Acquisition of multiple copies of a mutant topoisomerase II alpha allele by chromosome 17 aneuploidy is associated with etoposide resistance in human melanoma cell lines

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 II alpha were cloned from both FEM-X and FVP3, the most resistant mutant. Deletion of nucleotides 1320-1322 (or Ala429 from the resulting topoisomerase II alpha 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 II alpha 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 II alpha 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 topoisomerase II alpha alleles in FVP3 is approximately 2-fold that in the parental cells. Fluorescence in situ hybridization with a chromosome 17 paint reveals that amplification of the topoisomerase II alpha 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 II alpha locus. In this subline it is likely that three copies of the topoisomerase II alpha 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 II alpha locus that correlate with observed cytogenetic changes in FVP3. These results suggest that the acquisition of the mutant allele of topoisomerase II alpha 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 II alpha mRNA over that of the wild-type which is associated with a concomitant increase in the number of mutant topoisomerase II alpha alleles.