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Cell Growth & Differentiation Vol. 13, 335-341, August 2002
© 2002 American Association for Cancer Research

Retinoic Acid Receptor {alpha}2 Is a Growth Suppressor Epigenetically Silenced In MCF-7 Human Breast Cancer Cells1

Eduardo F. Farias, Alice Arapshian, Ira J. Bleiweiss, Samuel Waxman, Arthur Zelent and Rafael Mira-y-Lopez2

Departments of Medicine [E. F. F., A. A., S. W., R. M. L.] and Pathology (I. J. B.), Mount Sinai School of Medicine, New York, New York 10029-6574, and Leukaemia Research Fund Centre, Institute of Cancer Research, London SW3 6JB, United Kingdom [A. Z.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Retinoic acid (RA) receptor (RAR) ß2 has been shown to be underexpressed in human breast cancer cells, including MCF-7 cells, and recent reports have suggested that hypermethylation of the RARß2 promoter and 5'-UTR is the underlying cause. Here we show that RAR{alpha}2 is also underexpressed in MCF-7 breast cancer cells, at both the message and the protein level, relative to normal or nontumorigenic breast epithelial cells. Bisulfite sequencing of the CpG island in the RAR{alpha}2 promoter revealed highly penetrant and uniform cytosine methylation in MCF-7 cells. Pretreatment with the DNA methyltransferase inhibitor, azacytidine, followed by treatment with RA and a histone deacetylase inhibitor, trichostatin A, resulted in partial promoter demethylation and RAR{alpha}2 induction, which strongly suggested that promoter hypermethylation is responsible for RAR{alpha}2 underexpression. We compared the outcome of ectopic expression in MCF-7 cells of matched levels of RAR{alpha}2 and RARß2. On the basis of a clonogenic assay, RAR{alpha}2 displayed ligand-dependent growth-suppressive activity similar to that of RARß2; thus, 10 and 20 nM RA inhibited clonogenic growth by 52 and 80%, respectively, in RAR{alpha}2-transfected cells compared with 75 and 77%, respectively, in RARß2-transfected cells. We conclude that the silencing of the RAR{alpha}2 promoter by hypermethylation may play a contributory role in the dysregulation of RA signaling in mammary tumorigenesis.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Most of the biological effects of vitamin A are mediated by vitamin A metabolites that bind and activate members of the all-trans-RA3 and "retinoid X" (9-cis-RA) receptors (RARs and RXRs, respectively), which belong to the superfamily of ligand-dependent transcription factors. RA regulation of gene transcription is optimally mediated by heterodimers formed between any of the three known RARs ({alpha}, ß, {gamma}) and any of the three known RXR genes ({alpha}, ß, {gamma}). Biological processes dependent on the activity of RXR-RAR heterodimers include mammalian development, as demonstrated by extensive gene targeting studies in the mouse (reviewed in Ref. 1 ), and cancer, as demonstrated by the role of RAR{alpha} in the pathogenesis and therapy of acute promyelocytic leukemia (reviewed in Ref. 2 ) and as suggested by the chemopreventive activity of retinoids toward solid tumors (reviewed in Ref. 3 ). Particularly interesting in connection to the possible role of RARs in regulating epithelial carcinogenesis is the recent observation that RARß regulates branching morphogenesis in the lung (4) .

Another line of investigation suggestive of a role of RARs in the regulation of epithelial carcinogenesis is the down-regulation of RAR gene expression in cancer. In particular, the expression of RARß2, which is normally induced by RA through the DR5 response element in its promoter (5) , has been shown to be down-regulated in a wide range of cancer cell types and tissues (6, 7, 8, 9, 10) . Several recent reports have argued that the down-regulation of RARß2 in cancer is secondary to hypermethylation of the gene (11, 12, 13, 14) . The finding that cancer cells that display no detectable RARß message usually retain RAR{alpha} and RAR{gamma} expression underlies the interest in the loss of RARß expression in cancer. However, the growth-suppressive activity of RARß2 (15, 16, 17) is not unique, because other RARs, such as RAR{alpha}1, are also growth suppressive (18 , 19) . Furthermore, several authors have demonstrated that the selective activation of RAR{alpha} is growth inhibitory (20, 21, 22) .

RAR{alpha}2 expression, like RARß2, is induced by RA through the DR5 element in the RAR{alpha}2 promoter, which is remarkably conserved with that of RARß2 (23) . We showed earlier that RAR{alpha}2 expression is down-regulated in several human breast cancer cell lines relative to normal breast fibroblasts (24) . However, the possible significance of this observation has been masked by the fact that most breast cancer cells maintain RAR{alpha}1 expression and by our use of normal mesenchymal cells as reference.

We have now analyzed RAR{alpha}1, RAR{alpha}2, and RARß2 expression in MCF-7 cells relative to immortalized but nontumorigenic MTSV1–7 human breast ductal epithelial cells. Furthermore, we studied the growth effects of ectopic RAR{alpha}2 as compared with RARß2 and analyzed the methylation status of the endogenous RAR{alpha}2 promoter. In aggregate, our results suggest that RAR{alpha}2 down-regulation contributes to the impaired RA responsiveness of cancer cells.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
RAR Expression in MCF-7 Cells Relative to Established Nontumorigenic Breast Epithelial Cells.
We compared and contrasted RAR{alpha}2 expression in tumorigenic MCF-7 and nontumorigenic MTSV1–7 breast epithelial cells with the expression of RAR{alpha}1 and RARß2 in the same cell pair. MTSV1–7 cells expressed both RAR{alpha}2 and RARß2 under basal conditions, and expression levels increased on treatment with 1 µM RA, as expected (Fig. 1A)Citation . Similar results were obtained for HMEC cultures generated from reduction mammoplasty tissue and enriched in luminal cells (Fig. 1B)Citation . In contrast, neither RAR{alpha}2 nor RARß2 expression could be detected in MCF-7 cells even after treatment with 1 µM RA (Fig. 1A)Citation . Thus, RAR{alpha}2 mirrors the expression pattern of RARß2. On the other hand, both MTSV1–7 and MCF-7 cells expressed RAR{alpha}1, but expression levels were clearly higher in MCF-7 cells (HMECs expressed RAR{alpha}1 at levels comparable with those of MTSV1–7 cells, data not shown). This is because MTSV1–7 cells, like most of the normal breast epithelium but unlike MCF-7 cells, do not express ER{alpha} (25) , which is known to up-regulate RAR{alpha}1 transcription (26) . These results suggest that the total RAR{alpha} mRNA concentration ({alpha}1 + {alpha}2) in MCF-7 cells is comparable with that of MTSV1–7 cells. However, when considering only RAR{alpha}2 expression, the pattern is analogous to the loss of RARß2 expression in MCF-7 breast cancer cells.



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Fig. 1. RA receptor expression in human breast epithelial cells. A, semiquantitative RT-PCR analysis of RAR{alpha}1, RAR{alpha}2, and RARß2 expression in immortalized nontumorigenic MTSV1–7 cells and MCF-7 breast carcinoma cells under basal conditions (Lanes labeled C) and after treatment with 1 µM RA for 24 h (Lanes labeled RA). The results shown are representative of at least three experiments. The first and second panels from the top, ethidium bromide staining and RT-PCR Southern data, respectively, for RAR{alpha} 1 and RAR{alpha}2 (see "Materials and Methods" for details of coamplification and unambiguous identification of each amplicon). The low but detectable RAR{alpha}1 expression in MTSV1–7 cells was more readily apparent in experiments in which RAR{alpha}1 was amplified separately (not shown). The third and fourth panels, RARß2 and GAPDH RT-PCR Southern data, respectively. B, semiquantitative RT-PCR Southern analysis of RAR{alpha}2 (RAR{alpha}1 primer not included) and RARß2 expression in primary normal HMECs enriched in luminal cells; the ethidium bromide signal for GAPDH is shown to demonstrate even loading. RA treatment was as in A. Expression of RAR{alpha}2 and RARß2 was confirmed in a second cell preparation. C, Western blot analysis of RAR{alpha} and RARß expression in MTSV1–7 and MCF-7 cells under basal conditions and after RA treatment as in A (see "Materials and Methods" and "Results" for details).

 
We next performed Western blot analysis to compare RAR{alpha} and RARß protein expression in MTSV1–7 and MCF-7 cells. Antibodies against the F domain of RAR{alpha} (recognizing both RAR{alpha}1 and {alpha}2) or RARß (likewise recognizing multiple subtypes) were used. The slightly shorter RAR{alpha}2 protein migrates sufficiently ahead of RAR{alpha}1 to allow unambiguous identification of each receptor subtype (27) . Interestingly, despite the RT-PCR differences described above, RAR{alpha}1 protein levels were essentially the same in MTSV1–7 and MCF-7 cells (Fig. 1C)Citation . In sharp contrast, RAR{alpha}2 was expressed in RA-treated MTSV1–7 but not in RA-treated MCF-7 cells, and its expression level was comparable with that of RAR{alpha}1. These results suggest that the total RAR{alpha} protein concentration ({alpha}1 + {alpha} 2) in MCF-7 cells is ~50% lower than in nontumorigenic cells. Like RAR{alpha}2, RARß was detected only in MTSV1–7 cells; its expression was evident under basal conditions but increased after RA treatment (Fig. 1C)Citation . On the basis of the lack of RARß1 expression in the breast and the good agreement between relative RARß2 mRNA (Fig. 1A)Citation and RARß protein expression levels (Fig. 1C)Citation , we suspect that the band detected by the anti-RARß antibody corresponds to RARß2.

In sum, nontumorigenic MTSV1–7 cells express both RAR{alpha}2 and RARß2 mRNA and RAR{alpha}2 and RARß protein, whereas MCF-7 tumor cells fail to express these inducible receptors.

Mechanism Underlying RAR{alpha}2 Silencing in MCF-7 Cells.
The RAR{alpha}2 promoter and 5'-UTR region are highly CpG rich. This is actually in contrast to the same region of the RARß2 gene, which is CpG poor. Nevertheless, there is good evidence that hypermethylation of the RARß2 promoter and 5'-UTR is the proximal cause of RARß2 underexpression in MCF-7 cells (11) . We, therefore, analyzed the methylation status of the RAR{alpha}2 promoter by bisulfite sequencing. The results (Fig. 2A)Citation demonstrated uniform and highly penetrant methylation of the region studied. This includes three CpG sites near the DR5 response element (highlighted in Fig. 2ACitation ), which are conserved and also methylated in the RARß2 promoter in MCF-7 cells. We have proposed that methylation of these sites may be particularly relevant to promoter silencing (28) .



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Fig. 2. The human RAR{alpha}2 promoter is hypermethylated in MCF-7 breast carcinoma cells. A, bisulfite sequencing of the RAR{alpha}2 promoter. The region analyzed is sketched (top), showing CpG sites (tick marks), the approximate position of the primers used (horizontal arrows) and the three CpG sites in the RARE (bracket) region. The RAR{alpha}2 sequence (gi:9392669) was numbered relative to the transcription start site. Bisulfite sequencing results (bottom) are shown for each clone analyzed ({bullet}, methylated CpGs; {circ}, unmethylated CpGs). Top panel, results for untreated cells; bottom panel, results for cells pretreated with azaC and treated with RA and TSA (see below); cells not pretreated with azaC and treated with RA and TSA yielded results similar to those for untreated cells (data not shown). B, semiquantitative RT-PCR analysis of RAR{alpha}2 expression in untreated MCF-7 cells (Lane 1), cells treated with 1 µM RA and 100 nM TSA for 24 h (Lane 2), and cells pretreated with 5 µM azaC for 72 h and then treated with 1 µM RA and 100 nM TSA for 24 h (Lane 3).

 
To test whether hypermethylation contributes to RAR{alpha}2 silencing, we pretreated exponentially growing MCF-7 cells with azaC, which is incorporated into DNA and inhibits DNA methyltransferase. Three days later, we treated the cells with a combination of RA and TSA, a histone deacetylase inhibitor; control cultures were pretreated with growth medium alone and then treated as above. As shown in Fig. 2BCitation , MCF-7 cells, pretreated with azaC and then treated with RA and TSA, expressed readily detectable amounts of RAR{alpha}2 message, whereas the same treatment had no effect in cells not pretreated with azaC. Bisulfite sequencing of DNA isolated from cultures pretreated with azaC and then treated with RA and TSA revealed a modest and variable degree of RAR{alpha}2 promoter demethylation (Fig. 2A)Citation . We suspect that more extensive demethylation occurred in a small fraction of cells and that RAR{alpha}2 reexpression in these cells accounted for the RT-PCR signal detected in Fig. 2BCitation . Alternatively, a modest degree of demethylation may be sufficient to initiate chromatin remodeling. These results implicate RAR{alpha}2 hypermethylation as a proximal cause of RAR{alpha}2 silencing in MCF-7 cells.

RAR{alpha}2 Is a Growth Suppressor.
We wished to compare the effect on cell growth of the ectopic expression of matched levels of RAR{alpha}2 and RARß2. To this end, we constructed a bicistronic mammalian expression vector encoding RAR{alpha}2 or RARß2 and EGFP (pRAR{alpha}2-IRES-2-EGFP and RARß2-IRES-2-EGFP, respectively). Our goal was to use EGFP to infer RAR expression levels rather than rely on the use of antibodies with different titer and affinity. Preliminary experiments demonstrated that transfection of MCF-7 cells with a fixed amount of vector (2 µg) resulted in the same overall expression level of EGFP and, thus, by inference, of RAR{alpha}2 and -ß2 (Fig. 3A)Citation . For the experiment proper, we transfected MCF-7 cells with the same fixed dose of each vector and, as a control, 2 µg of an "empty" (no RAR insert) bicistronic vector (pIRES-2-EGFP). Fig. 3BCitation shows the pattern of EGFP fluorescence of parallel cultures transfected as above and selected in G418 in the absence of RA; comparable fluorescent signals were obtained for each cell group. The experimental cultures were placed in selection medium with or without RA supplementation and colonies allowed to grow for a period of 3 weeks. All of the groups of transfected cells formed multiple colonies in selection medium without RA. Treatment with 10 and 20 nM RA had a minimal effect on the clonogenic potential of control, pIRES-2-EGFP-transfected cells (Fig. 4A)Citation . In contrast, the same treatment had a marked inhibitory effect on the clonogenic potential of pRAR{alpha}2-IRES-2-EGFP- and RARß2-IRES-2-EGFP-transfected cells. More importantly, the magnitude of this effect, as can be seen visually (Fig. 4A)Citation and after colorimetry (Fig. 4B)Citation , was comparable for RAR{alpha}2 and -ß2. We conclude that RAR{alpha}2, like RARß2, is a growth suppressor. This conclusion is based on the interpretation that the results obtained were attributable to the transfected RAR{alpha}2 and -ß2 receptors per se and not to secondary effects on other nuclear receptors.



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Fig. 3. Transgene expression in transfected MCF-7 cells. MCF-7 cells were transfected with bicistronic expression vectors [RAR and EGFP under the control of the cytomegalovirus (CMV) promoter], stable transfectants selected in the absence of RA, and EGFP protein assayed to infer RAR expression levels. A, Western blot analysis showing similar overall EGFP expression levels after stable transfection with equal amounts (2 µg) of pRAR{alpha}2-IRES-2-EGFP and pRARß2-IRES-2-EGFP. To verify that the CMV promoter was not RA dependent, EGFP expression was also monitored after 24-h treatment with 1 µM RA; this had no effect on EGFP expression, as expected. B, fluorescence microscopy (EGFP autofluorescence) of MCF-7 cultures transfected as in A and, as a control, transfected with 2 µg of pIRES-2-EGFP vector. The fluorescent signals produced by the three cell groups are similar.

 


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Fig. 4. Comparable inhibition of MCF-7 clonogenic growth by RAR{alpha}2 and RARß2. MCF-7 cells were transfected with 2 µg of pIRES-2-EGFP, pRAR{alpha}2-IRES-2-EGFP, or pRARß2-IRES-2-EGFP and then plated at low density in selection medium supplemented with charcoal-stripped serum and the indicated concentration of RA. A, crystal violet-stained cultures after 3 weeks in selection medium. For each plate, wells within the same column represent triplicates. B, densitometric evaluation of cell number in the cultures shown in A. Similar results to those shown were obtained in two additional experiments.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Our findings need to be discussed within the context of the prevailing idea that down-regulation of RARß2 may specifically contribute to cancer progression. Although this is an attractive idea, recent developments have revealed a more complex scenario. For instance, breast cancer cells can express the RARß4 isoform, which may act as a dominant negative RAR, and appears to be translated preferentially over RARß2 (29) . A recent study of RARß2 mRNA as a prognostic indicator in non-small cell lung cancer actually demonstrated decreased disease-free survival in RARß2-expressing cases (30) . These findings reflect the complexity of RAR signaling. In the context of breast cancer, we have now introduced an additional element of complexity by demonstrating that the thus-far-overlooked RARß2 congener, RAR{alpha}2, can function as an RA-dependent growth suppressor. Our results suggest that RAR{alpha}2, besides being RA-inducible, shares three properties with RARß2: it is underexpressed in MCF-7 cells relative to normal and nontumorigenic cells, it is epigenetically silenced, and its product acts as a growth suppressor. We, therefore, suggest that RAR{alpha}2 should be considered as a factor of equal importance to RARß2 in studies of aberrant RAR expression and function in breast cancer and perhaps other cancers as well.

Although homozygous deletion of RARß2 has been shown to impair RA-induced growth arrest in F9 cells (31) , and mice harboring an RARß2 antisense transgene have been shown to develop lung tumors (32) , similar studies with RAR{alpha}2 have not been reported. On the other hand, several studies have shown that ectopic RAR{alpha}1 is growth suppressive (18 , 19) and that selective RAR{alpha} ligands are growth inhibitory (20, 21, 22) . Interestingly, it has been proposed that the ability of RAR{alpha}1 to restore RA growth regulation in ER{alpha}-nonexpressing breast cancer cells is secondary to the induction of RARß2 (17) . However, induction of RAR{alpha}2 likely occurred under the same conditions because they have conserved promoters (23) . In this context, it is interesting to point out that the recent demonstration that constitutively expressed RAR{alpha} protein (i.e., RAR{alpha}1) is rapidly down-regulated after RA activation (33 , 34) suggests that the induction of RAR{alpha}2 and RARß2 may be critical for sustaining a RA response. Undoubtedly, the best way to dissect the relative contribution of each isoform would be to extend the targeted gene deletion approach undertaken in F9 cells (31) to a diploid breast cell line that is sensitive to RA-induced growth arrest.

An in situ hybridization study by Xu et al. (8) demonstrated that RAR{alpha} (total) mRNA is undetectable in 16% of invasive human breast cancers as compared with 2% of normal breast tissues. Taking into account that RAR{alpha}1 mRNA levels in nontumorigenic ER{alpha}-nonexpressing cells (MTSV1–7) approximate those in ER{alpha}-nonexpressing breast cancer cells (i.e., they are detectable but lower than the levels expressed by ER{alpha}-expressing cells; Fig. 1ACitation ; Ref. 21 ), we hypothesize that the RAR{alpha} down-regulation detected by Xu et al. represents down-regulation of RAR{alpha}2 (the true extent of RAR{alpha}2 down-regulation may in fact be greater). An in situ hybridization study using RAR{alpha}1- and RAR{alpha}2-specific riboprobes is needed to test this hypothesis and reveal the extent, if any, of in situ RAR{alpha}2 down-regulation in breast cancer.

The evidence implicating aberrant RAR expression in cancer remains largely circumstantial, and this report is no exception. Further progress might be achieved, for instance, through a study of RAR methylation and expression in existing mouse models of breast cancer, which afford the opportunity to sample tissues at linear time points. RAR isoform and subtype-specific knockout mice represent another opportunity for progress.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cells.
The MCF-7 breast cancer cell line was obtained from the American Type Culture Collection (Rockville, MD) and grown in DMEM supplemented with 10% FBS, 5 µg/ml insulin, and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin). MTSV1–7 is a SV40 T-antigen-immortalized, nontumorigenic human breast ductal epithelial cell line that was generated, kindly donated, and maintained as described, by Joyce Taylor-Papadimitriou (The Imperial Cancer Research Fund, Guy’s Hospital, London, England) and colleagues [Bartek et al. (35) ]. Because late-passage MTSV1–7 cells exhibit properties of transformed cells (data not shown), we used early-passage cells in the present studies. Both of the cell lines were tested for Mycoplasma by DAPI staining and shown to be free of contamination.

Normal HMECs were derived from reduction mammoplasty material that was residual to pathologic analysis and bore no identifiers. After obtaining epithelial organoids as described previously (36) , enrichment in luminal cells was performed by negative immuno-selection (37) and the cells cultured in collagen I-coated dishes in mammary epithelium growth medium (Clonetics, San Diego, CA) supplemented with 5 µg/ml transferrin and 10 µM isoproterenol (cells were used after 0–1 passages).

RT-PCR Analysis.
Aliquots of 1 µg of total RNA were reverse transcribed in 20-µl reaction volumes using the SuperScript Preamplification System (Life Technologies, Inc. Gaithersburg, MD). The PCR mixture consisted of 10x PCR buffer [200 mM Tris-HCl (pH 8.4), 500 mM KCl], 1 mM MgCl2, 200 µM dNTPs, 0.4 µM each primer, 2 µl cDNA, and 5 units of Taq (Promega) in a total volume of 50 µl. Primers for the amplification of RAR{alpha}1 and -{alpha}2 were as described previously (38) , and cycling parameters were: 94°C for 5 min; 30 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 1 min; and 72°C for 5 min. RAR{alpha}1 and -{alpha}2 were amplified, separately or in the same reaction, using sense primers that anneal to the A1 or A2 region, respectively, and an antisense primer that anneals to the common B region, yielding products of 220 bp and 287 bp, respectively. PCR products (25 µl aliquots) were separated in 1.5% agarose gels, blotted, and hybridized to probes labeled with [{alpha}32P]dCTP by random priming. The RAR{alpha}1 amplification product was detected using an autologous probe (this was validated by probing with full-length RAR{alpha}1 cDNA) and the RAR{alpha}2 product was detected using full-length RAR{alpha}2 cDNA. When RAR{alpha}1 and -{alpha}2 were amplified in the same reaction, the two products were detected by hybridization to full-length RAR{alpha}2 cDNA and could be unambiguously identified based on their different sizes (separate amplification reactions validated the results obtained after coamplification). RARß2 and GAPDH RT-PCR were performed as described previously (28) .

Western Blot Analysis.
Cells were lysed on ice for 30 min with radioimmunoprecipitation assay buffer (1x PBS, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS) plus proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin) and phosphatase inhibitors (1 mM Na3VO4 and 1 mM NaF); for more efficient cell lysis, the samples were frozen (-80°C) and thawed once. Protein was quantitated by the method of Bradford (39) and 50 µg of total protein/lane were resolved by SDS-PAGE (10% polyacrylamide). Rabbit polyclonal antibodies raised against the F region of RAR{alpha} (number 115) was a generous gift from Pierre Chambon (Institut de Genetique et de Biologie Moleculaire et Cellulaire, Strasbourg, France); rabbit polyclonal antibodies raised against the F region of RARß (C-19) were obtained from Santa Cruz Biotechnology. Polyvinylidene difluoride membranes were blocked with TBST plus 5% milk for 1 h and incubated with first antibody diluted to 1 µg/ml in TBS plus 1% milk for 1 h at room temperature. The membranes were washed five times with TBST and once with TBS and treated with secondary antirabbit antibody diluted to 0.3 µg/ml in TBS plus 1% milk for 45 min at room temperature. The membranes were then washed as above and reaction products visualized using Amersham’s chemiluminiscence detection system.

For EGFP expression, total cell lysates were resolved by SDS-PAGE and probed with EGFP antibodies obtained from Clontech (Palo Alto, CA).

Bisulfite Sequencing.
Briefly, 5 µg of XbaI-digested genomic DNA was denatured with 0.5 M NaOH at 75°C for 15 min, deaminated with freshly prepared 4.0 M (pH 5.0) sodium metabisulfite (BDH Laboratory Supplies, Poole, Dorset, England) and incubated at 55°C for 8 h in a total volume of 1.2 ml under mineral oil. Desalting, desulfonation by alkali, neutralization, and ethanol precipitation were as described previously (40) ; the DNA was resuspended in 50 µl of H2O. First-round PCR was carried out in a 50-µl reaction mixture of 10x PCR buffer, 1.2 mM MgCl2, 160 µM dNTPs, 0.4 µM each primer, 10 µl bisulfite-treated DNA, and 5 units of Taq (Promega). Subsequently, two rounds of seminested PCR were carried out using the same conditions as above and 5 µl of the reaction product generated in the preceding round as template. Primers and parameters were: 5'-TTAGGGATTTATTTAAGTTAGGTTTTTT-3' (sense) and 5'-AACTCCCCCAAAATTTAATCAATCCCTAAC-3' (antisense); 94°C for 5 min; 40 cycles of 94°C for 1 min, 48°C for 1 min, 72°C for 1 min; 72°C for 5 min. Primers and parameters for the second round were: 5'-TTTTTGTTTGTGTTTGTGTTAATAGTATT-3' (sense) and the same antisense as above with parameters as above, except annealing temperature was 46°C, and the reaction was terminated after 20 cycles. Third round primers and parameters were: 5'-TATATATATGTTATAGATAATGATAT-3' (sense) and the same antisense as above; parameters were the same as for second round parameters but for 40 cycles. The purified PCR fragment was subcloned into pCR2.1, and individual clones were submitted for sequencing (T7 primer).

Cell Transfection.
Monolayers of MCF-7 cells at 70–80% confluence were transfected (FuGene system, Roche) with 2 µg of pIRES-2-EGFP vector alone or carrying full-length RAR{alpha}2 or RARß2 cDNA inserts. Two days later, the cells were trypsinized and plated at the desired concentration in selection medium (800 µg/ml Geneticin). Mock-transfected MCF-7 cells were used to follow cell selection and transfection efficiency was monitored by fluorescence microscopy.

RA Treatment.
For testing the effect of 1 µM RA on RAR expression, cells in regular growth medium were treated with RA for 24 h and harvested for analysis. For testing the effect of low RA doses on MCF-7 clonogenicity, FBS was charcoal stripped as follows: 1.25 g of activated charcoal and 0.125 g of dextran T-70 were added to a 500-ml FBS bottle followed by incubation at 55°C for 30 min and centrifugation at 3000 rpm at 4°C for 20 min. The resulting single-stripped serum was collected into a fresh 500-ml flask and the procedure repeated with a second round of charcoal-dextran, but incubated at 37°C for 30 min and, finally, sterile-filtered. MCF-7 breast cancer cells transfected as above were seeded in 6-well (5 x 104 cells/well) or 12-well (2 x 104 cells/well) plate dishes in medium supplemented with 10% charcoal-stripped FBS, 5 µg/ml insulin, 800 µg/ml Geneticin, and antibiotics. After 3 weeks, Geneticin-resistant colonies were fixed in 10% formalin in PBS and stained with 0.05% crystal violet. After photography, the stain was solubilized with 10% acetic acid-10% methanol, and the A595 was measured to quantitate adherent cell mass.


    Acknowledgments
 
We thank Silvina Bertran for expert technical assistance and Joyce Taylor-Papadimitriou (MSTV1–7 cells) and Pierre Chambon (anti-RAR antibodies and RAR cDNAs) for reagent gifts.


    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 Supported by NIH Grant CA54273 (to RML), a grant from the Leukemia Research Fund of Great Britain (to AZ), The Samuel Waxman Cancer Research Foundation and The Norman and Rosita Winston Foundation. Back

2 To whom requests for reprints should be addressed, at Department of Medicine, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029-6574. Back

3 The abbreviations used are: RA, retinoic acid; RAR, all-trans-RA receptor; RXR, 9-cis-RA receptor; DR5, direct repeat separated by five basepairs; HMEC, human mammary epithelial cell; ER{alpha}, estrogen receptor {alpha}; RT-PCR, reverse transcription-PCR; azaC, 5-azacytidine; TSA, trichostatin A; EGFP, enhanced green fluorescent protein; FBS, fetal bovine serum; TBS, Tris-buffered saline; TBST, TBS with Tween 20; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back

Received for publication 1/ 7/02. Revision received 4/26/02. Accepted for publication 6/17/02.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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