Selective Promoter Usage of the Human Estrogen Receptor-{alpha} Gene and Its Regulation by Estrogen

C. Donaghue, B. R. Westley and F. E. B. May

University Department of Pathology Royal Victoria Infirmary Newcastle upon Tyne, NE1 4LP United Kingdom


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Three promoters have been identified for the human estrogen receptor-{alpha} gene. The positions of promoters A and B are known whereas that of the recently identified promoter C is not. Cloning and hybridization experiments demonstrated that promoter C is located more than 21 kb upstream of promoter A. The use of the three promoters was examined in estrogen receptor-positive breast cancer cell lines, cell lines derived from other malignancies, and some normal tissues by RT-PCR and transient transfection. All estrogen-responsive breast cancer cell lines used all three promoters, apart from ZR-75 cells, which did not use promoter B in one of two sublines examined. Cell lines derived from other malignancies and other normal tissues that express lower levels of estrogen receptor-{alpha} showed more selective promoter usage. This suggests that the level of expression of estrogen receptor-{alpha} is determined by the number of promoters used, rather than the selective use of specific promoters. We also show that promoter C is used more widely than suggested by others. Analysis of a series of estrogen receptor-positive primary breast tumors showed that all three promoters were used in all the tumors. All three promoters were modulated by estrogen in estrogen-responsive breast cancer cell lines: all three promoters were down-regulated by estrogen in MCF-7 cells in which estrogens reduce receptor expression whereas all promoters used were up-regulated in T47D, ZR-75, and EFM-19 cells in which estrogens increase receptor expression. This suggests that it is the repertoire of transcription factors present within a cell rather than the selective use of a specific promoter that determines whether estrogen receptor-{alpha} expression is increased or decreased by estrogen.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogens play an important role in normal physiological functions such as pregnancy, development of female secondary sexual characteristics, and the maintenance of bone density (1), but are also involved in pathological processes in the breast (2) and endometrium (3). Estrogens exert their effects through the estrogen receptor, a member of the nuclear steroid-thyroid hormone receptor superfamily (4), which is expressed in a cell- and tissue-specific manner. This specific pattern of estrogen receptor expression enables estrogens to direct their effects to target tissues; however, the mechanisms involved in the regulation of estrogen receptor expression are poorly defined.

Two forms of the human estrogen receptor gene have been identified, estrogen receptor-{alpha} (5), which was the first to be identified, and estrogen receptor-ß (6). Estrogen receptor-{alpha} seems to be the predominant form in breast cancer (7), although estrogen receptor-ß has been detected at a low level in some breast cancers (8). The estrogen receptor-{alpha} gene is transcribed from three promoters. The proximal promoter was the first to be characterized and was termed promoter A. The single site of transcription initiation from promoter A was identified by primer extension and S1 nuclease mapping (9), and this promoter contains a TATA box and CAAT element. Subsequently, sequencing of upstream genomic DNA revealed a region at position -1.9 kb, which was homologous to the 5'-end of the rat and mouse estrogen receptor-{alpha} mRNAs, and RT-PCR experiments demonstrated that this region was contained within human estrogen receptor-{alpha} RNA transcripts (10). This suggested that an additional exon, denoted exon 1', was located upstream of exon 1 and identified an additional promoter, denoted promoter B. Several sites of transcription initiation from this promoter have been suggested. Position -1978 was proposed on the basis of homology to the mouse estrogen receptor-{alpha} promoter (10), position -2120 due to the presence of an initiation response element (INR) located in a region shown to be the beginning of exon 1' by primer extension experiments (11), position -3251 due to the presence of a CAP site and a nearby sequence with similarity to a TATA element (12), position -3006 from RT-PCR and primer extension analysis (13), and most recently positions -2008, -1992, -1978, and -1947 from rapid amplification of cAMP ends (RACE)-PCR experiments (14). The last study also identified a novel estrogen receptor-{alpha} transcript containing a distinct 5'-exon (exon 2') in human liver whose transcription must be initiated from a previously unidentified upstream promoter (promoter C). The location of this novel exon and its promoter was not identified, but Southern hybridization experiments on uncloned DNA suggested that it was located more than 10 kb upstream of promoter A (14).

The factors controlling the level of expression of estrogen receptor-{alpha} are not well characterized; however, cell- and tissue-specific expression may be regulated by differential promoter usage, i.e. the use of a strong estrogen receptor-{alpha} promoter in one cell type and the use of a different, weaker estrogen receptor-{alpha} promoter in another. This possibility has been investigated (15, 16, 17), and it was concluded that promoter A is used in breast cancer cell lines and in endometrium but not in bone or liver; promoter B is used in some breast cancer cell lines, endometrium, and bone, but not in liver; and promoter C is used exclusively in liver.

The levels of estrogen receptor-{alpha} mRNA are regulated by estrogen in breast cancer cell lines; estrogen has been shown to decrease estrogen receptor-{alpha} mRNA levels in MCF-7 cells (18, 19) and increase estrogen receptor-{alpha} mRNA levels in EFM-19 (18, 20), ZR-75 (18), and T47-D cells (18, 21). The differential regulation by estrogen may also be due to different promoter usage, i.e. the use of one promoter that is up-regulated by estrogen in one cell type and use of another promoter that is down-regulated in another.

In this report, we have investigated promoter usage in breast cancer cell lines as well as in cell lines from other malignancies and some normal tissues. We report the cloning and sequencing of estrogen receptor-{alpha} DNA further 5' to that previously isolated and demonstrate that promoter C is located further upstream than realized previously. Using RT-PCR and reporter gene assays we show that estrogen receptor-positive breast cancer cells use more estrogen receptor-{alpha} promoters than other cell types and that all three promoters are regulated in a coordinate way by estrogen.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation of the 5'-End of the Estrogen Receptor-{alpha} Gene
A genomic library was constructed from MCF-7 cell DNA and was screened with a radiolabeled probe corresponding to promoter B of the estrogen receptor-{alpha} gene. Recombinants were characterized by a combination of restriction mapping and Southern analysis and were found to span estrogen receptor-{alpha} DNA from -21 to +14 kb, position 0 being the major start site of transcription from promoter A (9). Previously, only 4 kb of DNA upstream of promoter A had been isolated (22), and this is, therefore, the largest amount of estrogen receptor-{alpha} promoter DNA cloned to date. The position of the {lambda}-recombinants in the estrogen receptor-{alpha} genomic DNA and their restriction maps are shown in Fig. 1AGo.



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Figure 1. Isolation of the 5'-End of the Estrogen Receptor-{alpha} Gene and Investigation of Promoter C

A, The estrogen receptor genomic DNA is schematically represented with the recognition sites for the enzymes EcoRI (E), BamHI (B), HindIII (H), and XbaI (X). The size and position of each of the {lambda}-DASH clones is also shown. B, The estrogen receptor genomic DNA is represented and the three estrogen receptor promoters are shown. The position and orientation of four 22-bp oligonucleotides (ER1, ER2, ER3, and ER4) are indicated by solid arrows. C, The {lambda}-DASH clones {lambda}9a and {lambda}13 were restricted with XbaI, and the resultant DNA fragments were separated by gel electrophoresis on a 0.8% agarose gel, transferred to nylon membrane, and hybridized with the indicated 32P-labeled probes. The lefthand panel shows the ethidium bromide-stained gel. The following three panels show the Southern hybridization with the ER4, ER3, and ER1 probes.

 
These recombinants were used to investigate the location of promoter C of estrogen receptor-{alpha}. The two furthest 5'-clones ({lambda}9a and {lambda}13) were digested with XbaI, and the fragments were separated by gel electrophoresis. The hybridization of three radiolabeled probes to Southern transfers is shown in Fig. 1CGo. The three probes were 22-bp oligonucleotides corresponding to exon 1 (ER4), exon 1' (ER3), and exon 2' (ER1) (Fig. 1BGo). Figure 1CGo shows that the ER4 probe (exon 1) did not hybridize to any restriction fragments of clone {lambda}9a as this clone terminates upstream of promoter A, but did hybridize to a 4.5-kb fragment from clone {lambda}13. The ER3 probe (exon 1') hybridized to a 3.4-kb fragment from clone {lambda}9a and a 4.5-kb fragment from clone {lambda}13. The ER1 probe (exon 2') did not hybridize to any of the restriction fragments from either of the clones. This suggested that exon 2' was not located within clone 9a or clone 13. To substantiate this conclusion, an additional hybridization experiment was carried out with a longer cDNA probe containing 60 bp of exon 2'. The probe was synthesized by RT-PCR using oligonucleotides ER1 from exon 2' and ER5 from exon 1 (see Fig. 1BGo). The resultant 143-bp PCR product contains 60 bp of exon 2' and 83 bp of exon 1 and should hybridize to restriction fragments that contain exon 2' or exon 1. The PCR product hybridized to fragments containing exon 1 only in {lambda}9a (data not shown). Collectively, these results demonstrated that exon 2', and therefore promoter C, is not located within clones {lambda}9a or {lambda}13 and must therefore be located more than 21 kb upstream of promoter A.

Sequence Analysis of the 5'-End of the Estrogen Receptor-{alpha} Gene
To analyze the estrogen receptor-{alpha} promoter region in detail, DNA between -3280 and -5330, which had not been sequenced previously, was subcloned from the {lambda}-clones and sequenced. The data were then combined with previously published sequence data from -3 to +2 kb and analyzed for the presence of transcription and enhancer elements using GCG software (Genetics Computer Group, Madison, WI) (23). The results of this analysis are shown schematically in Fig. 2.

The area around and upstream of promoter B is shown in Fig. 2AGo, along with the proposed transcription initiation sites for this promoter. There are few putative TATA and no putative CAAT elements located near the proposed initiation sites, whereas a number of INR elements are present (Fig. 2AGo). It is likely, therefore, that transcription initiation is positioned via INR elements at this promoter. An INR element can often direct transcription initiation from several closely positioned start sites, and this is in keeping with the findings of Grandien (14), who detected four clustered initiation sites for promoter B. There are INR elements either at or just upsteam of the two initiation sites proposed by Keaveney et al. (10, 11) and three of the four initiation sites proposed by Grandien (14). There are no INR elements at the two upstream initiation sites proposed by Piva et al. (12, 13); instead, there are putative TATA boxes. In general, the putative TATA sequences are evenly spaced throughout the promoter B region, whereas the INR elements are clustered in the 5'-half of exon 1' around the proposed downstream initiation sites (Fig. 2AGo). There are several Sp1 and AP1 sites in the vicinity of promoter B, which may augment transcription from this promoter. There are no perfect palindromic estrogen response elements (EREs) in the vicinity of the promoter or in the 3-kb region upstream of the promoter. However, there are several palindromic ERE-like elements containing 3 mismatched base pairs, and several consensus half-ERE sites that may confer estrogen responsiveness on the promoter. There are also numerous half-ERE sites with 1 mismatched base pair present throughout the estrogen receptor-{alpha} promoter region. Palindromic and mismatched half-sites, either alone, in tandem, or in conjunction with adjacent Sp1 or AP1 sites, have been shown to elicit an estrogen response (24, 25, 26, 27, 28, 29, 30, 31).



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Figure 2. Sequence Analysis of the 5'-End of the Estrogen Receptor-{alpha} Gene

Approximately 2.5 kb of previously uncharacterized estrogen receptor promoter DNA was sequenced by automated fluorescent sequencing. The sequence was combined with previously published sequence data (22 34 35 ) and analyzed for transcriptional and regulatory sequence elements using GCG software (12 ). A, The estrogen receptor upstream DNA encompassing promoter B is represented schematically, from position -5300 to -1500. B, The estrogen receptor DNA encompassing promoter A is represented schematically, from position -1500 to +2070. The proposed sites of transcription initiation for both promoters are shown. The recognition sites for the enzymes EcoRI (E), BamHI (B), HindIII (H), and XbaI (X) are shown. The positions of putative TATA, INR, Sp1, AP1, and ERE elements are indicated by single lines. Putative CAAT sequences are represented by a vertical dashed line. Thicker lines represent multiple elements. Half-ERE sites with no mismatched base pairs (0) and one mismatched base pair (1 ) are shown. Palindromic ERE sites [ERE(x)] are also shown with either two (2 with an asterisk above it) or three (3 with an asterisk above it) mismatched base pairs, as indicated.

 
Figure 2BGo represents the sequence analysis in the region of promoter A. The TATA and CAAT elements that direct transcription from this promoter are indicated. Five Sp1 sites and one AP1 site that may enhance transcription initiation are located within 1 kb of promoter A. As with promoter B, there are no palindromic EREs. There are, however, several ERE-like elements with 2 or 3 mismatched base pairs, several consensus half-ERE sites, and numerous half-ERE sites with 1 mismatched base pair.

Use of the Three Estrogen Receptor-{alpha} Promoters in Different Cell Types
Estrogen receptor-{alpha} expression was first asessed by RT-PCR, using the universal primers ER7 and ER8 that amplify mRNA transcribed from all promoters (Fig. 3AGo) (33) in a panel of estrogen receptor-positive (MCF-7, T47D, ZR-75, EFM-19, and EFF-3) and negative (SKBR, Hs578T, BT20, MDA-MB231, and HBL-100) breast cancer cell lines and in a variety of cell lines derived from other malignancies. These included AGS, Kato III, and HGT-1 (gastric carcinoma), Ishikawa (endometrial carcinoma), CaCo2, HCT-116, T84, HT29 (colon carcinoma), HepG2 (hepatocellular carcinoma), HCT-8 (ileocecal adenocarcinoma), A431 (vulval epidermoid carcinoma), HeLa (cervical carcinoma), and HC12 (small cell lung carcinoma). A 223-bp fragment corresponding to the predicted PCR product was amplified using RNA from all the estrogen receptor-positive cell lines as well as the BT20 estrogen receptor-negative cell line but not the SKBR, Hs578T, MDA-MB231, or HBL-100 cells (data not shown). BT20 cells have been reported by others (33) to produce a variant mRNA, and this would be amplified by RT-PCR using the universal primers. Small amounts of PCR product were detected using RNA from Kato III, CaCo2, and Ishikawa cell lines, indicating that estrogen receptor-{alpha} is expressed in these cells but in lower amounts than in the estrogen receptor-positive breast cancer cell lines. Very small amounts of PCR product were produced from A431, HGT-1, and HCT-8 cell RNA (data not shown), and these were not analyzed further. RT-PCR was also used to examine estrogen receptor mRNA expression in normal stomach and liver, and it was detected in all samples examined.



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Figure 3. Use of the Three Estrogen Receptor-{alpha} Promoters in Different Cell Types

A, The structure of the 5'-end of the estrogen receptor-{alpha} gene is shown. The position of oligonucleotides ER1-ER8 is marked. The splicing events are shown by broken lines, and the position of the resultant RT-PCR products is marked. The names given to the RT-PCR products are shown in italics. Exon 1' is divided into two regions. The 3'-region harbors the transcription initiation sites identified originally by homology to the mouse gene (21 ), by sequence analysis (22 ) and recently by RACE-PCR (13 ). The 5'-region harbors the transcription initiation sites identified by other studies (34 35 ). B, cDNA was prepared from the breast cancer cell lines MCF-7, T47-D, ZR-75 (two sublines a and b), EFM-19, EFF-3, and BT20, from normal liver and two samples of normal stomach (A and B), and from the cell lines Kato III, CaCo2, and Ishikawa. PCR reactions were carried out with the cDNA and primer pairs for the Universal, PrC, PrB.1, PrB.2, PrA.1, and PrA.2 PCR products. The products were separated by gel electrophoresis on a 3% agarose gel and stained with ethidium bromide. C, The relative amount of PCR product was estimated from the intensity of the product on the stained gel using UVP image analysis software (UVP Inc, San Gabriel, CA). The highest level of expression for each promoter is defined as being 100%, and the levels of expression in the other cell lines are shown as a percentage of the maximum value. The data in the table are based on product PrC for promoter C, product PrB.2 for promoter B, and PrA.2 for promoter A. Different exposure times were used to photograph the agarose gels of the PCR products shown in Fig. 3BGo, and these have been taken into account in the semiquantitative assessment of the amount of PCR product shown in the table. The figures in the table are the average values of between 3 and 10 experiments.

 
Estrogen receptor-{alpha} promoter usage was then analyzed in the cells that express significant levels of estrogen receptor-{alpha} mRNA by RT-PCR using the primers shown in Fig. 3AGo. The cell lines analyzed were the MCF-7, ZR-75, T47-D, EFM-19, and EFF-3 estrogen receptor-positive breast cancer cell lines, the BT-20 breast cancer cell line that produces variant estrogen receptor RNA (33), the Kato III gastric carcinoma, CaCo2 colonic carcinoma, Ishikawa endometrial carcinoma cell lines, and normal liver and stomach. Two sets of primers specifically amplify transcripts originating from promoter A: primers ER4 and ER5 amplify 108 bp within exon 1 (PrA.1 product) while primers ER4 and ER6 amplify 611 bp that crosses the exon1/exon2 splice site (PrA.2 product). Two sets of primers were designed to amplify promoter B transcripts: primers ER2 and ER5 amplify 406 bp across the exon 1'/exon 1 splice site (PrB.1 product) from transcripts that would be generated from the two most 5' promoter B transcription initiation sites (12, 13), whereas primers ER3 and ER5 amplify a shorter region of 183 bp across the exon 1'/exon 1 splice site (PrB.2 product) from transcripts generated from any of the proposed promoter B transcription initiation sites (10, 11, 12, 13, 14, 34). Primers ER1 and ER5 were designed to amplify a 143-bp promoter C transcript that crosses the exon 2'/exon 1 splice site (PrC product).

Agarose gels of the PCR products are shown in Fig. 3BGo, and a summary of the relative amounts of PCR product generated for each promoter from all the cell lines analyzed is given in Fig. 3CGo. In MCF-7 cells, PCR products were generated using primers ER7 and ER8, which would amplify DNA from RNA transcribed from all three promoters. A 143-bp PCR product was amplified using primers ER1 and ER5, demonstrating that estrogen receptor-{alpha} is transcribed from promoter C. A 183-bp product was amplified by primers ER3 and ER5, whereas no product was amplified using primers ER2 and ER5, suggesting that in this cell line transcription initiation from promoter B occurs from the sites proposed by Keaveney et al. (10, 11) and Grandien et al. (14, 15), but not from the sites proposed by Piva et al. (12, 13). Promoter A products of 108 bp (primers ER4 and ER5) and 611 bp (primers ER4 and ER6) were detected.

The pattern of estrogen receptor-{alpha} promoter usage in the MCF-7 cell line was mirrored in the T47-D, EFM-19, and EFF-3 estrogen receptor-{alpha}-positive breast cancer cell lines. Comparison of the amounts of PCR products produced by the various sets of primers suggested that promoter usage was similar in the MCF-7, EFM-19, and T47D cells. Promoter usage in the ZR-75 estrogen receptor-{alpha}-positive breast cancer cell line differed somewhat from that of the other ER{alpha}-positive cell lines in that no transcripts originating from promoter B were detected in one of two ZR-75 sublines tested [the negative subline (a) is shown in Fig. 3BGo whereas data for both (a) and (b) sublines are summarized in Table 1Go]. The amount of PCR product derived from mRNA transcribed from promoter A in the ZR-75 cell line was similar to the MCF-7 cell line, while less was generated from promoter C transcripts. In the BT-20 cell line, which has been classed as an estrogen receptor-{alpha}-negative cell line, but which expresses low levels of an estrogen receptor-{alpha} splice variant (17, 33), low levels of PCR product derived from transcripts from promoter A only were detected.


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Table 1. Expression of Estrogen Receptor-{alpha} in Human Cell Lines and Tissues

 
In contrast to breast cancer cells, greater differences in promoter usage were observed in other cell types. Promoter C transcripts only were detected in the normal liver and one of the two samples of normal stomach (sample B), while in the other sample of normal stomach (sample A), both promoter B and C transcripts were detected. The Kato III cell line contained transcripts from promoter B only, and the CaCo2 cell line contained transcripts from promoters B and C only. Transcripts from promoters A and B only were detected in the Ishikawa cell line. Longer exposure times were required to photograph the agarose gels for the tissues and KATO III and CaCo2 cell lines (Fig. 3Go) compared with the breast cancer cell lines. The table in Fig. 3CGo shows the relative abundance of the PCR products taking into account the differences in exposure times.

Location of Estrogen Receptor-{alpha} Gene Promoter Activity
Transient transfection of reporter constructs was then used to directly demonstrate estrogen receptor-{alpha} promoter activity, to localize the regions that contain this activity, and to study directly differences in promoter activity in the ZR-75 (subline A) and the EFM-19 breast cancer cell lines which showed differences in the usage of promoters A and B by RT-PCR.

Fragments of the estrogen receptor-{alpha} promoter region containing promoters A and B were subcloned from {lambda}9a and 13 into the promoterless luciferase reporter vector pGL3B (Fig. 4AGo). X4.48 contains promoters A and B. The 4/4 fragment was subcloned from X4.48 and contains basic promoter elements (CAAT and TATA sequences), but no putative regulatory elements. Four subclones contained promoter B sequences. The BH1.4 fragment encompasses several INR elements and all of the proposed transcription initiation sites (10, 11, 14) apart from the 5'-site proposed by Piva et al. (13). The GV fragment encompasses the other transcription initiation site proposed by Piva et al. (12). The XB0.5 and B1.3 fragments span the 5'-transcription initiation site proposed by Piva et al. (13).



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Figure 4. Location of Estrogen Receptor-{alpha} Gene Activity

A, The estrogen receptor genomic DNA is represented from -5 kb to +2 kb, and the position of the recognition sites for the restriction enzymes EcoRI (E), BamHI (B), HindIII (H), XbaI (X), BglII (G), EcoRV (V), and SacII (S) are indicated, as are the positions of promoters A and B. The positions of the subcloned fragments are also shown. The promoterless pGL3B plasmid and the recombinants containing promoter fragments were cotransfected with the pJ7lacZ plasmid into ZR-75 (subclone a) cells (B) and EFM-19 cells (C) growing in normal culture medium. The cells were not cotransfected with the HEGO estrogen receptor expression construct. The resulting luciferase and ß-galactosidase activities were then measured. Luciferase results were corrected for transfection efficiency with the ß-galactosidase assay. The activities are shown as an increase over the background from the parent pGL3B promoterless plasmid. The bars show the mean values of two experiments. For both experiments, constructs were transfected in triplicate.

 
These promoter fragments were transiently transfected into the ZR-75 and EFM-19 breast cancer cell lines. In the ZR-75 cell line, The X4.48 fragment, which contains both promoters A and B (Fig. 4BGo), gave promoter activity of 16.8 ± 1.9 times background in ZR-75 cells and 3 times background in EFM-19 cells. Lower but significant promoter activity (2.4 ± 0.4 times that of background for ZR-75 cells and 1.8 times that of background for EFM-19 cells) was obtained with the 4/4 fragment that contains the CAAT and TATA sequences.

None of the four promoter B fragments produced promoter activity when transfected in ZR-75 cells. The BH1.4 fragment, but not the other three fragments, showed transcriptional activity in EFM-19 cells. The BH1.4 fragment therefore mirrors the activity of the promoters as measured by RT-PCR in that it shows promoter activity in EFM-19 but not ZR-75 cells.

Regulation of the Estrogen Receptor-{alpha} Promoters by Estrogen Using PCR
Estrogen receptor-{alpha} mRNA levels are regulated by estrogen in breast cancer cell lines; estrogen increases estrogen receptor-{alpha} mRNA levels in ZR-75 (18), EFM-19 (20), and T47-D cells (21) and decreases mRNA levels in MCF-7 cells (19). We have suggested previously that the differential regulation of estrogen receptor-{alpha} mRNA levels by estrogen may be due to the use of different promoters that are regulated in different ways by estrogen (18). The effect of estrogen on individual estrogen receptor-{alpha} mRNA promoters has not been studied, and we have therefore investigated the effect of estrogen on transcription from each promoter using quantitative RT-PCR.

In preliminary experiments, the products of a reverse transcription reaction of MCF-7 RNA were diluted 2-, 5-, 10-, and 30-fold, and the resulting cDNA was amplified using varying numbers of cycles and the primer pairs ER1/ER5 (promoter C), ER3/ER5 (promoter B), and ER4/ER6 (promoter A). Figure 5Go shows a representative experiment for promoter C. The amount of product was quantified by scanning densitometry and is plotted in Fig. 5BGo. The rate at which PCR product was generated was proportional to the amount of cDNA put into the PCR reaction. This assay could detect a 2-fold difference in RNA abundance and was therefore used to measure the effect of estradiol on the abundance of RNA transcribed from each promoter in the estrogen receptor-positive breast cancer cell lines. The effects of estrogen on the three promoters are illustrated and quantified for MCF-7 and EFM-19 cells in Fig. 6Go, and the results for four estrogen receptor-positive cell lines are summarized in the table. Estrogen affected the abundance of RNA transcribed from all three promoters in all the cell lines but to varying degrees. In MCF-7 cells, the amount of RNA transcribed from all three promoters was decreased, whereas it was increased in the other cell lines. In MCF-7 cells, the decrease was most marked for promoter B (6-fold) and least marked for promoter C (4- fold). In the other estrogen receptor-positive cell lines, the increase in the amounts of estrogen receptor RNAs was most marked in the EFM-19 cell line and least marked in the T47D cell line.



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Figure 5. Quantitation of Estrogen Receptor-{alpha} Promoter C Transcripts

Total MCF-7 RNA was reverse transcribed, and dilutions of the reverse transcription reaction were put into a series of PCR reactions containing primers ER3 and ER5 and amplified for varying numbers of cycles. The PCR products were electrophoresed, and the amount of PCR product was measured by densitometric analysis of the image of the ethidium bromide-stained gel. Panel A shows the ethidium bromide-stained gel, and panel B shows the amount of PCR product for each dilution plotted as a function of the number of PCR cycles.

 


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Figure 6. Effect of Estrogen on the Levels of Estrogen Receptor mRNA Transcribed from Estrogen Receptor-{alpha} Promoters A, B, and C

A, RNA was prepared from the MCF-7, T47-D, ZR-75, and EFM-19 cell lines grown in withdrawal medium in the absence (Control, •) or presence (Estrogen, {circ}) of 10-9 M 17ß-estradiol. cDNA was prepared and used in PCR reactions with the primers for the products PrC (promoter C), PrB.2 (promoter B), and PrA.2 (promoter A) for 25, 30, 35, 40, and 45 cycles. PCR products were separated by gel electrophoresis on a 3% agarose gel and stained with ethidium bromide. The change in the amount of RNA transcribed from the three promoters was estimated as described in Materials and Methods after densitometric analysis of the image of the ethidium bromide-stained gel. The results of the quantitation of all four estrogen receptor-positive cell lines is shown in the table. The results shown are the mean values from two experiments, and these did not vary by more than 20%.

 
These experiments showed, therefore, that in each cell line, all three estrogen receptor-{alpha} promoters were regulated by estrogen. Interestingly, all promoters used were either up-regulated (T47D, EFM-19, ZR-75) or down-regulated (MCF-7), demonstrating that the nature of the regulation varies in a cell-specific manner, but not in a promoter-specific manner.

Regulation of the Estrogen Receptor-{alpha} Promoters by Estrogen Using Reporter Gene Assays
The estrogen responsiveness of promoter A and B was then investigated using transient transfection either by cotransfecting with the HEGO ER{alpha} expression vector (Fig. 7BGo) or by treating transfected cells with estradiol (Fig. 7CGo). EFM 19 cells were used for both types of experiment. Reporter constructs were cotransfected with pJlacZ and with or without the estrogen receptor-{alpha} expression plasmid HEGO (35) into cells growing in maintenance medium. Two control constructs, pGL3PpS2 and pGL3PVit, were also transfected. These consist of the estrogen response element (ERE) sequences from the human pS2 gene and the Xenopus laevis Vitellogenin A2 gene cloned upstream of the SV40 promoter in the pGL3P vector. These ERE sequences confer estrogen responsiveness on a heterologous promoter, and the estrogen response from these control constructs was compared with that of the estrogen receptor-{alpha} promoter constructs.



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Figure 7. Investigation of the Effect of HEGO on the Estrogen Receptor Promoters Using Reporter Gene Assays

A, The estrogen receptor genomic DNA is represented from -5 kb to +2 kb, and the position of the recognition sites for the restriction enzymes EcoRI (E), BamHI (B), HindIII (H), XbaI (X), BglII (G), EcoRV (V), and SacII (S) are indicated, as are the positions of promoters A and B. The positions of the subcloned fragments are also shown. B, The pGL3B promoterless plasmid and recombinants containing promoter fragments were cotransfected with the pJ7lacZ plasmid into EFM-19 cells grown in maintenance culture medium with (solid bars) or without (hatched bars) cotransfection of the estrogen receptor-{alpha} expression plasmid HEGO. C, The pGL3B plasmid and the estrogen receptor luciferase subclones were cotransfected with the pJ7lacZ plasmid into EFM-19 cells that had been withdrawn from the effects of estradiol for 2 days and then either treated (solid bars) or not (open bars) with 10-9 M estradiol. Cell lysates were assayed for luciferase and ß-galactosidase activity, and the luciferase results were corrected for transfection efficiency with the ß-galactosidase plasmid. The luciferase activities are shown as a relative increase over the background, pGL3B plasmid in the absence of HEGO (b), or absence of estradiol (c). The bars show the mean values of two experiments. For both experiments, constructs were transfected into triplicate wells.

 
HEGO increased the activity of the the X4.48 fragment 35-fold, which was comparable to the increases of the pGL3PpS2 and pGL3PVit plasmids (31.9 and 37.3 times, respectively). To determine whether this large effect of HEGO was mediated through promoter A and/or B, expression constructs containing smaller fragments were used. The activity of the 4/4 promoter A fragment was not increased by HEGO to a greater extent than the control plasmid. In contrast, HEGO markedly increased the activity of some of the promoter B fragments. The activities of BH1.4 7/8 and 7/4 were all increased by HEGO to a similar extent. The 7/4 fragment is the smallest and suggests that the effect of HEGO is localized to a region between -2183 and -1905 that contains the transcription initiation sites identified by Keaveney et al. (10, 11) and Grandien (14). As for the experiment shown in Fig. 4Go, the fragments from further upstream (B1.3, XB0.5) and the small GV fragment showed no activity when compared with the control plasmid, and this was not increased by cotransfection with HEGO.

The effect of estrogen on the transient expression of ERE expression constructs was also examined (Fig. 7CGo) in the absence of HEGO. EFM-19 cells were cultured for 2 days in estrogen-free medium and transfected with an estrogen receptor construct or the control constructs (pGL3PpS2 or pGL3PVit), and the luciferase activity was measured after 2 days treatment with estradiol. Estrogen increased expression from the control plasmids pGL3PpS2 and pGL3PVit 5- and 8-fold, respectively. Estrogen dramatically increased expression from the X4.48 fragment that contains promoters B and A. Estrogen modestly increased expression from the 4/4 promoter A but had a much greater effect on the BH1.4, 7/8, and 7/4 promoter B fragments. Estrogen did not increase the expression of the B1.3, XB0.5, or GV to a greater extent than the pGL3B fragment. The direct effect of estrogen therefore paralled the cotransfection experiments using HEGO.

Use of Estrogen Receptor-{alpha} Promoters in Primary Breast Tumors
The above studies were all performed on breast cancer cell lines. To assess whether the findings in breast cancer cell lines are relevant to primary breast tumors, RNA was extracted from a series of 10 estrogen receptor-positive breast tumors, and the presence of transcripts from the three promoters was assessed by RT-PCR. Interestingly, all 10 tumors showed a similar pattern with minor variations in the amount of PCR product generated. All three promoters were used in all tumors, although as for the cell lines, no Pr.B1 was detected. Figure 8Go shows representative results for two tumors. The amount of PCR product generated suggested that the transcripts were more abundant in the tumors than in the estrogen-responsive breast cancer cell lines. These experiments therefore show that use of all three promoters is a characteristic of breast cancer cells.



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Figure 8. Analysis of Estrogen Receptor-{alpha} Promoter Usage in Primary Breast Tumors

Total RNA was extracted from primary breast tumors, and estrogen receptor-{alpha} mRNAs transcribed from promoters C, B, and A were amplified using the primer pairs for the universal, PrC, PrB.1, PrB.2, PrA.1, and PrA.2 PCR products shown in Fig. 3Go. Ethidium bromide-stained agarose gels of the PCR products for two tumors are shown.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The aim of this study was to investigate the importance of estrogen receptor-{alpha} promoters and their reglation by estrogen in the control of the expression of the human estrogen receptor-{alpha} gene in breast cancer and other cell types. This receptor is expressed in a limited repertoire of normal tissues including breast (36), uterus (3), ovary (37), liver (38), stomach (39), brain (40), and bone (15). For malignant tissues, high levels of expression are characteristic of breast tumors (41), but estrogen receptor can also be expressed in endometrial carcinoma (42) and a variety of sarcomas (43). There is considerable fundamental and clinical interest in understanding the factors controlling estrogen receptor expression, at least in part, because treatment with agents that act directly or indirectly through the estrogen receptor has become a popular and effective therapeutic option for patients with tumors that express the estrogen receptor.

Three human estrogen receptor promoters (A, B, and C) have now been identified, and the control of estrogen receptor expression could be exerted through one, a combination, or all of these promoters. While several studies have mapped promoters A and B, promoter C has been identified only recently by RACE-PCR, and it was concluded that it must lie at least 10 kb upstream of the other two promoters. We have now cloned 21 kb of estrogen receptor-{alpha} upstream of DNA and conclude that promoter C does not lie within this region and must be a considerable distance from the protein-coding region of the gene. This is comparable to the mouse glucocorticoid receptor, which has at least three promoters, one of which is located more than 30 kb upstream of the other two (44).

We first used RT-PCR to determine estrogen receptor-{alpha} promoter usage in a selection of cell lines and tissues. These experiments complement and extend the findings of others (14, 15, 16, 17), and the results are summarized in Table 1Go. Promoter A transcripts were detected in all breast cancer cell lines with limited variation in the amount of PCR product produced. This is similar to the findings of Grandien et al. (15, 16) and Weigel et al. (17), although we have examined a larger number of cell lines. Promoter B usage was more variable. No products were detected from the most 5'-start site (-3090) within promoter B, and this in contrast to the study of Piva et al. (13), who originally reported transcription initiation at -3090 in MCF-7 cells. Transcripts originating from more 3'-start sites in promoter B were readily detected in the majority of breast cancer cell lines with the exception of the ZR-75 (a) subline and BT20. Three other studies (15– 17) have failed to detect promoter B transcripts in ZR-75 cells, and Weigel et al. (17) did not detect promoter B transcripts in BT20 cells. Promoter C transcripts were detected in all estrogen receptor-positive breast cancer cell lines, and this is the first demonstration of its use in these cells. Our data, however, are in contrast to the study of Grandien (14), who reported promoter C usage in liver but not MCF-7 cells. A study published while this manuscript was in revision (45) reported that there are five transcription initiation start sites for the human estrogen receptor. Quantitation using S1 nuclease mapping suggested that the RNA transcribed from promoter A was most abundant in breast cancer cell lines, whereas RNA transcribed from promoter C was most abundant in liver.

The surprising conclusion of our experiments was that, with the exception of the ZR-75(a) cell line, all the well characterized estrogen-responsive breast cancer cell lines use all three promoters. This largely excludes the possibility that a single promoter controls the expression of estrogen receptor mRNA levels in breast cancer cells and is uniquely responsible for the high level of expression. Rather, it suggests that the high levels of expression result from an additive effect of the contributions from the three promoters. This conclusion is substantiated by the findings that all three promoters are used in estrogen receptor-positive primary breast tumors. This is the first report of promoter usage in a series of primary tumors. Although it was not possible to examine the effects of estrogen on the expression of the various RNAs in vivo, it would be interesting to analyze the effects of antiestrogens on the various promoters, particularly in the light of reports that antiestrogens can affect estrogen receptor expression in vivo.

Estrogen receptor expression is thought to be low in normal breast epithelial cells and to increase during the progression to malignancy. Although the reasons for the differences in expression are not known, the lower levels of expression in normal breast epithelial cells could result from the use of fewer promoters or a lower level of activity of all promoters. The use of promoters A and B, but not C, has been assessed in normal breast epithelial cells. Weigel et al. (17) showed that promoter B was not used in a sample of normal breast epithelial cells, whereas Grandien et al. (15) found that transcripts from promoter A and B were present in approximately equal abundance. Changes in promoter usage during malignant transformation, therefore, while remaining an attractive concept, remains an open question.

Estrogen receptor expression is lower in normal tissues and in gastric and colonic cancer cells than is generally found in estrogen-responsive breast cancer cells. Our data suggest that non-breast cancer cells use fewer promoters than the estrogen-responsive breast cancer cell lines and that the promoter(s) used vary between tissues. For example, only promoter B was used in the gastric (Kato III) cell line, only promoters B and C were used in the CaCo2 colon cancer cell lines and, in agreement with Grandien et al. (15) and Flouriot et al. (45), we found that promoter C is used exclusively in the liver. Grandien et al. (15) also reported that primary osteoblasts use promoter B exclusively. It seems probable, therefore, that tissue-specific promoter usage could allow tissue-specific control of estrogen receptor expression in some tumor types and in normal tissues.

The factors that control the expression of the estrogen receptor gene are poorly defined. Comparison of DNA from estrogen receptor-positive and -negative tumors have identified differences in methylation in a CpG island located toward the 3'-end of exon 1, and treatment of estrogen receptor-negative cells with the demethylating agent 5-aza-2'-deoxycytidine results in the production of receptor protein (46). Methylation at the 5'-end of the gene does, therefore, appear to control receptor expression, but the extent to which it could influence the activity of specific promoters is unknown. Others have identified specific elements in the estrogen receptor promoter that are important in receptor expression. ERF-1 is expressed preferentially in estrogen receptor-positive cells and binds to a DNA sequence in the region from +135 to +210 in the 5'-leader sequence (47, 41). ERF-1 is thought to be important for expression from promoter A, but its importance in controlling expression from promoters B and C has not been addressed. Tang et al. (22) have identified a functionally important enhancer (ER-EH0) that includes an AP-1 site at position -3778 to -3744. This region, which is upstream of promoter B, is active in estrogen receptor-positive but not negative cells, and mutational analysis has suggested that it, rather than the ERF-1 site, is responsible for the high level of estrogen receptor expression in breast cancer cells (42, 47). However, as for ERF-1, the spectrum of estrogen receptor-{alpha} promoter(s) regulated by ER-EH0 is not known (22).

DNA fragments from around the transcription start sites were cloned into a promoterless luciferase plasmid to assess their promoter activity after transfection into recipient cells. The small fragment containing the TATA and CAAT elements and encompassing the transcriptional start site of promoter A (-142 to +112) conferred transcriptional activity. This fragment did not contain the ERF-1 recognition site, indicating that this sequence is not absolutely required for promoter activity. Interestingly, transfection with a fragment that included the ERF-1 recognition sequence gave only marginally greater activity (data not shown), which tends to support the view of others (22) that this element may not be of major functional significance.

Fragments encompassing promoter B showed little transcriptional activity when transfected into ZR-75(a) cells, which is consistent with the PCR data showing that no promoter B transcripts could be detected in this ZR-75 subline. This suggests that either there is a lack of transcriptional activators or that transcriptional inhibitors specific for this promoter are present in these ZR-75 cells. When transfected into EFM-19 cells, the BH1.4 fragment was transcriptionally active. This fragment encompasses all the transcriptional start sites identified from this promoter apart from the most 5' site (46). Interestingly, this fragment does not extend sufficiently 5' to include the ER-EH0 enhancer sequence, and this suggests that there are sufficient elements to allow transcription in the absence of this enhancer. The B1.3 fragment was the only fragment to encompass this enhancer, but the low activity may have resulted from the fact that it contained only the transcriptional start site described by Piva et al. (13), and our PCR experiments had shown that we were unable to detect RNA initiated from this site.

Estrogens have been reported to up-regulate and down-regulate estrogen receptor-{alpha} expression under different circumstances. For breast cancer cell lines, the majority of studies have concluded that estrogen down-regulates estrogen receptor expression in the MCF-7 cell line (18) but up-regulates it in other cell lines such as T47D (18, 21) ZR-75 (18), and EFM-19 (18, 20). Regulation of estrogen receptor-{alpha} mRNA levels in vivo may be important in the response of tumors to antiestrogens, and if the responses in tumors reflected the diversity of responses observed in tumor cell lines, this could have major implications for the understanding of the control of estrogen responsiveness by antiestrogens.

Little is known about the mechanisms involved in the regulation of estrogen receptor-{alpha} expression by estrogen. In the livers of the trout (48) and X. laevis (38) in which receptor levels are increased by estrogen, functional estrogen response elements have been identified in the protein-coding region of the estrogen receptor-{alpha} gene (48, 49). Only one study has attempted to identify the sequences responsible for controlling estrogen receptor expression by estrogens in human cells. Treilleux et al. (42), in a study that focused entirely on the regulation of promoter A, concluded that this promoter is regulated by estrogens on the basis of transfection experiments in which reporter constructs were transfected into MCF-7 cells. Surprisingly, Treilleux et al. (42) showed that the expression of promoter A reporter constructs were increased by estrogen despite the fact that most studies concur that estrogen receptor is down-regulated by estrogen in MCF-7 cells. In addition, mutational analysis suggested that the regulation involved the interaction with three ERE half-sites lying between -420 and -892. Our experiments have shown that regulation by estrogen is not promoter specific, as transcripts from the three promoters are all increased in ZR-75, T47D, and EFM-19 cells and all decreased in MCF-7 cells. The half-EREs that are scattered throughout the estrogen receptor promoter could be responsible for regulating all three promoters in concert. However, the observation that all promoters are down-regulated in MCF-7 cells but up-regulated in the three other cell lines suggests that there are additional cell-specific factors that determine the way in which the three human estrogen receptor-{alpha} promoters are regulated by estrogen.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation of Estrogen Receptor-{alpha} Promoter DNA
DNA from MCF-7 cells was partially digested with MboI. Fragments of 15–23 kb were isolated by sucrose density gradient centrifugation and ligated to the lambda-DASH vector, restricted with BamHI. The library was screened with two 32P-radiolabeled 22-bp oligonucleotide probes corresponding to position -2181 to -2142 and position -1897 to -1858 in the estrogen receptor-{alpha} promoter region. Positive clones were characterized by restriction with BamHI, XbaI, EcoRI, HindIII, XhoI, and SalI and subsequently hybridized with three 32P-radiolabeled probes; an estrogen receptor cDNA probe corresponding to clone OR3 from Green et al. (9), an estrogen receptor promoter A probe corresponding to position -142 to +112, and an estrogen receptor promoter B probe corresponding to position -2181 to -1858.

Cell Culture
MCF-7 (50), ZR-75B (51), EFM-19 (52), T47-D (34), BT-20 (53), MDA MB231 (54), Ishiykawa (55), Kato III (56), and CaCo 2 cell lines were maintained in DMEM supplemented with 10% FCS and 1 µg/ml insulin. To remove the effects of estrogen, cells were cultured in withdrawal medium consisting of phenol red-free MEM supplemented with 10% newborn calf serum that had been treated with dextran-coated charcoal, 20 mM HEPES, 0.075% sodium bicarbonate, and 1 µg/ml insulin.

Transfection
Cells (2 x 105 cells/well) were plated in 24-well plates in normal medium and incubated for 24 h. For experiments in which the effects of estradiol were examined, cells were plated in normal growth medium, allowed to attach overnight, and washed with PBS (1 ml), and the medium was replaced with phenol red-free MEM supplemented with 10% newborn calf serum that had been treated with dextran-coated charcoal, 20 mM HEPES, 0.075% sodium bicarbonate, and 1 µg/ml insulin. Cells were washed again with PBS and the medium was replaced 6 h later. The following day the medium was changed 1 h before transfection. An adapted transfection method was used (55). For each well, 0.9 µg of the parent promoterless expression vector pGL3B (Promega Corp., Madison, WI) or a recombinant containing an estrogen receptor promoter fragment was mixed with 50 ng of the ß-galactosidase coding plasmid pJ7lacZ and made up to 45 µl. For some experiments, 62.5 ng HEGO (35) or the control CAT4 reporter vector DNA were included. The HEGO expression vector encodes the normal human estrogen receptor under the control of an SV40 promoter and produces a ligand-dependent estrogen receptor. The DNA was precipitated by the addition of 50 µl 2 x BBS (50 mM N,N-bis[2-hydroxyethyl]-2-aminoethane sulfonic acid, pH 6.95, 280 mM NaCl, and 1.5 mM Na2HPO4) and 5 µl 2.5 M CaCl2, incubated at room temperature for 15 min before being added to the well. The plates were incubated for 18 h and washed twice with 1 ml medium to remove the remaining precipitate, and the cells were then incubated in 1 ml normal medium, 1 ml withdrawal medium, or 1 ml withdrawal medium containing 10-9 M 17ß-estradiol. Medium was then changed every day for 2 days. Cells were washed twice with 1 ml PBS and then lysed by incubation in 120 µl lysis buffer (Triton-100 0.2% wt/vol, 60 mM KH2PO4, pH 7.8) for 15 min. The lysates were centrifuged at 12,000 rpm for 5 min.

Luciferase Assays
Luciferase assays were performed using the Promega Corp. luciferase extended glow kinetic system following the manufacturers instructions. Chemiluminescence was measured over 100 sec in a Berthold 9501 luminometer.

ß-Galactosidase Assays
Cell extracts were incubated at 48 C for 50 min to inactivate endogenous ß-galactosidase activity and then centrifuged at 12,000 rpm for 5 min. Supernatant (10 µl) was mixed with 40 µl reaction buffer (Galacto-Light kit, Tropix, Inc., Bedford, MA) and incubated at room temperature for 1 h. Accelerator solution (60 µl) was added, and light emission was measured for 5 sec in a Berthold 9501 luminometer.

Oligonucleotides
Oligonucleotides were synthesized on a 381A DNA synthesizer (Applied Biosystems, Foster City, CA) and purified using oligonucleotide purification cartridges. The sequence of the oligonucleotides and their positions in the estrogen receptor-{alpha} promoter region are as follows. ER1, position 146 to 168 of estrogen receptor-{alpha} promoter C cDNA (14) (5'-GCACAGCACTTCTTGAAAAAGG-3'); ER2, position -2181 to -2142 of genomic DNA (5'-TACAGCTTTCTCTGGCTGTGCCACACTGCT CCCTGTGAGC-3'); ER3, position -1958 to -1937 of genomic DNA (5'-CACATGCGAGCA CATTCCTTCC-3'); ER4, position +140 to +161 of ER{alpha} promoter A cDNA (5'-CCTCGGGC TGTGCTCTTTTTCC-3'); ER5, position +228 to +247 of ER{alpha} promoter A cDNA (5'-AGGG TCATGGTCATGGTCCG-3'); ER6, position +749 to +727 of ER{alpha} promoter A cDNA (5'-TT CCCTTGTCATTGGTACTGGC-3'); ER7, position +882 to +904 of ER{alpha} promoter A cDNA (5'-ACGACTATATGTCCAGCC-3'); and ER8, position +1104 to +1081 of ER{alpha} promoter A cDNA (5'-AGGTTGGCAGCTCTCATGTCTCC-3'). In addition, 18 S ribosomal RNA (rRNA) was amplified in some control experiments. The primers were 5'-CAATAACAGGTCTGTGATGCCC-3' and 5'-AACCATCCAATCGGTAGTAGCG-3', which amplify a 247-bp fragment between nucleotide 1482 and 1729 in human 18S rRNA.

RT-PCR
RNA was prepared from cultured cells and tissue samples as described by Auffray and Rougeon (58). Before reverse transcription, an aliquot of the RNA was reprecipitated twice with 150 mM KCl and 75% ethanol. The precipitated RNA was redissolved in water, and the concentration was adjusted to 1 mg/ml. RNA (0.5 µg) was mixed with 0.5 µg of random primers in 5.9 µl, incubated at 70 C for 10 min, cooled on ice, and then incubated in 1 x buffer (10 mM Tris-HCl, pH 8.8, 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100), 10 mM DTT, 0.5 mM dNTPs, 50 µg/ml random primers, and 100 ng RNAguard (Promega Corp.) in a total volume of 10 µl for 3 min at 37 C. Moloney murine leukemia virus reverse transcriptase (9 U, Pharmacia Biotech, Piscataway, NJ) was added and the incubation continued for 1 h. Part of the reverse transcription reaction (0.1 µl) was made up to 1 x PCR buffer (composition as recommended by the supplier of the thermostable DNA polymerase), 250 µM of each dNTP, 100 µg/ml BSA, and 0.31 U of Taq DNA polymerase (DynaZyme II, Finnzymes), Red Hot Polymerase (Advanced Biotechnologies Ltd, Epsom, Surrey, UK) or Vent Polymerase (New England Biolabs, Inc., Beverly, MA) was added, and the mixture was then added to 40 ng of each primer. A drop of mineral oil was added, and the tube was placed in the thermal cycler preset at the denaturing temperature.

Quantitative PCR
The effects of estrogen on specific estrogen receptor-{alpha} promoters were estimated from the rate of accumulation of PCR products. Standard curves for each promoter were generated in each experiment from dilutions of the reverse transcription reaction, which contained cDNA containing the highest levels of estrogen receptor RNA, i.e. RNA extracted from control MCF-7 cells and estrogen-treated T47D, ZR-75, and EFM-19 cells. Each dilution was amplified for different numbers of cycles, and the number of cycles required to give 50% maximal amplification was plotted against the dilution of the RT-PCR. The number of cycles required to give 50% maximal induction of the test samples was then used to assess the dilution to which it was equivalent. For example, if the number of cycles required to generate 50% of the maximum amount of PCR product in the test sample was the same as the 6-fold dilution of the sample containing the highest level of RNA, it was concluded that there was a 6-fold difference in RNA abundance between the two samples. No differences in the accumulation of rRNA PCR products from RNA extracted from control and estrogen-treated cells were observed. This assay could detect less than a 2-fold difference in mRNA concentrations.

Subcloning
One microgram of {lambda}DNA was digested with 4 U of enzyme in the appropriate buffer with 100 µg/ml BSA in a total volume of 20 µl and incubated for 3 h at 37 C. Digests were separated on 0.8% agarose gels, and fragments of DNA were isolated using the Qiaex II Gel Extraction Kit (Qiagen, Chatsworth, CA). Fragments were ligated to the appropriately digested and dephosphorylated pGL3B vector (Promega Corp.) at a molar ratio of 3:1 (insert to vector) with 0.3 U T4 DNA ligase in the appropriate buffer in a total volume of 10 µl.

Sequencing
Automated fluorescence sequencing was carried out by the University Central Facility using PE Applied Biosystems (Norwalk, CT) 373A and 377 DNA sequencers and an ABI 877 Molecular Biology Workstation (ABI Advanced Biotechnologies, Inc., Columbia, MD).


    ACKNOWLEDGMENTS
 
We thank Professor P. Chambon (Strasbourg, France) for the HEGO estrogen receptor expression vector.


    FOOTNOTES
 
Address requests for reprints to: B. R. Westley, University Department of Pathology, Royal Victoria Infirmary, Newcastle upon Tyne, NE1 4LP United Kingdom. e-mail: B.R.Westley@ncl.ac.uk

C. Donaghue was the recipient of a studentship from the Medical Research Council, United Kingdom.

Received for publication July 7, 1998. Revision received June 18, 1999. Accepted for publication July 19, 1999.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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