An RPE cell line as a useful in vitro model for studying retinoic acid receptor beta: expression and affinity.
Retinoids mediate their biological effect by interacting with specific nuclear receptors. Of the several known RAR (retinoic acid receptor) subtypes, RAR-beta 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-beta 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-beta-ligand interaction. RT-PCR (reverse transcription-PCR) and Western blot analyses show that HRPE cells specifically express only RAR-beta and none of the other receptor subtypes. In addition, we show that the expression of RAR-beta increases with increasing passage number of the cells. Interestingly, the increase in RAR-beta 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-beta-ligand interactions using nuclear extract from late passage HRPE cells as a source of endogenous RAR-beta. Using [(3)H]CD367 as the ligand, RAR-beta in HRPE cells showed an affinity of 9.6 +/- 0.6 nM and a B(max) of 780 +/- 14 fmol/mg of protein. We have confirmed the feasibility of using this assay to detect the interaction of ligands with RAR-beta by investigating the ability of certain flavonoids to inhibit the binding of [(3)H]CD367 to nuclear extracts from HRPE cells. The inhibition constant of the flavonoids for RAR-beta was between approx. 1-30 microM, showing that the flavonoids interact with RAR-beta with low affinity.
Pavan
Barbara
B
General Physiology section, Department of Biology, University of Ferrara, via L. Borsari, 46-44100 Ferrara, Italy.
Dalpiaz
Alessandro
A
Biondi
Carla
C
Nieddu
Marzia
M
De Luca
Antonella
A
Prasad
Puttur D
PD
Paganetto
Guglielmo
G
Favaloro
Bartolo
B
D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D016922:Cellular Senescence / Q000187:drug effects / Q000502:physiology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D008024:Ligands; D008954:Models, Biological; D018168:Receptors, Retinoic Acid / Q000819:agonists / Q000096:biosynthesis*; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*; D012176:Retinoids / Q000494:pharmacology; D016615:Telomere / Q000378:metabolism*
D015415:Biomarkers; D008024:Ligands; D018168:Receptors, Retinoic Acid; D012176:Retinoids; C082925:retinoic acid receptor beta; C064545:4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)benzoic acid