MOLECULAR-CLONING AND IDENTIFICATION OF A POINT MUTATION IN THE TOPOISOMERASE-II CDNA FROM AN ETOPOSIDE-RESISTANT CHINESE-HAMSTER OVARY CELL-LINE

Topoisomerase II (Trop II) is the target enzyme for many antineoplastic drugs such as epipodophyllotoxins, anthracyclines, and acridines. Cell lines with alterations in Top II are resistant to drugs that interact with the enzyme. Studies of the Top II from a Chinese hamster ovary line, Vpm(R)-5, that is resistant to VP-16 and VM-26, demonstrated that it is very similar, qualitatively and quantitatively, to its normal counterpart except that DNA cleavage by the Vpm(R)-5 enzyme is not stimulated by VP-16 or VM-26. To understand the basis for the drug-resistant phenotype, the Top II cDNAs were isolated from both Chinese hamster ovary (CHO) and Vpm(R)-5 cells by cDNA cloning with lambdagt22, and the entire cDNAs were sequenced. A mutation of G --> A at nucleotide 1478 was the only alteration observed in the Vpm(R)-5 Top II cDNA compared with the wild-type gene. The mutation in Vpm(R)-5 was confirmed by sequencing DNA fragments amplified from the genomic DNA by the polymerase chain reaction. Southern blot hybridization analysis of genomic DNA demonstrated loss of a Top II allele in Vpm(R)-5 probably occurred during the development of resistance to etoposide. The mutation in Vpm(R)-5 changes amino acid 493 from arginine to glutamine and is located adjacent to a putative ATP binding site of Top II. Mutations in an analogous region have been identified in two human leukemia cell lines by amplification of segments of Top II cDNA with Taq DNA polymerase. Taken together, these observations suggest that mutations in this region of the gyrase B domain of mammalian topoisomerase II may be capable of conferring resistance to antineoplastic agents that interact with this enzyme.