth factor β-like (TGFβ-like) molecules as well as the biological effect of basic FGF (bFGF) and TGFβ1 on high (NDP) and low (LTP) metastatic variants of B16BL6 melanoma. Both NDP and LTP cells expressed bFGF-like and TGFβ-like polypeptides as detected by Western 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 cells. 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 extracts of LTP, but not NDP cells, contained an M_r 47,000 monomeric bFGF-like form that was not retained on heparin-Sepharose. Three major specific immunoreactive forms of M_r 44,000, 36,000, and 29,000 were present in conditioned medium from NDP cells. The M_r 29,000 form present in the conditioned medium of NDP cells was retained on heparin-Sepharose. 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-TGFβ antibody that recognized both TGFβ1 and TGFβ2 detected 3 different TGFβ-like forms (M_r 25,000, 23,000 and 22,000) in NDP and LTP cell extracts. Conditioned medium from NDP cells contained an M_r 38,000 form of TGFβ; however, no immunoreactive forms were found in conditioned medium from LTP cells. Thus, the NDP-LTP differences in this melanoma system were primarily in growth factor secretion, not expression. The effect of exogenous bFGF and TGFβ1 on proliferation of LTP and NDP cells was determined by [methyl-³H]thymidine uptake. bFGF stimulated proliferation of NDP cells; whereas, LTP cells exhibited no increase in proliferation. Both NDP and LTP cells responded to TGFβ1. Proliferation of NDP cells was inhibited more by this growth factor than was proliferation of LTP cells. When NDP and LTP cells were incubated with 5 ng/ml TGFβ1 and various amounts of bFGF, the effect of TGFβ1 was masked. Antibody depletion of bFGF-like molecules from NDP conditioned medium resulted in the decreased proliferation of NDP cells but not LTP cells. Depletion of TGFβ-like molecules resulted in increased proliferation of LTP cells but did not affect NDP cells. These data suggest (a) that bFGF-like and/or TGFβ-like molecules are important markers of the metastatic phenotype of murine B16BL6 melanoma and (b) that the inability of B16BL6 melanoma cells to secrete FGF-like and TGFβ-like molecules may contribute to a decreased metastatic capability.