RPE cell line as a useful in vitro model for studying retinoic acid receptor beta: expression and affinity
Résumé
Retinoids mediate their biological effect by interacting with specific nuclear receptors. Of the several known retinoic acid receptor subtypes, RARβ is of particular interest since its expression is silenced in many cancers and is believed to be a tumor suppressor. Specific ligands of RARβ can potentially be used in anti-cancer therapy. Here we have investigated the feasibility of using HRPE cells as an experimental model for characterizing RARβ-ligand interaction. RT-PCR and western blot analyses show that HRPE cells specifically express only RARβ and none of the other receptor subtypes. In addition, our studies show that the expression of RARβ increases with increasing passage number of the cells. Interestingly, the increase in RARβ expression is not associated with shortening of the telomere length, a typical biomarker of cellular senescence. We also describe here a protocol to characterize RARβ-ligand interaction using nuclear extract from late passage HRPE cells as the source of endogenous RARβ. Using [3H]CD367 as the ligand, RARβ in HRPE cells showed an affinity of 9.6±0.6 nM and a Bmax of 780±14 fmol/mg protein. We have confirmed the feasibility of using this assay to detect interaction of ligands with RARβ by investigating the ability of certain flavonoids to inhibit the binding of [3H]CD367 to HRPE cell nuclear extract. The inhibition constant of the flavonoids for RARβ ranged between ~1-30 μM, showing that the flavonoids interact with RARβ with low affinity
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