Detergent extraction and characterization of tumor hemolytic factor from plasma membranes of oncogene transformed fibroblasts

Abstract

Cancer cells have the capacity to lyse erythrocytes by a cell-contact-requiring phenomenon. Subcellular fractionation procedures have revealed that the hemolytic principle resides in the cancer cell plasma membrane. In this study we report the detergent extraction of a potent hemolytic factor from the plasma membranes of ras-oncogene-transformed fibroblasts. Ammonium-sulfate partitioning (60-100%) of detergent-extracted proteins was used to enrich hemolytic activity. Tumor membrane Hemolytic Factor (mTHF) was inactivated by treatment with papain, suggesting that it is a protein. mTHF was inhibited by serum, but was unaffected by extremes of temperature and pH, also by metal chelation with EDTA. Surface radio-iodination of tumor cells and isolation of cell organelles was used to characterize the outer plasma membrane localization of mTHF. mTHF retained hemolytic activity when reconstituted into stable phospholipid vesicles. Pre-incubation of mTHF with red cell ghosts led to an abrogation of hemolytic activity. mTHF-induced hemolysis consists of a 2-stage phenomenon: an early binding step, followed by hemolysis after 4 hr.