ISOLATION OF A NITRIC-OXIDE INHIBITOR FROM MAMMARY-TUMOR CELLS AND ITS CHARACTERIZATION AS PHOSPHATIDYL SERINE

Macrophages from mice bearing large D1-DMBA-3 mammary tumors have a de creased capacity to kill tumor targets. This effect is due to an impai red ability to produce nitric oxide (NO) in response to lipopolysaccha ride (LPS) stimulation. Here we report that the DA-3 tumor cell line, derived from the in vivo adenocarcinoma D1-DMBA-3, produces a factor t hat inhibits both NO production/release and cytotoxicity of LPS-activa ted peritoneal exudate macrophages (PEM). However, other complex macro phage functions such as phagocytosis, superoxide production, mitochond rial dehydrogenase activity, and synthesis of proteins were not reduce d by this factor. The NO inhibitor has been found to be lipid in natur e. Lipid extracts from DA-3 cell culture supernatants were purified by repeated silica gel column chromatography. The active molecule was un ambiguously characterized as phosphatidyl serine (PS) by fast atom bom bardment tandem mass spectrometry. Preliminary results indicate a lack of induced NO synthase (iNOS) activity in the lysates of LPS-activate d PEM pretreated with PS. The ubiquity of PS in the inner leaflet of b iological membranes and its NO inhibitory property, suggest that this phospholipid may be one of the long elusive molecules responsible for regulating physiological levels of NO in the host and hence preventing cellular dysfunction and/or tissue damage. Furthermore, the possible overexpression and shedding of PS by DA-3 tumor cells may represent a novel mechanism to impair macrophage cytotoxicity, a host function tha t contributes to the protection against developing neoplasms.