An RPE cell line as a useful in vitro model for studying retinoic acid receptor β: expression and affinity

Abstract

Retinoids mediate their biological effect by interacting with specific nuclear receptors. Of the several known RAR (retinoic acid receptor) subtypes, RAR-β is of particular interest, since its expression is silenced in many cancers and it is believed to be a tumour suppressor. Specific ligands of RAR-β can potentially be used in anti-cancer therapy. In the present study, we have investigated the feasibility of using HRPE cells (human retinal pigment epithelial cells) as an experimental model for characterizing RAR-β–ligand interaction. RT–PCR (reverse transcription–PCR) and Western blot analyses show that HRPE cells specifically express only RAR-β and none of the other receptor subtypes. In addition, we show that the expression of RAR-β increases with increasing passage number of the cells. Interestingly, the increase in RAR-β expression is not associated with telomere shortening, a typical biomarker of cellular senescence. In the present study, we also describe a protocol for characterizing RAR-β–ligand interactions using nuclear extract from late passage HRPE cells as a source of endogenous RAR-β. Using [3H]CD367 as the ligand, RAR-β in HRPE cells showed an affinity of 9.6±0.6 nM and a Bmax of 780±14 fmol/mg of protein. We have confirmed the feasibility of using this assay to detect the interaction of ligands with RAR-β by investigating the ability of certain flavonoids to inhibit the binding of [3H]CD367 to nuclear extracts from HRPE cells. The inhibition constant of the flavonoids for RAR-β was between approx. 1–30 μM, showing that the flavonoids interact with RAR-β with low affinity.