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 Citations (Scopus)

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

Fingerprint

Retinoic Acid Receptors
Cells
Cell Line
Ligands
Flavonoids
In Vitro Techniques
Telomere Shortening
Neoplasms
Retinal Pigments
Cell Aging
Retinoids
Biomarkers
Transcription
Cytoplasmic and Nuclear Receptors
Cell Extracts
Reverse Transcription
Tumors
Assays

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

Cite this

An RPE cell line as a useful in vitro model for studying retinoic acid receptor ß :Expression and affinity. / Pavan, Barbara; Dalpiaz, Alessandro; Biondi, Carla; Nieddu, Marzia; De Luca, Antonella; Prasad, Puttur D; Paganetto, Guglielmo; Favaloro, Bartolo.

In: Bioscience Reports, Vol. 28, No. 6, 01.12.2008, p. 327-334.

Research output: Contribution to journalArticle

Pavan, B, Dalpiaz, A, Biondi, C, Nieddu, M, De Luca, A, Prasad, PD, 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, vol. 28, no. 6, pp. 327-334. https://doi.org/10.1042/BSR20080103
Pavan, Barbara ; Dalpiaz, Alessandro ; Biondi, Carla ; Nieddu, Marzia ; De Luca, Antonella ; Prasad, Puttur D ; Paganetto, Guglielmo ; Favaloro, Bartolo. / An RPE cell line as a useful in vitro model for studying retinoic acid receptor ß :Expression and affinity. In: Bioscience Reports. 2008 ; Vol. 28, No. 6. pp. 327-334.
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