University Department of Pathology Royal Victoria Infirmary Newcastle upon Tyne, NE1 4LP United Kingdom
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ABSTRACT |
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INTRODUCTION |
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Two forms of the human estrogen receptor gene have been identified,
estrogen receptor- (5), which was the first to be identified, and
estrogen receptor-ß (6). Estrogen receptor-
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-
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-
mRNAs, and RT-PCR experiments demonstrated that this
region was contained within human estrogen receptor-
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-
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-
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- 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-
promoter in one cell type and the use of a different, weaker estrogen
receptor-
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- mRNA are regulated by estrogen in
breast cancer cell lines; estrogen has been shown to decrease estrogen
receptor-
mRNA levels in MCF-7 cells (18, 19) and increase estrogen
receptor-
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- 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-
promoters than other cell types and that all three promoters are
regulated in a coordinate way by estrogen.
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RESULTS |
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Sequence Analysis of the 5'-End of the Estrogen Receptor-
Gene
To analyze the estrogen receptor- promoter region in detail,
DNA between -3280 and -5330, which had not been sequenced previously,
was subcloned from the
-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. 2A, 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. 2A
). 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. 2A
). 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-
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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Use of the Three Estrogen Receptor- Promoters in Different Cell
Types
Estrogen receptor- expression was first asessed by RT-PCR,
using the universal primers ER7 and ER8 that amplify mRNA transcribed
from all promoters (Fig. 3A
) (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-
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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Agarose gels of the PCR products are shown in Fig. 3B, and a summary of
the relative amounts of PCR product generated for each promoter from
all the cell lines analyzed is given in Fig. 3C
. 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-
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- promoter usage in the MCF-7
cell line was mirrored in the T47-D, EFM-19, and EFF-3 estrogen
receptor-
-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-
-positive
breast cancer cell line differed somewhat from that of the other
ER
-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. 3B
whereas data for both (a) and
(b) sublines are summarized in Table 1
].
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-
-negative cell line,
but which expresses low levels of an estrogen receptor-
splice
variant (17, 33), low levels of PCR product derived from transcripts
from promoter A only were detected.
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Location of Estrogen Receptor- Gene Promoter Activity
Transient transfection of reporter constructs was then used to
directly demonstrate estrogen receptor- 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- promoter region containing
promoters A and B were subcloned from
9a and 13 into the
promoterless luciferase reporter vector pGL3B (Fig. 4A
). 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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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- Promoters by Estrogen Using
PCR
Estrogen receptor- mRNA levels are regulated by estrogen in
breast cancer cell lines; estrogen increases estrogen receptor-
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-
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-
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 5 shows a
representative experiment for promoter C. The amount of product was
quantified by scanning densitometry and is plotted in Fig. 5B
. 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. 6
, 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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Regulation of the Estrogen Receptor- 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 expression vector (Fig. 7B
) or by treating
transfected cells with estradiol (Fig. 7C
). EFM 19 cells were used for
both types of experiment. Reporter constructs were cotransfected with
pJlacZ and with or without the estrogen receptor-
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-
promoter constructs.
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The effect of estrogen on the transient expression of ERE expression
constructs was also examined (Fig. 7C) 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- 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 8 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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DISCUSSION |
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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- 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- 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 1
. 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- 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- 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-
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- 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-
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-
promoters are regulated by
estrogen.
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MATERIALS AND METHODS |
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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-
promoter region are as follows. ER1, position 146 to 168 of estrogen
receptor-
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
promoter A cDNA (5'-CCTCGGGC TGTGCTCTTTTTCC-3');
ER5, position +228 to +247 of ER
promoter A cDNA (5'-AGGG
TCATGGTCATGGTCCG-3'); ER6, position +749 to +727 of ER
promoter A
cDNA (5'-TT CCCTTGTCATTGGTACTGGC-3'); ER7, position +882 to +904 of
ER
promoter A cDNA (5'-ACGACTATATGTCCAGCC-3'); and ER8, position
+1104 to +1081 of ER
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-
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 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).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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