MODULATION OF CELL-CYCLE ASSOCIATED ANTIGEN EXPRESSION BY THE B16 MELANOMA: MULTIPARAMETER ANALYSIS USING MONOCLONAL ANTIBODIES AND FLOW CYTOMETRY (LYMPHOKINES, DIMETHYL SULFOXIDE)

We have developed two monoclonal antibodies, designated 152 E12 D7 (D7) and 153 C7 A6 (A6), which have reactivity with cell surface antigens expressed on the B16 mouse melanoma. These monoclonal antibodies are produced by hybridomas resulting from the fusion of splenocytes taken from C57BL/6 mice bearing the B16-F10 tumor. These monoclonal antibodies are of the IgM class and have been shown to react with three variants of the B16 and another mouse melanoma but not normal murine tissues. Multiparameter flow cytometric analysis revealed the expression of antigens recognized by D7 and A6 to be cell-cycle related in that cells in the later stages of the cycle had greater antigen density and a greater percentage were antigen positive. Exposure of B16 melanoma cells to a Con-A stimulated spleen cell mixed lymphokine preparation (LK) and to DMSO enhanced the expression of the cell-surface antigens recognized by these monoclonal antibodies. The cultures stimulated with LK or DMSO contained a greater proportion of cells expressing the antigens recognized by monoclonal antibodies D7 and A6 than did unstimulated controls. In addition to increasing the proportion of antigen positive cells, the antigen density per cell, as measured by fluorescence intensity, was substantially increased following exposure to LK and DMSO. The increase in antigen density occurred in cells analyzed from each phase of the cell cycle, moreover, the cells in the later phases continued to express antigen at higher densities than cells in the early phases. The effects of treatment with LK or DMSO were apparant after 24 hours exposure but did not persist after the agent was removed from the cultures, suggesting that the enhancement of antigen expression was a transient event rather than a permanent differentiation of the melanoma cells. Differences in the kinetics of antigen expression and loss as well as antibody competitive binding studies suggest that monoclonal antibodies D7 and A6 recognize different epitopes found on two separate cell surface molecules. The effect of LK or DMSO on antigen expression was blocked by the protein synthesis inhibitor cycloheximide. Treatment of the cells with LK or DMSO also reduced the incorporation of H-3TdR and S-35 methionine indicating that exposure to these agents decreased both cell proliferation and overall protein synthesis.