Human small cell lung cancer NYH cells selected for resistance to the bisdioxopiperazine topoisomerase II catalytic inhibitor ICRF-187 demonstrate a functional R162Q mutation in the Walker A consensus ATP binding domain of the alpha isoform

Bisdioxopiperazine drugs such as ICRF-187 are catalytic inhibitors of DNA t opoisomerase II, with at least two effects on the enzyme: namely, locking i t in a closed-clamp form and inhibiting its ATPase activity. This is in con trast to topoisomerase II poisons as etoposide and amsacrine (m-AMSA), whic h act by stabilizing enzyme-DNA-drug complexes at a stage in which the DNA gate strand is cleaved and the protein is covalently attached to DNA, Human small cell lung cancer NYH cells selected for resistance to ICRF-187 (NMP/ 187) showed a 25% increase in topoisomerase II alpha level and no change In expression of the beta isoform, Sequencing of the entire topoisomerase II alpha cDNA from NYH/187 cells demonstrated a homozygous G-->A point mutatio n at nucleotide 485, leading to a R162Q conversion in the Walker A consensu s Am binding site (residues 161-165 in the cu isoform), this being the firs t drug-selected mutation described at this site, Western blotting after inc ubation with ICRF-187 showed no depletion of the alpha isoform in NYH/187 c ells in contrast to wild-type (wt) cells, whereas equal depletion of the be ta isoform was observed in the two sublines, Alkaline elution assay demonst rated a lack of inhibition of etoposide-induced DNA single-stranded breaks in NYH/187 cells, whereas this inhibition was readily apparent in NYH cells . Site-directed mutagenesis ia human topoisomerase II alpha! introduced int o a yeast Saccharomyces cerevisiae strain with a temperature-conditional ye ast TOP2 mutant demonstrated that R162Q conferred resistance to the bisdiox opiperazines ICRF-187 and -193 but not to etoposide or m-AMSA, Both etoposi de and m-AMSA induced more DNA cleavage with purified R162Q enzyme than wit h the wt, The R162Q enzyme has a 20-25% decreased catalytic capacity compar ed to the wt and was almost inactive at <0.25 mM ATP compared to the wt. Ki netoplast DNA decatenation by the R162Q enzyme at I mM ATP was not resistan t to ICRF-187 compared to wt, whereas it was clearly less sensitive than wt to ICRF-187 at low ATP concentrations. This suggests that it is a shift in the equilibrium to an open-clamp state in the enzyme's catalytic cycle cau sed by a decreased ATP binding by the mutated enzyme that is responsible fo r bisdioxopiperazine resistance.