Taxol is a novel anticancer agent with activity against a broad range of tumors. It has a unique ability to stabilize polymerized tubulin into microtubule bundles within the cell. We have established a taxol‐resistant human small‐cell lung cancer cell line (H69/Txl) by exposing H69 cells to stepwise increases in taxol concentration. The resistance of H69/Txl cells to taxol was 4.7‐fold that of the original H69 cells: the IC50 values for H69 and H69/Txl were 113.7 ± 56.54 nM and 538.7 ± 214.7 nM by the tetrazolium dye assay, respectively. Removal of the drug from the medium resulted in a 38% decrease in the growth rate of H69/Txl as compared with that in the presence of 30 nM taxol, suggesting that the growth of H69/Txl was partially dependent on taxol. H69/Txl showed higher sensitivity to vinca alkaloids such as vindesine, vincristine and vinblastine than the parental H69. There was no significant difference in intracellular [3H]taxol content between H69 and H69/Txl cells. No MDR‐1 mRNA was detected in H69/Txl by the reverse transcription polymerase chain reaction. There was no significant difference of total and polymerized tubulin content between H69 and H69/Txl cells. Altered mobility of one of the α‐tubulin isoforms in H69/Txl was revealed by using isoelectric focusing and Western blotting with anti‐α‐tubulin antibody. In H69, two α‐tubulin isoforms were observed, whereas three were evident in H69/Txl, two of them comigrating with the isoforms of H69 and the other being more acidic. We observed the increased acetylation of α‐tubulin in H69/Txl cells as compared with that in H69 cells. The acetylation of α‐tubulin may be responsible for the taxol resistance and/or taxol‐dependent growth of H69/Txl.
A cisplatin‐resistant non‐small cell lung cancer cell line, PC‐14/CDDP, was established from PC‐14 by stepwise escalation of CDDP concentrations in vitro. PC‐14/CDDP cells were 11.4‐fold more resistant to CDDP compared with PC‐14 cells. This resistant cell line was cross‐resistant to platinum analogues, such as carboplatin (CBDCA) (X 3.5), cis‐diammine(glycolate‐O, O′)platinum(II) (254‐S) (X 5.6) and cis‐dichloro(ethylenediammine)platinum(II) (cis‐DEP) (X 4.2). On the other hand, relative resistance value to ormaplatin was only 1.4‐fold. To elucidate the mechanism(s) of CDDP resistance and of its circumvention by ormaplatin, we investigated the characteristics of this cell line. Total sulfhydryl content was slightly elevated in PC‐14/CDDP cells compared with PC‐14 cells. There was no significant difference in the DNA repair ability between the two cell lines. Cellular accumulations of CDDP, CBDCA, 254‐S, and cis‐DEP in PC‐14/CDDP cells were markedly decreased to 23%, 27%, 29%, and 32% of those in PC‐14 cells, respectively. However, the accumulation of ormaplatin in PC‐14/CDDP was almost the same as that in PC‐14. To elucidate the mechanisms of uptake of these platinum analogs in the cells, we studied the effects of ouabain, an Na+, K+ ‐ATPase inhibitor, on cellular drug uptake in both cell lines. Preincubation with 300 nM ouabain for 1 h inhibited approximately 60% of CDDP accumulation in PC‐14. However ouabain preincubation at any concentration up to 300 nM did not affect CDDP accumulation in PC‐14/CDDP. The accumulation of ormaplatin was not inhibited by ouabain in either of the cell lines. These data suggest that the mechanism of the uptake of ormaplatin is different from that of CDDP, and that ormaplatin exerts a cytotoxic effect in CDDP‐resistant cells which have defective cisplatin accumulation.
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