title: Novel mutation of topoisomerase I in rendering cells resistant to camptothecin.
abstract: To identify mechanisms of camptothecin (CPT) resistance and the relationship between CPT-resistant cells and other anticancer agents, a CPT-resistant cell line (CPT30) and its partial revertant cell line (CPT30R) were established from a human nasopharyngeal carcinoma cell line (HONE-1). CPT30 and CPT30R cells displayed a 14- and 3.5-fold resistance to CPT compared with HONE-1 cells, respectively. The resistant and partial revertant cell lines showed cross-resistance to topotecan and increased sensitivity to cisplatin, carboplatin, and 1,3-bis(chloroethyl)-1-nitrosurea. The topoisomerase (Top) I catalytic activity of CPT30 and CPT30R cells was 30% and 200%, respectively, compared with that of HONE-1 cells. The expression of Top I protein and mRNA levels in CPT30 cells was 40% and 30% less than that in HONE-1 cells, respectively, whereas in CPT30R cells, the levels of Top I protein and mRNA were 50% and 20% higher, respectively, than that in HONE-1 cells. Both the resistant and revertant cell line whole-cell lysates demonstrated different levels of sensitivity to CPT in in vitro assays in comparison with that of HONE-1 cells. Furthermore, CPT exhibited 15- and 7-fold better binding affinity in stabilizing protein-linked DNA breaks in HONE-1 cells than in CPT30 and CPT30R cells, respectively. Direct DNA sequencing of the reverse transcription-PCR product and genomic DNA revealed a point mutation resulting in E418K mutation in the Top I of both CPT30 and CPT30R cells. Wild-type Top I RNA and genomic DNA were also detected in these two cell lines. A yeast system was used to examine whether this mutation could be responsible for CPT resistance. Our results showed that a single amino acid change (E418K) resulted in CPT resistance. Therefore, quantitative and qualitative changes in Top I were responsible for CPT resistance in CPT30 cells. CPT resistance in CPT30R cells was caused by mutation of Top I.
authors.affiliation: Division of Cancer Research, National Health Research Institutes, 100 Taipei, Taiwan, Republic of China. jychang@nhri.org.tw
authors.forename: Jang-Yang
authors.initials: JY
authors.lastname: Chang
authors.forename: Jin-Fen
authors.initials: JF
authors.lastname: Liu
authors.forename: Shin-Hun
authors.initials: SH
authors.lastname: Juang
authors.forename: Tsang-Wu
authors.initials: TW
authors.lastname: Liu
authors.forename: Li-Tzong
authors.initials: LT
authors.lastname: Chen
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000493:pharmacokinetics / Q000494:pharmacology*; D002166:Camptothecin / Q000493:pharmacokinetics / Q000494:pharmacology*; D016190:Carboplatin / Q000494:pharmacology; D002330:Carmustine / Q000494:pharmacology; D002384:Catalysis; D002455:Cell Division / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology; D004249:DNA Damage; D004264:DNA Topoisomerases, Type I / Q000235:genetics* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D004791:Enzyme Inhibitors / Q000493:pharmacokinetics / Q000494:pharmacology*; D015870:Gene Expression; D006801:Humans; D009303:Nasopharyngeal Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics; D017354:Point Mutation; D012334:RNA, Neoplasm / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D004791:Enzyme Inhibitors; D012334:RNA, Neoplasm; D016190:Carboplatin; D004264:DNA Topoisomerases, Type I; D002945:Cisplatin; D002330:Carmustine; D002166:Camptothecin