Cytotoxic properties of eosinophil granule major basic protein for tumor cells

Background: Eosinophil granule major basic protein (MBP) mediates many eosinophil-associated immune functions and it adheres eosinophils to parasite targets. Methods: We compared the toxicities of MBP and melittin to K562 and HL-60 cells using five cytotoxicity methods. Results: Trypan blue staining, propidium iodide/CellTracker(TM)Green staining and incorporation of C-14-leucine assays indicated that MBP damages most cells by 1 h. In contrast, Cr-51 and lactic dehydrogenase (LDH) release assays indicated that MBP damages most cells only at 20 h. All five methods indicated that melittin damages nearly all cells by 1 h. To resolve these discrepancies, the procedures were modified. Without cell transfer, dye staining methods showed that MBP produces very little cytotoxicity at 4 h. Cr-51 and LDH assays, modified to mimic cell transfer, showed increased cytotoxicities at 4 h. The C-14-leucine assay modified by solubilization of cells with SDS and by trichloroacetic acid precipitation showed increased recovery of labeled protein and, thus, lower cytotoxicity, about 50%, at 4 h. Conclusion: Overall, MBP's ability to cause molecular and cellular adhesion confounds cytotoxicity measurements. A modified C-14-leucine assay overcame MBP's adhesiveness and provided an accurate measure of cytotoxicity.