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[Cancer Research 60, 5479-5487, October 1, 2000]
© 2000 American Association for Cancer Research

Experimental Therapeutics

Activation of Retinoic Acid Receptor {alpha} Is Sufficient for Full Induction of Retinoid Responses in SK-BR-3 and T47D Human Breast Cancer Cells1

Sonja M. Schneider, Martin Offterdinger, Heinz Huber and Thomas W. Grunt2

Laboratory for Cell Growth and Differentiation [S. M. S., M. O., T. W. G.], Division of Oncology [H. H., T. W. G.], Department of Internal Medicine I, University of Vienna, A-1090 Vienna, Austria


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Retinoid signaling via retinoic acid (RA) and retinoid X receptors (RARs and RXRs) regulates mammary epithelial cell growth and differentiation. Loss of RAR-ß might represent an early event during breast carcinogenesis. Higher differentiated, estrogen-dependent, estrogen receptor (ER)-positive (ER+) mammary carcinoma cells have been found to contain relatively high levels of RAR-{alpha} and to be responsive to retinoids, whereas most undifferentiated, estrogen-independent, ER-negative (ER-) cells are characterized by low RAR-{alpha} expression and by retinoid resistance. In contrast, RAR-{gamma} is detectable at equal levels in both ER+ and ER- cells. In the present investigation, we directly examined the relative contribution of the distinct retinoid receptors to the retinoid response of breast cancer cells by comparing the effects of low concentrations of specific retinoids, which selectively activate individual receptor subtypes, on growth, cell cycle distribution, apoptosis, and on the autoregulation of RAR-{alpha} and RAR-{gamma} in ER- SK-BR-3 and ER+ T47D breast cancer cells. In vitro growth activity was determined by using a colorimetric cell viability assay and analysis of cell cycle distribution, and apoptosis was performed by flow cytometry of propidium iodide-stained or fluorescent Annexin V-labeled cells, respectively, whereas expression of RAR-{alpha} and RAR-{gamma} was determined by Northern blotting. Both cell lines are retinoid sensitive and express high amounts of RAR-{alpha}, RAR-{gamma}, and RXR-{alpha}. RAR-{alpha}-selective compounds (AM80 and AM580) inhibit cell growth, induce G1 arrest, stimulate apoptosis, and up-regulate RAR-{alpha} and RAR-{gamma} mRNA as efficiently as RAR/RXR-pan-reactive (9-cis RA) and RAR-pan-reactive retinoids (all-trans RA, TTNPB). Remarkably, an RAR-{alpha} antagonist (Ro 41-5253) not only blocks the RAR-{alpha}-selective agonists but also the pan-reactive compounds. In contrast, RAR-ß-selective (CD417), RAR-{gamma}-selective (CD437/AHPN), and RXR-{alpha}-selective (Ro 25-7386) retinoids exert no effects on the examined parameters. Thus, our results support the idea that RAR-{alpha} is the crucial receptor mediating the biological effects during retinoid signaling in both ER- SK-BR-3 and ER+ T47D human breast cancer cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Retinoids, the natural and synthetic vitamin A analogues, exert profound effects on cell proliferation, differentiation, morphogenesis, metabolism, and apoptosis and are considered promising agents for the prevention and treatment of human breast cancer. Retinoids act via two families of nuclear receptors: RARs3 and RXRs, which belong to the steroid/thyroid/retinoid superfamily of ligand-inducible transcription factors that interact with each other, forming heterodimers, and bind to RAREs and/or retinoid X response elements to control the expression of responsive target genes in the presence of retinoids. Both families consist of three subtypes: {alpha}, ß, and {gamma}. RAR-{alpha}, RAR-ß, RAR-{gamma}, and RXR-{gamma} are known to be expressed in different isoforms that arise from alternative splicing at the 5' end and from the use of multiple promoters in the corresponding gene (1) . RAR subtypes and isoforms are expressed differentially, both temporally and spatially (2, 3, 4) . RAR-{alpha} is expressed ubiquitously, whereas RAR-ß and RAR-{gamma} display more distinct patterns of distribution, with RAR-{gamma} being predominantly expressed in the skin (5) . tRA and 9cRA are natural vitamin A derivatives and differ in their abilities to activate RARs and RXRs. Whereas RARs can be activated by both tRA and 9cRA, RXRs are activated by 9cRA only. The unselective affinity of natural retinoids to various retinoid receptor subtypes appears to be responsible for the broad spectrum of biological activities and might contribute to a number of undesirable side effects of these compounds. Recently, synthetic retinoids have become available, which preferentially activate one or more of the RAR or RXR subtypes and which can elicit a more tissue- or cell type-selective response. These compounds have greatly improved our understanding of the mechanisms of retinoid action, and some of them might be of great clinical use because of the lack of adverse side effects. Mammary carcinoma cells have been demonstrated to be growth inhibited by retinoids (6 , 7) . The antiproliferative effects of retinoids are frequently associated with cell differentiation and/or programmed cell death (8 , 9) . Several studies demonstrated that RAR-{alpha} mediates the growth-inhibitory effect of tRA in ER+ breast cancer cell lines and that the loss of tRA sensitivity in ER- cells may be attributable to low levels of RAR-{alpha} (6 , 7 , 10) .

In this study, we investigated the effects of natural and synthetic retinoid agonists and antagonists with distinct selectivity for retinoid receptor types on proliferation, cell cycle distribution, apoptosis, and on the autoregulation of RAR expression in SK-BR-3 and T47D mammary carcinoma cells. SK-BR-3 cells are ER-, estrogen independent, and antiestrogen resistant. In contrast, T47D cells are positive for ER and respond to (anti)estrogens. Both cell lines express high amounts of RAR-{alpha}, RAR-{gamma}, and RXR-{alpha} and are sensitive to retinoids. SK-BR-3 cells contain equal levels of all three receptor subtypes (~53 fmol/mg total soluble protein). T47D cells, on the other hand, contain ~34 fmol/mg RAR-{alpha} and RAR-{gamma}, respectively, and 83 fmol/mg RXR-{alpha}. RXR-ß can also be found at low levels in both cell lines, whereas RAR-ß and RXR-{gamma} could not be detected (11, 12, 13, 14) .4 These two cell lines thus represent good model systems to study the relative contribution of RAR-{alpha}, RAR-{gamma}, and RXR-{alpha}/-ß activation to the retinoid response of both ER- and ER+ breast cancer cells. Our data demonstrate that RAR-{alpha} might be the major receptor that mediates retinoid-dependent growth inhibition, G1 cell cycle arrest, apoptotic cell death, and autostimulatory up-regulation of RAR-{alpha} and RAR-{gamma} mRNA expression in SK-BR-3 and T47D breast cancer cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
9cRA (ICN, Costa Mesa, CA), tRA (Calbiochem, San Diego, CA), TTNPB, Ro 25-7386, Ro 41-5253 (obtained from Dr. M. Klaus, F. Hoffmann-La Roche Ltd., Basel, Switzerland), AM80, AM580 (kind gifts from Dr. K. Shudo, Faculty of Pharmaceutical Sciences, Tokyo, Japan), and CD417 and CD437 (AHPN; provided by Dr. B. Shroot, Galderma R&D, Sophia Antipolis, Valbonne, France) were reconstituted in DMSO. The final concentrations of the solvents never exceeded 0.2% (v/v) in the cell cultures.

Hybridization probes were isolated from plasmids containing: 0.6-kb PstI cDNA fragment from human RAR-{alpha} (clone p63) in pTZ19R (American Type Culture Collection, Rockville, MD); 0.6-kb EcoRI cDNA fragment from human RAR-ß in pSG5 (donated by Dr. P. Chambon, INSERM, Illkirch, France); 1.3-kb EcoRI/AvaI cDNA fragment from human RAR-{gamma} in pSG5 (kind gift from Dr. P. Chambon); and a 1.3-kb EcoRI/HindIII cDNA fragment from rat GAPDH in pSP65. Isolation of plasmids and inserts were performed as described previously (15) .

Cell Culture and Treatment.
SK-BR-3 and T47D cells were obtained from the American Type Culture Collection and were cultured in a humidified 5% CO2 atmosphere at 37°C in {alpha}-MEM containing 10% FCS, 2 mM L-glutamine, and 1000 IU penicillin/streptomycin (Life Technologies, Inc., Karlsruhe, Germany; standard medium). The cells were subcultured weekly at a ratio of 1:5 (SK-BR-3) or 1:10 (T47D) for a maximum of 30 passages. Experimental cultures were usually grown in phenol red-free RPMI 1640 (Life Technologies, Inc.) supplemented with 5% charcoal-stripped FCS (HyClone Laboratories, Logan, UT), 2 mM L-glutamine, and 1000 IU penicillin/streptomycin (depleted medium) to minimize the interference of the experimental agents with contaminating hormones, growth factors, and signaling molecules. Generally, the natural and synthetic retinoids were used at low concentrations that specifically bind and activate only the cognate receptor subtype(s), thus avoiding nonspecific activation of the other subtype receptors. Table 1Citation summarizes published (16, 17, 18) and unpublished data5 on the binding and activation concentrations of the retinoids used in this investigation.


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  		Table 1 Accumulated data on RAR and RXR binding and transactivation of the used retinoids

 
Cell Proliferation Assay.
Cells were plated in depleted medium at 500 cells/well into 96-well plates (Costar, Cambridge, MA). After 3 days in depleted medium, the cells were exposed for 1–7 days to the indicated concentrations of the respective retinoids, and the media with or without drugs were renewed every second day. Cell growth was determined using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega Corp., Madison, WI) according to the manufacturer’s instructions. Absorbance values of experimental cultures were determined in a MR 7000 microplate reader (Dynatech Laboratories, Inc., Chantilly, VA). Results represent mean values of triplicate determinations. SD was always <15% of the mean.

Cell Cycle Analysis.
SK-BR-3 cells plated in depleted medium into 24-well plates (Costar) at 4 x 104 cells/well were treated for 4 days with the indicated concentrations of retinoids and were then washed twice with sample buffer (PBS containing 1 g/l glucose). After trypsinization, the cells were centrifuged at 1500 rpm for 5 min at 4°C, washed with PBS, fixed overnight in 70% ice-cold ethanol, centrifuged again, and then stained for 30 min at room temperature in 1 ml of sample buffer containing 50 µg/ml propidium iodide (Sigma Chemical Co., St. Louis, MO) and 100 units/ml RNaseA (Roche Diagnostics GmbH, Vienna, Austria). Analysis was performed with a FACScan flow cytometer (Becton Dickinson, San Jose, CA) using Cell Fit software and the SOBR model with eight S-phase peaks.

Annexin V Apoptosis Assay.
SK-BR-3 cells were seeded at 5 x 104 cells/well into 12-well plates (Costar). After 3 days in depleted medium, cells were treated for 4 days with 0.1% DMSO, 10 µM Taxol (Sigma), or the indicated concentrations of retinoids. The adherent cells were trypsinized and pooled with the spontaneously detached cells. Annexin V binding was determined by flow cytometry using the Annexin V/FITC kit (Bender MedSystems, Vienna, Austria). Propidium iodide was used to exclude necrotic cells from the analysis. Two marker regions were defined for quantitation. Low fluorescent region 1 comprised >95% of all solvent treated cells (nonapoptotic, "Annexin V negative"), whereas high fluorescent region 2 contained the remaining apoptotic cells ("Annexin V positive"). Treatment-induced apoptosis was estimated by the amount of cells that were shifted from region 1 to region 2 (see histograms in Fig. 5BCitation ).



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  		Fig. 5. The effects of different retinoids on apoptotic cell death of SK-BR-3 cells demonstrated by Annexin V labeling and flow cytometry as described in "Materials and Methods." Cultures were exposed for 96 h to DMSO, Taxol, or different retinoids at the indicated concentrations. For quantitative analysis, two marker regions were defined. Low fluorescent region 1 (M1) comprises >95% of all solvent-treated cells (nonapoptotic), whereas high fluorescent region 2 (M2) contains the apoptotic cells. Mean values (n >=6) of apoptotic cells from M2 (A) and representative histograms (B) are given; bars, SD.

 
Northern Blotting.
Cells were plated in depletion medium at 3 x 106 cells per T75 tissue culture flask (Costar) and incubated for 3 days under these conditions. Subsequently, the cells were treated for 6–48 h with the indicated concentrations of the test compounds. Cells were incubated for another 1 or 2 h with 1 µg/ml actinomycin D (Sigma) for examination of the effects of retinoids on RNA stability. Total RNA was extracted by a one-step acid guanidinium isothiocyanate/phenol procedure using RNazol (Biotecx, Houston, TX), and 20 µg of RNA were loaded onto each lane and were electrophoresed at 50 V for 4 h in a 1% formaldehyde-containing agarose gel. The separated RNA was then diffusion-blotted overnight onto nylon membranes (Schleicher & Schuell, Dassel, Germany) and immobilized by UV-cross-linking. RAR- and GAPDH-specific cDNA probes were labeled with [32P]dCTP (New England Nuclear, Vienna, Austria) using the random oligonucleotide primer extension kit (Stratagene, La Jolla, CA). The membranes were hybridized overnight at 65°C, washed twice with 2x SSC, and 0.1% SDS at 37°C and twice with 0.1 x SSC and 0.1% SDS at 65°C and were exposed at -80°C to Kodak X-OMAT AR film (Eastman Kodak, Rochester, NY) between two intensifying screens. The blots were then stripped and reprobed (overnight, 65°C) with the GAPDH cDNA as an internal control to monitor the quantity and integrity of the loaded RNA samples.

Semiquantitative Evaluation of Autoradiographs.
Autoradiographs were evaluated by densitometry using a pdi model DNA 35 densitometer (Huntington Station, NY) and Quantity One software. The peak absorbances of the two combined RAR-{alpha}-specific mRNA bands (3.4 and 2.8 kb), of the RAR-ß-specific band (3.7 kb), or of the RAR-{gamma}-specific mRNA band (3.3 kb) were related to the peak absorbance of the GAPDH mRNA band (1.6 kb). The ratio values obtained from each experimental culture were related to the ratios obtained from untreated control cells and were expressed in percentage of control.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
RAR-pan-reactive and RAR-{alpha}-selective Retinoids Inhibit Cell Growth with Equal Efficiency.
Growth of the human breast cancer cell line SK-BR-3 was inhibited by most of the used retinoids. The natural RAR/RXR-pan-reactive retinoid 9cRA, the natural and synthetic RAR-pan-reactive compounds tRA and TTNPB, the RAR-{alpha}-agonists AM80 and AM580, and the RAR-{gamma}-selective ligand CD437 induced growth arrest in a time- and dose-dependent manner. Both RA isomers caused a drop of cell number after 24 h of exposure, which might indicate an initial cytotoxic effect (Fig. 1, A and B)Citation . 9cRA, tRA, and TTNPB exerted their antiproliferative effect at a concentration of 10 nM, AM80 and AM580 already at a tenth of this dose (1 nM), whereas CD437 was effective on growth only at a 100 times higher concentration (100 nM). CD417, an RAR-ß-agonist, and Ro 25-7386, an RXR-{alpha}-selective compound, did not inhibit proliferation of SK-BR-3 cells (Fig. 1)Citation . In this particular experiment, small increases in cell numbers were seen after exposure to 100 nM CD417 (Fig. 1F)Citation , 10 nM CD437 (Fig. 1G)Citation , and 10 nM Ro 25-7386 (Fig. 1H)Citation . However, in all other cell growth experiments (n = 2–9), neither a consistent increase nor a decrease in cell number at various times of exposure to these retinoid concentrations was seen, suggesting that these stimulating effects were not biologically significant. Similar results were obtained in T47D cells (Fig. 2)Citation . The observed exquisite sensitivity of both cell lines against RAR-{alpha}-selective retinoids argues for a primary role of RAR-{alpha} in the growth control of these cells.



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  		Fig. 1. Dose- and time-dependent effects of different retinoids on the in vitro growth of SK-BR-3 breast cancer cells demonstrated by a colorimetric cell viability assay as described in "Materials and Methods." The cells were exposed to the indicated concentrations of 9cRA (A), tRA (B), TTNPB (C), AM80 (D), AM580 (E), CD417 (F), CD437 (G), or Ro 25-7386 (H). The absorbances (OD) were measured at 490 nm after 1–7 days of treatment. Results represent mean values of triplicate determinations. SD was always <12% of the mean.

 


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  		Fig. 2. The effects of different retinoids on the in vitro growth of T47D breast cancer cells. Treatment was performed for 7 days, and cell growth was determined as described in Fig. 1Citation . Results are derived from one of two separate experiments and represent means of triplicate determinations; bars, SD.

 
An RAR-{alpha}-selective Antagonist Reverts Growth Inhibition Caused by an RAR-pan-reactive Retinoid.
AM80 and AM580 were even more potent than TTNPB in a 7-day assay. To determine whether the growth reduction of SK-BR-3 and T47D cells was RAR-{alpha}-mediated, we concurrently applied TTNPB, AM80, or AM580 with the RAR-{alpha} antagonist Ro 41-5253. Ro 41-5253 alone had no effect on proliferation of SK-BR-3 cells, but it was able to block the growth-inhibiting effects not only of AM80 and AM580 but also of TTNPB in a dose-dependent manner (Fig. 3, A and B)Citation . The RAR-{alpha}-antagonist achieved up to 100% efficiency in blocking the effect of all three ligands on cell growth inhibition, implying that TTNPB requires RAR-{alpha} to exhibit its growth-inhibiting potential. Essentially similar effects were obtained in T47D cells, although these cells responded to 1 µM Ro 41-5253 with a 20% growth reduction and with only a partial reversal of the agonist-induced antiproliferative effects (Fig. 3C)Citation .



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  		Fig. 3. The effect of AM80, AM580, or TTNPB alone or in combination with different concentrations of the RAR-{alpha} antagonist Ro 41-5253 on the proliferation of SK-BR-3 cells after 4 (A) or 7 (B) days or of T47D cells after 7 days (C) of treatment. Values represent means of triplicate determinations expressed as a percentage of solvent control; bars, SD.

 
G1 Arrest Is Induced Only by RAR-pan-reactive and RAR-{alpha}-selective Retinoids.
Cell cycle analysis revealed that the pan-reactive retinoids 9cRA, tRA, and TTNPB and the RAR-{alpha}-ligands AM80 and AM580 induce cell cycle arrest in SK-BR-3 cells after 96 h treatment. Table 2Citation demonstrates an increase in the percentage of cells in G1 and a decrease in the percentage of cells in S-phase, relative to untreated control cells. The results show that the treatment with these retinoids elevated the G1:(S+G2) ratio from 3 to ~16 in a dose-dependent manner (Fig. 4)Citation . CD417 (RAR-ß-agonist), CD437 (RAR-{gamma}-agonist), Ro 25-7386 (RXR-{alpha}-agonist), and Ro 41-5253 (RAR-{alpha}-antagonist) were found to be ineffective on cell cycle distribution in SK-BR-3 cells. These results demonstrate that G1 arrest is a major mechanism by which RAR-pan-reactive and RAR-{alpha}-selective retinoids inhibit proliferation and that this effect is mediated primarily by activation of RAR-{alpha}.


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  		Table 2 Effects of 96-h treatment with different retinoids on the cell cycle distribution of SK-BR-3 cells

 


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  		Fig. 4. The effects of different retinoids on the cell cycle of SK-BR-3 mammary carcinoma cells. Cultures were exposed for 96 h to the compounds, and the cell cycle distribution was expressed as the ratio between the G0/G1-phase and the sum of the S- and the G2M-phase [G1/(S+G2)]. Means of one representative experiment done in triplicate are shown; bars, SD.

 
RAR-pan-reactive and RAR-{alpha}-selective Retinoids Induce Apoptosis Most Efficiently.
The Annexin V binding assay was used to determine whether the treatment-induced antiproliferative effects on SK-BR-3 cells could partially be explained by apoptotic cell death. A 96-h exposure of the cells to the RAR-pan-reactive retinoids and RAR-{alpha}-agonists increased the proportion of Annexin V-positive (apoptotic) cells from 5% (control) to ~13–18%. Moreover, a clear dose dependence of apoptosis was found in cells that were treated with the pan-reactive retinoid 9cRA (Fig. 5A)Citation , which probably relates to the drop in cell counts at day 1 and to the persistent dose-dependent reduction of cell numbers at day 4 (see Fig. 1ACitation ). Under the same conditions, CD417 (RAR-ß), CD437 (RAR-{gamma}), and Ro 25-7386 (RXR-{alpha}) did not show a clear apoptosis-inducing effect, except that a slight induction of apoptotic cells was observed after treatment with CD417 and CD437. In comparison, Taxol (10 µM), representing a well-known inducer of apoptosis, elevated the proportion of apoptotic cells from 5 to 22% (Fig. 5)Citation . Thus, all pan-reactive and RAR-{alpha}-selective retinoids were found to cause apoptosis to a substantial extent, once again suggesting that RAR-{alpha} is crucial for mediation of biological responses during retinoid signaling in SK-BR-3 cells.

Activation of RAR-{alpha} Is Crucial for Up-Regulation of RAR-{alpha} and RAR-{gamma} Expression.
The regulation of RAR-{alpha}, RAR-ß, and RAR-{gamma} gene expression was examined after 6, 24, and 48 h exposure to retinoids in SK-BR-3 cells. As shown in Fig. 6Citation , the RAR/RXR pan-agonist 9cRA, the RAR-pan-reactive retinoids tRA and TTNPB, and the RAR-{alpha} agonists AM80 and AM580 efficiently induced mRNA expression of RAR-{alpha} and RAR-{gamma} in a time-dependent manner, whereas neither agonists such as RAR-ß-selective CD417, RAR-{gamma}-selective CD437, and RXR-{alpha}-reactive Ro 25-7386 nor RAR-{alpha} antagonists such as Ro 41-5253 markedly affected RAR-{alpha} and RAR-{gamma} expression. Quantitation of gene expression by densitometry of Northern blot autoradiographs demonstrated a >100% increase of RAR-{alpha} mRNA after 48 h exposure of the cells to TTNPB, AM80, and AM580. RAR-ß mRNA was neither spontaneously expressed nor was it inducible with retinoids in SK-BR-3 cells. To estimate a possible contribution of retinoid-mediated alteration of mRNA stability to the observed ligand-induced up-regulation of RAR-{alpha} and RAR-{gamma} mRNA, we incubated retinoid-treated cultures for 1 and 2 h with 1 µg/ml actinomycin D. Retinoids generally slightly reduced RNA stability. This effect decreased during prolonged incubation with actinomycin D. In some instances, a weak stabilization could be observed after 2 h exposure to actinomycin D (Table 3)Citation . However, this response could not account for the elevated RAR-{alpha}/RAR-{gamma} mRNA levels seen in Fig. 6Citation . These data suggest that pan-reactive and RAR-{alpha}-selective retinoids efficiently stimulate RAR-{alpha} and RAR-{gamma} gene transcription. A similar effect of retinoid treatment on RAR-{alpha} and RAR-{gamma} expression was obtained in T47D cells (Table 4)Citation .



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  		Fig. 6. The effects of retinoids on the expression of RAR-{alpha} and RAR-{gamma} mRNA in SK-BR-3 cells. The cells were exposed for 6, 24, or 48 h to vehicle (0.1% DMSO) alone or to different retinoids. Preparation of total RNA and Northern blotting was performed as described in "Materials and Methods." The autoradiographs were evaluated by densitometry, and the peak absorbances of both RAR-{alpha}-specific mRNA bands were combined and related to the peak absorbance of the GAPDH mRNA band. The single RAR-{gamma} mRNA band was also related to GAPDH, and the obtained ratios were expressed in percentage of control. Means of up to four different experiments are given. SD was always <22% of control (A). Comparison of the effects of a 48-h exposure of SK-BR-3 cells to pan-reactive or subtype-selective retinoids on the expression of RAR-{alpha} and RAR-{gamma} mRNA. Means of up to four different experiments are given (B); bars, SD.

 

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  		Table 3 Effects of 1 and 2 h of 1 µg/ml actinomycin D (ActD) on the stability of RAR-{alpha} and RAR-{gamma} mRNA in untreated or retinoid-treated (48 h) SK-BR-3 cells

 

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  		Table 4 Effects of a 48-h exposure of pan-reactive and subtype-selective retinoid agonists and antagonists on the expression of RAR-{alpha} and RAR-{gamma} mRNA in T47D cells as demonstrated by Northern blotting

 
An RAR-{alpha}-selective Antagonist Reverts RAR-{alpha} and RAR-{gamma} Up-Regulation Caused by an RAR-pan-reactive Retinoid.
We then wondered whether retinoid-dependent up-regulation of RAR-{alpha} and RAR-{gamma} mRNA was mediated by RAR-{alpha}. To that effect, SK-BR-3 and T47D cells were treated for 48 h with 1 or 10 nM of RAR-{alpha} agonists AM80 or AM580 or with 10 nM of RAR-pan-reactive retinoid TTNPB alone or in combination with 1 or 10 µM of the RAR-{alpha} antagonist Ro 41-5253. Northern blot analysis revealed that Ro 41-5253 reverted the agonist-induced elevation of RAR-{alpha} and RAR-{gamma} (Fig. 7Citation ; Table 4Citation ). For instance, a 100-fold excess of the antagonist reduced the AM80- or AM580-mediated up-regulation of RAR-{alpha} and RAR-{gamma} mRNA, whereas a 1000-fold antagonist concentration completely blocked the increasing effects. More importantly, Ro 41-5253 also antagonized TTNPB-induced up-regulation of RAR-{alpha} and RAR-{gamma}, indicating that activation of RAR-{alpha} is necessary for induction of RAR-{alpha} and RAR-{gamma} mRNA expression in both cell lines.



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  		Fig. 7. Competitive inhibition of up-regulation of RAR-{alpha} and RAR-{gamma} mRNA expression by cotreatment of SK-BR-3 cells for 48 h with RAR-pan-reactive (TTNPB) or RAR-{alpha}-selective agonists (AM80 and AM580) and RAR-{alpha} antagonists (Ro 41-5253). Northern blotting and quantitative evaluation of autoradiographs were performed as described in Fig. 6Citation . Bars, SD.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Retinoids or vitamin A derivatives are crucial regulators of many biological processes including growth, differentiation, and apoptosis of epithelial cells and therefore represent promising chemopreventive and therapeutic antitumor drugs. The identification of RAR-{alpha}, RAR-ß, RAR-{gamma}, and RXR-{alpha}, RXR-ß, RXR-{gamma} as the cognate receptors for retinoids and the elucidation of receptor-dependent, RARE-mediated transactivation of retinoid target genes and of the transrepressive anti-AP-1 activity greatly enhanced our understanding of the mechanisms of retinoid action. Yet, the ultimate molecular pathways that mediate retinoid-induced growth inhibition, differentiation, and/or apoptosis are still largely unknown. It has been shown that malignant transformation of mammary epithelial cells is associated with diminished RAR-ß expression, which in some systems correlates with the development of retinoid resistance (10 , 19) . In addition, data obtained from human breast tumor specimens suggest that loss of RAR-ß might represent an early event during mammary carcinogenesis (20) . Furthermore, differentiated, ER+, estrogen-responsive breast cancer cells usually express relatively high levels of RAR-{alpha} and are sensitive to retinoids, whereas most poorly differentiated, ER-, estrogen-independent cells have low RAR-{alpha} expression and reveal retinoid resistance (6 , 7) . In contrast, RAR-{gamma}, RXR-{alpha}, and RXR-ß appear to be stably expressed during mammary carcinogenesis and progression, whereas RXR-{gamma} is not detectable in breast cancer cells (6 , 12 , 21, 22, 23) . Although these data provide circumstantial evidence that RAR-{alpha} and RAR-ß might exert crucial functions during the regulation of normal and malignant mammary epithelial cell growth, differentiation, and apoptosis, direct proofs of this hypothesis are still very sparse (14 , 20 , 24) .

In recent years, synthetic agonistic and antagonistic retinoids have become available that reveal distinct RAR- and RXR-subtype selective transactivating and transrepressive activities, respectively. In addition, "dissociating" retinoids have been identified, which exert potent repression of AP-1-dependent transcription but are devoid of the RARE transactivating function. Using such selective retinoid agonists and antagonists, we set out to directly examine the relative contribution of the distinct receptor subtypes to the mediation of the biological effects of retinoid signaling in ER- SK-BR-3 and ER+ T47D breast cancer cells. Both cell lines are sensitive to retinoids and express high amounts of RAR-{alpha} (SK-BR-3, 55 fmol/mg total soluble protein; T47D, 35 fmol/mg), RAR-{gamma} (SK-BR-3, 54 fmol/mg; T47D, 33 fmol/mg) and RXR-{alpha} (SK-BR-3, 51 fmol/mg; T47D, 83 fmol/mg), and low levels of RXR-ß. RAR-ß and RXR-{gamma} were not detectable in these cells (11, 12, 13, 14 , 23) . These cell lines thus reveal expression patterns that together are representative of many breast cancers and represent good model systems to study the biological significance of the individual RAR/RXR subtypes in mammary carcinomas. Collectively, our data strongly support the idea that activation of RAR-{alpha} is sufficient for mediation of retinoid responses in these two cell lines.

9cRA represents a natural pan-reactive retinoid, which activates all RAR and RXR subtypes, whereas tRA interacts with RARs only. However, stereoisomerization of tRA to 9cRA has been observed to a minor extent in cell culture (25) , suggesting that residual activation of RXRs cannot be excluded if tRA is applied to the cells. Therefore, TTNPB was used in parallel, which specifically activates RAR-{alpha}, RAR-ß, and RAR-{gamma} but leaves all RXRs unaffected (26 , 27) . Selective activation of RAR-{alpha}, RAR-ß, or RAR-{gamma} or of RXR-{alpha} was achieved by applying the synthetic agonists AM80 (RAR-{alpha}), AM580 (RAR-{alpha}), CD417 (RAR-ß), CD437 (RAR-{gamma}), or Ro 25-7386 (RXR-{alpha}), respectively, to the cultures (28, 29, 30, 31) , whereas RAR-{alpha} transcriptional activity was inhibited by the RAR-{alpha}-selective antagonist Ro 41-5253 (32) . Most importantly, all compounds have been applied at concentration ranges for which receptor selectivity has been proven, thus avoiding nonspecific interaction with other receptor subtypes (see Table 1Citation and references therein). We observed that SK-BR-3 and T47D in vitro cell growth was abolished by 10 nM of RAR/RXR-pan-reactive 9cRA, of RAR-pan-reactive tRA or TTNPB, and by 1 nM of RAR-{alpha}-selective AM80 or AM580. In contrast, RAR-ß-selective (CD417), RAR-{gamma}-selective (CD437), and RXR-{alpha}-selective (Ro 25-7386) retinoids show no (SK-BR-3) or only very weak (T47D) growth-inhibiting activity at 10 nM (Figs. 1Citation and 2)Citation . In SK-BR-3, a concentration of 100 nM of CD437 was required for growth arrest (Fig. 1G)Citation . Some doses of these less active retinoids even caused slight growth stimulation (Fig. 1FCitation 1GCitation 1HCitation , and Fig. 2Citation ). However, growth activation was not consistently found in all experiments, arguing against a biological significance of this effect. The antiproliferative responses to RAR-{alpha}-selective and to RAR-pan-reactive agonists could be reverted by coadministration of a RAR-{alpha}-selective antagonist (Ro 41-5253), indicating that the growth inhibition imposed by the RAR-pan-reactive and RAR-{alpha}-selective compounds is mediated solely by activation of RAR-{alpha} and not of RAR-ß (which is not detectable in these cells) or RAR-{gamma}. Fitzgerald et al. (14) recently identified one breast cancer cell line (MDA-MB-435) that expresses large and equal amounts of RAR-ß and RAR-{gamma} protein but only low levels of RAR-{alpha} protein. In contrast to others (33) , these workers reported that DNA replication was not inhibited in MDA-MB-435 cells by retinoids. In agreement with Fitzgerald et al. (14) , Ro 41-5253 alone at 0.1 and 1 µM did not markedly affect SK-BR-3 cell proliferation and cell cycle distribution, whereas a very slight growth reduction has been seen with 1 µM in T47D cells (Fig. 3)Citation . Interestingly, Toma et al. (34) described some antiproliferative and apoptosis-inducing effects of this compound in two other ER+ cell lines at concentrations >=1 µM. These responses have been attributed to its residual anti-AP-1 transrepressing activity (34) . Data in Fig. 3Citation thus suggest that T47D cell growth might be under closer control of AP-1 than SK-BR-3 cell proliferation. This would also explain why growth arrest by 10 nM AM580 can be reverted in SK-BR-3 cells by 1 µM Ro 41-5253 but not in T47D cells. Furthermore, this hypothesis would also provide some clue as to why 1 µM Ro 41-5253 cannot completely relieve retinoid-dependent growth arrest in T47D cells, whereas it can lift the block in SK-BR-3 cells. Our data also show that growth inhibition by pan-reactive and RAR-{alpha}-selective retinoids was caused mainly by cell cycle arrest in G1 and to a lesser extent by induced cell death. It has to be noted that a subtype-selective concentration (10 nM) of CD417 revealed low degree apoptotic activity, suggesting that the cells might still express some minute amounts of RAR-ß that are not detectable by the methods used here. Yet, this activity did not translate into an overt growth effect. A similar response was obtained with RAR-{gamma}-selective CD437. This retinoid agonist has recently gained much attention from cancer researchers. Although CD437 selectively binds RAR-{gamma} and activates RARE-dependent transactivation (Table 1Citation and references therein), it was found to inhibit the proliferation not only of retinoid-sensitive, RAR-positive cancer cells but also of retinoid-resistant, RAR-negative cancer cells (35) . In addition, CD437 does not interact with RXRs, nor does it induce transactivation through retinoid X response elements. Thus, CD437 appears to act via unique, nuclear receptor-independent pathways. CD437-induced G1 arrest and apoptosis have been described for several cancer cell lines including mammary carcinoma. Significant effects have usually been obtained with CD437 concentrations ~1 µM (35, 36, 37) . In the present study, CD437 was used at doses two orders of magnitude lower (10 nM) to avoid interference with these RAR/RXR-independent pathways, while selectively stimulating RAR-{gamma} activity. Although CD437 has been found to control the expression and function of a variety of molecules including the tumor suppressor p53, the cyclin-dependent kinase inhibitor p21WAF1/Cip1, the cyclin-dependent kinases cdk2 and cdk4, the Rb retinoblastoma protein, the transcription factor c-Jun, the nuclear orphan receptor nur77, and some other regulatory proteins, its definite mechanisms of action are still elusive (35, 36, 37, 38) . Furthermore, RXR-{alpha}-selective Ro 25-7386 did not affect SK-BR-3 and T47D cell growth, cell cycle distribution, and apoptosis. This is in agreement with previous work done in cell culture, which demonstrated that retinoid-sensitive breast cancer cells are resistant or only very weakly responsive to RXR ligands, also known as rexinoids (14 , 39 , 40) . Surprisingly, Bischoff et al. (40) reported recently that the rexinoid LGD1069 exerts significant in vivo efficacy as a chemopreventive and a therapeutic agent in an ER+ rat mammary carcinoma model. These effects might be caused by additional paracrine mechanisms originating from the surrounding stroma tissue (40) . Nevertheless, in cultured breast cancer cells an inverse relationship exists between direct RXR-specific rexinoid versus RAR-specific retinoid sensitivity, the latter being usually associated with high levels of RAR-{alpha} and ER and hence with estrogen dependence. Moreover, data from Wu et al. (39) suggest that RAR-{alpha} may provide a signaling switch between RAR- and RXR-mediated growth inhibition of breast cancer cells; high levels of RAR-{alpha} trigger signaling by RAR-reactive retinoids via RAR-{alpha}/RXR heterodimers, whereas low amounts of RAR-{alpha} enable RXR agonists to effectively inhibit cell growth via RXR/nur77 heterodimers (39) .

Northern blot analysis of total RNA revealed that all retinoids causing growth inhibition, cell cycle arrest, and apoptosis up-regulate RAR-{alpha} and RAR-{gamma} mRNA in a time-dependent way. This was not associated with increased mRNA stability, arguing for a retinoid-dependent induction of gene transcription. The RAR-{alpha}-selective agonists, which were active already at 10-fold lower concentrations than the pan-reactive agents (1 nM AM80 and AM580 versus 10 nM 9cRA, tRA, and TTNPB), were the most potent inducers. By contrast, biologically inactive compounds such as RAR-ß-selective, RAR-{gamma}-selective, and RXR-{alpha}-selective ligands caused no or only very weak alterations in RAR-{alpha} and RAR-{gamma} mRNA expression. The RAR-{alpha} antagonist Ro 41-5253 alone had no effect on RAR-{alpha} and RAR-{gamma} levels. It could, however, revert the agonist-induced elevations. Strikingly, this antagonist not only blocked the up-regulation induced by RAR-{alpha} ligands but also that caused by RAR-pan-reactive TTNPB, demonstrating that autoregulation of RAR expression is controlled primarily by RAR-{alpha}. Conflicting results have been published concerning expression and inducibility of RAR-ß. For example, Swisshelm et al. (19) demonstrated that normal human mammary epithelial cells reveal both baseline and retinoid-inducible expression of RAR-ß, whereas in transformed cells, RAR-ß is neither detectable nor inducible by retinoids, which is in accordance with our data. In contrast, Liu et al. (10) reported that ER+ tumor cells respond to tRA by elevated expression of RAR-ß. This discrepancy might be caused by different sensitivities of the detection methods used and/or by different culture conditions and cell lines. According to these workers, ligand-dependent activation of RAR-{alpha} is responsible for the induction of RAR-ß, which then mediates the biological response, suggesting that RAR-{alpha} would represent the crucial receptor for ligand interaction, whereas RAR-ß would fulfill effector functions. Our results are partially consistent with this hypothesis in that an RAR-ß agonist was not able to trigger a cell response, whereas RAR-{alpha} retinoids were. Generally, the effects were more pronounced in SK-BR-3 cells when compared with T47D and required 48 h of drug exposure. This is in accordance with a previous report by Roman et al. (6) , who did not find altered levels of RAR-{alpha} and RAR-{gamma} after treating ER+ and ER- mammary carcinoma cells for 6 h with 1 µM tRA. Unfortunately, however, these authors did not examine longer periods of drug exposure. Available evidence suggests that the mechanism responsible for the RA sensitivity of breast cancer cells does not involve transcriptional modulation of the RXRs. The reason for this suggestion is that both RA-sensitive and RA-resistant breast cancer cell lines express RXR-{alpha} and RXR-ß mRNAs. Thus, the presence of these transcripts does not correlate with the RA response (12) .

In conclusion, our results demonstrate that only pan-reactive and RAR-{alpha}-selective, but not RAR-ß-selective, RAR-{gamma}-selective, or RXR-selective retinoids inhibit SK-BR-3 and T47D breast cancer cell growth, induce G1 arrest and apoptosis, and cause up-regulation of RAR-{alpha} and RAR-{gamma} mRNA. Thus, in both cell lines, irrespective of the ER status, growth, cell cycle, apoptosis, and RAR expression appear to be regulated primarily by activation of RAR-{alpha}. Clinically, RAR-{alpha} compounds may represent promising drugs for breast cancer management, because much of the retinoid-associated toxicity is mediated by the action of RAR-{gamma} (41 , 42) .


   ACKNOWLEDGMENTS
 
We thank Dr. M. Klaus (F. Hoffmann-La Roche Ltd., Basel, Switzerland) for TTNPB, Ro 25-7386 and Ro 41-5253, Dr. K. Shudo (Faculty of Pharmaceutical Sciences, Tokyo, Japan) for AM80 and AM580, Dr. B. Shroot (Galderma R&D, Sophia Antipolis, Valbonne, France) for CD417 and CD437, and all of them for valuable technical information on these compounds. Dr. P. Chambon (INSERM, Illkirch, France) is acknowledged for supplying us with human RAR-ß and RAR-{gamma} cDNAs. We are also indebted to Dr. H. Beug (Research Institute of Molecular Pathology, Vienna, Austria) for valuable suggestions.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grant P10777-Med from the Austrian Science Fund (FWF), Grant 7503 from the Austrian National Bank (ONB), and by an unrestricted research grant from Boehringer Ingelheim Austria (to T. W. G.). Back

2 To whom requests for reprints should be addressed, at Laboratory for Cell Growth and Differentiation, Division of Oncology, Department of Internal Medicine I, University of Vienna, Waehringer Guertel 18–20, A-1090 Vienna, Austria. Phone: 43(1)40400-4429; Fax: 43(1)40400-5465; E-mail: thomas.grunt{at}akh-wien.ac.at Back

3 The abbreviations used are: RAR, retinoic acid receptor; RXR, retinoid X receptor; RARE, retinoic acid response element; tRA, all-trans retinoic acid; 9cRA, 9-cis retinoic acid; ER, estrogen receptor; ER+, ER-positive; ER-, ER-negative; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RA, retinoic acid. Back

4 Unpublished data. Back

5 K. Shudo, B. Shroot, and M. Klaus, personal communications. Back

Received 12/28/99. Accepted 8/ 2/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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