title: Characterization of Abcc4 gene amplification in stepwise-selected mouse J774 macrophages resistant to the topoisomerase II inhibitor ciprofloxacin.
abstract: Exposure of J774 mouse macrophages to stepwise increasing concentrations of ciprofloxacin, an antibiotic inhibiting bacterial topoisomerases, selects for resistant cells that overexpress the efflux transporter Abcc4 (Marquez et al. [2009] Antimicrob. Agents Chemother. 53: 2410-2416), encoded by the Abcc4 gene located on Chromosome 14qE4. In this study, we report the genomic alterations occurring along the selection process. Abcc4 expression progressively increased upon selection rounds, with exponential changes observed between cells exposed to 150 and 200 µM of ciprofloxacin, accompanied by a commensurate decrease in ciprofloxacin accumulation. Molecular cytogenetics experiments showed that this overexpression is linked to Abcc4 gene overrepresentation, grading from a partial trisomy of Chr 14 at the first step of selection (cells exposed to 100 µM ciprofloxacin), to low-level amplifications (around three copies) of Abcc4 locus on 1 or 2 Chr 14 (cells exposed to 150 µM ciprofloxacin), followed by high-level amplification of Abcc4 as homogeneous staining region (hsr), inserted on 3 different derivative Chromosomes (cells exposed to 200 µM ciprofloxacin). In revertant cells obtained after more than 60 passages of culture without drug, the Abcc4 hsr amplification was lost in approx. 70% of the population. These data suggest that exposing cells to sufficient concentrations of an antibiotic with low affinity for eukaryotic topoisomerases can cause major genomic alterations that may lead to the overexpression of the transporter responsible for its efflux. Gene amplification appears therefore as a mechanism of resistance that can be triggered by non-anticancer agents but contribute to cross-resistance, and is partially and slowly reversible.
authors.affiliation: Université catholique de Louvain, Louvain Drug Research Institute, Pharmacologie cellulaire et moléculaire, Brussels, Belgium.
authors.forename: Béatrice
authors.initials: B
authors.lastname: Marquez
authors.forename: Geneviève
authors.initials: G
authors.lastname: Ameye
authors.forename: Coralie M
authors.initials: CM
authors.lastname: Vallet
authors.forename: Paul M
authors.initials: PM
authors.lastname: Tulkens
authors.forename: Hélène A
authors.initials: HA
authors.lastname: Poirel
authors.forename: Françoise
authors.initials: F
authors.lastname: Van Bambeke
mesh_terms: D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology; D002460:Cell Line; D002875:Chromosomes / Q000187:drug effects; D002939:Ciprofloxacin / Q000627:therapeutic use*; D003582:Cytogenetics; D004250:DNA Topoisomerases, Type II / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D015971:Gene Expression Regulation, Enzymologic; D057809:HEK293 Cells; D050956:HSP40 Heat-Shock Proteins / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007621:Karyotyping; D051379:Mice; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics* / Q000378:metabolism; D059003:Topoisomerase Inhibitors / Q000627:therapeutic use*
chemical_list: D000890:Anti-Infective Agents; D002939:Ciprofloxacin; D004250:DNA Topoisomerases, Type II; D050956:HSP40 Heat-Shock Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D059003:Topoisomerase Inhibitors; C489540:Abcc4 protein, mouse; C500846:Dnajc3 protein, mouse