Efflux Protein Expression in Human Retinal Pigment Epithelium Cell Lines
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The objective of this study was to characterize efflux proteins (P-glycoprotein (P-gp), multidrug resistance proteins (MRP1–6) and breast cancer resistance protein (BCRP)) of retinal pigment epithelium (RPE) cell lines.
Expression of efflux proteins in two secondary (ARPE-19, D407) and two primary (HRPEpiC and bovine) RPE cell lines was measured by quantitative RT-PCR and western blotting. Furthermore, activity of MRP1 and MRP5 of ARPE-19 cell line was assessed with calcein-AM and carboxydichlorofluorescein (CDCF) probes.
Similar efflux protein profile was shared between ARPE-19 and primary RPE cells, whereas D407 cell line was notably different. D407 cells expressed MRP2 and BCRP, which were absent in other cell lines and furthermore higher MRP3 transcript expression was found. MRP1, MRP4 and MRP5 were identified from all human RPE cell lines and MRP6 was not expressed in any cell lines. The pattern of efflux protein expression did not change when ARPE-19 cells were differentiated on filters. The calcein-AM and CDCF efflux tests provided evidence supporting MRP1 and MRP5 activity in ARPE-19 cells.
MRP1, MRP4 and MRP5 are the main efflux transporters in RPE cell lines. There are differences in efflux protein expression between RPE cell lines.
KEY WORDSblood retinal barrier cell model drug transport efflux proteins retinal pigment epithelium
We are grateful to Dr P. Borst (Netherlands Cancer Institute) for providing MDCKII and HEK293-MRP4 cell lines, Dr J. Mönkkönen (University of Kuopio) for his support in the completion of this work and Dr M. Suhonen (University of Kuopio) for providing compounds for calcein-AM studies. M.Sc. Sanna Siissalo is acknowledged for her help with the CDCFDA/CDCF-assay and M.Sc. Mika Reinisalo for his advices regarding qRT-PCR standards. We also thank Dr J. Taipalensuu and Lucia Lazorova (Uppsala University, Sweden) for providing the plasmids for quantitative RT-PCR measurement and original authors for the permissions to use plasmids: Dr. Branimir Sikic (Stanford University School of Medicine, USA), Dr. Susan Cole (Cancer Research Institute, Queen’s University, Kingston, Canada), Dr. Piet Borst (Netherlands Cancer Institute, The Netherlands), Dr. John Schuetz (St. Jude Children’s Research Hospital, Memphis, USA), Astellas Pharma Inc. (former Yamanouchi Pharmaceutical Co., Ltd), Dr. Andras Varadi and Dr. Attila Iliás (Hungarian Academy of Sciences, Hungary), Dr. Douglas Ross (University of Maryland, Marlene and Stewart Greenebaum Cancer Center, Baltimore, USA).
This work was supported by the Finnish Eye Foundation (EM, KK), the Finnish Eye and Tissue Bank Foundation (EM), the Evald and Hilda Nissi’s Foundation (EM), Sokeain Ystävät ry/De Blindas Vänner rf (EM), the Finnish Cultural Foundation of Northern Savo (EM), the Finnish Funding Agency for Technology and Innovation (TEKES) (KK, AU).
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