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. Thi s cell line is 974-fold more resistant to camptothecin than parental c ells. Resistance is only partially explained by 2-fold reductions in t opoisomerase I protein and mRNA levels. We further investigated bioche mical and molecular features of topoisomerase I in the resistant cell line. Sequence analyses of the top1 cDNA from CEM/C2 identified mutati ons corresponding to two amino acid substitutions, Met370Thr and Asn72 2Ser. Asn722Ser is next to the catalytic Tyr723 in a region highly con served among type I eukargotic DNA topoisomerases. Recombinant top1 wi th the corresponding substitution was found to be catalytically active and resistant to camptothecin. These results indicate that camptothec in resistance of CEM/C2 is due to the mutation Asn722Ser and strongly suggest that the asparagine immediately flanking the catalytic tyrosin e is important for the camptothecin action.