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Molecular and Cellular Biology, August 2000, p. 5828-5839, Vol. 20, No. 16
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
TEL, a Putative Tumor Suppressor, Modulates Cell Growth and Cell
Morphology of Ras-Transformed Cells While Repressing the
Transcription of stromelysin-1
Randy
Fenrick,1,2
Lilin
Wang,1,2
John
Nip,1,2
Joseph M.
Amann,1,2
Robert J.
Rooney,3
Jennifer
Walker-Daniels,4
Howard C.
Crawford,2,5
Diana L.
Hulboy,2,5
Michael S.
Kinch,4
Lynn M.
Matrisian,2,5 and
Scott W.
Hiebert1,2,*
Departments of Biochemistry1 and
Cell Biology5 and
Vanderbilt-Ingram Cancer Center,2
Vanderbilt University School of Medicine, Nashville, Tennessee
37232; Department of Genetics, Duke University Medical School,
Durham, North Carolina 27715-30543; and
Department of Basic Medical Sciences, Purdue University,
West Lafayette, Indiana 479074
Received 8 December 1999/Returned for modification 14 February
2000/Accepted 12 May 2000
 |
ABSTRACT |
TEL is a member of the ETS family of transcription factors that
interacts with the mSin3 and SMRT corepressors to regulate transcription. TEL is biallelically disrupted in acute
leukemia, and loss of heterozygosity at the TEL locus has
been observed in various cancers. Here we show that expression of TEL
in Ras-transformed NIH 3T3 cells inhibits cell growth in soft agar and
in normal cultures. Unexpectedly, cells expressing both Ras and TEL
grew as aggregates. To begin to explain the morphology of Ras-plus TEL-expressing cells, we demonstrated that the endogenous matrix metalloproteinase stromelysin-1 was repressed by TEL. TEL
bound sequences in the stromelysin-1 promoter and repressed
the promoter in transient-expression assays, suggesting that it is a
direct target for TEL-mediated regulation. Mutants of TEL that removed a binding site for the mSin3A corepressor but retained the ETS domain
failed to repress stromelysin-1. When BB-94, a matrix
metalloproteinase inhibitor, was added to the culture medium of
Ras-expressing cells, it caused a cell aggregation phenotype similar to
that caused by TEL expression. In addition, TEL inhibited the
invasiveness of Ras-transformed cells in vitro and in vivo. Our results
suggest that TEL acts as a tumor suppressor, in part, by
transcriptional repression of stromelysin-1.
| |
INTRODUCTION |
The TEL (for
"translocation-ETS-leukemia," also referred to as ETV6)
transcription factor is a target for disruption by chromosomal translocations in several forms of acute leukemia (24-27, 38, 50,
51, 54, 57, 63). TEL was originally identified as the
gene on chromosome 12 that is disrupted by t(5;12) in patients with
chronic myelomonocytic leukemia (25). This translocation fuses the N-terminal homodimerization domain of TEL to the tyrosine kinase domain of the platelet-derived growth factor receptor 
. The N
terminus of TEL is also fused to the majority of the AML-1B (Runx-1)
transcription factor by t(12;21), which is the most frequent translocation in pediatric B-cell acute lymphoblastic leukemias (23, 26, 57, 61).
TEL is a member of the ETS family of transcription factors. ETS factors
bind heterogenous sequences centered around a core GGA sequence and
cooperate with other transcription factors to regulate the
transcription of a diverse set of genes (28, 52, 74).
Several ETS factors are downstream effectors of oncogenic Ras proteins
and are phosphorylated by mitogen-activated protein kinases (73,
80). Aberrant expression of these ETS factors induces cellular
transformation (73, 74). By contrast, TEL acts as a
transcriptional repressor. In t(12;21), fusion of the TEL N-terminal
domain to AML-1 creates a dominant transcriptional repressor (18,
19, 32). This observation led to the identification of an
association between TEL and the mSin3A and SMRT corepressors (13,
20).
The TEL gene maps to chromosome 12 region p13. Loss of heterozygosity
in this region of chromosome 12 is found in many types of cancer
including leukemias and tumors of the breast and ovary (25, 31,
58, 62, 66, 79). For example, in more than 90% of cases
associated with t(12;21), the second TEL allele also is deleted
(26, 54, 57, 63). These findings suggest that the widely
expressed TEL protein may function as a tumor suppressor (12, 54,
64). However, there is no direct evidence to support this
hypothesis, because targeted disruption of the TEL gene in mice is lethal in utero at embryonic day 10.5 (E10.5) (71).
TEL knockout mice die of an inability to maintain the developing
vascular network in the yolk sac (71). However,
hematopoietic progenitors from these embryos are capable of
differentiating along the various blood cell lineages in vitro
(71). Therefore, TEL is not intrinsically required for
the growth or differentiation of hematopoietic cells. However, in
chimeric mice, TEL
/ embryonic stem cells contributed to
fetal liver hematopoiesis but not to bone marrow-derived hematopoiesis
and were unable to colonize the stromal microenvironment
(72). This phenotype was hypothesized to reflect defects in
cell adhesion or in pathways responsive to cell adhesion
(72).
Matrix metalloproteinases (MMPs) are a family of secreted,
zinc-dependent proteinases that degrade various components of the extracellular matrix (ECM). MMPs are required for cell migration, ECM
organization, tissue remodeling, and tumor cell invasion
(2). Cross talk between the signal transduction pathways
that are regulated by cell-cell and cell-ECM adhesion may lead to
coordinate regulation of these pathways (9, 17, 30, 37, 40, 47,
48, 60, 76). Consequently, alterations in MMP expression may
affect cell-cell interactions as well as cell-ECM adhesion. MMPs are also linked to cell growth. The expression of MMPs is induced by growth
factors, and the promoters of the MMP genes contain Ras-responsive
elements, which contain binding sites for the ETS family of
transcription factors and AP-1 (3, 16, 49, 55). Moreover,
MMP expression may directly contribute to tumor development. For
example, expression of stromelysin-1 (MMP-3) in
transgenic mice promoted spontaneous premalignant cellular
changes and stimulated oncogenic transformation in mammary glands
(65).
We present evidence showing that the TEL transcriptional repressor
affects cell growth and causes cell aggregation. TEL inhibited the
growth of Ras-transformed cells both in soft agar and in normal cultures. Expression of TEL in Ras-transformed NIH 3T3 cells resulted in a pronounced aggregation of these cells. The aggregation phenotype coincided with a reduction in the expression of the Ras-responsive gene
stromelysin-1 (MMP-3). TEL-mediated
repression of stromelysin-1 may be critical for the
aggregation phenotype because an MMP-specific chemical
inhibitor produced a similar phenotype. Finally, MMPs are required for
tumor invasion, and we demonstrated that TEL inhibits tumor invasion.
These results add biological support for the role of TEL as a tumor suppressor.
| |
MATERIALS AND METHODS |
Plasmids.
The pBabePuro and pCMVTEL constructs have been
described elsewhere (32, 46). pCMVTEL
ETS was made by
replacing nucleotides 1143 to 1179 (25) with a
BamHI restriction site. The C-terminal BamHI-SalI fragment was amplified using standard
PCR techniques, sequenced for verification, and reinserted into
BamHI-SalI-cleaved pCMVTEL. This mutation deletes
amino acids 373 to 385, which corresponds to the 
2 helix in the ETS
DNA-binding domain as described previously (52). The
TEL and TELETS cDNAs were inserted into
pBabePuro as EcoRI-SalI fragments. The rat
stromelysin-1 reporter from pG7-754TR CAT (43) was inserted
into the pGL2 Luciferase vector (Promega) as an
Acc65I-BglII fragment. The deletion mutants of
the stromelysin-1 promoter were made as follows. pTR334 was
made by truncating pGL2-754TR at the unique BsrGI site.
pTR182 and pTR127 were made by PCR using oligonucleotides starting at
residue 
182 or 127 (5'-TAGGTACCCACCATTCGCTTTGCAAA-3' or
5'-CATGAGCTCTCACTCTTCTGATTT-3' with
5'-GCAGATCTCGAGCTAGCACGCGTAAG-3'). The fragment was then
cloned into the pGL2 vector. pTR89 was made by truncating pGL2-754TR at
the unique BstXI site. pTREBS was made by cutting out the
BsrGI-BstXI fragment from pGL2-754TR.
Gel mobility shift assay.
Conditions for the gel shift assay
were basically as described previously (70). Proteins and
radiolabeled probes were incubated in 10 µl of buffer (25 mM HEPES
[pH 7.9], 50 mM KCl, 5% glycerol, 0.5 mg of bovine serum albumin per
ml, 5 µM zinc sulfate, 0.5 mM dithiothreitol) for 30 min at room
temperature and then loaded onto an 8% polyacrylamide gel prepared
with Tris-glycine buffer. Poly(dI-dC) was used at 500 ng/ml as a
nonspecific competitor. Glutathione S-transferase (GST) and
GST-TEL fusion proteins were purified with glutathione-Sepharose 4B
beads (Pharmacia). Double-stranded consensus ETS factor binding-site
oligonucleotides used for the gel shift assay were (sense strand)
5'-AATTCATAAACAGGAAGTGGCTT-3' (wild type) and
5'-AATTCATAAACACTGAGTGGCTT-3' (mutant).
Oligonucleotides from the stromelysin-1 promoter were as
follows: 213 to 183, 5'-CTAAGGCAGGAAGCATTTCCTGGAGATTAA-3'
(wild type) and 5'-CTAAGGCACTGAGCATTGACTGTCGATTAA-3' (mutant); 111 to 88, 5'-TAATTTTTGGAAATGGTCCCATTT-3';
and 93 to 74, 5'-CCATTTGGATGGAAGCAATT-3'. For the
mutant oligonucleotides from the stromelysin-1 promoter, the
GGA sequences were changed to TCG in each case.
Cell culture, transfection, retroviral infection, RNA analysis,
and cell fractionation.
NIH 3T3 cells were maintained in
Dulbecco's modified Eagle's medium (DMEM; BioWhittaker) supplemented
with 10% calf serum (BioWhittaker), 2 mM L-glutamine
(BioWhittaker), and 10 µg of gentamicin (Sigma) per ml. v-Harvey
Ras-transformed NIH 3T3 cells were the generous gift of Stephen J. Brandt.
Retroviral packaging 
NX cells were maintained in DMEM containing
10% fetal calf serum, 2 mM L-glutamine, and 10 µg of
gentamicin per ml. Plates (10% confluent) were transfected for 6 to
8 h with 25 µl of Lipofectamine reagent (Gibco) and 5 µg of
the pBabePuro retroviral vectors. The medium was replaced 24 h
later, and virus-containing supernatants were harvested at 60 h.
Supernatants were filtered through a 0.45-µm-pore-size acrodisk (VWR)
and added to a plate of NIH 3T3 cells (10% confluent). Ras-expressing
cells were infected with retrovirus to eliminate clonal variations.
Infected cells were split 1:10 48 h later and selected for 72 h with 1 µg of puromycin (Sigma) per ml. Based on the minimal cell
death observed upon selection, we concluded that more than 90% of the
cells were infected. The cells were used within the first 10 passages
after infection, with the critical experiments being performed within the first few passages. However, we observed the phenotypes (e.g., morphology and soft agar) immediately, and they did not change with
cell passage. In addition, we used at least five independent batches of
retrovirus and obtained similar results. The expressed proteins were
detected by immunoblot analysis as described previously (42). stromelysin-1 mRNA was detected by RNA blot
analysis as described previously (1). Affymetrix gene chip
analysis was performed by the Duke University gene analysis core using
poly(A)+ mRNA from NIH 3T3 cells expressing Ras or Ras plus TEL.
Cell photographs were recorded using a Zeiss Axiophot microscope with a
Leaf Lumina digital camera (magnification, ×62.5).
Images were
recorded as TIFF files in Adobe Photoshop. The images
were captured
with the camera in either the forward or back position,
which yielded
subtle differences in
magnification.
For cell fractionation, cell pellets were resuspended in Iso-Hi buffer
(10 mM Tris-HCl [pH 8.4], 140 mM NaCl, 1.5 mM MgCl
2,
complete protease inhibitor [Roche], 1 mM EDTA) containing 0.5%
NP-40. After 5 min on ice, the nuclei were collected by centrifugation
at 2,000 rpm for 5 min in a Beckman GS-6R. Nuclei were reextracted
to
ensure purity. Equal proportions of the supernatant (cytoplasmic)
and
nuclear fractions were analyzed by immunoblot analysis with
mSin3A, TEL
C-terminal, and Ras
antisera.
Transcription assays.
Cells and supernatants were harvested
44 to 48 h posttransfection. The cells were washed twice with
phosphate-buffered saline and lysed in 100 to 200 µl of reporter
lysis buffer (Promega). Aliquots (10 to 80 µl) were assayed for
luciferase activity using the luciferase reagent assay (Promega) as
specified by the manufacturer. Cytomegalovirus immediate early
(CMV)-SEAP (secreted alkaline phosphatase) plasmids were included as
internal controls for transfection efficiency (32, 78). SEAP
activity was quantitated as described previously (5), except
that the incubations were performed at room temperature. Luciferase
activities were then normalized with respect to SEAP activity.
Soft-agar assays.
For the bottom agar, 2.5 ml of 1.6%
agarose was diluted with 2.5 ml of 2× medium (2× DMEM, 20% calf
serum, 4 mM L-glutamine, 20 µg of gentamicin per ml, 2 µg of puromycin per ml). A 4-ml volume of bottom agar was plated in a
60-mm tissue culture dish and allowed to harden. Cells were trypsinized
and resuspended at 1.5 × 105 cells/ml in normal
medium. The top agar cell suspensions were composed of 300 µl of cell
suspension, 3.6 ml of 2× medium, and 2.4 ml of 0.75% agarose.
Aliquots (2 ml) of this mixture were overlaid on each of two dishes
containing bottom agar. The final plating concentration was 1,500 cells
per dish.
Matrigel invasion assay.
Biocoat Matrigel invasion chambers
(Becton Dickinson) were used to assess the invasiveness of TEL-infected
NIH 3T3 cells as described previously (8). Briefly, 5 × 104 cells were resuspended in 100 µl of serum-free
DMEM containing 0.2% bovine serum albumin and added to the cell
culture inserts of the invasion chambers. A chemoattractant, 5 µg of
human fibronectin (Gibco-BRL) per ml, was added to the lower wells. The
cells were incubated at 37°C and allowed to invade through the matrix
and the pores (8 µm) of the attached lower membrane for 48 h.
The filters were fixed by immersion in 1% glutaraldehyde and stained with 0.2% crystal violet. The upper surface of the membrane was cleaned with a cotton swab to remove all noninvasive cells. The stained, invasive cells were then photographed using a digital camera.
Mouse tumor formation assays.
A single-cell suspension of
5 × 104 cells in phosphate-buffered saline was
injected subcutaneously into the right lateral flank of 7-week-old
athymic female nude (nu/nu) mice (Harlan Sprague-Dawley). The mice were housed in microisolator cages, given food and water ad
libitum, and handled in a sterile laminar-flow hood. Tumor sizes were
measured every 3 days using Vernier calipers along two perpendicular
axes. The mice were sacrificed after 14 days. Primary tumors were
excised, fixed overnight in 4% paraformaldehyde in PBS, and stored in
70% ethanol at 4°C. For histologic testing, the tumors were embedded
in paraffin and 5-µm sections were stained with hematoxylin and
eosin. Statistical comparison was performed using an analysis of
variance followed by a one-tailed t test.
| |
RESULTS |
TEL regulates cell growth.
Based on genetic evidence from
various cancers, TEL is postulated to be a tumor suppressor in both
leukemias and solid tumors (12, 54, 64). Because oncogenic
ETS factors are downstream effectors of Ras-signaling pathways
(73, 80), we tested whether TEL could directly inhibit the
growth of Ras-transformed cells. To express TEL but avoid nonspecific
effects of high-level overexpression of ETS domain-containing proteins
and clonal variation, we infected normal and Ras-transformed NIH 3T3
cells with recombinant retroviruses expressing TEL. For each assay in
this study, several different infected-cell populations were used, to
reduce the possibility that the results were affected by viral
integration site effects. As controls, we tested TEL mutants lacking
the N-terminal pointed domain (122 and P) or containing a small
deletion in the ETS domain (TELETS) for their ability to inhibit
Ras-dependent growth. Immunoblot analysis using a C-terminal TEL
antibody confirmed that the mutant proteins were expressed at similar
levels (Fig. 1A). To ensure that the
mutant proteins were appropriately transported into the nucleus, we
performed cell fractionation experiments. Compared to nuclear (mSin3A)
and cytoplasmic (Ras) controls, the majority of the proteins expressed
for each mutant were found in the nuclear fractions (Fig. 1B).
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FIG. 1.
TEL inhibits growth in soft-agar assays. (A) Immunoblot
analysis showing the expression of TEL and TEL mutant proteins in the
stable cell lines used in panel B. The right-hand panel shows the level
of the endogenous (Control) and expressed TEL using anti-N-terminal
TEL. (B) Cellular localization of TEL and TEL mutants. Cells expressing
TEL or the indicated TEL mutants were incubated with 0.5% NP-40 in an
isotonic buffer for 5 min before being subjected to low-speed
centrifugation to collect the nuclei. Equal amounts of cytoplasmic (C)
and nuclear (N) fractions were analyzed by immunoblotting with
antibodies directed to mSin3A as a nuclear protein control (upper
panel), TEL (middle panel), or Ras as a cytoplasmic protein (bottom
panel). (C) TEL inhibits soft-agar colony formation of Ras-transformed
cells. Cells were grown for 10.5 days in medium containing 0.375%
agarose, as overlays on 0.8% agarose beds. A total of 1,500 cells of
the indicated cell lines were plated per dish. Values shown are the
mean and range of duplicate samples.
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One hallmark of the transformed phenotype is the ability of cells to
grow in semisolid medium (
56). Ras-transformed NIH
3T3 cells
grew efficiently in soft agar (Fig.
1C). TEL expression
inhibited the
ability of Ras-transformed NIH 3T3 cells to grow
in an
anchorage-independent manner in these assays (Fig.
1C).
This appeared
to be due to slower growth, since more colonies
became apparent with
longer culture times. Deletion of an mSin3-binding
domain of TEL,
either with a specific internal deletion (
P) or
by truncation of the
N-terminal 122 amino acids, ablated TEL-mediated
growth inhibition
(Fig.
1C). Thus, simple overexpression of the
TEL DNA-binding domain
did not inhibit growth, as observed for
ETS-2 (
21,
34,
39,
75). As a further control, we expressed
a TEL mutant with a
12-amino-acid deletion within the DNA-binding
domain.
Unexpectedly, the TEL
ETS mutant cooperated with Ras to
stimulate
growth in soft agar (Fig.
1C). Because TEL forms homodimers
(
35,
44), it is possible that this mutant acts as an
inhibitory
protein to block endogenous TEL functions. In addition, TEL
inhibited
the growth of Ras-transformed Rat 1A cells in soft agar (data
not
shown).
To determine whether TEL could also influence anchorage-dependent cell
growth, the growth rates of normally cultured cells
were determined.
TEL did not affect the growth of control NIH
3T3 cells in culture (Fig.
2A). However, TEL did slow the growth
of
Ras-transformed NIH 3T3 cells (Fig.
2B). As observed in the
soft-agar
colony assays, TEL
ETS cooperated with Ras to cause
Ras-expressing
cells to grow faster (Fig.
2B). Although TEL slowed
the growth of
Ras-transformed cells, there were no signs of apoptosis
under normal
growth conditions, as determined by morphology or
by propidium iodide
staining of DNA (data not shown). Also, TEL
expression did not
dramatically disrupt a specific cell cycle
phase as judged by DNA
content analysis (data not shown). However,
it is possible that the
TEL-expressing cells were cycling more
slowly but with a proportional
lengthening of each phase of the
cell cycle.
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FIG. 2.
TEL inhibits cell growth. A total of 8 × 105 cells of each cell line were plated in 100-mm tissue
culture dishes containing 20 ml of medium. Cells were counted every
24 h for 3 days. Values shown are the mean and standard deviation
of quadruplicate samples (A) and the mean and range of duplicate
samples (B).
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TEL causes Ras-transformed NIH 3T3 cells to grow as
aggregates.
In culturing the TEL-expressing cells, it was readily
apparent that TEL was affecting the morphology of these
cells. At lower densities (i.e., subconfluent growth),
Ras-transformed NIH 3T3 cells grew as monolayers (Fig.
3). TEL did not appear to have any
morphological effect on wild-type NIH 3T3 cells (data not shown). By
contrast, enforced expression of TEL in Ras-transformed NIH 3T3 cells
resulted in an aggregation of these cells under subconfluent conditions
(Fig. 3). As Ras-plus TEL-expressing (Fig. 3, Ras+TEL) cells continued
to grow, most of them piled up rather than spread over the plate.
Distinct lanes of cells could also be seen bridging the large clusters
of cells (Fig. 3, Ras+TEL LP). In many cases, these connecting strands
consisted of a string of single cells oriented end to end. With
extended culturing times, the aggregates grew into large
"spheroids" that remained attached to the plate only at the initial
point of cell aggregation (data not shown). Unlike control
Ras-transformed cells, Ras-plus TEL-expressing cells did not fill the
culture dish and did not form transformed "foci" (data not shown).
This effect was specific to wild-type TEL, since deletion of the
pointed domain or the small deletion in the DNA-binding domain of TEL
abolished the phenotype (Fig. 3).
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FIG. 3.
TEL causes aggregation of Ras-transformed NIH 3T3 cells.
Ras-transformed NIH 3T3 cells infected with recombinant retroviruses
expressing TEL and the indicated TEL mutants were plated and allowed to
grow for 72 h. Cells were visualized by bright-field microscopy
(magnification, ×62.5) and recorded as digital images using Adobe
Photoshop. The panel labeled Ras+TEL (LP) contains cell photographed at
threefold-lower power (LP, lower power).
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TEL represses transcription of the stromelysin-1
gene.
The phenotype caused by TEL overexpression could be due to
alterations in cell-cell or cell-ECM adhesion or to a disruption of
cell migration due to changes in the signaling pathways that regulate
these processes. Because of the large number of genes involved in these
processes that could be directly or indirectly affected by TEL, we
performed microarray analysis to define changes in gene expression. RNA
extracted from NIH 3T3 cells expressing Ras and from cells expressing
Ras plus TEL were compared using Affymetrix gene chip arrays. However,
of the nearly 50 genes whose expression was significantly altered, none
are directly involved in cell adhesion or migration (data not shown).
Because microarray analysis may miss changes in gene expression when
genes are expressed at low levels, we asked whether genes that are
involved in cell adhesion and that are regulated by Ras and oncogenic
ETS factors were repressed by TEL (73). MMPs are stimulated
by Ras through the activation of ETS and AP-1 factors and repressed by
negatively acting ETS factors such as Erg (15, 16, 29, 33, 59, 80). RNA blot analysis indicated that the levels of the MMPs expressed in NIH 3T3 cells, including MMP1, MMP7, and MMP9, were low in
these cells and were not affected by TEL (data not shown). While the
levels of stromelysin-1 (MMP3) mRNA were also low
in control NIH 3T3 cells, these levels were further suppressed by expression of TEL (Fig. 4A, middle
panel). As expected, expression of oncogenic Ras increased
stromelysin-1 mRNA levels (Fig. 4A). However, coexpression
of TEL with Ras reduced the amounts of stromelysin-1 mRNA
expressed, whereas TEL mutants that lack an mSin3A binding site failed
to repress stromelysin-1 (Fig. 4A). Consistent with the
ability of the TELETS mutant to cooperate with Ras to stimulate growth, this mutant cooperated with Ras to increase the amount of
stromelysin-1 transcript (Fig. 4A). Thus, TEL regulates the level of stromelysin-1 mRNA.
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FIG. 4.
TEL inhibits transcription from the
stromelysin-1 promoter. (A) Total cellular RNA was isolated
from the cell lines described in Fig. 1. Samples (40 µg) were
electrophoresed through a 2.5 M formaldehyde-1% agarose gel. (Upper
panel) The RNA was transferred to a nylon membrane, UV cross-linked,
and probed with a radiolabeled stromelysin-1 cDNA. (Lower
panel) RNA stained with ethidium bromide. The third panel (left side)
contains the same blot probed for actin expression. (B) NIH 3T3 cells
(left panel) or Ras-expressing NIH 3T3 cells (right panel) were
transfected with 1 µg of the rat stromelysin-1 luciferase
plasmid and 100 ng of the pCMV-TEL expression constructs shown.
Luciferase activity was determined 44 h later. Values were
normalized for transfection efficiency by using CMV-SEAP activity as an
internal control. Values shown are the fold repression (mean and range)
of duplicate samples.
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To determine whether TEL directly represses the
stromelysin-1 promoter, we tested whether TEL could repress
expression from
a plasmid containing
stromelysin-1
regulatory sequences linked
to a luciferase reporter gene. As little as
20 ng of TEL-expressing
plasmid repressed transcription from the rat
stromelysin-1 promoter
by more than 15-fold in NIH 3T3 cells
(Fig.
4B). This inhibition
was specific, since TEL had no effect on the
CMV promoter used
as an internal control in these experiments. TEL also
failed to
repress the ETS factor-regulated multidrug resistance gene 1 (
MDR-1)
promoter (reference
41 and data
not shown). In addition, mutations
of an mSin3-binding domain (
122
and
P) or the DNA-binding domain
(TEL
ETS) impaired TEL-mediated
repression (Fig.
4B, left panel).
However, at 10-fold-higher levels of
input TEL
P expression plasmid,
we did observe two- to threefold
repression, suggesting competition
for DNA-binding sites with
endogenous ETS factors (data not shown).
When these experiments were
repeated in Ras-expressing cells,
the basal levels of expression from
the
stromelysin-1 promoter
were higher (data not shown) and
TEL-mediated repression was even
more dramatic (Fig.
4B, right
panel).
To define the sequences in the
stromelysin-1 promoter that
are required for TEL-mediated repression, a deletion analysis was
performed (Fig.
5A). Deletion of over 400 bp upstream of a previously
mapped ETS factor-binding site (
3,
10,
11,
16) had no
effect on repression, since the
334 construct
was repressed by
over 15-fold (Fig.
5A and B). However, deletion to
nucleotide
182, removing the known ETS-binding site, reduced
repression
to only five- to sixfold (Fig.
5B, TR182), indicating that
these
sequences contributed to TEL-mediated repression. TEL-mediated
repression was significantly impaired by deletion to residue
89
(Fig.
5B, TR89). In addition, an internal deletion of sequences
from
334 to
89 was only marginally responsive to repression
by TEL (Fig.
5B,
TR
EBS). Although both of these latter deletions
reduced the basal
expression from the promoter, each promoter
was still active, thus
allowing us to determine the effects of
TEL.
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FIG. 5.
Mapping TEL-responsive elements in the
stromelysin-1 promoter. (A) Schematic diagram of the
stromelysin-1 promoter and deletion mutants used in panel B. The known ETS factor-binding site is labeled EBS, and a putative
TEL-binding site is labeled TBS. (B) Deletion analysis of the
stromelysin-1 promoter. The ability of TEL to repress the
full-length stromelysin-1 promoter and the deletion mutants
shown in panel A is demonstrated. RLU, relative luciferase activity.
Assays were performed with NIH 3T3 cells, as in Fig. 4, using 20 ng
(left panel) or 100 ng (right panel) of TEL-expressing plasmid. (C) Gel
mobility shift assays using GST-TEL and a consensus ETS factor-binding
site as the probe or the TEL-binding site (111 to 88) from the
stromelysin-1 promoter as the probe and bacterially produced
GST-TEL DNA-binding domain fusion protein (right panel).
Oligonucleotides used for competition are designated above each lane
(205, 213 to 183; 93, 93 to 74). The sequences of these
oligonucleotides are provided in Materials and Methods. W, wild type;
M, mutant. The numbers above the lanes in the left panel are the
amounts of the wild-type competitor added to each sample.
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Within the region from
182 to
89, there are two putative ETS
factor-binding sites at
103 and
95 (labeled TBS in Fig.
5A).
Therefore, we deleted sequences upstream of residue
127 to further
isolate the second TEL-responsive sequence. The
127 promoter
was
nearly as active as the full-length
754 promoter, and it
was
repressed five- to sixfold by TEL expression (Fig.
5B, right
panel).
Once again, the
89 promoter was only weakly inhibited
by TEL. Thus,
two regions of the
stromelysin-1 promoter that contain
putative ETS factor-binding sites are responsive to
TEL.
To determine whether TEL could directly interact with the
TEL-responsive sites in the
stromelysin-1 promoter, we asked
if
TEL could bind these sequences in gel mobility shift assays. A
GST-TEL DNA-binding domain fusion protein formed a specific complex
with an oligonucleotide encompassing the two ETS factor-binding
sites
(GGAA at residue
103 and TCC at residue
95; the probe
contains
residues
111 to
88 [Fig.
5C]). This complex was competed
by
oligonucleotides containing a wild-type but not a mutant consensus
ETS
factor-binding site that TEL recognizes (
52,
53,
77)
(Fig.
5C). Wild-type double-stranded oligonucleotides containing
the known
ETS-binding sites from the
stromelysin-1 promoter (at
nucleotides
205 and
196) also competed for TEL binding, whereas
oligonucleotides containing mutations in the GGA sequences did
not
compete (Fig.
5C, lanes
205 W and
205 M). Although the TR89
promoter was slightly repressed at higher levels of input TEL
expression plasmid (Fig.
5B, right panel), the two GGA putative
ETS
factor-binding sites that remain in this promoter failed to
compete for
TEL binding (nucleotides
93 to
74, labeled
93 in
Fig.
5C).
However, we cannot rule out the possibility that TEL
binds these
sequences weakly to mediate the reduction in expression
observed in
Fig.
5B. Therefore, TEL can bind at least two sites
in the
stromelysin-1 promoter.
In several of the assays described above, the expression of TEL
ETS
had the opposite effect to the expression of TEL. Because
NIH 3T3 cells
express endogenous TEL, TEL
ETS might act to inhibit
endogenous TEL
activity or might sequester or titrate a TEL cofactor.
However, in
these transient-expression assays, the ratio of reporter
plasmid to
endogenous TEL is likely to be too high for the endogenous
TEL to
repress transcription and thus allow TEL
ETS to "activate"
transcription by blocking its action. Consequently, we asked whether
TEL
ETS could inhibit TEL function when TEL was expressed exogenously
in the transcription assays (Fig.
6). An
equimolar amount of TEL
ETS
plasmid was sufficient to block
TEL-mediated repression, but the
mutant did not further activate
transcription. Taken together
with the biological data (Fig.
1 to
3),
these results suggest
that TEL
ETS can act as an inhibitor of TEL
function.
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 6.
TELETS is an inhibitor of TEL-mediated repression.
NIH 3T3 cells were transfected with the rat stromelysin-1
luciferase plasmid and the pCMV-TEL or pCMV-TELETS expression
constructs shown. The numbers below the graph are the amounts of each
expression plasmid transfected. Luciferase activity was determined
44 h later. Values were normalized for transfection efficiency
using CMV-SEAP as an internal control. Values shown are the mean and
standard deviation of triplicate samples. Fold repression represents
the promoter activity from cells transfected with expression plasmids
compared to those transfected with the empty vector.
|
|
An MMP inhibitor yields the same phenotype as TEL expression.
Although we thought it unlikely that a single target gene could result
in the observed TEL-induced aggregation phenotype, MMPs are critical
regulators of cell adhesion and metastasis (14). Therefore,
we used a specific MMP inhibitor, BB-94 (67), to determine
whether inhibition of MMPs is important for TEL-mediated cellular
aggregation. BB-94 (2 µM) was added to the culture medium at the time
when the cells were plated, and the cells were allowed to grow for 3 days. BB-94 had no effect on NIH 3T3 morphology (data not shown). By
contrast, when added to the culture medium of Ras-transformed NIH 3T3
cells, BB-94 caused the cells to grow in clusters (Fig.
7), with lanes of cells extending from
one cluster to another. This morphology was similar to that observed
when TEL was expressed (Fig. 3).
View larger version (79K):
[in this window]
[in a new window]
|
FIG. 7.
An MMP inhibitor produces a phenotype similar to TEL
expression. Ras-transformed NIH 3T3 cells were cultured in the presence
or absence of BB-94, a specific MMP inhibitor (67). Cells
were grown for 72 h and photographed as described in Materials and
Methods.
|
|
TEL inhibits Ras-dependent tumor cell invasion.
MMPs are
critical regulators of cell migration and adhesion. For tumor growth,
MMPs are required for angiogenesis and for invasion of surrounding
tissue (other than adipose tissue). Therefore, we asked whether TEL
expression in Ras-transformed cells could inhibit tumor cell invasion
in vitro and in vivo. First, we compared the ability of cells to
invade through a reconstituted three-dimensional basement membrane gel
(Matrigel) (45). Control NIH 3T3 cells or TEL-expressing
cells invaded the reconstituted matrix only poorly (Fig.
8 upper panel). Cells expressing
oncogenic Ras or cells expressing Ras and TELETS, TELP, or
TEL122 invaded through and adhered to the underside of the
polycarbonate filter (the invading cells are indicated as dark regions
in these stained cultures in Fig. 8). By contrast, when TEL was
coexpressed with Ras, it inhibited the ability of Ras-transformed cells
to invade the three-dimensional matrix (Fig. 8).
View larger version (79K):
[in this window]
[in a new window]
|
FIG. 8.
TEL inhibits Ras-dependent cell invasion in vitro. NIH
3T3 cells (control) or cells expressing TEL, Ras, Ras plus TEL, or Ras
plus the indicated TEL mutants (lower panel) were cultured in Matrigel
chambers for 3 days using fibronectin in the lower chamber as a
chemoattractant. The filters were fixed by immersion in 1%
glutaraldehyde and then stained with 0.2% crystal violet. The upper
surface of the membrane was cleaned with a cotton swab to remove all
noninvasive cells. The stained, invasive cells were then photographed
using a digital camera. The two rows are duplicate samples.
|
|
Based on these in vitro results, we injected Ras-expressing NIH 3T3
cells and Ras-plus TEL-expressing cells into nude mice
to determine if
TEL could inhibit tumor invasion in vivo. The
Ras-transformed cells
formed tumors readily, and nearly every
tumor invaded the surrounding
muscle tissue (Table
1), as determined
by
gross morphology and by histologic analysis (data not shown).
In
general, the Ras-plus TEL-expressing cells formed somewhat
smaller
tumors (Table
1). However, when tumors of similar size
were compared,
the TEL-expressing tumors failed to invade the
surrounding muscle. At
necropsy, three of the TEL-expressing tumors
showed the first signs of
invasion and were scored as partially
positive (Table
1). This small
number of partially invading tumors
could be due to our use of
populations of cells that express different
amounts of TEL rather than
using single-cell clones expressing
only large amounts of TEL. However,
we conclude that TEL inhibits
Ras-mediated tumor cell invasion in vitro
and in vivo.
| |
DISCUSSION |
Loss of heterozygosity in tumor samples suggests that TEL acts as
a tumor suppressor in both leukemias and solid tumors. We have provided
biochemical and biological evidence that supports this hypothesis.
Expression of TEL inhibited the growth of Ras-transformed cells in soft
agar and slowed their growth in normal cultures (Fig. 1 and 2). The
ability of TEL to induce cell aggregation suggested that TEL-mediated
growth inhibition may be due to alterations in cell adhesion (Fig. 3).
Analysis of stromelysin-1 mRNA levels and the ability of TEL
to regulate the stromelysin-1 promoter suggested that TEL
acted as a transcriptional repressor to regulate MMP levels. The
aggregation phenotype appeared to be partially due to the repression of
stromelysin-1 because an MMP inhibitor also caused these
cells to grow as aggregates (although the phenotype was not identical
to that of TEL-expressing cells [compare Fig. 2 and 7]). Finally, TEL
was shown to inhibit tumor invasion. Therefore, we propose that at
least a portion of the action of TEL as a tumor suppressor is to
regulate cell growth and tumor cell invasion by repressing target genes
such as stromelysin-1.
The repression of both the stromelysin-1 promoter and the
endogenous gene suggests that stromelysin-1 is a direct
target of TEL-mediated repression. TEL binds a canonical ETS
factor-binding site (52, 77), and it also binds the
previously identified consensus ETS-binding site and at least one other
site in the stromelysin-1 promoter (Fig. 5). Our
promoter analysis identified two regions containing TEL-binding sites
as being important for TEL-mediated repression. Although a link between
modulation of stromelysin-1 transcription and Ras-dependent
transformation has been established by expressing wild-type Ets-2 or
dominant inhibitory mutants of ETS factors (21, 34, 39, 75),
the morphological phenotype associated with TEL expression is distinct.
Although the morphological effects of an MMP inhibitor are similar to
the effects observed with TEL, TEL probably represses the transcription of other genes involved with growth control.
When an mSin3-binding domain of TEL was deleted, TEL was
transcriptionally and biologically inactivated (Fig. 1 to 4 and 8). Given that these TEL mutants retain the ETS domain, the biological effects of TEL are due to active repression and not competition for DNA
binding with endogenous ETS factors. This is in contrast to the effects
of a dominant inhibitor of ETS-2 or the expression of isolated ETS
factor-binding domains, which competed for ETS factor-binding sites to
inhibit Ras-dependent transformation (21, 34, 39, 75).
Interestingly, the TEL mutants lacking an mSin3A-binding motif retain a
domain capable of interacting with the SMRT corepressor (13). However, in transcription assays, the mSin3A-binding
mutant failed to repress transcription (Fig. 4), suggesting that
multiple corepressor contacts are required for TEL-mediated repression.
Although the TELETS mutant was designed to be a nonfunctional
protein, its overexpression had some physiological consequences. When
expressed alone, TELETS had little or no effect on cell aggregation,
cell growth, or transcriptional regulation of stromelysin-1. However, in conjunction with Ras, TELETS stimulated cell growth both
in exponentially growing cultures and in soft-agar assays. TELETS
also cooperated with oncogenic Ras to activate stromelysin-1 expression. The effects of this mutant on cell growth but not cell
adhesion suggest that TEL also regulates growth independently of its
effects on cell adhesion. We will need to define other TEL target genes
to fully understand how TEL regulates these complex cell phenotypes.
In chimeric mice derived using TEL/ embryonic stem
cells, the TEL-null cells failed to contribute to the developing bone
marrow, possibly due to a defect in stem cell migration or adhesion
(72). In NIH 3T3 cells, TEL altered the morphology of the
cells, possibly by affecting cell adhesion (Fig. 3 and 7). The results
obtained with these two experimental systems suggest that the
regulation of stromelysin-1 levels by TEL may be important
during development. In addition, TEL null mice die in utero at E10.5
due to defects in angiogenesis and MMPs are critical for the tissue
remodeling required for angiogenesis (6, 14). Tumors formed
by injecting Ras-plus TEL-expressing cells into nude mice (Table 1)
appeared to have less vasculature than did tumors derived from
Ras-transformed cells, but this was difficult to quantitate (data not
shown). Further work must be performed to establish a direct role for TEL in regulating angiogenesis.
There are several precedents for tumor suppressors that regulate cell
adhesion. Perhaps the best characterized is E-cadherin. E-cadherin
mediates cell-cell interactions and is frequently deleted in metastatic
tumors (4, 22, 69). In fact, altering the expression of
stromelysin-1 changed the proteolytic cleavage of E-cadherin
(40). The observation that TEL can inhibit tumor invasion
(Fig. 8 and Table 1) may suggest that TEL acts at a late stage of tumor
development and that its loss may be associated with metastasis. In at
least one case, loss of TEL was a late event in the progression of a
t(12;21)-containing leukemia (36). For leukemogenesis, the
ability of immature hematopoietic progenitors to survive in the absence
of stromal cell contacts is critical (7, 68). It is possible
that loss of TEL may promote leukemogenesis by affecting cell growth
and/or by altering cell adhesion. Future investigations will be
necessary to identify additional targets of TEL that may directly or
indirectly affect cell adhesion and growth.
| |
ACKNOWLEDGMENTS |
Randy Fenrick and Lilin Wang contributed equally to this paper.
Peter Brown, British Biotech, Ltd., Oxford, England, kindly supplied
the BB-94. We also thank Helena Abushamaa, Duke University Genome Core
Facility, for assistance with the Affymetrix GeneChip analysis. We
thank Yue Hou and Jonathon Sheehan for technical assistance and members
of the Hiebert, Kinch, and Matrisian laboratories for helpful discussions.
The experiments described here were performed in part through the
use of the VUMC Cell Imaging Resource and the VCC sequencing facility,
which are supported by NIH grants CA68465 and DK20593.R01. This
work was also supported by NIH/NCI grants RO-1 CA46843 (to L.M.M.) and
RO1-CA64140 and RO1-CA77274 (to S.W.H.), by American Cancer Society
grants JFRA-591 (to S.W.H.) and RPG CSM-86522 (to M.S.K.), by a Center
grant from the National Cancer Institute (CA68485), and by the
Vanderbilt Cancer Center. R.F. and J.A. were funded by NIH training
grant T32 DK07186-22. J.N. is a Special Fellow of the Leukemia Society
of America (3827-99).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Rm 512 Medical Research Building II, Nashville, TN
37232. Phone: (615) 936-3582. Fax: (615) 936-1790. E-mail: scott.hiebert{at}mcmail.vanderbilt.edu.
| |
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Molecular and Cellular Biology, August 2000, p. 5828-5839, Vol. 20, No. 16
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Abstract
Introduction
