Abstract
Abstract: :
Purpose: P–Glycoprotein (PGP) is an active efflux transporter encoded by the MDR1 gene in humans. PGP expression has recently been demonstrated in the human retinal pigment epithelium in–situ1, suggesting that it plays a role the protection of the retina against naturally occurring toxins or xenobiotics. We wished to establish whether human RPE cell lines expressed PGP under standard culture conditions. Methods:Three cell lines were studied: D407 and ARPE–19 are well–characterised cell lines that arose spontaneously. H1–RPE–7 is the result of SV40 T–antigen transfection into native human RPE cells2. All cell lines were grown to confluence under the standard conditions as recommended by the original authors. Reverse Transcriptase–Polymerase Chain Reaction (RT–PCR) was used to establish the presence of PGP in RPE cells of different passage numbers. MDCK cells transfected with the MDR1 gene, and showing functional PGP expression, were used as positive controls. Immunocytochemistry was carried out using an anti–PGP antibody (C219), with a FITC–conjugated anti–mouse secondary antibody. Cell nuclei were counterstained with Propridium Iodide. Results:We were unable to detect MDR1 mRNA in any of the RPE cell lines studied, and immunocytochemistry confirmed the absence of PGP protein. By contrast, transcript and protein were identified in MDR1–transfected positive controls. Conclusions:The absence of PGP in these cell lines, whilst being reported to be present in–situ, means that these cell lines are not suitable for modelling the transport of PGP substrates across the RPE. 1Kennedy and Mangini, Mol Vis 8, 422. (2002). 2Kanunga et al, Invest Ophthalmol Vis Sci. 43, 546 (2002).
Keywords: retinal pigment epithelium • drug toxicity/drug effects