Characterization of a Taxol‐resistant Human Small‐cell Lung Cancer Cell Line

Abstract

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.

Paclitaxel-resistant Human Ovarian Cancer Cells Have Mutant β-Tubulins That Exhibit Impaired Paclitaxel-driven Polymerization

“…PTX-dependent for growth (18,31). Their growth rate is similar in the absence and presence of PTX (data not shown).…”

“…These include reduced total tubulin levels (10,24), increased tubulin content (15), differential migration of ␣-or ␤-tubulin on two-dimensional polyacrylamide gel electrophoresis (11,13), acetylation of ␣-tubulin (18), and increased expression of specific tubulin isotypes (14 -17). In the present study, the total tubulin content of the resistant cells was similar to that of the parental cells (data not shown), and no differences in the acetylation of purified ␣-tubulin were observed (data not shown).…”

“…Although precise molecular alterations in MTs have not been identified, several mechanisms have been proposed. These include decreased tubulin (10), point mutations as evidenced by altered migration of ␣-or ␤-tubulin on two-dimensional SDS-polyacrylamide gel electrophoresis (11)(12)(13), differential expression of ␤-tubulin isotypes (14 -17), and acetylation of ␣-tubulin (18).…”

Enhancement of in vivo Antitumor Activity of a Novel Antimitotic 1‐Phenylpropenone Derivative, AM‐132, by Tumor Necrosis Factor‐cc or Interleukin‐6

“…Growth inhibition assay To determine the growth-inhibitory effects of AM compounds, we utilized the tetrazolium dye assay developed by Mosmann with some modifications. 19,28) Briefly, 180 µl of exponentially growing cell suspensions containing 10 4 H69, H69/Txl and H69/VDS, 10 3 PC-14 and PC-14/TXT, or 10 3 P388, P388/4.0r-M and LLC/IL2, LLC/IL6, LLC/TNF, LLC/GM-CSF, LLC/IGIF, and LLC/IL12, were seeded in 96-well microtiter plates (Becton Dickinson & Co., Lincoln Park, NJ), and incubated for about 12 h, then 20 µl aliquots of drug solutions at the indicated concentrations were added. After a 72-h exposure to the drug, 20 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma Chemical, St. Louis, MO) solution (5 mg/ml in phosphate-buffered saline, PBS) was added to each well.…”

“…We suggest that there may be a combination effect of cytokines and AM compounds on murine lung cancer cells ectopically expressing several cytokines. 16,[18][19][20][21][22] 5 To whom reprint requests should be sent. …”