Leukoregulin-increased plasma membrane permeability and associated ionic fluxes

Abstract

The role of ion fluxes in the increased plasma membrane permeability of tumor cells exposed to the anticarcinogenic and tumor cell proliferation inhibitory lymphokine, leukoregulin, was examined by flow cytometic analysis of cell surface perturbations indicative of membrane destabilization. The pI 5.3 form of leukoregulin was isolated by ion exchange, isoelectric focusing, and molecular sizing chromatography of lymphokines from phytohemagglutinin-stimulated normal human lymphocytes. Membrane permeability increased within 5 min of leukoregulin exposure of tumor cells and was quantified by following the efflux of fluorescein or influx of propidium iodide. Membrane permeability increased in proportion to leukoregulin concentration. By 2 h, 0.25-30 units/ml induces a 10-90% change in K562 erythroleukemia cell permeability. Similar changes are effected by Ca2+ ionophores A23187 and X-537A but not by the Na+ ionophore monensin or the K+ ionophore valinomycin. The Ca2+ ionophores and the intracellular Ca2+ mobilizers ouabain and amphotericin B enhance, whereas calmodulin inhibits leukoregulin action. Ca2+ channel blockers nifedipine and verapamil and the Na+ and K+ ion transport inhibitors amiloride and atractyloside, respectively, neither alter membrane permeability nor influence leukoregulin activity. Further evidence for the role of increased Ca2+ flux in leukoregulin's action is provided by detection of increased intracellular free Ca2+ in leukoregulin-treated cells with the Ca2+-sensitive fluorescent probe 2-[2-bis(carboxymethyl)amino-5-methylphenoxy]methyl-6-methoxy-8-bis (carboxymethyl)aminoquinoline. Kinetic analysis of cell volume, forward and right angle light scatter, fluorescein efflux, and propidium iodide influx, moreover, reveals that the action of leukoregulin is unique. Membrane perturbations may be critical initial steps in the ability of leukoregulin to directly prevent the development of carcinogenesis as well as inhibit the continued proliferation of neoplastic cells.