A Fluorescent Cell-Based Technique for Monitoring Efflux of MRP4  

作　　者：Tombari Pius Monsi Adline Erinma Ben-Chioma Donaltus Onukwufor Onwuli Tombari Pius Monsi;Adline Erinma Ben-Chioma;Donaltus Onukwufor Onwuli(Microbiology Unit, Department of Medical Laboratory Science, Rivers State University, Port Harcourt, Nigeria;Department of Biomedical Science, Life and Health Science, Aston University, Birmingham, UK;Chemical Pathology Unit, Department of Medical Laboratory Science, Rivers State University, Port Harcourt, Nigeria)

机构地区：[1]Microbiology Unit, Department of Medical Laboratory Science, Rivers State University, Port Harcourt, Nigeria [2]Department of Biomedical Science, Life and Health Science, Aston University, Birmingham, UK [3]Chemical Pathology Unit, Department of Medical Laboratory Science, Rivers State University, Port Harcourt, Nigeria

出　　处：《American Journal of Molecular Biology》2020年第3期188-199,共12页美国分子生物学期刊（英文）

摘　　要：<strong>Background:</strong> 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. <strong>Aim:</strong> 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). <strong>Methods:</strong> DH5α competent <em>E. coli</em> 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. <strong>Results:</strong> 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. <strong>Conclusion:</strong> The higher levels of fluorescence detected in the control in both the fluorescent microscopy and spectrophotometer showed that MRP4-tran<strong>Background:</strong> 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. <strong>Aim:</strong> 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). <strong>Methods:</strong> DH5α competent <em>E. coli</em> 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. <strong>Results:</strong> 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. <strong>Conclusion:</strong> The higher levels of fluorescence detected in the control in both the fluorescent microscopy and spectrophotometer showed that MRP4-tran

关 键 词：Efflux Pump Drug Resistance ABC-TRANSPORTER HEK 293 Fluorescence Assay 

分 类 号：R73[医药卫生—肿瘤]