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 increasing concentrations of the camptothecin derivative, 9-nitro-20(S)camptothecin (9-NC). The isolated single cell clone, designated U-937/CR, was approximately 20-fold resistant to 9-NC. Analysis of topoisomerase I (topo I) gene expression in U-937/CR cells demonstrated similar mRNA levels as compared with U-937 cells. Immunoblotting with an anti-topo I serum revealed reactive proteins at 100, 75, and 67 kDa which were expressed at the same level in the parental and 9-NC-resistant clones. These cell lines also demonstrated similar levels of topo I catalytic activity as determined by assaying nuclear extracts for relaxation of supercoiled plasmid DNA. In contrast, catalytic assays performed in 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 cells. 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 the mutant topo I protein in Escherichia coli demonstrated resistance of this enzyme to 9-NC in catalytic assays. Taken together, these findings identify a novel mutation in topo I which confers resistance to 9-NC and support the involvement of this region in the interaction between topo I and 9-NC.
Rubin
E
E
Division of Cancer Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
Pantazis
P
P
Bharti
A
A
Toppmeyer
D
D
Giovanella
B
B
Kufe
D
D
D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000633:toxicity*; D001483:Base Sequence; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000633:toxicity; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D002470:Cell Survival / Q000187:drug effects; D002999:Clone Cells; D003062:Codon / Q000235:genetics; D017931:DNA Primers; D004264:DNA Topoisomerases, Type I / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D004351:Drug Resistance / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D007951:Leukemia, Myeloid; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D003062:Codon; D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D004264:DNA Topoisomerases, Type I; C079905:rubitecan; D002166:Camptothecin