On the Mechanism of Toxin Resistance in Cell Variants Resistant to Abrin and Ricin

Abstract

Variants of HeLa cells tolerating 10-200 times more abrin and ricin than the parent line have been isolated by growing cells in the presence of increasing concentrations of toxin with and without previous irradiation. The resistant cell lines showed morphological differences from the parent cells, but their growth rate and plating efficiency were almost the same as those of the parent cells. All variants were resistant to abrin as well as to ricin, but not to modeccin and diphtheria toxin. The sensitivity of the various cell lines to abrin and ricin was evaluated by measuring either inhibition of colony formation or inhibition of protein synthesis 3 h after addition of the toxins. The resistant variants proved to be less resistant in the colony assay system than when inhibition of protein synthesis was measured. The sensitivity of isolated ribosomes from HeLa cells and from the ricin-resistant variants was measured by incubating the ribosomes with ricin A chain and measuring after various periods of time their residual ability to support polymerization of [14C]phenylalanine. The ribosomes from the ricin-resistant variants were as sensitive to the A chain as those from the parent cell line. Similar data were obtained with ribosomes from baby hamster kidney cells and from three ricin-resistant variants. Cell-surface binding sites for abrin and ricin were labelled by treating the cells with galactose oxidase and then with boro[3H]hydride. Polyacrylamide gel electrophoresis of the cell extracts showed a variety of labelled proteins. Pretreatment of the cells with neuraminidase resulted in a strong increase in labelling. Fluorography of the gels revealed some minor differences in the moving rates of two labelled glycoproteins in the parent HeLa cells and the ricin-resistant variants. The reason for the lack of cross-resistance to modeccin and diphtheria toxin is discussed.