Gene amplification and expression in lung cancer cells with acquired paclitaxel resistance.
A paclitaxel-resistant subline was generated from the non-small lung cancer cell line NCI-H460 by stepwise selection in paclitaxel from 0.032 to 250 nmol/L. The resulting subline, designated NCI-H460/PTX250, showed 792-fold resistance against paclitaxel compared with the parental cell line NCI-H460. The chemosensitivity analysis revealed the cross-resistance phenotype against various anticancer drugs including docetaxel, vinblastine, and doxorubicin, but not against camptotecin, cisplatin, and 5-fluorouracil. The addition of 5 mumol/L verapamil or reversin 121 reversed the resistance against paclitaxel, vinblastine, and doxorubicin. The gene expression profile, examined using oligonucleotide microarrays, demonstrated that the expression of 332 and 342 genes was significantly increased and decreased, respectively, in NCI-H460/PTX250 compared with NCI-H460. The most highly upregulated gene was MDR1/ABCB1 with a 1,092-fold increase. The overexpression was confirmed at the protein level by Western blot and flow cytometry analyses. The copy number profile, examined using microarray-based comparative genomic hybridization, revealed amplification of the q11.21 approximately q21.12 region on chromosome 7. In particular, the entire q21.12 region displayed 11- to 13-fold higher copy number in NCI-H460/PTX250 than in NCI-H460. Most of the genes within the region were highly expressed, and the increased expression of these genes could be explained by the amplification in the gene copy number. However, the increase in MDR1/ABCB1 expression greatly exceeded the genomic copy number increase of the gene, suggesting the existence of one or more additional factors, such as transcriptional enhancement or mRNA stabilization, associated with the elevated MDR1/ABCB1 expression. In conclusion, both chromosomal region-specific copy number amplification and gene-specific activation are probably involved in the overexpression of MDR1/ABCB1, resulting in acquisition of the drug resistance phenotype in NCI-H460/PTX250.
Yabuki
Nami
N
Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd., 200-Kajiwara, Kamakura, Kanagawa 247-8530, Japan.
Sakata
Kiyoaki
K
Yamasaki
Tomoaki
T
Terashima
Hiromichi
H
Mio
Toshiyuki
T
Miyazaki
Youko
Y
Fujii
Toshihiko
T
Kitada
Kunio
K
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D018432:Drug Resistance, Multiple / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005784:Gene Amplification; D020869:Gene Expression Profiling; D015894:Genome, Human / Q000235:genetics; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000473:pathology; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D017239:Paclitaxel / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D000970:Antineoplastic Agents; D012333:RNA, Messenger; D017239:Paclitaxel