1. PLoS One. 2011;6(12):e28368. Epub 2011 Dec 5.

Characterization of Abcc4 Gene Amplification in Stepwise-Selected Mouse J774
Macrophages Resistant to the Topoisomerase II Inhibitor Ciprofloxacin.

Marquez B, Ameye G, Vallet CM, Tulkens PM, Poirel HA, Van Bambeke F.

Université catholique de Louvain, Louvain Drug Research Institute, Pharmacologie 
cellulaire et moléculaire, Brussels, Belgium.

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.

PMID: 22162766  [PubMed - in process]