Enzymatic susceptibility and spontaneous release of human melanoma tumor-associated antigens.

A chimpanzee anti-human melanoma antiserum was used to study the enzymatic susceptibility and spontaneous release into tissue culture medium of human melanoma tumor-associated antigens (TAA). Limited proteolytic digestion of melanoma cells with trypsin or with pronase rendered these cells refractory to lysis by the chimpanzee antiserum and complement. Longer periods of incubation of higher concentrations of enzyme caused an increased sensitivity to lysis. Digestion of melanoma cells with neuraminidase apparently exposed antigens reactive with natural antibodies in rabbit complement because cells so treated had a marked increase in sensitivity to cytolysis. Absorption of the complement with either neuraminidase-treated human melanoma cells or washed human spleen cells prior to its use in the cytotoxicity assay removed this activity. When absorbed complement was used, neuraminidase had no noticeable effect on the expression of malanoma TAA. These results suggest that proteolytic digestion of melanoma cells may prove to be a useful means of solubilizing TAA. The spontaneous release of melanoma cell membrane TAA was studied. Protein precipitated by (NH4)2SO4 from four of six samples of tissue culture medium used to feed malanoma cell lines contained significant antigenic activity compared to a control "antigen" preparation, whereas one preparation contained only limited TAA activity. One melanoma cell line that apparently failed to release TAA into the culture medium had previously become nonreactive with the chimpanzee antiserum. From these data, we conclude that melanoma cells growing in tissue culture rapidly release large amounts of TAA into the culture media and, as a result, the spent culture medium may be a good source for obtaining TAA for further study. The significance of these results is discussed.