Abstract
Retinoids mediate their actions via RARs (retinoic acid receptors) and RXRs (retinoid X receptors). Each class of these nuclear retinoid receptors is further subdivided into three species, namely alpha, beta, and gamma. Recent studies demonstrate that estrogen receptor (ER)-positive human breast carcinoma (HBC) cell lines and tumor samples exhibit significantly higher levels of RAR alpha than their ER-negative counterparts. ER-positive HBC cell lines are sensitive to, and ER-negative cell lines are resistant to, growth inhibitory effects of retinoic acid (RA). We previously demonstrated that the expression of functional ERs in an established ER-negative cell line resulted in higher levels of RAR alpha and sensitivity to growth inhibition by RA. To further investigate the major role of RAR alpha in retinoid-mediated inhibition of growth, we transfected RAR alpha cDNA in two RA-resistant ER-negative HBC cell lines. Analyses of different clonal populations of RAR alpha transfectants from each cell line revealed growth inhibition by retinoids. Utilizing RAR- and RXR-class selective retinoids, we further demonstrated that only the RAR alpha-selective retinoids mediated the growth inhibition in these cells, while the RXR-selective retinoids were biologically inert. We thus provide evidence that the molecular mechanisms of retinoid inhibition of HBC proliferation predominantly involve RAR alpha.
Highlights
From the $Department of Medicine, Division of Oncology and Cancer Center, University of Maryland School of Medicine and the VeteransAdministration Medical Center, Baltimore, Maryland 21201, the §University of Montpellier I, Faculty of Medicine, INSERM Unite‘ 148, 60 rue de Navacelles, 34090 Montpellier, France, the Ibife Sciences Division,SRI International, Menlo Park, California 94025, and the **Cell Biology Section, Laboratory of Pulmonary Pathobiology, NIEHS, National Institutes of Health, Research Diangle Park, North Carolina 27709
(141, utilizing a Drosophila SL-3cell line that does not respond to retinoids, does not contain any known nuclear retinoid re
Our results demonstrated that theER transfectants not MDA-MB-231and MDA-MB-468cells were initially platedat a density only exhibited sensitivity to growth inhibition by RA and of 1x lo6celld100-mm dish 24behfore transfection in regular medium
Summary
N g of genomic DNA was used i n a final reaction volume of 100 pl t h a t contained 20pmol of each primer p~ of each dNTP, and 2 unitosf Taq. Ham's F-12 medium(1:l)supplemented with 5% fetal bovine serum as cised from pSG1-cathepsin D plasmid witEhcoRI and gel-purified[39]. IFansientDansfection and CAT Assays-Transient transfections VHIRA (55.7 CUmmol) waspurchased from DuPontNEN.(E)-4-[2- were performed as we have previously described [26] andthe plasmids (5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)propenylJbpeBnL-CAT2 @RARE-tk-CAT), CRBPII-tk-CAT (kindly provided by Dr. zoic acid (Ro13-7410; TTNPB) was prepared according tohe methodof X-k. 4-[2-Methyl-1-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphtshtara-nded sequence, containing a direct repeat (DR5) RARE from the lenyl)propenyl]benzoic acid (SRI1217) was prepared by Friedel-Crafts RAR& promoter inserted upstream of the thymidine kinase (tk) proacylation of 1,2,3,4-tetrahydro-l,1,4,4-tetramethylnaphthalewneith moter a t position -105, as shown below. Assay of RA Binding Activity-The logarithmically growing RARa transfectants and the mock transfectants were harvested and washed several timeswith 1x phosphate-buffered saline; nuclearextracts were prepared as described previously [44]. The relative amount (%) of each analyte was determined as thepercentage of the analyte peak area ratio compared to sum of peak area ratios for all analytes
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