A Microarray Comparison of the Molecular Phenotypes of the RPE Cell Lines ARPE-19 and D407

Abstract

Abstract: : Purpose: ARPE-19 and D407 are two commonly used retinal pigment epithelial cell lines. Both exhibit, to varying degrees, features of the RPE in vivo. The aim of this investigation is to analyze whether the pattern of genes expressed by these cell lines matches the expected molecular phenotype of the RPE and to determine how differential gene expression between the two cell lines correlates with their observed in vitro phenotypes. Methods: The human ARPE-19 cell line was obtained from the ATCC (Rockville, MD, USA) and maintained in DMEM/F12 (Life Technologies) with 2% FCS. The human D407 RPE cell line (Dr Richard Hunt) was maintained in DMEM (Life Technologies) with 2% fetal calf serum (FCS). Total RNA was isolated with Tri-reagent (Sigma), followed by DNase treatment and column purification (Qiagen). RNA quality was assessed using a 2100 Bioanalyzer (Agilent). To measure changes in gene expression RNA was extracted, reverse transcribed and hybridized to a cDNA microarray containing 12,000 expressed human sequences (Agilent human 1). Fluorescent labelling was performed with a 3DNA Submicro Kit (Genisphere) and spot intensities measured using ScanAlyze. Fold changes below 2.0 were disregarded. Quantitative RT-PCR was performed using a LightCycler (Roche). Results: The ARPE-19 and D407 cells both display morphological features of the RPE in vivo and were demonstrated to also express genes characteristic of the RPE. In keeping with the contact-inhibited, differentiated nature of ARPE-19 cultures a number of proteins implicated in cell attachment/matrix interactions were among the most preferentially expressed relative to D407. These included thrombospondin 1 precursor (~7 fold higher), matrilin-2 precursor (~7 fold), Cadherin-related tumor suppressor homolog precursor (~6 fold) and SPARC precursor (~4 fold). In contrast, the most highly differentially expressed protein in D407 was the S100P calcium binding protein (~13 fold) which has been associated with transformed cells. Other genes associated with greater cell turnover, such as histones and the MCM2 DNA replication licensing factor, were also expressed at higher levels in D407. Conclusions: Microarray analysis of different cell lines has revealed differential gene expression which will contribute to our understanding of RPE biology and role in pathology.

Keywords: gene microarray • retinal pigment epithelium • proliferative vitreoretinopathy