ALBERT

Notes: Abstract Mutants of the human melanoma cell line, FEM-X, selected in multiple steps with VP-16 (etoposide), are cross resistant to the epipodophyllotoxins and doxorubicin. Complementary DNA's for topoisomerase IIα were cloned from both FEM-X and FVP3, the most resistant mutant. Deletion of nucleotides 1320–1322 (or Ala429 from the resulting topoisomerase IIα protein) was unique to the cDNA from the drug resistant cell line. Expression of the mutant mRNA increases in parallel with VP-16 resistance in this series of cell lines. Restriction analysis and Southern analysis with allele-specific oligonucleotide probes were used to quantify the ratio of wild-type to mutant topoisomerase IIα alleles present in DNA amplified by PCR from both FEM-X and the drug resistant sublines. This analysis shows that in cell lines of increasing drug resistance, the number of mutant topoisomerase IIα alleles increases incrementally along with a concomitant decrease in the number of wild-type alleles. By quantitative Southern analysis of genomic DNA the total number of topoosomerase IIα alleles in FVP3 is approximately 2-fold that in the parental cells. Fluorescencein situ hybridization with a chromosome 17 paint reveals that amplification of the topoisomerase IIα locus in FVP3 correlates with an increase in the number of chromosome 17's, specifically the long arm. Cytogenetic analysis demonstrates that FEM-X contains three copies of chromosome 17, two of which are morphologically normal. During drug selection, FVP3 has gained 2–3 additional copies of the long arm of chromosome 17, the chromosomal location of the topoisomerase IIα locus. In this subline it is likely that three copies of the topoisomerase IIα gene are found on normal chromosome 17's and two on an isochromosome of the long arm of 17. By pulsed field gel electrophoresis, we were able to detect changes in the restriction pattern of the region of the long arm of chromosome 17 around the topoisomerase IIα locus that correlate with observed cytogenetic changes in FVP3. These results suggest that the acquisition of the mutant allele of topoisomerase IIα confers a selective advantage to cells in the presence of VP-16. As the drug concentration increased during the selection process, surviving sublines show preferential expression of the mutant topoisomerase IIα mRNA over that of the wild-type which is associated with a concomitant increase in the number of mutant topoisomerase IIα alleles.