Identification of Vinca alkaloid acceptors in P388 murine leukemia cells with a photoactive analogue of vinblastine.
The cytotoxic, antimitotic, and growth inhibition properties of a photoactive analogue of vinblastine, N-(p-azidobenzoyl)-N'-beta-aminoethylvindesine (NABV), and vinblastine on P388 murine leukemia cells were compared. After 72-h exposure, the 50% drug-inhibitory concentrations for exponentially growing P388 leukemic cells were 1.2 nM for NABV and 0.6 nM for vinblastine. The ultrastructural effects of NABV and vinblastine on P388 cells were similar: formation of tubulin paracrystals; mitotic arrest (C-mitosis); increased post-C-mitotic multinucleated cells; increased number of annulate lamellae; and the appearance of intracytoplasmic paired cisternae. [3H]NABV was used to identify Vinca alkaloid binding sites in P388 cells by photoaffinity labeling. After irradiation at 302 nm, radioactive Vinca alkaloid binding components were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified in 1-mm gel slices. The most prominent photolabeled species were Mr 44,000, 54,000, and 75,000 polypeptides located in the 100,000 X g supernatant fraction. The Mr 54,000 component was also observed in the membrane fraction. Specific photolabeling of Mr 54,000 and 44,000 polypeptides was blocked in the presence of 20 microM excess of vinblastine and was saturable with half-maximal saturation concentrations of 0.18 and 0.4 microM [3H]NABV, respectively. The Mr 54,000 component was identified as a tubulin subunit by immunoprecipitation with antitubulin monoclonal antibodies. Since NABV and vinblastine have similar pharmacological and biological properties, this photoactive analogue may be useful for identifying important Vinca alkaloid cellular acceptors which may be responsible for drug cytotoxic and antineoplastic activities.
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000378:metabolism* / Q000494:pharmacology; D007941:Leukemia P388 / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D007942:Leukemia, Experimental / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D011955:Receptors, Drug / Q000032:analysis*; D014747:Vinblastine / Q000378:metabolism / Q000494:pharmacology; D014748:Vinca Alkaloids / Q000378:metabolism*; D014751:Vindesine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology
D000345:Affinity Labels; D001386:Azides; D011955:Receptors, Drug; D014748:Vinca Alkaloids; C048694:N-(4-azidobenzoyl)-N'-beta-aminoethylvindesine; D014747:Vinblastine; D014751:Vindesine