Retinoids mediate their biological effect by interacting with specific nuclear receptors. Of the several known RAR (retinoic acid receptor) subtypes, RAR-ß 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-ß 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-ß-ligand interaction. RT-PCR (reverse transcription-PCR) and Western blot analyses show that HRPE cells specifically express only RAR-ß and none of the other receptor subtypes. In addition, we show that the expression of RAR-ß increases with increasing passage number of the cells. Interestingly, the increase in RAR-ß 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-ß-ligand interactions using nuclear extract from late passage HRPE cells as a source of endogenous RAR-ß. Using [3H]CD367 as the ligand, RAR-ß 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-ß 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-ß was between approx. 1-30 μM, showing that the flavonoids interact with RAR-ß with low affinity.
- Culture passage
- Human retinal pigment epithelial cell (HRPE cell)
- Retinoic acid receptor ß (RAR-ß)
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology