A highly cytotoxic human transferrin-ricin A chain conjugate used to select receptor-modified cells.
The toxic A chain of ricin was linked to human transferrin via a disulfide bond and the resulting conjugate was shown to bind to cell membrane transferrin receptors. Surface-localized transferrin A chain (TF-A chain) gained access to the cytoplasm and inactivated ribosomes as witnessed by a rapid curtailment of cellular protein synthesis (t1/2 = 6 h) and subsequent cytolysis. The intact conjugate produced potent cytotoxic effects on human leukemia CEM cells, (ID50 = 3 X 10(-11) M), while a 10,000-fold higher concentration of uncoupled transferrin plus A chain was required for comparable action. TF-A chain cytotoxicity was totally blocked by native transferrin or by antibodies directed against ricin A chain and human transferrin. CEM cells gradually acclimated to grow in the presence of TF-A chain displayed 1000-fold resistance to the conjugate while their sensitivity to whole ricin was undiminished. The level of transferrin receptor expressed by these cells was 1/20 the amount present on the parent line and their capacity to bind human transferrin was below the limits of detectability using fluorescent probes. This receptor-deficient cell line was unresponsive to the low levels of transferrin which stimulated proliferation of control CEM cells, but their growth was supported by greatly elevated concentrations of ligand. Receptor variant CEM cells, together with the specific TF-A chain toxin, will be useful for studying the mechanisms for transmembrane delivery of both Fe3+ and ricin A chain into cells and will aid in understanding the growth regulatory functions of the receptor-ligand interaction.
D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D006801:Humans; D007930:Leucine / Q000378:metabolism; D007938:Leukemia / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011990:Receptors, Transferrin; D012276:Ricin / Q000633:toxicity*; D013997:Time Factors; D014168:Transferrin / Q000378:metabolism*
D011956:Receptors, Cell Surface; D011990:Receptors, Transferrin; D014168:Transferrin; D012276:Ricin; D007930:Leucine