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

Barbara Pavan, Alessandro Dalpiaz, Carla Biondi, Marzia Nieddu, Antonella De Luca, Puttur D. Prasad, Guglielmo Paganetto, Bartolo Favaloro

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)327-334
Number of pages8
JournalBioscience Reports
Volume28
Issue number6
DOIs
StatePublished - Dec 1 2008

Keywords

  • Culture passage
  • Human retinal pigment epithelial cell (HRPE cell)
  • Retinoic acid receptor ß (RAR-ß)
  • Senescence
  • Telomere

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Pavan, B., Dalpiaz, A., Biondi, C., Nieddu, M., De Luca, A., Prasad, P. D., Paganetto, G., & Favaloro, B. (2008). An RPE cell line as a useful in vitro model for studying retinoic acid receptor ß :Expression and affinity. Bioscience Reports, 28(6), 327-334. https://doi.org/10.1042/BSR20080103