A tumor-specific membrane phosphoprotein marker in human cell hybrids

Abstract

Fusion of normal human fibroblasts with HeLa results in hybrids with completely suppressed tumorigenic potential, which still behave as transformed cells in culture. From these stably suppressed hybrids, rare tumorigenic segregants have been isolated. We prepared antiserum against one tumorigenic segregant, ESH5T, by injecting live cells into rabbits, then absorbed this antiserum extensively against the corresponding nontumorigenic hybrid, ESH5, to remove any cross-reacting antigenic specificities. Immunoprecipitation of iodinated cell surface proteins with this absorbed antiserum revealed a 75 kilodalton (kd) protein on the cell surface of tumorigenic HeLa-fibroblast hybrids and HeLa, but not on normal fibroblasts or any nontumorigenic hybrids examined. Under nonreducing conditions in SDS-polyacrylamide gel electrophoresis, the 75 kd protein migrated as a 150 kd dimer. Immunoprecipitation of cells metabolically labeled with 35S-methionine confirmed that the 75 kd protein is synthesized in tumorigenic cells but not in nontumorigenic hybrids or normal fibroblasts. Metabolic labeling with 32PO 4 revealed that the 75 kd protein is phosphorylated, and immunoprecipitation of galactose-oxidase/ 3NaBH 4-labeled cells with absorbed antiserum identified it as a glycoprotein. This protein does not appear to be a major cell surface iodinated component and represents only 0.03% of the total cellular protein. The exclusive correlation of the expression of this cell surface phosphoglycoprotein with tumorigenicity in HeLa-fibroblast hybrids suggests that it may be an important determinant for in vivo growth potential.