Novel missense mutation of the DNA topoisomerase I gene in SN-38-resistant DLD-1 cells.
Irinotecan hydrochloride, a camptothecin derivative, is one of the most effective drugs for colorectal cancer, and SN-38 is its main active metabolite. Development of resistance is a major obstacle to the clinical application of this drug. We established an SN-38-resistant subline from DLD-1 human colon cancer cells by continuous exposure to SN-38 and studied the mechanisms of resistance. The resistant subline (designated as DLDSNR6) had 10- to 100-fold higher resistance to camptothecin derivatives but showed no cross-resistance to doxorubicin, mitomycin C, and etoposide. DLDSNR6 cells carried a missense mutation in one allele of the DNA topoisomerase I gene that substituted glycine for serine at amino acid residue 365 accompanied by loss of the latter part of the remaining wild-type allele. Topoisomerase I expression was equal in DLDSNR6 and DLD-1 cells, but the nuclear extract of DLDSNR6 cells showed lower topoisomerase I catalytic activity. Moreover, exposure to camptothecin caused less accumulation of topoisomerase I-DNA complexes in DLDSNR6 cells than in DLD-1 cells. These findings suggest that the mutation interfered with both the catalytic activity of topoisomerase I and the stability of the ternary complex between topoisomerase I, DNA, and SN-38. This SN-38-resistant DLDSNR6 cell line may be useful for understanding the mechanisms of topoisomerase I function and drug-enzyme interactions.
Department of Molecular Genetics, Institute of DNA Medicine, Jikei University School of Medicine, Nishi-Shimbashi 3-25-8 Minato-ku, Tokyo 105-8461, Japan. yarakawa@jikei.ac.jp
Yasuhiro
Y
Arakawa
Hideaki
H
Suzuki
Shinobu
S
Saito
Hisashi
H
Yamada
D019943:Amino Acid Substitution / Q000235:genetics; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000494:pharmacology; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000201:enzymology / Q000235:genetics*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005998:Glycine / Q000737:chemistry / Q000235:genetics; D006801:Humans; D000077146:Irinotecan; D020125:Mutation, Missense; D011487:Protein Conformation; D012694:Serine / Q000737:chemistry / Q000235:genetics
D000972:Antineoplastic Agents, Phytogenic; D012694:Serine; D000077146:Irinotecan; D004264:DNA Topoisomerases, Type I; D005998:Glycine; D002166:Camptothecin