A NOVEL POINT MUTATION IN THE 3'-PLANKING REGION OF THE DNA-BINDING DOMAIN OF TOPOISOMERASE-II-ALPHA ASSOCIATED WITH ACQUIRED-RESISTANCE TO TOPOISOMERASE-II ACTIVE AGENTS

V511 and V513 are Chinese hamster cell lines with acquired resistance to topoisomerase II (topo II) directed agents. These cell lines were obtained by mutagenizing Chinese hamster V79 cells with N-methyl-N'-nitro-N-nitrosoguanidine and subsequently selecting in etoposide (VP-16). We have previously shown that this resistance is not associated with alterations in drug uptake. To elucidate whether any alterations in the functionally important domains of topo II alpha were associated with VP-16 resistance, we used reverse transcriptase-polymerase chain reaction, single-strand conformational polymorphism analysis, and subsequent sequencing of topo II alpha from V79, V511, and V513 to search for mutations in five major functional domains including the regions of the consensus ATP binding sequences (Motif A and Motif B/dinucleotide binding site), the DNA binding domain, and the 5' and 3' flanking regions of the DNA binding position. The V511 cells showed no mutational changes in these regions. However, the topo II alpha gene from V513 showed a point mutation at nucleotide 2552 that resulted in a glycine-to-aspartate mutation at amino acid position 851 in the 3' flanking region of the DNA binding site. This mutation at amino acid position 851 in V513 cells is associated with reduced VP-16-induced cleavable complex formation demonstrated by potassium-sodium dodecyl sulfate assay and band-depletion analysis. Our results suggest that the mutation at amino acid position 851 may play a role in drug resistance, presumably by interfering with enzyme-DNA binding.