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/ CellTrackertrade markGreen staining and incorporation of 14C-leucine assays indicated that MBP damages most cells by 1 h. In contrast, 51Cr 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. 51Cr and LDH assays, modified to mimic cell transfer, showed increased cytotoxicities at 4 h. The 14C-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.


CONCLUSIONS
Overall, MBP's ability to cause molecular and cellular adhesion confounds cytotoxicity measurements. A modified 14C-leucine assay overcame MBP's adhesiveness and provided an accurate measure of cytotoxicity.
Kubo
H
H
Departments of Immunology and Medicine, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
Loegering
D A
DA
Adolphson
C R
CR
Gleich
G J
GJ
D001798:Blood Proteins / Q000276:immunology*; D003594:Cytoplasmic Granules / Q000276:immunology; D003602:Cytotoxicity, Immunologic; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000276:immunology* / Q000648:ultrastructure; D018922:HL-60 Cells; D006801:Humans; D020014:K562 Cells; D009369:Neoplasms / Q000276:immunology*; D012260:Ribonucleases
D001798:Blood Proteins; D047091:Eosinophil Granule Proteins; D012260:Ribonucleases