title: Disulfide spacer between methotrexate and poly(D-lysine). A probe for exploring the reductive process in endocytosis.
abstract: Poly(D-lysine) is taken up avidly by cultured cells through adsorptive endocytosis and can serve as a carrier to increase cellular uptake of other molecules. While direct conjugation of methotrexate to poly(D-lysine) yields a conjugate devoid of cytotoxic effects because poly(D-lysine) is not digested in lysosomes, the indirect conjugation using a triglycine spacer or a disulfide spacer strongly inhibits the growth of both the wild type and the methotrexate transport-defective Chinese hamster ovary cells. Cell treatment with 3 mM NH4Cl or 50 micrograms/ml leupeptin prevents the effect of conjugate with the triglycine spacer, but not of conjugate with the disulfide spacer. On the other hand, preincubation with 2-mercaptoethanol abolishes the effect of the drug-disulfide conjugate in the methotrexate transport-defective mutant, but not the effect of the drug-triglycine conjugate. The disulfide conjugate shows an identical cytotoxic effect in alpha-minimal essential medium and RPMI 1640 media, even though cells grown in the latter have only half the glutathione content as cells grown in the former medium. We conclude that the reductive process through which methotrexate is released from the disulfide spacer (a) occurs inside cells and not at the cell surface, (b) requires neither acid pH nor lysosomal enzymes, and (c) is not mediated by a glutathione-disulfide exchange reaction requiring high glutathione concentrations. Although the cellular compartment in which this reductive process occurs is not yet identified, there are reasons to assume that it is prelysosomal.
mesh_terms: D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media; D004220:Disulfides / Q000378:metabolism; D004705:Endocytosis; D005260:Female; D005978:Glutathione / Q000378:metabolism; D008623:Mercaptoethanol / Q000494:pharmacology; D008727:Methotrexate / Q000378:metabolism* / Q000494:pharmacology* / Q000031:analogs & derivatives; D009154:Mutation; D010053:Ovary; D010084:Oxidation-Reduction; D010455:Peptides / Q000378:metabolism*; D011107:Polylysine / Q000378:metabolism* / Q000031:analogs & derivatives
chemical_list: D003470:Culture Media; D004220:Disulfides; D010455:Peptides; D011107:Polylysine; D008623:Mercaptoethanol; C030305:methotrexate-polylysine; D005978:Glutathione; D008727:Methotrexate