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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

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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.

Original languageEnglish
JournalCancer Research
Volume59
Issue number14
Pages (from-to)3442-50
Number of pages9
ISSN0008-5472
Publication statusPublished - 15 Jul 1999

    Research areas

  • Adenosine Triphosphate/metabolism, Amino Acid Sequence, Amino Acid Substitution, Amsacrine/pharmacology, Animals, Antineoplastic Agents/chemistry, Binding Sites, CHO Cells, Carcinoma, Small Cell/drug therapy, Catalysis/drug effects, Consensus Sequence, Cricetinae, Cricetulus, DNA Damage, DNA Mutational Analysis, DNA Topoisomerases, Type II/genetics, DNA, Neoplasm/genetics, DNA, Single-Stranded/genetics, Drug Resistance, Neoplasm/genetics, Enzyme Inhibitors/pharmacology, Etoposide/pharmacology, Humans, Lung Neoplasms/drug therapy, Molecular Sequence Data, Mutagenesis, Site-Directed, Point Mutation, Protein Isoforms/antagonists & inhibitors, Razoxane/chemistry, Recombinant Fusion Proteins/metabolism, Saccharomyces cerevisiae/genetics, Structure-Activity Relationship, Thiobarbiturates/pharmacology, Topoisomerase II Inhibitors, Tumor Stem Cell Assay

ID: 59178626