Resistance to microtubule-targeted cytotoxins in a K562 leukemia cell variant associated with altered tubulin expression and polymerization.

A vinblastine resistant cell line, KCVB2, was established by co-selecting the parental erythroleukemic cell line K562 with step-wise increased concentrations of vinblastine (Velban) in the presence of the cyclosporin D analogue PSC 833 (2 microM), a potent modulator of the multidrug resistance phenotype. KCVB2 cells are 8-fold resistant to the selecting agent, vinblastine, but also exhibit significant resistance to other vinca alkaloids, including 14-fold resistance to vinorelbine, as well as 6-fold cross-resistance to paclitaxel. Doubling time and morphology were similar to the parental K562 cells. Rt-PCR analysis revealed no alterations in the expression of the mdr1 and MRP genes. Intracellular vinblastine accumulation was unchanged. Disruption of the mitotic spindles and multiple mitotic asters occurred in both cell lines but required higher concentrations of vinblastine in KCVB2 cells than in K562 cells. Significant differences were observed in the tubulin content of KCVB2 cells: reduction of total tubulin content, increased polymerized fraction of total tubulin, and overexpression of class III beta-tubulin which is expressed at very low levels in the parental K562 cells. K562 cells transfected with murine class III beta-tubulin did not display the resistance pattern observed in KCVB2 cells. Revertants of KCVB2 manifested reversion to parental drug sensitivity, an increase in total tubulin level, and a decrease in polymerized tubulin. In conclusion, the KCVB2 cell line displays a novel mechanism of resistance to both depolymerizing and stabilizing microtubule-targeted cytotoxins which does not involve altered cellular drug accumulation, but corresponds to alterations in the total tubulin content and polymerization status, and may involve an effect on microtubule dynamics.

Conformational changes in the cyclic undecapeptide cyclosporin induced by interaction with metal ions. An FTIR study.

Infra-red spectra have been measured for the cyclic undecapeptide cyclosporin A (CsA) and three analogues CsC, CsD and CsH in acetonitrile and in the presence of 10:1 molar excess of Mg2+, Ca2+, Na+ or Li+ in the same solvent. Interaction with each of these ions is suggested by marked changes in band positions over the amide I region (1600-1700 cm-1). The formation of complexes of cyclosporin with calcium and magnesium ions is indicated by the presence of C = O stretching bands well outside the range normally expected for the amide I absorptions of free peptides. Although they share this characteristic, the spectra indicate that the mode and/or strength of Ca2+ binding is quite different from that of Mg2+ binding. In contrast, the two monovalent ions interact with CsA, CsC and CsD to yield spectra that are very similar to one another. The spectra are consistent with binding of the monovalent ions simultaneously to several carbonyl groups of the loop structure.