Multiparameter flow cytometric analysis of a novel cytotoxin (Factor 2) induced tumor cell membrane permeability

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Abstract
An improved twin‐probe multiparameter flow cytometric technique was applied to examine a novel cytotoxin, Factor (F2), induced tumor cell permeability. Ability to retain preloaded intracellular bis‐carboxyethyl carboxyfluorescein (BCECF, green fluorescence) and to exclude extracellular propidium (red fluorescence) was measured simultaneously with forward and right‐angle scatter. In addition to the two expected cell populations which were stained green negative, red positive (“membrane‐damaged” and “non‐viable”, Region 2), and green positive, red negative (“membrane intact” and “viable”, Region 3), a third population was seen which fluoresced neither green nor red and displayed intermediate light scatter characteristics (Region 1). K562 cells progressed from Region 3 to Region 1, and then from Region 1 to Region 2 after treatment with F2. These results suggest that sequential changes in membrane structure lead to increased permeability, first with respect to intracellular BCECF and then in turn to extracellular propidium. Flow cytometric changes caused by F2 were detectable 10 min after treatment with 2.5 U/ml of F2, and 5 min after 10 or 40 U/ml of F2. Flow cytometric analysis showed that F2‐induced tumor cell lysis and growth inhibition were accompanied by rapid alternations in tumor cell membrane permeability. Flow cytometric analysis also distinguished F2 cytotoxicity from phorbol myristate acetate (PMA) associated cytotoxicity to K562 cells and determined that F2 produced spontaneously or induced by PMA and/or ciprofloxacin had a similar ability to induce tumor cell membrane permeability change.