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.