From Axys Pharmaceuticals, Inc., South San Francisco, CA 94080 and the g Department of Biology, University of California at San Diego, La Jolla, California 92093
Received for publication, December 4, 2000, and in revised form, February 23, 2001
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ABSTRACT |
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Exon trapping and
cDNA selection procedures were used to search for novel genes at
human chromosome 11p13, a region previously associated with loss of
heterozygosity in epithelial carcinomas. Using these approaches,
we found the ESE-2 and ESE-3 genes, coding for
ETS domain-containing transcription factors. These genes lie in close
proximity to the catalase gene within a ~200-kilobase genomic
interval. ESE-3 mRNA is widely expressed in human
tissues with high epithelial content, and immunohistochemical analysis with a newly generated monoclonal antibody revealed that ESE-3 is a
nuclear protein expressed exclusively in differentiated epithelial cells and that it is absent in the epithelial carcinomas tested. In
transient transfections, ESE-3 behaves as a repressor of the Ras- or
phorbol ester-induced transcriptional activation of a subset of
promoters that contain ETS and AP-1 binding sites. ESE-3-mediated repression is sequence- and context-dependent and depends both on the presence of high affinity ESE-3 binding sites in combination with AP-1 cis-elements and the arrangement of these sites
within a given promoter. We propose that ESE-3 might be an important determinant in the control of epithelial differentiation, as a modulator of the nuclear response to mitogen-activated protein kinase signaling cascades.
Intracellular signaling through mitogen-activated protein
(MAP)1 kinases is a universal
mechanism controlling multiple aspects of cell division, migration, and
differentiation (1). Among the known nuclear effectors downstream of
MAP kinase signaling cascades are members of the ETS family of
transcription factors, a group of proteins that share a conserved DNA
binding domain, known as the ETS domain, and that are only found in
metazoans. ETS transcription factors have been implicated as positive
and negative regulators in signal transduction pathways that control a
number of cellular processes, including differentiation, proliferation, cellular migration or invasion, and inflammation, and some of these
factors are indeed direct targets of MAP kinases (2-5).
The biological role of ETS proteins in the integration of signals from
MAP kinases has been best characterized in Drosophila melanogaster. Genetic and biochemical studies have led to the identification of two ETS proteins, Pointed and Anterior Open (Aop)/Yan, as pivotal components in the nuclear integration of MAPK
signaling cascades (6-10). The activity of Pointed P2, a product of
the pointed gene (11), and a putative ortholog of the
vertebrate ETS-1 and ETS-2 proteins (12, 3), is dependent upon direct
phosphorylation by MAP kinases. On the other hand, Aop/Yan has been
described as a repressor of Pointed-responsive genes (6-10) and is
also a downstream target of MAP kinase cascades. No mammalian
epithelial ortholog of Aop/Yan has been identified yet. It has been
proposed that phosphorylation of Aop/Yan by MAP kinases results in its
inactivation and nuclear export, thus alleviating repression and
allowing the transcriptional activation of Pointed target genes (6).
The balance between the activity of these two transcription factors
appears to modulate the specificity of the nuclear response to MAPK
signaling cascades.
ETS proteins have also been shown to interact with each other and with
other structurally unrelated transcription factors (see Refs. 3, 4, 13,
and 14 and references therein), thereby providing additional complexity
to their role as transcriptional regulators. In particular, the
interaction of ETS proteins with Jun has significant relevance, since
ETS binding sites appear in the vicinity of AP-1
cis-elements in the promoters of a number of genes involved
in cell proliferation and migration, including those of extracellular
proteases. This functional interaction results in the transcriptional
activation of these genes in response to both extracellular
signal-regulated kinase and Jun N-terminal kinase/stress-activated
protein kinase MAP kinase signaling cascades (3, 15-17).
Paradoxically, similar regulatory elements are present in the promoters
of protease inhibitors and genes expressed during epithelial cell
differentiation (18-23), raising the question of how specificity is
achieved at the nuclear level. It is plausible that members of the ETS
family may be important elements in the generation of target gene
specificity. In this regard, transcription factors belonging to a
specific group within the ETS family of proteins, such as
ESE-1/ESX/JEN/ERT/ELF-3, ELF-5/ESE-2, and more recently EHF/ESE-3, have
been implicated in the control of the transcription of epithelial
differentiation genes (21, 24-32). The presence of various members
with diverse binding specificities and the distinct expression patterns
within the ESE group could be important for the selective control of
the expression of epithelial target genes by these ETS transcription factors.
Elevated levels of ETS proteins have been also associated with cellular
transformation (30, 33). Cancer is the result of a multistep process
that is commonly associated with the loss of function of genes required
for the maintenance of nonproliferative, differentiated cell fates and
whose loss would facilitate tumor survival and/or metastasis. One of
the mechanisms that may lead to the haploinsufficiency of such
regulatory genes is the deletion of genomic regions containing these
genes as a result of the increased genomic instability associated with
the transformation process, a mechanism known as allelic loss of
heterozygosity (LOH) (34).
We have performed an extensive search for genes at human chromosome
11p13, in a region that has been previously associated with allelic
loss of heterozygosity in epithelial carcinomas (see Refs. 34-37 and
references therein). In particular, LOH at this region is associated
with increased metastatic potential, thus suggesting that this region
contains genes required for the control of cell migration (36). As a
result of this search, we have found the genes coding for two
epithelium-specific ETS transcription factors, which we initially
identified as I and J in our study (38) and have been later reported as
ELF5/ESE-2 and EHF/ESE-3, respectively (26-28, 31). The analysis of
EHF/ESE-3 revealed that the expression of this transcription factor is
restricted to differentiated epithelial cells, and remarkably, it is
absent in epithelial carcinomas originated from the same tissues where this factor is expressed normally. EHF/ESE-3 is a transcriptional repressor of a subset of ETS/AP-1-responsive genes, including the
interstitial collagenase gene (matrix metalloproteinase 1; MMP-1) promoter. We suggest that EHF/ESE-3 might be an important regulatory factor in the maintenance of a differentiated phenotype in
epithelial cells by modulating the nuclear response to MAP kinase
signaling cascades.
Cloning of Genes in Chromosome 11p13--
To generate a physical
map for the 11p13 region, we began with searches of publicly available
information sources for yeast artificial chromosome (YAC) clones that
best represented the region. Queries of the Whitehead Institute Genome
Center's physical map data base yielded multiple CEPH mega-YAC clones
that were detected by genetic markers from the region. Each clone was
analyzed by STS content mapping with all available markers by pulsed
field gel electrophoresis and blotting and by fluorescent in
situ hybridization to metaphase chromosomes. This extensive
analysis led to the establishment of a minimal tiling path of clones
that appeared to be nonchimeric, stable, and free of deletions. The
resulting ~4-Mb YAC contig encompasses an 8-9-centimorgan
interval defined by D11S935 and D11S1776. To increase the map's
resolution and utility for gene identification experiments, the minimal
set of YACs was then used to identify plasmid-based genomic clones
corresponding to the region. DNA samples from YACs purified by pulsed
field gel electrophoresis were radiolabeled and used to screen high
density filter grids of a human total genomic DNA BAC library (Research
Genetics, Huntsville, AL), as well as a human chromosome 11-specific
cosmid library (39). A subset of the detected clones could subsequently
be ordered and oriented by STS content mapping. Overlap relationships between these clones were defined using DIRVISH, and gaps in the contigs were closed by end walking.
BAC clones covering the region were subjected to exon trapping (40) and
cDNA selection rounds using a commercial kit
(CLONTECH, Palo Alto, CA) to search for coding
sequences. Full-length cDNAs corresponding to exon-trapped products
or selected cDNAs were cloned, sequenced, and assigned letters as
they were identified. cDNA clones containing the full-length J ORF
were obtained from a human prostate pDR2 cDNA library
(CLONTECH).
Computer-aided Sequence Analysis--
Partitioning of protein
sequences into globular and nonglobular segments was performed using
the local compositional complexity measures implemented in the SEG
algorithm run with the following optimized parameters: window length,
45; trigger complexity, 3.4; extension complexity, 3.8 (41). Protein
data base searches were performed using the PSI-BLAST program (42).
Phylogenetic analysis was performed on a continuous
127-amino acid data set derived by concatenating the conserved ETS and SAM/SPM domains. Positions containing gaps within these domains were
deleted sitewise. Hypotheses of phylogenetic relationships were
constructed using neighbor joining, a distance-based method, and the
maximum likelihood methods. Support for neighbor joining topologies was
evaluated using bootstrap analysis with 1000 replicates, and support
for maximum likelihood tree interference utilized quartet puzzling
reliability values from 10,000 puzzling steps (analogous to bootstrap
replicates) (43). The distance matrix in the neighbor joining tree
construction was calculated by two methods: the simple proportional
difference and Lake's paralinear distance because of its robustness of
interference on data sets having significantly unequal rate effects
(44). The calculation of the distance matrices, neighbor joining
interference of tree topology, and the subsequent bootstrap analysis
were implemented in DAMBE (45). The neighbor joining tree derived from
the maximum likelihood distance matrix was used to compute the
parameters for the models of substitution and rate heterogeneity. A
model of rate heterogeneity with one invariant and eight
DNA Clones--
A cDNA clone containing the full-length J
(EHF/ESE-3b) ORF, was subjected to PCR to construct different clones
containing a FLAG epitope either at the 5' (clone C7132) or at the 3'
(clone F5) ends, that were subcloned into pRC/CMV (Invitrogen,
Carlsbad, CA). To generate a full-length GST fusion bacterial
expression construct (clone pGEX B4), a PCR fragment containing the
full-length ORF was subcloned into the PGEX-20T vector, a derivative of
PGEX-2T (Amersham Pharmacia Biotech). A similar approach was used to
generate a His-tagged full-length construct in the pProEx-HTB vector
(Life Technologies, Inc.). An ESE-1a/ESX expression vector was
constructed by performing PCR on an expressed sequence tag containing
the full-length ESE1a/ESX ORF (expressed sequence tag 78004;
GenBankTM accession number AA 3667460), also inserting a
FLAG epitope at the 5'-end, and subcloned into pRC/CMV. The murine
ETS-2 and the oncogenic Ras 61L expression vectors, the human
collagenase (MMP-1) ( Cell Culture, Transfections, and Luciferase Assays--
The
colon HCT 15, COLO 205, prostate LNCaP FGC 10, and lung NCI H-1299
adenocarcinoma cell lines (ATCC, Manassas, VA) were cultured in RPMI
1640, supplemented with 10% heat-inactivated fetal bovine serum, 10 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin (Life Technologies). COS-7 cells (ATCC) were
cultured in Dulbecco's modified Eagle's medium (Life Technologies) supplemented as above. Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma. Cells were kept at 37 °C, in a humidified atmosphere containing 5% CO2, 95% air.
Plasmids for transfections were isolated by alkaline lysis, RNase A
treatment, and polyethyleneglycol precipitation (51), followed by a
single centrifugation through a CsCl gradient. HCT-15 cells were
transfected by using LipofectAMINE (Life Technologies) as recommended
by the manufacturer. After 5 h, the transfection mixture was
removed, and RPMI 1640 containing 0.5% fetal bovine serum was added to
the cells. After 24 h, cells were treated with 10 ng/ml PMA for
12 h, and the luciferase activity of the cell extracts was
analyzed in a 3010 Luminometer (Analytical Bioluminiscence, Ann Arbor,
MI) using a commercially available reagent kit (Analytical Bioluminiscence). For the Ras 61L co-transfections, cell lysates were
collected 24 h after the addition of fresh medium containing 0.5%
fetal bovine serum. Relative luciferase units were normalized according
to the protein concentration of the extracts. The transfection experiments shown were done in triplicate and repeated at least three
different times.
Northern Analysis--
A full-length J cDNA fragment was
radiolabeled with 32P using the Prime-It kit as recommended
by the manufacturer (Stratagene, La Jolla, CA). Membranes containing
polyadenylated human tissue mRNAs (CLONTECH) or
20 µg of total RNA extracted from epithelial cell lines were
hybridized to the 32P-labeled DNA probe by using RapidHyb
(Amersham Pharmacia Biotech) as recommended by the manufacturer.
Filters were washed up to 0.1× SSC, 0.1% SDS at 65 °C (1× SSC:
0.15 M NaCl, 15 mM sodium citrate), analyzed in
a Storm PhosphorImager using ImageQuant software (Molecular Dynamics,
Inc., Sunnyvale, CA), and digital images were finally processed in
Adobe Photoshop software (Adobe Systems, San Jose, CA) to elaborate the figures.
Recombinant Protein Production--
Bacterial expression
constructs were transformed into the Escherichia coli strain
BL21(DE3), and cultures at A600 = 0.5-0.6 were induced with 0.5 mM
isopropyl- Generation of Monoclonal Antibodies--
Harlan Sprague-Dawley
rats immunized with denatured purified His-tagged ESE-3 were tested for
ESE-3-reactive antisera using Western blots. Rat 362, whose serum
showed the highest immunoreactivity, was sacrificed, and spleen cells
were fused to the SP2 mouse myeloma (ATCC) using standard procedures
(52). Positive cloned hybridomas were selected by enzyme-linked
immunosorbent assay and were further tested for specificity by
performing sequential immunoprecipitations and Western blots (52) with
the monoclonal antibodies (mAbs) and the anti-FLAG antibody (Sigma) on
cell extracts from COS7 cells transfected with EHF/ESE-3b, ESE1a/ESX,
and ETS-2 and by using immunocytochemical analysis on the prostate cell
line LNCaP FGC 10. The mAb 5A5.5 was shown to detect EHF/ESE-3b with
high specificity and did not show any cross-reactivity with the closest human homolog ESX/ESE-1. The epitope recognized by the 5A5.5 mAb is not
included in either conserved domain (ETS or SAM) of the EHF/ESE-3b
protein (not shown).
Immunohistochemistry--
Neutral buffered formalin-fixed,
paraffin-embedded human tissues were sectioned (6 µm), baked on
Superfrost glass slides (Fisher) at 65 °C for 2 h, rehydrated,
and treated with antigen unmasking solution as recommended by the
manufacturer (DAKO, Carpinteria, CA). Immunohistochemistry was carried
out by using either the mAb 5A5.5-containing culture supernatant or the
rat myeloma SP2 culture supernatant as a control, followed by
incubation with a biotinylated mouse anti Rat mAb (DAKO) and a
streptavidin-horseradish peroxidase conjugate (DAKO). Detection was
performed with the Immunopure Metal Enhanced DAB Substrate Kit
(Pierce), and after the detection reaction was completed (10 min to
2 h), slides were subjected to a light hematoxilin stain (Harris
hematoxilin 1:10 in water for 20 s), rinsed in tap water,
dehydrated, and mounted with DPX (Fluka, Buchs, Switzerland). Digital
images were captured with a Polaroid DMC1 digital camera (Polaroid,
Cambridge, MA) and imported into Adobe Photoshop software (Adobe
Systems) to elaborate the figures.
Electrophoretic Mobility Shift Assays--
Nuclear protein
extracts were prepared, and electrophoretic mobility shift assays were
performed essentially as described (53). After electrophoresis, gels
were dried and analyzed as described above for Northern blots. The
sequences for the electrophoretic mobility shift assay probes
containing the ETS sites on the ESE-3 promoter and their variants are
shown in Fig. 5D. The full sequence of the ETS-AP-1 tandem
on the human TIMP-1 gene promoter (HT1) is
TGGGTGGATGAGTAATGCATCCAGGAAGCCTGGAGGCCTGTGGTTT
(sites are underlined) (18), and the ESE1a/ESX binding site at the
neu/erb2 gene (ESX) was as described (30).
Isolation of ETS-related Genes at Human Chromosome 11p13--
As
part of a genomic mapping and sequencing study (38), we used a
combination of exon trapping and cDNA selection to isolate novel
genes at 11p13 within a genomic interval centered on the catalase gene and flanked proximally by CD44 and
distally by CD59. Two of the genes identified, initially
termed I and J (38), potentially coded for ETS
domain proteins. Because of the role of ETS domain transcription
factors in the regulation of multiple processes including cell
division, migration, and transformation (2-5, 33) and the known
association of this region to LOH in several adenocarcinomas (see Refs.
34-37 and references therein), these genes were investigated in more
detail. During the course of this study, I has been reported as ELF5
(31) and as ESE-2 (26), and J has been reported as EHF and ESE-3b (27,
28). For simplicity, we will refer to I as ESE-2, and to J as
ESE-3.
ESE-2 and ESE-3 are transcribed in opposite
directions ("head-to-head") and are located ~110 Kb apart, with
ESE-3 being more proximal (Fig.
1). Their close proximity and sequence
similarity suggest that these two genes arose as a result of ancestral
gene duplication. The 3'-end of the catalase gene is located
~7 kb from the 3'-end of ESE-2. The microsatellite markers
that define the ESE-3 locus are D11S2008, situated ~2.5
kilobase pairs upstream of exon A, and D11S907, on the second intron
and at 0.3 kilobase pairs from exon C. Our sequence differs slightly
from those reported previously, and it is available from
GenBankTM under accession number AF 212848.
Expression of the ESE-3-specific mRNAs--
Northern blot
analysis showed two major ESE-3 mRNA transcripts of 5.6 and 1.3 Kb that presumably arise from alternative utilization of two
different consensus polyadenylation signals. An additional transcript
of 4.6 kb could also be detected. ESE-3 mRNAs were strongly expressed in a variety of tissues with significant epithelial content including trachea, colon, pancreas, prostate, and lung and were
absent or below the level of detection in nonepithelial tissues (Fig.
2A), showing a wider
distribution than that previously reported for EHF or
ESE-3 (27, 28). From the tissues analyzed, ESE-3
mRNAs appeared to be more widely expressed in tissues with high
epithelial content than ESE-I/ELF5/ESE-2 (not shown).
Expression analysis in epithelial cell lines showed that the prostate
carcinoma cell line LNCaP FGC 10 expressed high levels of
ESE-3 mRNAs, followed by the colon adenocarcinoma cell
lines COLO 205 and HCT 15. ESE-3 mRNA transcripts were
absent or below the level of detection in the lung adenocarcinoma cell
line NCI H-1299 (Fig. 2B).
ESE-3 Is a Nuclear Protein Expressed in Differentiated Epithelial
Cells--
To evaluate the expression of the ESE-3 protein, a
mAb was generated (see "Experimental Procedures" for
details). Immunoprecipitation and Western blot analyses showed that the
ESE-3 protein migrated as a Mr = 33,000 single
polypeptide in both LNCaP FGC 10 and ESE-3-transfected COS-7 cell
protein extracts, and it was absent in mock-transfected COS 7 cell
extracts (not shown). Immunohistochemistry analysis on several human
tissues revealed that ESE-3 expression was restricted to the nuclei of
highly differentiated epithelial cells, particularly those with
secretory functions (Fig. 3). Expression
was highest in the serous and mucous glands, and secretory ducts of the
airways (Fig. 3, A and B), the secretory
epithelium of the seminal vesicle (Fig. 3C), the salivary
glands (Fig. 3D), and the mucus-producing cells in
the crypts of Lieberkuhn in the colon (Fig. 3, E and F). Moderately high levels of expression were also
observed in the basal cells and to a lesser extent in the secretory
epithelium of the prostate (Fig. 3G).
In the squamous nonkeratinized epithelium of the esophagus, the
expression of ESE-3 was evident in the differentiating layers, while
the basal cells, undergoing active proliferation, were negative (Fig.
3H). Expression of ESE-3 could be also detected in the
columnar epithelial lining of the airways (Fig. 3A), and it
was absent or below the level of detection in the lining epithelium of
the colon (Fig. 3E).
ESE-3 Expression Is Lost during Epithelial
Carcinogenesis--
Previous reports have shown an association between
oncogenesis and elevated levels of expression of ETS proteins (see
Refs. 3, 30, 33, and references therein). However, ESE-3 expression was
shown to be restricted to differentiated epithelial cells and absent in
proliferating cells. This was clearly evident in the expression pattern
observed in the squamous epithelium of the esophagus (Fig.
3H). Additionally, expression of ESE-3 in the prostate was
highest in the basal cells, a cell type that is lost in malignant
prostate carcinomas (54). Consistently, we observed loss of ESE-3
positive cells in malignant prostate carcinomas (not shown).
Based these observations, ESE-3 immunoreactivity was monitored in other
epithelial carcinomas. Using the ESE-3-specific mAb, we analyzed
protein expression in samples of bladder adenocarcinoma (Fig.
4A), oral epithelium carcinoma
(Fig. 4B), and breast ductal carcinoma (C and
D). As shown, ESE-3 protein expression was restricted to the
normal epithelium and was clearly excluded from the transformed epithelium. Both normal and transformed epithelium were evident in all
samples, allowing us to exclude sample quality or processing as an
explanation for the loss of immunoreactivity.
The ESE-3 Protein Binds Selectively to a Small Subset of ETS
Binding Sites--
Since a number of transcription factors regulate
their own transcription, we examined the ESE-3 promoter for
putative ETS binding sites. Two potential ETS binding sequences were
found: one at
A number of natural known ETS binding sites were compared with the
To assess the binding of native ESE-3 protein, nuclear extracts from
the LNCaP FGC 10 prostate epithelial adenocarcinoma were tested for
binding to the ESE-3 Is a Context-dependent Transcriptional
Repressor--
The polyoma virus enhancer ETS site contains a very
similar sequence to the one that we experimentally determined as a high affinity binding site for ESE-3 and is contained within a regulatory cis module termed the oncogene response element (ORE).
Transcription from the ORE is synergistically stimulated through the
cooperation of ETS transcription factors and AP-1 in response to Ras
signaling (48). To analyze the transcriptional role of ESE-3 in the
ORE-mediated transcription, HCT-15 colon adenocarcinoma cells were
transfected with an ESE-3 expression vector and the (Py)2
The human interstitial collagenase (MMP-1) promoter has been also shown
to be a Ras-responsive promoter by virtue of the presence of ETS and
AP-1 cis-elements, which act synergistically to activate Ras-dependent transcription of the gene (15, 16). Similarly to what was observed previously with the synthetic ORE reporter, ESE-3
was able to repress the
Similar results were obtained when the tumor promoter PMA was used as
an activator of the transcription of the reporter constructs. Phorbol
esters activate protein kinase C, which in turn is able to
phosphorylate Raf leading to activation of the MAPK cascade (56). ESE-3
repressed, in a dose-dependent manner, the PMA-induced transcriptional activation of both reporters (Fig. 6, C and
D). Co-transfection of ETS-2 in this setting resulted
neither in an increase nor in a decrease of luciferase activity (not shown).
To determine the specificity of the transcriptional repression mediated
by ESE-3, other ETS/AP-1-responsive promoters were analyzed. Expression
of ESE-3 did not repress transcription stimulated by the tandem
ETS/AP-1 from the human TIMP-1 gene promoter cloned upstream of the
minimal
The epithelium-specific metalloprotease matrilysin (MMP-7) also
contains ETS and AP-1 cis-elements in its promoter, and its expression, like that of MMP-1, is also induced by PMA, tumor necrosis
factor-
The context-dependent repression by ESE-3 is further
confirmed by deletion of one tandem of ETS/AP-1 sites or by mutation of
the AP-1 cis-elements in the context of the ORE reporter.
Because it is impossible to eliminate the AP-1 site in the polyoma ORE without affecting the ETS site, since they overlap, we used the (Py + 6F)2 reporter containing the ETS/AP-1 tandem of the
polyoma ORE, where the ETS and AP-1 binding sites are separated by 6 nucleotides (48). As shown, ESE-3 was still able to repress the
PMA-mediated trans-activation of the (Py + 6F)2 reporter (Fig. 6G). Although (Py + 6F)2 reporter is, on average,
10-20-fold less active than the parental(Py)2
Previous reports indicate that overexpression of ETS-2 may lead to
transcriptional repression (58). In our experiments, co-transfection of
ETS-2 did not lead to a decrease of transcription in any of the cases
analyzed (not shown). Western analysis also revealed that both the
ETS-2 and the ESE-3 expression vectors were expressed at comparable
levels (not shown), and therefore we excluded the possibility that the
repression observed upon co-transfection with ESE-3 is due to
nonspecific repression.
The genes coding for ELF5/ESE-2 and EHF/ESE-3 were found during an
extensive search for genes localized at human chromosome 11p13 (38), in
a region that has been previously associated with LOH in a subset of
epithelial carcinomas (see Refs. 34-37 and references therein). In the
case of some lung carcinomas analyzed, LOH at this locus has been
associated with a higher patient mortality rate, a direct measurement
of the tumors' metastatic potential (36), thus suggesting that this
region may contain genes required for the control of cellular
migration. Genetic mapping of this LOH has been mostly associated with
the Catalase locus (35-37), which we found in close
proximity to ESE-2, and ESE-3.
The expression of ESE-3 is widespread in tissues with high
epithelial content. We show that ESE-3 mRNA has a much
wider expression profile than previously reported (27, 28), and
contains an additional 1.3-Kb mRNA species. We did not isolate the
short splice variant reported by others, indicating that it was
underrepresented in the library used. Using a newly generated specific
monoclonal antibody, we show that the ESE-3 protein is specifically
expressed in differentiated epithelial cells. This is most evident in
the stratified squamous epithelium of the esophagus, where ESE-3 is absent in the basal proliferating stem cells, and appears as soon as
they differentiate. Importantly, ESE-3 expression is lost in a number
of epithelial carcinomas, including bladder, oral squamous, breast
ductal (Fig. 4), and prostate epithelial carcinomas (not shown).
Certainly, it can be argued that the decreased expression of ESE-3 is
not an essential pathogenic event and that other oncogenic mechanisms
lead to the loss of ESE-3 expression. Indeed, although our data are
suggestive altogether, we do not have direct evidence correlating the
loss of ESE-3 expression with cancer progression. Nonetheless, the
restricted association of ESE-3 with differentiated epithelial cells
indicates that, at least, could be a marker for malignancy, since the
cell fate associated with ESE-3 expression appears to be no longer
determined. Others have reported variable EHF/ESE-3 mRNA expression
in several epithelial carcinomas analyzed (27), although increased
mRNA expression does not necessarily have to correlate with
elevated protein levels. This seems to be the case of the prostate
LNCaP FGC 10 adenocarcinoma cell line, in which the ESE-3 mRNA
levels are the highest among the cell lines tested, but the protein is
barely detectable both in Western blots (not shown) and in mobility
shift assays (Fig. 5). More tumor samples will have to be analyzed to
clarify this issue.
Functionally, ESE-3 is a transcriptional repressor of a subset of genes
that contain ETS and AP-1 cis-elements within their promoters and have been shown to be downstream targets that are positively regulated by MAP kinase signaling cascades. ESE-3-mediated repression of these promoters is dependent on the presence of high
affinity binding sites, which we have defined in good agreement with
those described by others (28). The restricted binding affinity of this
repressor to a limited number of ETS cis-elements, combined
with specific promoter requirements, indicates that ESE-3 is only a
repressor for a specific subset of ETS/AP-1-responsive genes. These
include the interstitial collagenase gene (MMP-1) promoter, also
observed by others (27), and a dimer of the oncogene response element
of the polyoma virus enhancer, both well characterized Ras-responsive
genes. Alternatively, ESE-3 may also modulate the transcription of
genes downstream of other pathways that are activated via phorbol
esters and that are only secondarily linked to the main Ras pathway.
The restricted expression pattern of ESE-3 in the squamous stratified
epithelium of the esophagus may provide insights into the
physiological role of this repressor. Stratified epithelia have served
as a paradigm for the study of epithelial differentiation (59). In this
dynamic system, a population of self-renewing basal stem cells give
rise to committed, nonproliferating precursors that undergo progressive
differentiation as they are pushed toward the lumen, where they finally
delaminate (60). The molecular mechanisms that govern the transition
from a proliferating basal cell to a committed nondividing precursor
are of great importance for deciphering the etiology of cancer. A
failure to adopt the committed fate by the basal cell may lead to the
development of adenomas or carcinomas, giving rise to most solid tumors.
Both autocrine and paracrine factors are known to maintain the
proliferative potential of the basal cells. Some of these factors, such
as transforming growth factor- As mentioned earlier, transcription factors of the ETS protein family
are good candidates as modulators of the nuclear response to MAP kinase
signaling cascades. Among these, factors belonging to the ESE subgroup
seem to play an important role in the activation of epithelium-specific
genes (21, 24-29). Indeed, a number of genes whose expression is
associated with epithelial cell differentiation contain ETS
cis-elements within their promoters, thereby supporting this
view (19-23). Both ESE-1 and ESE-2 are expressed at later stages
of keratinocyte differentiation (21, 26), indicating that they are
unlikely to play a role in the determination of the early
differentiated spinous precursor in stratified squamous epithelia.
Thus, it appears that, among the ESE family members described to date,
ESE-3 is expressed the earliest in the differentiated precursors. Based
on the functional analysis and the expression profile for this gene, as
presented herein, we hypothesize that ESE-3 could play a role in the
determination of the early differentiated fate in stratified squamous
epithelia. Since ESE-3-mediated repression may only occur at a specific
set of MAP kinase target promoters, while allowing the
transcription of others, we conclude that ESE-3 might be an important
factor in the nuclear specific response to MAP kinase signaling cascades.
The molecular mechanisms underlying the differentiation of secretory
epithelial cells is less known, although recent reports indicate that
epidermal growth factor receptor signaling is also involved in the
determination of goblet cells in the airways and subsequent mucus
production (61). The epithelium-specific ETS factors
ESE-1/ESX/JEN/ERT/ELF3 and ELF5/ESE-2 and more recently also EHF/ESE-3
have also been implicated in the selective transcriptional activation
of genes specific to glandular epithelia (26, 28). Further work will be
needed to understand how the interplay between these genes orchestrates
gene expression in secretory epithelia and their role (if any) in the
induction of secretory fates.
The role of ESE-3 as a repressor resembles that described for Aop/Yan,
an ETS protein in D. melanogaster (6). Additionally, ESE-3
and Aop/Yan not only share a similar structure but also recognize the
same DNA sequences, since the polyomavirus enhancer in the context of
the interstitial collagenase gene promoter has been used to demonstrate
repression by Aop/Yan (62). However, ESE-3 does not have any consensus
extracellular signal-regulated kinase phosphorylation sites; nor is it
phosphorylated by extracellular signal-regulated kinase or Jun
N-terminal kinase 1 in
vitro,3 suggesting that,
unlike Aop/Yan, ESE-3 may not be regulated post-translationally by
direct MAP kinase phosphorylation.
The ESE family has been shown to contain two distinguishable domains,
namely the ETS DNA binding domain at the carboxyl terminus and
an additional domain that has been recognized in some ETS-domain proteins as the A domain, or the Pointed domain (after the
Drosophila ETS protein Pointed; reviewed in Ref. 3), located
at the amino terminus. Recent detailed analysis of conserved motifs and
comparison of three-dimensional structures established the similarity
of the A domain to the SAM/SPM (sterile The semiautonomous nature of the SAM/SPM domain and its apparent rapid
and diverse evolution (68) impose a challenge in defining the
phylogenetic relationship between ESE-3 and related ETS proteins in
other species. In particular, the ETS domain sequence in
Drosophila with highest similarity to that of ESE-3 is found in E-74 (GenBankTM accession number AAA28493), which does
not contain a SAM/SPM domain. It is for this reason that we do not
favor the ELF nomenclature (E-74-like
factor) for this group. Phylogenetic analysis of the ETS
family members that contain ETS and SAM/SPM domains using both domains
in the analysis clearly indicates that Aop/Yan and the TEL proteins are
members of a monophyletic group (66, 28) also containing the ESE and
the PDEF/ETS 98B families. This clade of ETS proteins clusters to the
exclusion of a second monophyletic group containing
Drosophila Pointed P2 and its vertebrate orthologs c-ETS-1
and c-ETS-2 (Fig. 7).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-distributed rate categories was used.
1200 to +63), the (Py)2
56
dE Fos, (PyEts)2
56 dE Fos, and the (Py + 6F)2
56 dE Fos luciferase reporter
constructs have been described previously (46-48). To make the TIMP-1
56 dE Fos luciferase reporter, the HT-1 sequence (see
electrophoretic mobility shift assay probes below) was subcloned
upstream of the
56 dE Fos minimal promoter (48) driving the
luciferase reporter gene. A
635 to +5 human matrilysin promoter
fragment (49) was obtained through PCR amplification of total human
genomic DNA and subcloned into a high copy number derivative of p19Luc
(50).
-thiogalactoside, for 4 h at 37 °C. His tag
full-length ESE-3 was purified using the His·Bind resin and buffer
kit, following the manufacturer's protocol (Novagen, Madison, WI).
GST-full-length ESE-3 was purified on glutathione-Sepharose 4B resin
according to the manufacturer's protocol (Amersham Pharmacia Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Physical map of the ESE-3 genomic
region. Microsatellite markers are listed across the
top. The lines immediately
below the markers depict YAC clones. Below the
YAC clones are BAC and cosmid clones. Genes are shown at the
bottom, and the arrows indicate the direction of
transcription.
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Fig. 2.
Expression of the ESE-3 mRNA. The
expression of ESE-3 mRNAs was analyzed by Northern blot
hybridization on human tissues (A) and the epithelial cell
lines COLO 205, LNCaP FGC 10, HCT-15, and NCI-H1299 (B). The
migration of the molecular size standards is shown on the
left. The arrows indicate the three major
mRNA transcripts observed.
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Fig. 3.
ESE-3 is a nuclear protein expressed in
differentiated epithelial cells. Human tissues were subjected to
immunohistochemical analysis by using the ESE-3-specific monoclonal
antibody 5A5.5. Brown staining reveals immunoreactivity, and blue
staining corresponds to hematoxilin nuclear stain.
A, bronchial columnar lining epithelium (top) and
secretory ducts; B, bronchial secretory ducts and mucous and
serous glands; C, seminal vesicle; D, salivary
glands; E, colon lining and crypts of Lieberkuhn;
F, crypts of Lieberkuhn (transversal section); G,
prostate gland; H, esophagus stratified epithelium.
Bar, 100 µm.
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Fig. 4.
ESE-3 immunoreactivity is lost during
carcinogenesis. Sections from bladder (A) and oral
epithelial (B) adenocarcinomas and breast ductal carcinoma
(C and D) containing normal tissue adjacent to
the carcinoma cells were subjected to immunohistochemical analysis by
using the ESE-3-specific monoclonal antibody 5A5.5. The
black arrows indicate the normal ESE-3-expressing
epithelium, while the red arrows show the
transformed epithelium, which does not show ESE-3 immunoreactivity.
Bar, 100 µm in A and B and 50 µm
in C and D.
240 and another at
730 nucleotides from the major
transcription start site.2
Both His tag (Fig. 5A,
lane 1) and GST-full-length ESE-3 (Fig. 5A, lane 3) could bind the
730 site
with high affinity, as evidenced by a competition profile using cold
oligonucleotide as competitor (not shown). The mobility of these
nucleoprotein complexes was affected by the addition of an anti-ESE-3
polyclonal antiserum (
362; see "Experimental Procedures" for
details) (Fig. 5A, lanes 2 and
4).
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Fig. 5.
DNA binding specificity of the ESE-3
protein. A, electrophoretic mobility shift assays were
used to monitor the binding of E. coli expressed His-tagged
full-length ESE-3 (His-ESE-3; lanes 1 and
2) or GST-full-length ESE-3 (GST-ESE-3; lanes
3 and 4) fusion proteins (10-100 fmol) to a
32P-labeled double-stranded oligonucleotide (40 fmol; 3 nM final concentration) containing the ETS consensus
binding site present at position 730 on the ESE-3 gene
promoter. Specificity of the nucleoprotein complexes was determined by
using the 362 rat antiserum against ESE-3 (362
S; lanes 2 and 4).
B, binding of E. coli expressed and purified
GST-ESE-3 to double-stranded oligonucleotides containing the following:
the ETS consensus sites at
240 (240; lane
1) and at
730 (730; lanes
2 and 6) on the ESE-3 gene promoter; a
variant of the
730 site (730 m1; lane 3); the
ETS and AP-1 sites on the human TIMP-1 gene promoter
(HT-1; lane 4); the ESE1a/ESX binding
site at the human NEU gene (ESX; lane
5); and the variants of the 730 sequence (730 m2
(lane 7), 730 m3 (lane 8),
730 m4 (lane 9), and 730 m5 (lane
10)). C, detection of ESE-3 binding activity in
the prostate LNCaP FGC 10 epithelial cell line. Twenty µg of nuclear
proteins were incubated with the 730 m2 32P-labeled
oligonucleotide in the presence of the 362 rat anti ESE-3 antiserum
(362
S, lane 2),
preimmune serum (362 PI; lane
1), or a 50-fold molar excess of cold competitor
oligonucleotide (lane 3). The arrows
show the nucleoprotein complexes that are reactive to the anti-ESE-3
antiserum. D, sequences of the probes used, relative binding
affinities, and proposed consensus.
730 site and with several sequences derived from the
730 site by
single nucleotide substitutions. Full-length GST-ESE-3 displayed
different relative affinities for the sites tested (Fig. 5B), suggesting the consensus A > G:C > G:A:G:G:A:A:G:T, as a high affinity binding site for ESE-3 (Fig.
5D).
730 m2 probe (see below) and showed a number of
nucleoprotein complexes (Fig. 5C, lane
1). The addition of the anti-ESE-3 polyclonal serum to the
binding reactions selectively altered the migration of two
nucleoprotein complexes (Fig. 5C, lane
2). The migration profile of these DNA-protein complexes was
similar to that of purified His-tagged ESE-3 (Fig. 5A,
lane 1), indicating that both bands arose from a
single polypeptide. The slower migrating complexes, which were not
recognized by the anti ESE-3 serum, were shown to bind specifically to
the
730 m2 sequence, and they did not appear when the
730 m1
oligonucleotide was used as a probe in the binding reactions (not
shown). Thus, these complexes may result from the binding of other ETS
proteins present in the extracts. The band near the top of the gel only appeared in the lanes that contained preimmune serum and not in those
containing antiserum or no serum at all (not shown), indicating that it
is an artifact caused by the preimmune serum.
56 dE Fos luciferase reporter, either in the presence or the absence
of an oncogenic Ras (Ras 61L) expression construct. The
(Py)2
56 dE Fos luciferase reporter contains two ORE
modules upstream of a minimal Fos promoter driving the luciferase gene
(48). HCT-15 cells were used in this study due to their low, albeit
detectable, endogenous levels of ESE-3 mRNA expression
(see Fig. 2B). As shown (Fig.
6A), co-expression of ESE-3
repressed the oncogenic Ras-mediated transcriptional activation of the
polyoma virus ORE-driven reporter. Similar results were obtained when a
constitutively active membrane-targeted form of the nucleotide exchange
factor SOS (55) was co-transfected instead of the oncogenic Ras mutant
(not shown).
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Fig. 6.
ESE-3 represses the Ras- and the phorbol
ester-dependent trans-activation of
ETS/AP-1-responsive genes. HCT-15 colon adenocarcinoma cells were
transfected with either (Py)2 56 dE Fos luciferase
(A) or the human collagenase Luc (B) luciferase
reporter constructs (1 µg/30 mm dish) and with eucaryotic expression
constructs encoding murine ETS-2 (hatched bar)
(0.5 µg/30-mm dish) or human ESE-3 (clone pRC/C7132; 0.5 µg/30-mm
dish) (gray bar) in the presence of a Ras 61L
expression construct (0.5 µg/30-mm dish) where indicated. HCT 15 cells were also transfected with increasing amounts of the
ESE-3 (C-I) or ESE-1/ESX expression construct
(J) (0.1 µg, hatched bar, and 0.5 µg, gray bar) as indicated, together with the
(Py)2
56 dE Fos luciferase (C), human
intersticial collagenase (MMP-1) (D), TIMP-1
56 dE Fos
luciferase (E), matrilysin (F), (Py + 6F)2
56 dE Fos luciferase (G), Py + 6F
56 dE Fos luciferase (H), and
(PyEts)2
56 dE Fos luciferase (I and
J) luciferase reporter constructs and treated with PMA where
indicated. Luciferase activity was measured from cell lysates, and
values were normalized to the protein concentration of the extracts.
Experiments shown are representative of three independent experiments,
all of them performed in triplicate. Error bars,
S.D.
1200 to +63 collagenase promoter response to
Ras (Fig. 6B). In both cases, expression of ETS-2 resulted
in enhanced expression of the reporter in these cells (Fig. 6,
A and B).
56 dE Fos promoter (Fig. 6E). This is consistent
with the low affinity of ESE-3 for the ETS cis-element present in the TIMP-1 promoter (see Fig. 5).
, interleukin-1, and epidermal growth factor (16, 49). The
activity of matrilysin has been associated with normal epithelial cell
migration, although it is also expressed at high levels in nonmigrating
glandular epithelial cells (57), which also express ESE-3 (this study).
In agreement with their tissue co-localization, we found that ESE-3 was
unable to repress transcription from the matrilysin promoter (Fig.
6F) despite the presence of a high affinity binding site for
ESE-3, suggesting that repression might be
context-dependent.
56 dE Fos
luciferase reporter, we feel that, qualitatively, they both reflect
similar results. It is also possible that ESE-3 repressed less
efficiently in this setting. In any case, ESE-3 was unable to repress a
reporter that contained a single copy of the Py + 6F module
(Fig. 6H). Mutation of the AP-1 cis-elements in
this context, represented by the (PyEts)2
56 dE Fos
luciferase reporter (48), showed that repression was dependent on the
presence of functional ESE-3 binding sites in combination with AP-1
cis-elements. In fact, ESE-3 then behaved as an activator at
low concentrations (Fig. 6I), although this activity was
very moderate in comparison with the related polypeptide ESE-1/ESX
(Fig. 6J). On the other hand, synthetic promoters where the
ETS binding sites were eliminated in this context, leaving multimers of
the AP-1 cis-elements (3×AP-1, and 6×AP-1; see Ref. 48), were not
repressed by ESE-3 (not shown), indicating that DNA binding to cognate
cis-elements is essential to mediate this activity.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, keratinocyte growth
factor, and fibroblast growth factors, are ligands for
tyrosine kinase receptors and thus trigger the activation of signaling
cascades involving MAP kinases. These signaling cascades are
reiteratively used throughout the differentiation of these cells, since
they activate the expression of differentiation markers in the upper layers of the epidermis (59). This implies that additional factors are
required to modulate appropriate response (e.g.
proliferation versus differentiation) to MAP kinase
signaling cascades.
motif/scm-polyhomeotic motif) domains, also involved in protein-protein interactions (3, 63).
In the case of the ETS proteins belonging to the TEL subgroup, this
domain has been shown to mediate homotypic interactions (14, 64), which
are required for repression (65-67), but we have not observed such a
role in the case of ESE-3.3
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Fig. 7.
Evolutionary relationship among ETS
transcription factors structurally related to ESE-3. Phylogenetic
analysis on ETS family members that contain both ETS and SAM/SPM
domains was performed, including both domains as described under
"Experimental Procedures." A star phylogeny for the maximum
likelihood tree is shown. All three methods of phylogenetic
interference used (see "Experimental Procedures" for details)
recovered the same topology. The numbers close to the
internal nodes represent the quartet puzzling support values for the
branching relationships. The human sequences used were as
follows: ESE-1a/ESX/JEN/ERT/ELF3 (AAB65824), ESE-2a/ELF5 (AAD22960.1),
TEL-1 (NP 001978), TEL-2b (AAD43251), c-ETS-1 p54 (P 27577), c-ETS-2 (P
15036), FLI-1 (Q 01543), human GABP (Q 06546), and the human
epithelial prostate-specific PDEF (AAC 95296). Our ESE-3/EHF
cDNA sequence data are available (as ESE-J) from
GenBankTM under accession number AF 212848. The tree also
includes all the Drosophila ETS proteins that contain both
the ETS and SAM domains: Aop/Yan/Pok (Q 01842), Pointed P2 (P 51023),
ETS 21C (AAF51484.1), ELG/ETS 97D (Q 04688), and ETS 98B (AAF
56746).
Functionally, TEL-1, TEL-2b, ESE-1, ESE-2, and EHF/ESE-3 may all behave as repressors (Refs. 14, 26-28, 32, and 65-67 and this study). It is tempting to speculate that the regulatory role of Aop/Yan may have diversified in vertebrates and could be represented by these genes. This regulatory role may have acquired additional functions during evolution, since some of these proteins also behave as activators, although to date, it has not been reported whether Aop/Yan is only a repressor. Since TEL-1 appears to be only expressed in mesoderm, where it appears to have an important role during hematopoiesis and angiogenesis (69), it seems plausible that the ESE subgroup could represent the function of Aop/Yan in vertebrate epithelia. Furthermore, we hypothesize that the function of these genes could be partially redundant, as evidenced by the fact that deletion of ESE-3 coding sequences in mice did not result in any clearly apparent phenotype.4
As Aop/Yan, transcription factors belonging to the ESE subgroup might
be also involved in the election of alternative cell fates, such as the
choice between proliferation and migration versus
differentiation, in response to complex environmental cues. We propose
that the role of these proteins has been conserved throughout evolution
as a nuclear switch necessary for the modulation of MAPK signaling
cascades in epithelial cells.
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ACKNOWLEDGEMENTS |
---|
The contributions of the Physical Mapping, Sequencing, and Bioinformatics groups at Sequana were fundamental to this work. We thank Javier Fraga for advice and Michael Karin, William McGinnis, John K. Westwick, José Javier García Ramírez, and David A. Brenner for valuable suggestions on the manuscript. J. Fraga, Craig Hauser, and M. Karin kindly provided materials used in this study.
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FOOTNOTES |
---|
* This work was supported by Boehringer Ingelheim Pharma KG, and Axys Pharmaceuticals Inc. (formerly Sequana Therapeutics, Inc.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF212848.
a To whom correspondence should be addressed: Dept. of Biology, 0349, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093. Tel.: 858-822-0461; Fax: 858-822-0460; E-mail: atugores@yahoo.com.
b Present address: Structural Genomics, Inc., 10505 Roselle St., San Diego, CA 92121.
c Present address: Highwire Press, 1454 Page Mill Rd., Palo Alto, CA 94304-1124.
d Present address: Ardais Corp., 128 Spring St., Lexington, MA 02421.
e Present address: Maxim Pharmaceuticals, 6650 Nancy Ridge Dr., San Diego, CA 92121.
f Present address: Division of Molecular Genetics/H5, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
h Present address: Stowers Institute for Medical Research, 1000 E. 50th St., Kansas City, MO 64110.
i Present address: Biocept Inc., 2151 Las Palmas Dr., Suite C, Carlsbad, CA 92009.
j Present address: GenProbe Inc., 10210 Genetic Center Dr., San Diego, CA 92121.
Published, JBC Papers in Press, March 19, 2001, DOI 10.1074/jbc.M010930200
2 A. Tugores, unpublished observations.
3 P. S. Reddy, unpublished results.
4 L. Carlée, and M. Duyao, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: MAP, mitogen-activated protein; MAPK, MAP kinase; AP-1, activator protein 1; BAC, bacterial artificial chromosome; LOH, loss of heterozygosity; mAb, monoclonal antibody; ORE, oncogene response element; PCR, polymerase chain reaction; PMA, phorbol myristate acetate; YAC, yeast artificial chromosome; MMP, matrix metalloproteinase; ORF, open reading frame; kb, kilobase; contig, group of overlapping clones.
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