Mechanism of resistance to ricin toxin in selected mouse lymphoma cell lines

The role of impaired toxin uptake in conferring cellular resistance to the plant toxin RCA II (ricin) has been examined using a murine BW5 147 lymphoma line and a toxin‐resistant variant (BW5 147 Ric R .3) selected by repeated exposure to RCA II . The toxin‐resistant variant is 250 times more resistant to RCA II in long‐term growth experiments and 1,000 times more resistant in short‐term protein synthesis assays. Experiments with ferritin‐conjugated 125 I‐labeled RCA II (ferritin‐ 125 I‐RCA II ) indicated that toxin binding to sensitive and resistant cells is similar at low toxin concentrations where maximum differential cytotoxicity occurs but that major difference exist with respect to toxin uptake. In sensitive cells toxin is internalized via endocytosis, and as seen previously in other systems subsequent rupture of some of the toxin‐containing endocytotic vesicles releases toxin into the cytoplasm, where it inhibits protein synthesis. The process of toxin transfer to the cytoplasm is presumed to account for the one‐hour lag before toxin‐induced inhibition of protein synthesis can be detected. Endocytotic uptake of toxin is impaired in resistant BW5147Ric R .3 cells, and they are unaffected by toxin concentrations that inhibit protein synthesis and kill sensitive parental cells. Killing of resistant cells at low toxin concentrations was accomplished by encapsulating RCA II into lipid vesicles capable of fusing with the plasma membrane. Direct introduction of toxin into resistant cells using lipid vesicles as carriers produced rapid inhibition (< 15 min) of protein synthesis and eliminated the lag in toxin action seen in sensitive cells exposed to free toxin. These findings are discussed in relation to the mechanism of toxin action and proposals that toxin activity requires structural modification of the toxin molecule at the cell surface before transport into the cell.