A NOVEL MUTANT TOPOISOMERASE II-ALPHA PRESENT IN VP-16-RESISTANT HUMAN-MELANOMA CELL-LINES HAS A DELETION OF ALANINE-429

The human melanoma cell line FEM-X was selected in multiple steps with VP-16 (etoposide) and an inhibitor of P-glycoprotein (Campain ef al., 1993). The resulting clones, FVP1b and FVP3, are highly resistant to the nonintercalative epipodophyllotoxins and exhibit moderate levels of resistance to doxorubicin. The topoisomerase II activity present in crude nuclear extracts from mutant and wild-type cells is similar in amount and equally sensitive to VP-16. However, in live cells, the topoisomerase II from FVP1b and FVP3 is much less susceptible to drug-induced cleavable complex formation than is that from FEM-X. Using reverse transcription followed by the polymerase chain reaction (RT-PCR), we have cloned and sequenced the entire cDNA for topoisomerase II alpha from FEM-X and FVP3. The only sequence change unique to the cDNA from drug-resistant cells is a 3 bp deletion of nucleotide 1320-1322, resulting in a deletion of Ala429. Three FEM-X sublines of increasing resistance were tested, and the prevalence of the mutant RNA over wild-type increases in these cells in parallel with their resistance to VP-16. In FVP3, the most highly resistant line, expression of the wild-type allele is barely detectable. Analysis of genomic DNA shows that FEM-X is homozygous for the wild-type topoisomerase II alpha sequence and that each of the drug-resistant clones possesses both wild-type and mutant alleles. Although not definitive, these genetic results suggest that the deletion of Ala429 from topoisomerase II alpha makes the enzyme less susceptible to drug-induced cleavable complex formation and confers a growth advantage upon cells in the presence of VP-16. Since topoisomerase II alpha activity in extracts from these drug-resistant FEM-X lines is normally sensitive to drugs, the deletion of an Ala at residue 429 may alter intracellular localization of the enzyme or change its interaction with other factors, which could then decrease the DNA-topoisomerase II alpha complexes trapped in the presence of VP-16. However, proof that the mutant topoisomerase II alpha is responsible for drug resistance requires its successful expression in drug-sensitive cells.