Mutation at the catalytic site of topoisomerase I in CEM/C2, a human leukemia cell line resistant to camptothecin.
We developed previously a resistant cell line, CEM/C2, from the human leukemia cell line CCRF-CEM by stepwise selection in camptothecin. This cell line is 974-fold more resistant to camptothecin than parental cells. Resistance is only partially explained by 2-fold reductions in topoisomerase I protein and mRNA levels. We further investigated biochemical and molecular features of topoisomerase I in the resistant cell line. Sequence analyses of the top1 cDNA from CEM/C2 identified mutations corresponding to two amino acid substitutions, Met370Thr and Asn722Ser. Asn722Ser is next to the catalytic Tyr723 in a region highly conserved among type I eukaryotic DNA topoisomerases. Recombinant top1 with the corresponding substitution was found to be catalytically active and resistant to camptothecin. These results indicate that camptothecin resistance of CEM/C2 is due to the mutation Asn722Ser and strongly suggest that the asparagine immediately flanking the catalytic tyrosine is important for the camptothecin action.
Fujimori
A
A
Laboratory of Molecular Pharmacology, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255.
Harker
W G
WG
Kohlhagen
G
G
Hoki
Y
Y
Pommier
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002166:Camptothecin / Q000494:pharmacology*; D006224:Cricetinae; D003412:Cricetulus; D004264:DNA Topoisomerases, Type I / Q000235:genetics* / Q000378:metabolism; D004351:Drug Resistance; D006801:Humans; D007938:Leukemia / Q000188:drug therapy* / Q000201:enzymology / Q000473:pathology; D008969:Molecular Sequence Data; D017354:Point Mutation; D011994:Recombinant Proteins / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin