IDENTIFICATION OF A MUTANT HUMAN TOPOISOMERASE-I WITH INTACT CATALYTIC ACTIVITY AND RESISTANCE TO 9-NITRO-CAMPTOTHECIN

Human U-937 myeloid leukemia cells were selected for resistance to inc reasing concentrations of the camptothecin derivative, 9-nitro-20(S)ca mptothecin (9-NC). The isolated single cell clone, designated U-937/CR , was approximately 20-fold resistant to 9-NC. Analysis of topoisomera se I (topo I) gene expression in U-937/CR cells demonstrated similar m RNA levels as compared with U-937 cells. Immunoblotting with an anti-t opo I serum revealed reactive proteins at 100, 75, and 67 kDa which we re expressed at the same level in the parental and 9-NC-resistant clon es. These cell lines also demonstrated similar levels of topo I cataly tic activity as determined by assaying nuclear extracts for relaxation of supercoiled plasmid DNA. In contrast, catalytic assays performed i n the presence of 9-NC demonstrated that topo I activity from U-937/CR cells was approximately 10-fold more resistant than that from U-937 c ells. Nucleotide sequencing of topo I cDNAs revealed the substitution of phenylalanine (TTC) at residue 361 in U-937 cells with serine (TCC) in the 9-NC-resistant clone. Expression and partial purification of t he mutant topo I protein in Escherichia coli demonstrated resistance o f this enzyme to 9-NC in catalytic assays. Taken together, these findi ngs identify a novel mutation in topo I which confers resistance to 9- NC and support the involvemenT of this region in the interaction betwe en topo I and 9-NC.