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 o f resistance to doxorubicin. The topoisomerase II activity present in crude nuclear extracts from mutant and wild-type cells is similar in a mount and equally sensitive to VP-16. However, in live cells, the topo isomerase II from FVP1b and FVP3 is much less susceptible to drug-indu ced 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 f rom FEM-X and FVP3. The only sequence change unique to the cDNA from d rug-resistant cells is a 3 bp deletion of nucleotide 1320-1322, result ing in a deletion of Ala429. Three FEM-X sublines of increasing resist ance were tested, and the prevalence of the mutant RNA over wild-type increases in these cells in parallel with their resistance to VP-16. I n FVP3, the most highly resistant line, expression of the wild-type al lele 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 le ss susceptible to drug-induced cleavable complex formation and confers a growth advantage upon cells in the presence of VP-16. Since topoiso merase II alpha activity in extracts from these drug-resistant FEM-X l ines 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-topo isomerase 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 .