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 a Isoform
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چکیده
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 IIa level and no change in expression of the b isoform. Sequencing of the entire topoisomerase IIa cDNA from NYH/187 cells demonstrated a homozygous G3A point mutation at nucleotide 485, leading to a R162Q conversion in the Walker A consensus ATP binding site (residues 161–165 in the a isoform), this being the first drug-selected mutation described at this site. Western blotting after incubation with ICRF-187 showed no depletion of the a isoform in NYH/187 cells in contrast to wild-type (wt) cells, whereas equal depletion of the b 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 IIa 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.
منابع مشابه
Mutation in the Walker A Consensus ATP Binding Domain of Catalytic Inhibitor ICRF-187 Demonstrate a Functional R162Q Resistance to the Bisdioxopiperazine Topoisomerase II Human Small Cell Lung Cancer NYH Cells Selected for
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...
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