Differential growth factor production, secretion, and response by high and low metastatic variants of B16BL6 melanoma
Blanckaert, V.D.; Schelling, M.E.; Elstad, C.A.; Meadows, G.G.
Cancer Research 53(17): 4075-4081
1993
ISSN/ISBN: 0008-5472 PMID: 8358737 Document Number: 421004
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 beta-like (TGF-beta-like) molecules as well as the biological effect of basic FGF (bFGF) and TGF-beta-1 on high (NDP) and low (LTP) metastatic variants of B16BL6 melanoma. Both NDP and LTP cells expressed bFGF- like and TGF-beta-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 37,000 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-TGF-beta antibody that recognized both TGF-beta-1 and TGF-beta-2 detected 3 different TGF-beta-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 TGF-beta; 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 TGF-beta-1 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 TGF-beta-1. 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 TGF-beta-1 and various amounts of bFGF, the effect of TGF-beta-1 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 TGF-beta-like molecules resulted in increased proliferation of LTP cells but did not affect NDP cells. These data suggest (a) that bFGF-like and/or TGF-beta-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 TGF-beta-like molecules may contribute to a decreased metastatic capability.