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 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.
Department of Pathology, Laboratory Center, Rigshospitalet, Copenhagen, Denmark.
I
I
Wessel
L H
LH
Jensen
P B
PB
Jensen
J
J
Falck
A
A
Rose
M
M
Roerth
J L
JL
Nitiss
M
M
Sehested
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000677:Amsacrine / Q000494:pharmacology; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D001665:Binding Sites; D016466:CHO Cells; D018288:Carcinoma, Small Cell / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D002384:Catalysis / Q000187:drug effects; D016384:Consensus Sequence; D006224:Cricetinae; D003412:Cricetulus; D004249:DNA Damage; D004252:DNA Mutational Analysis; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism; D004273:DNA, Neoplasm / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D020033:Protein Isoforms / Q000037:antagonists & inhibitors* / Q000235:genetics; D011929:Razoxane / Q000737:chemistry / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013329:Structure-Activity Relationship; D013858:Thiobarbiturates / Q000494:pharmacology; D059005:Topoisomerase II Inhibitors; D014410:Tumor Stem Cell Assay
D000970:Antineoplastic Agents; D004273:DNA, Neoplasm; D004277:DNA, Single-Stranded; D004791:Enzyme Inhibitors; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D013858:Thiobarbiturates; D059005:Topoisomerase II Inhibitors; D000677:Amsacrine; D011929:Razoxane; D005047:Etoposide; D000255:Adenosine Triphosphate; D004250:DNA Topoisomerases, Type II; C045459:merbarone