Cytotoxicity of CD3-ricin A chain immunotoxins in relation to cellular uptake and degradation kinetics.
The cytotoxicity of WT32 (CD3)-ricin A immunotoxin (IT) to the acute lymphoblastic leukemia T-cell line Jurkat was compared with the rate of internalization and intracellular degradation of WT32 and WT32-ricin A during continuous exposure. Moreover, the influence of NH4Cl and monensin on these processes was studied. Based on protein synthesis inhibition ([3H]leucine incorporation), it appeared that cytotoxicity was not fully expressed directly after exposure to IT due to a delay in either the internalization of membrane-bound IT or the action of intracellular ricin A. Varying the duration of incubation and postponing [3H]leucine addition for up to 24 h after initiation showed that cytotoxicity occurred in two phases, rapid internalization of initially bound IT followed by a continuous but slower uptake, possibly due to reexpression of the CD3 antigen. No differences were found in the rate of internalization and degradation of 125I-labeled WT32 and WT32-ricin A. Internalization started rapidly after binding at 37 degrees C, was fastest during the first 12 h (+/- 360,000 molecules/cell), and continued for at least 24 h (+/- 420,000 molecules/cell). Exocytosis of intracellularly degraded molecules became measurable after 1 to 2 h of incubation at 37 degrees C and increased to approximately 400,000 molecules/cell in 24 h. After 4 h of incubation at 37 degrees C the number of internalized molecules exceeded the amount of WT32 that could maximally bind to the cell membrane (+/- 150,000 molecules/cell), confirming reexpression of antigen. The addition of NH4Cl and monensin enhanced the cytotoxicity of WT32-ricin A, probably due to an increased intracellular amount of IT. These agents appeared to reduce strongly the degradation of internalized WT32, resulting in an accumulation of intracellular molecules. NH4Cl was most effective during the first 12 h of incubation, whereas monensin increased the amount of intracellular WT32 molecules after 2 to 24 h. Our observations suggest that incubation conditions for the optimal cytotoxicity of IT treatment can be predicted by studying the internalization and degradation of the IT or respective monoclonal antibody.
van Oosterhout
Y V
YV
Department of Hematology, University Hospital St. Radboud, Nijmegen, The Netherlands.
Preijers
F W
FW
Wessels
H M
HM
de Witte
T
T
D000643:Ammonium Chloride / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000627:therapeutic use; D004347:Drug Interactions; D006801:Humans; D000922:Immunotoxins / Q000378:metabolism* / Q000627:therapeutic use; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000378:metabolism* / Q000628:therapy; D008985:Monensin / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000096:biosynthesis; D012276:Ricin / Q000378:metabolism* / Q000627:therapeutic use; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000922:Immunotoxins; D009363:Neoplasm Proteins; D000643:Ammonium Chloride; D012276:Ricin; D008985:Monensin