NOVEL MECHANISM OF RESISTANCE TO PACLITAXEL (TAXOL(R)) IN HUMAN K562 LEUKEMIA-CELLS BY COMBINED SELECTION WITH PSC-833

A paclitaxel-resistant cell line, KPTA5, was established by co-selecti ng the parental erythroleukemic cell line K562 with stepwise increased concentrations of paclitaxel (Taxol(R)) in the presence of the cyclos porin D analogue PSC 833 (2 mu M), a potent modulator of the multidrug resistance phenotype. KPTA5 cells are 9-fold resistant to paclitaxel and taxotere, but do not exhibit significant resistance to Vinca alkal oids, etoposide, anthracyclines, antimetabolites, or alkylating agents . Doubling time and morphology were similar to the parental K562 cells . Reverse transcriptase-polymerase chain reaction (rt-PCR) analysis re vealed no alterations in the expression of the mdr1 and MRP genes. Cel lular paclitaxel accumulation was unchanged. Cell cycle analyses showe d that at 20 nM there was a significantly higher proportion of K562 ce lls blocked in G(2)/M, in comparison with KPTA5 cells. In both cases, disruption of the mitotic spindles and the presence of multiple mitoti c asters were comparable but occurred at lower paclitaxel concentratio ns in K562 cells than in KPTA5 cells. There was no difference in total tubulin content between K562 and KPTA5 cells as analyzed by immunoblo tting with an anti-beta-tubulin monoclonal antibody. However, we found that KPTA5 cells presented a 2-fold increase both in 5 beta-tubulin m RNA expression and in the corresponding tubulin protein Class IV isoty pe content, as evaluated by rt-PCR and immunostaining. In conclusion, the KPTA5 cell line displays a novel mechanism of resistance to paclit axel which does not involve altered cellular drug accumulation. The da ta presented suggest that alterations in expression of the 5 beta-tubu lin gene may be involved in paclitaxel resistance.