Temporal Synthesis and Presentation of Antigens by Cultured B16 Melanoma Cells

Abstract

Previous studies have demonstrated the presence of two distinct antigens, B700 and B50, which are unique to murine melanoma. One of these, B700 has been studied in detail, and is present on 5 different murine melanomas; it can function as a transplantation antigen in at least 3 of them (B16, JB/RH and K1735). The synthesis and presentation of these antigens has been studied as a function of cell culture conditions. Direct immunofluorescence studies of cells in serial culture indicate that the expression of B700 and B50 antigens at the cell surface and in the cytoplasm increases as a function of time in culture, over 1-5 days. By day 5, when the cells are confluent, all cells show some degree of antibody binding. Parallel 35S-methionine pulse chase labeling experiments show that incorporation into Triton soluble proteins, and Triton insoluble SDS soluble proteins, increases to a peak at 3.5 days after subculturing, then decreases as the cells reach confluence. Incorporation into proteins shed into the culture supernatant continued throughout the time course of cell growth to confluence. However, as the cells become confluent, total protein synthesis shifts towards greater production of the antigens (both cellular and shed). The sum of the results suggest that tumor growth may succeed in vivo by the wholesale production of "decoy" antigens.

Serologic Demonstration of the Albuminoid Nature of the B700 Murine Melanoma Antigen

“…The purity of the proteins were verified by sodium dodecyl sulfate-gel electrophoresis according to Laemmli (14). Rabbit antibodies to B700 (wB700) were from the same preparation used in our previous studies, where their specificities and titers are reported in detail (15)(16)(17). Rabbit antisera, and affinity-purified IgG, to MSA (a-MSA) and to AFP (a-AFP) were gifts from Dr. B. Ledford, Medical University of South Carolina, Charleston, SC (1 8).…”