Detection of topoisomerase I gene point mutation in CPT-11 resistant lung cancer cell line.
CPT-11, a recently developed topoisomerase I (Topo I) inhibitor, attracts the attention not only of basic researchers but also of clinicians because of its high antitumor activity. The CPT-11 resistant human lung cancer cell line, PC-7/CPT, showed 10-fold resistance compared to parental cell line, PC-7. The total activity of Topo I in the resistant cell line was one fourth that of the parental sensitive cell line. The Topo I from the resistant cells was also 5-fold more resistant to the inhibitory effect of CPT-11 than that of the parental cells. We speculated that the alteration of the Topo I gene may be responsible for the change in topoisomerase activity of the CPT-11 resistant cell line. Therefore, we analyzed the mutation of Topo I gene using the method of single strand conformation polymorphism of polymerase chain reaction and the reverse transcriptase. We divided Topo I cDNA into ten fragments which overlapped each other and covered whole coding sequences of the Topo I cDNA. We observed mobility shift of two fragments in the PC-7/CPT, suggesting the presence of some mutations in these fragments. We performed the direct-sequencing of these portions by the dideoxy chain termination method and observed an altered sequence having a G to A base change in PC-7/CPT. This base substitution results in replacement of the conserved threonine at 729 position with alanine. These results suggest that the point mutation of Topo I gene is related to the decreases of Topo I activity and the sensitivity to Topo I inhibitor in PC-7/CPT cells.
Kubota
N
N
Pharmacology Division, National Cancer Center Research Institute, Tokyo, Japan.
Kanzawa
F
F
Nishio
K
K
Takeda
Y
Y
Ohmori
T
T
Fujiwara
Y
Y
Terashima
Y
Y
Saijo
N
N
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D001483:Base Sequence; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000494:pharmacology; D004264:DNA Topoisomerases, Type I / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D004351:Drug Resistance / Q000235:genetics*; D006801:Humans; D000077146:Irinotecan; D008175:Lung Neoplasms; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D014407:Tumor Cells, Cultured
D000972:Antineoplastic Agents, Phytogenic; D004273:DNA, Neoplasm; D009838:Oligodeoxyribonucleotides; D012334:RNA, Neoplasm; D000077146:Irinotecan; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin