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Efflux Protein Expression in Human Retinal Pigment Epithelium Cell Lines

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Abstract

Purpose

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.

Methods

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.

Results

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.

Conclusions

MRP1, MRP4 and MRP5 are the main efflux transporters in RPE cell lines. There are differences in efflux protein expression between RPE cell lines.

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Acknowledgements

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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Authors and Affiliations

  1. Department of Pharmaceutics, University of Kuopio, Yliopistonranta 1C, 70210, Kuopio, Finland

    Eliisa Mannermaa, Katriina Kokkonen & Veli-Pekka Ranta

  2. Centre for Drug Research, University of Helsinki, Helsinki, Finland

    Kati-Sisko Vellonen & Arto Urtti

  3. Division of Biopharmacy and Pharmacokinetics, University of Helsinki, Helsinki, Finland

    Kati-Sisko Vellonen

  4. Department of Ophthalmology, University of Kuopio, Kuopio, Finland

    Tuomas Ryhänen & Kai Kaarniranta

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  1. Eliisa Mannermaa
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Correspondence to Eliisa Mannermaa.

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Mannermaa, E., Vellonen, KS., Ryhänen, T. et al. Efflux Protein Expression in Human Retinal Pigment Epithelium Cell Lines. Pharm Res 26, 1785–1791 (2009). https://doi.org/10.1007/s11095-009-9890-6

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