Purification and characterization of the cytotoxic factor in rat peritoneal exudate cells: its identification as the calcium binding protein complex, calprotectin

Abstract

We previously reported the existence of a growth inhibitory factor for mitogen-stimulated lymphocytes and murine tumor cell lines, MM46 and L-929, in inflammatory polymorphonuclear leukocytes. In this study, by using mouse MM46 mammary carcinoma as target, we purified the inhibitor from lysate of rat inflammatory peritoneal exudate cells by ammonium sulfate precipitation, gel filtration, isoelectrofocusing, and anion exchange chromatography. Although the in vitro inhibitory activity for MM46 growth was partitioned into three peaks in the final step, it was found that these inhibitory samples all consist of 8- and 13-kDa peptides. Analysis of amino acid sequences revealed that the partial sequences of the 8- and 13-kDa peptides completely agree with the smaller and larger components of rat calprotectin, which are predicted from cDNA, respectively, suggesting the cell growth inhibitory factor is calprotectin. In addition to MM46, the partially purified calprotectin inhibited the growth of a rat, three mice, and a human tumor cell line in similar dose-response relationships in vitro. Moreover, it exerted a cytolytic effect against all examined tumor cells. It was confirmed that the purified calprotectin induces growth inhibition and the lysis of MM46 cells and that the minimum effective concentration is between 50 and 100 micrograms/ml. The factor also inhibited the growth of bone marrow cells and macrophages. These results suggest that calprotectin is a negative regulatory factor for the growth and/or survival states of normal and tumor cells.