A NOVEL POINT MUTATION IN THE 3'-PLANKING REGION OF THE DNA-BINDING DOMAIN OF TOPOISOMERASE-II-ALPHA ASSOCIATED WITH ACQUIRED-RESISTANCE TOTOPOISOMERASE-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 o btained by mutagenizing Chinese hamster V79 cells with N-methyl-N'-nit ro-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 th e functionally important domains of topo II alpha were associated with VP-16 resistance, we used reverse transcriptase-polymerase chain reac tion, single-strand conformational polymorphism analysis, and subseque nt sequencing of topo II alpha from V79, V511, and V513 to search for mutations in five major functional domains including the regions of th e consensus ATP binding sequences (Motif A and Motif B/dinucleotide bi nding site), the DNA binding domain, and the 5' and 3' flanking region s of the DNA binding position. The V511 cells showed no mutational cha nges in these regions. However, the topo II alpha gene from V513 showe d a point mutation at nucleotide 2552 that resulted in a glycine-to-as partate mutation at amino acid position 851 in the 3' flanking region of the DNA binding site. This mutation at amino acid position 851 in V 513 cells is associated with reduced VP-16-induced cleavable complex f ormation demonstrated by potassium-sodium dodecyl sulfate assay and ba nd-depletion analysis. Our results suggest that the mutation at amino acid position 851 may play a role in drug resistance, presumably by in terfering with enzyme-DNA binding.