Characterization of a novel topoisomerase I mutation from a camptothecin-resistant human prostate cancer cell line.
In this study, we characterized the structure and function of topoisomerase I (top1) protein in the camptothecin (CPT)-resistant prostate cancer cell lines, DU-145/RC0.1 and DU-145/RC1 (RC0.1 and RC1, respectively). Both of the cell lines were previously selected by continuous exposure to 9-nitro-CPT. The RC0.1 and RC1 cells have high cross-resistance to CPT derivatives including SN-38 and topotecan, but are not cross-resistant to the non-top1 inhibitors etoposide, doxorubicin, and vincristine. Although the top1 protein levels were not decreased in the resistant cells compared with the parental cells, CPT-induced DNA cleavage was markedly reduced in the RC0.1 and RC1 nuclear extracts. The resistant-cell-line nuclear extracts also demonstrated top1 catalytic activity and resistance to CPT, in in vitro assays. Reverse transcription-PCR products from the resistant cell lines were sequenced, and revealed a point mutation resulting in a R364H mutation in the top1 of both RC0.1 and RC1. No wild-type top1 RNA or genomic DNA was detected in the resistant cell lines. Using a purified recombinant R364H top1, we found that the R364H mutant top1 was CPT resistant and fully active. In the published top1 crystal structure, the R364H mutation is close to the catalytic tyrosine and other well-known mutations leading to CPT resistance.
Urasaki
Y
Y
Division of Basic Sciences, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255, USA.
Laco
G S
GS
Pourquier
P
P
Takebayashi
Y
Y
Kohlhagen
G
G
Gioffre
C
C
Zhang
H
H
Chatterjee
D
D
Pantazis
P
P
Pommier
Y
Y
D000595:Amino Acid Sequence; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D001483:Base Sequence; D002166:Camptothecin / Q000494:pharmacology*; D002384:Catalysis; D004264:DNA Topoisomerases, Type I / Q000235:genetics* / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D011471:Prostatic Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013539:Simian virus 40 / Q000235:genetics; D059004:Topoisomerase I Inhibitors; D014407:Tumor Cells, Cultured
D000972:Antineoplastic Agents, Phytogenic; D004279:DNA, Viral; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D059004:Topoisomerase I Inhibitors; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin