Bisdioxopiperazine drugs such as ICRF-187 are catalytic inhibitors of DNA 
topoisomerase II, with at least two effects on the enzyme: namely, locking it in 
a closed-clamp form and inhibiting its ATPase activity. This is in contrast to 
topoisomerase II poisons as etoposide and amsacrine (m-AMSA), which 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 (NYH/187) showed a 25% 
increase in topoisomerase IIalpha level and no change in expression of the beta 
isoform. Sequencing of the entire topoisomerase IIalpha cDNA from NYH/187 cells 
demonstrated a homozygous G-->A point mutation at nucleotide 485, leading to a 
R162Q conversion in the Walker A consensus ATP binding site (residues 161-165 in 
the alpha isoform), this being the first drug-selected mutation described at 
this site. Western blotting after incubation with ICRF-187 showed no depletion 
of the alpha isoform in NYH/187 cells in contrast to wild-type (wt) cells, 
whereas equal depletion of the beta isoform was observed in the two sublines. 
Alkaline elution assay demonstrated 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 in human topoisomerase IIalpha 
introduced into a yeast Saccharomyces cerevisiae strain with a 
temperature-conditional yeast TOP2 mutant demonstrated that R162Q conferred 
resistance to the bisdioxopiperazines ICRF-187 and -193 but not to etoposide or 
m-AMSA. Both etoposide and m-AMSA induced more DNA cleavage with purified R162Q 
enzyme than with the wt. The R162Q enzyme has a 20-25% decreased catalytic 
capacity compared to the wt and was almost inactive at <0.25 mM ATP compared to 
the wt. Kinetoplast DNA decatenation by the R162Q enzyme at 1 mM ATP was not 
resistant 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 caused by 
a decreased ATP binding by the mutated enzyme that is responsible for 
bisdioxopiperazine resistance.