B16 melanoma sublines (B16-F10-BL6 and B16-F1) exhibited elevated adenosine 3',5'-cyclic monophosphate (cAMP) levels when cultured in Dulbecco's modified Eagle's medium (DMEM) in comparison to cells in RPMI-1640 medium. In parallel, cells cultured in DMEM had increased tyrosinase activity, melanization and dendrite formation, all markers of melanoma differentiation. Also, the proliferative rates of both cell lines were decreased by 80-85% when cultured in DMEM relative to cells maintained in RPMI-1640 medium. In these studies, elevated levels of the melanin precursors tyrosine (Tyr) and phenylalanine (Phe) found in DMEM were shown not to be solely responsible for the phenotypic changes observed with DMEM. Both BL6 and B16-F1 cell lines formed more experimental pulmonary tumor metastasis in syngeneic C57BL/6 mice when maintained in DMEM vs RPMI-1640 medium. Analysis of metastasis formation in nude mice with normal and depleted natural killer (NK) cell activity revealed that the enhanced lung colonizing capacity of the BL6 cells maintained in DMEM was independent of the function of T-cell or NK-cell-mediated immunity. A close association between metastatic ability of tested lines and the expression of the membrane-associated enzyme gamma-glutamyltranspeptidase (gamma-GTPase, EC 2.3.2.2) was observed. The highly metastatic BL6 cell line had 20-fold higher levels of gamma-GTPase activity than the weakly metastatic B16-F1 cell line. Both cell lines, when grown in DMEM, had elevated gamma-GTPase activity that paralleled augmentation of metastatic ability. The dramatic changes in lung-colonizing capacity of the variant B16 melanoma cells maintained in DMEM in contrast to those grown in RPMI-1640 medium may serve as a useful model in understanding certain steps involved in triggering cell differentiation as well as metastasis development.

The effect of class I H-2 antigen expression on the metastatic properties of BL6 melanoma cells was investigated. The BL6-8 clone isolated from the highly metastatic BL6 melanoma did not express H-2Kb gene. Following transfection with the H-2Kb gene, BL6-8 cells displayed a low metastatic potential in the immunocompetent as well as immunosuppressed (X-irradiated) or triple-immunodeficient mice with impaired T, B and natural killer (NK) cells function. The expression of H-2Kb gene and the low metastatic ability of transfected BL6 melanoma cells were associated with appearance of cell membrane soybean agglutinin (SBA) and Griffonia simplicifolia 1B4 (GS1B4) lectin-binding carbohydrates. These alterations in cell surface carbohydrates were found to be a result of reduction in sialylation of SBA binding sites and upregulation of the alpha 1.3 galactosyltransferase (alpha 1.3GT) gene. To assess the importance of H-2Kb-induced alterations in cell surface carbohydrates for metastasis formation, BL6-8 melanoma cells were transfected with H-2Kb gene without neor gene cotransfection and selected for adherence to SBA-lectin-conjugated agarose beads. The transfected clones that expressed SBA and GS1B4 lectin-binding carbohydrates were low metastatic. Further analysis of these clones showed that presence of SBA and GS1B4 lectin-binding carbohydrates rather than expression of H-2Kb molecules per se might be responsible for low metastatic potentials of H-2Kb-transfected cells in the immunocompromised mice. Studies of the possible mechanisms responsible for low metastatic ability of H-2Kb-transfected melanoma cells revealed that these cells displayed a reduced ability to adhere to murine pulmonary endothelial cells as well as to laminin and collagen IV. We hypothesized that the observed nonimmunological effects of H-2Kb gene in BL6 melanoma cells is a result of an interaction between the H-2Kb gene and B16 melanoma-specific ecotropic retrovirus. It results in inhibition of this retrovirus production with consecutive alteration in the expression of cellular genes controlling cell surface glycosylation and adhesion properties essential for the metastatic phenotype of BL6 melanoma.

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