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Blair, J.M., Bavro, V.N., Ricci, V., Modi, V., Cacciotto, P., Kleinekathofer, U., Ruggerone, P., Vargiu, A.V., Baylay, A.J. and Smith, H.E. (2015) AcrB Drug-Binding Pocket Substitution Confers Clinically Relevant Resistance and Altered Substrate Specificity. Proceedings of the National Academy of Sciences of the United States of America, 112, 3511-3516.
https://doi.org/10.1073/pnas.1419939112

has been cited by the following article:

• TITLE: A Fluorescent Cell-Based Technique for Monitoring Efflux of MRP4

  AUTHORS: Tombari Pius Monsi, Adline Erinma Ben-Chioma, Donaltus Onukwufor Onwuli

  KEYWORDS: Efflux Pump, Drug Resistance, ABC-Transporter, HEK 293, Fluorescence Assay

  JOURNAL NAME: American Journal of Molecular Biology, Vol.10 No.3, July 9, 2020

  ABSTRACT: Background: Overexpression of efflux pumps is the drug resistance and adaptation mechanism employed by some eukaryotes and bacteria to transport endogenous and chemotherapeutic compounds from the intracellular to the extracellular environment. Aim: The study aimed at establishing a fluorescent cell-based assay to monitor the efflux activities of an ABC-transporter, multi-drug resistance protein 4 (MRP4). Methods: DH5α competent E. coli cells were transformed with pcDNA-MRP4 by the heat-shock process. The presence of the MRP4 gene was analyzed by the digestion of plasmid using EcoRI and analyzed on a 1% agarose gel. HEK 293 cells were transfected with purified pcDNA-MRP4 under optimized conditions using a Polyethylenimine (PEI) protocol. The level of MRP4 in the HEK 293 cells was characterized by western blotting analysis using M4I-10 anti-MRP4 and anti-Rat IgG (whole molecule)-Alkaline phosphatase antibodies. The fluorescent uptake study was performed by the incubation of 0.02 mM 8-[fluo-cAMP] with the MRP4-transfected and control HEK 293 cells for 1 h. The level of fluorescence was analyzed using fluorescence microscopy and spectrometer. Results: The agarose gel analysis showed a plasmid of 9.4 kb and restriction product of 5 kb, which correspond with the pcDNA and MRP4 sizes respectively. The western blot results of the transfection showed 4 μg pcDNA-MRP4 and the N/P ratio of 9 was the optimized condition to transfect our HEK 293 cells as it showed the broadest band. In the efflux studies, the fluorescence images of the MRP4-transfected HEK 293 cells were very low compared to the untransfected control. The level of fluorescence accumulation was significantly (P ≤ 0.0001) higher 228.6 ± 13.1 RFU in the untransfected cells than the MRP4-transfected cells 8.6 ± 1.8 RFU. Conclusion: The higher levels of fluorescence detected in the control in both the fluorescent microscopy and spectrophotometer showed that MRP4-transfected cells had effluxed the 8-[fluo-cAMP] substrate out of the cell. This method could be employed in the detection of MRP4 functions in bacteria and cancer cells.