From the Department of Biochemistry and Molecular
Biology, Medical College of Ohio, Toledo, Ohio 43699-0008, the
Department of Molecular Genetics, Oncology Drug Discovery, Bristol
Myers Squibb, Princeton, New Jersey 08543-4000, the
Department of Medicine, Case Western Reserve
University/Ireland Cancer Center, Case Western Reserve University,
Cleveland, Ohio 44106, and the §§ Department of
Stomatology, University of California, San
Francisco, California 94143
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ABSTRACT |
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Autocrine transforming growth factor (TGF
)
is an important positive growth effector in malignant cells and plays a
significant role in generating the growth factor-independent phenotype
associated with malignant progression. However, the molecular
mechanisms by which TGF
confers a growth advantage in progression is
poorly understood. The highly tumorigenic cell line HCT116 up-regulates TGF
mRNA expression during growth arrest, whereas the poorly tumorigenic growth factor-dependent FET cell line down-regulates TGF
mRNA expression as it becomes quiescent. We have identified a 25-bp
sequence at
201 to
225 within the TGF
promoter which mediates
the differential regulation of TGF
expression during quiescence
establishment in these two cell lines. This same sequence confers
TGF
promoter responsiveness to exogenous growth factor or autocrine
TGF
. The abberant upregulation of TGF
mRNA in quiescent HCT116
cells may allow them to return to the dividing state under more
stringent conditions (nutrient replenishment alone) then quiescent FET
cells (requires nutrients and growth factors). Antisense TGF
approaches showed that the dysregulated TGF
expression in quiescent
HCT116 cells is a function of the strong TGF
autocrine loop (not
inhibited by blocking antibodies) in these cells.
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INTRODUCTION |
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Malignant progression is often associated with the loss of
dependence on exogenous growth factors for growth control. The production and utilization of endogenous autocrine growth factors underlies the development of autonomous growth (1-3). Transforming growth factor (TGF
)1
is a positive autocrine growth factor in colon carcinoma (4-7) and
contributes to malignant progression of colonic tumors (8). TGF
is a
member of the epidermal growth factor (EGF) family, and like EGF,
exerts its effects via interaction with the epidermal growth factor
receptor (EGFR) (9, 10). It is produced as a transmembrane precursor
which is cleaved to a 50-amino acid active peptide (11, 12). However,
larger forms are secreted from transformed cells (12, 13) and the
transmembrane form also appears to be active (14-16). TGF
was
subsequently shown to be an autocrine growth factor in normal cells
(17) but transformed cell lines appear to produce greater amounts of
the peptide than their normal counterparts (18, 19).
Previous studies in this laboratory have identified two distinct
phenotypes within our colon carcinoma cell lines which differ with
respect to tumorigenicity, growth factor dependence, and the
utilization of TGF (4, 5, 20). Well differentiated, poorly
tumorigenic FET cells express both TGF
and its cognate receptor EGFR
and are growth inhibited by antibodies to EGFR. However, FET cells
require exogenous EGF as well as transferrin and insulin for both
optimal growth and DNA synthesis. They are, therefore, growth
factor-dependent. This cell line expresses a classical
TGF
autocrine loop in which mature, proteolytically released ligand
interacts with EGFR on the cell surface. In contrast, HCT116 cells are
poorly differentiated and highly tumorigenic. While this cell line
expresses both TGF
and EGFR, it is not growth inhibited by blocking
antibodies to the EGFR and is growth factor independent as it does not
require any exogenous growth factors for DNA synthesis and growth (21,
22). However, transfection of a TGF
antisense expressing vector
results in dependence upon exogenous EGF for growth (20). Therefore,
the HCT116 cell line exhibits a highly active TGF
autocrine loop
which is sequestered from inhibition by extracellular blocking agents.
Both of these cell lines are typical members of previously documented
groups of colon carcinoma cell lines which have been subclassified on the basis of these growth regulatory phenotypes (5).
Further differences in TGF regulation between the FET and HCT116
cell lines become apparent when the cells are rendered quiescent (4, 5,
22). Both cell lines express equivalent levels of TGF
mRNA
during logarithmic growth (22). However, the FET cell line
down-regulates TGF
mRNA as it enters quiescence (22). It
requires both nutrients and growth factors for subsequent release through the cell cycle (4, 5, 22). In contrast, HCT116 cells
up-regulate TGF
mRNA as they become quiescent, and nutrient replenishment alone is sufficient for release from quiescence further
emphasizing the growth factor independence of these cells (4, 5, 22).
The underlying mechanism by which endogenous TGF
confers growth
factor independence, and so growth advantage, to the malignant
phenotype appears to be via enhanced movement of non-dividing,
quiescent cells back into the cell cycle due to inappropriate
expression rather than TGF
mRNA overexpression in logarithmic
phase growth. In support of this hypothesis, overexpression of TGF
in poorly tumorigenic colon carcinoma cells by stable transfection of a
constitutively active sense TGF
expression vector generates a more
tumorigenic phenotype, which shows enhanced clonal initiation and a
decreased lag phase rather than an altered exponential growth phase
doubling time (8). Therefore, the increased expression of TGF
in
quiescent HCT116 cells appears to "prime" them for re-entry into
the cell cycle in a less favorable, growth factor-deficient milieu than
FET cells, thereby imparting them with a growth advantage in limiting
growth conditions.
To understand the mechanisms regulating TGF expression in both the
growth factor-dependent and growth factor-independent state
of progression, we have studied TGF
promoter activity in quiescent
FET and HCT116 cell lines. We hypothesized that if TGF
autocrine
function plays a role in regulating TGF
promoter activity, then the
changes in endogenous TGF
expression during the acquisition of
quiescence should be reflected by similar changes in TGF
promoter activity, particularly as cells are not exposed to any exogenous growth
factors during the establishment of quiescence. We demonstrate that
regulation of transcriptional activation of the TGF
promoter in both
HCT116 and FET cells is an integral part of the differential expression
of TGF
in the HCT116 and FET cell lines during growth arrest and
quiescence. Moreover, this transcriptional regulation maps to a unique
25-bp element which also confers EGF/TGF
autoregulation on the
TGF
promoter. HCT116 cells with repressed TGF
expression following TGF
antisense expression become growth
factor-dependent (20). We now show TGF
expression is not
up-regulated during the transition from exponential growth to
quiescence in the TGF
antisense transfected cells, further
implicating TGF
autocrine function in the regulation of TGF
expression.
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MATERIALS AND METHODS |
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Cell Culture-- The FET and HCT116 cell lines are routinely maintained in a defined serum-free medium containing insulin (20 µg/ml, Sigma) transferrin (4 µg/ml; Sigma), and EGF (10 ng/ml; R + D Systems) as described previously (4, 5, 21). To render the cell lines quiescent, cells were grown to confluence and then changed to McCoy's medium without growth factors. The FET cells were deprived of further nutrients and growth factors for 6 days and the HCT116 cells for 5 days as described previously (22).
Cloning of the TGF Promoter--
A normal human leukocyte
genomic library (CLONTECH) was screened by plaque
hybridization using a probe consisting of a portion of the 5'-region of
the TGF
coding sequence (+104 to +129). A 2.8-kilobase
PstI-SnaBI fragment containing the TGF
promoter along with the first exon of TGF
was isolated (23, 24).
Following Bal3I exonuclease digestion to nucleotide
4 (to
remove the TGF
coding sequence), the resulting fragment was cloned
upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene
in the vector pGCAT, to give the largest construct designated
p2813-CAT. By use of appropriate restriction sites or polymerase chain
reaction, various deletion fragments of the TGF
promoter were
generated. These constructs are designated by the total number of base
pairs of TGF
promoter which they contain, e.g. p370-CAT,
p201-CAT.
Heterologous Constructs--
Heterologous constructs containing
the regions of interest of the TGF promoter were also generated.
Double-stranded oligomers spanning the sequences were synthesized with
BamHI "sticky ends" and cloned just upstream of the
heterologous thymidine kinase promoter in the pBLCAT2 vector.
Isolation of Stable Transfectants--
FET or HCT116 cells
(70-80% confluent) were harvested by trypsinization, washed once in
serum-free medium, and resuspended at 2 × 107 and
2 × 108 cells per 800 µl of serum-free medium,
respectively, and transferred to a 0.4-cm electrogap cuvette. Cells
were electroporated at 250 volts and 960 microfarads in a Bio-Rad gene
pulser with 30 µg of vector and 5 µg of an SV-40-Neomycin (NEO)
plasmid and plated at 1:20 to 1:50 dilutions. At 2 days following
plating, cells were switched to medium containing 600 µg/ml active
Geneticin (G418; Life Technologies, Inc.) to allow selection for NEO
resistant clones. These clones were expanded and tested for CAT
expression. The development of the TGF antisense clone (clone U) has
been described previously (20). Briefly, HCT116 cells were transfected with a vector containing 930 bp of TGF
cDNA in the antisense orientation relative to the cytomegalovirus (CMV) promoter. The plasmid
also contained a NEO selection marker to allow cloning in
Geneticin.
Transient Transfection Studies--
For the transient
transfection studies in FET cells, the TGF promoter-CAT constructs
(30 µg) were also transfected by electroporation into FET cells
maintained in serum-free medium minus EGF. A Rous-Sarcoma virus-driven
-galactoside vector (10 µg) was co-transfected to allow
normalization for transfection efficiency. Control and EGF-treated cells (6 h) were harvested at 30-48 h following transfection for CAT
assay.
CAT Assays--
Following 3 washes in phosphate-buffered saline,
cells were harvested in 1 ml of TEN buffer (40 mM Tris, pH
7.4, 1 mM EDTA, and 150 mM NaCl). After
centrifugation, cells were resuspended in 100 µl of 250 mM Tris-HCl, pH 7.8, containing 15% glycerol (v/v) and
lysed by three cycles of freeze-thawing. Protein concentration in the
cleared lysates was determined by microassay using BCA reagent
(Pierce). The -galactosidase activity was determined by following
the Promega protocol. The CAT assay was performed essentially as
described (25) but with the acetyl-CoA present at a concentration of
3.6 mM in the final reaction.
RNA Preparation and RNase Protection Assay--
Total RNA was
isolated using a cesium trifluoroacetic acid gradient (Pharmacia; Ref.
26). High specific activity TGF riboprobe was synthesized by Sp6-RNA
polymerase in the presence of [32P]UTP (3000 Ci/mmol; NEN
Life Science Products Inc.) from the 306-bp
EcoRI-SphI fragment of TGF
cDNA cloned
into the pGEM 3Z(
) vector (Promega; Ref. 20). Normalization of
loading was determined by simultaneous hybridization with an actin
probe which yields a 145-bp protected fragment when hybridized with
human actin (27). Both RNase protection and CAT assay data was
quantitated using the Molecular Analyst program (Bio-Rad).
Gel Retardation Assay--
Nuclei were prepared from log phase
and quiescent cells by hypotonic lysis (28). Nuclear proteins were
salt-extracted and concentrated following dialysis (29). Aliquots were
snap-frozen in liquid N2 and stored at 80 °C.
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RESULTS |
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Growth factor-independent HCT116 cells showed different regulation
of TGF mRNA from FET cells during the establishment of quiescence. HCT116 cells up-regulated TGF
mRNA during the
transition from exponential phase to the quiescent state, whereas FET
cells down-regulated TGF
mRNA upon entering quiescence (Fig.
1). The level of TGF
mRNA is
increased ~4-fold in the quiescent state compared with exponential
phase HCT116 cells (Fig. 1A). FET cells show a 2-3-fold
decrease in the level of TGF
mRNA in quiescent cells compared
with exponential phase cells (Fig. 1B). It should be noted
that the FET gel is overexposed relative to the HCT116 gel to emphasize
the decreased TGF
mRNA level, as these cells become
quiescent.
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The TGF mRNA level is approximately equivalent in these two cell
lines during exponential growth (22) but these cell lines differentially regulate TGF
mRNA expression during the
acquisition of quiescence. To begin to understand the mechanism
underlying this differential regulation, we next studied whether the
alterations in TGF
mRNA expression were transcriptionally
regulated. The HCT116 and FET cell lines were each stably transfected
with a TGF
promoter-reporter construct, p2813-CAT and several clones were isolated. Fig. 2 shows a typical
result with clones designated HCT116-8 and Fet 3. The cells were grown
in serum-free medium and harvested during exponential phase, at
confluence, and at various times after removal of growth factors for
induction of quiescence to assay for CAT activity as a function of
growth state. The CAT activity of the p2813-CAT construct in the
HCT116-8 clone progressively increases from exponential phase to
quiescence (starve day 6; approximately 10-15-fold increase), whereas
the CAT activity in FET-3 is decreased approximately 50% by starve day
6 (quiescence). Similar results were obtained with different clones of
the FET and HCT116 cells confirming that these results are not an
integration site effect. Therefore, it appears that transcriptional
regulation of the TGF
promoter does indeed play a role in
determining the level of TGF
expression as cells are rendered
quiescent. However, in the case of HCT116, the increase in promoter
activity is greater than the overall increase in the level of TGF
mRNA expression. This high level of CAT activity may reflect
accumulation of the relatively stable CAT protein over the course of
the assay. Alternatively, post-transcriptional mechanisms may also be
involved in the regulation of the TGF
mRNA in addition to
transcription in the quiescent HCT116 cells.
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To determine how much 5'-flanking sequence of the TGF promoter was
required for regulating TGF
transcription during the acquisition of
quiescence, additional stable clones were made. Stable clones of both
FET and HCT116 cells transfected with the TGF
promoter deletion
constructs p370-CAT and p201-CAT were isolated and several clones with
CAT activity identified. It is necessary to use stable clones rather
than transient transfections for these experiments because of the
length of time to complete the experiment (13-14 days) and because any
media changes in transient transfection protocols would disrupt the
establishment of quiescence. These clones were then made quiescent and
the CAT activity was assayed during the
transition from exponential phase to quiescence. Typical results are
shown in Figs. 3 and 4. The p370-CAT
construct showed increasing CAT activity as HCT116 cells were rendered
quiescent (Fig. 3A). By day 5 of quiescent induction, the
CAT activity was increased 7.4-fold compared with log phase. Thus the
activity of the p370-CAT construct, like the full-length p2813-CAT
construct, paralleled the changes in the level of endogenous TGF
mRNA during the transition from exponential growth to quiescence in
HCT116 cells. In contrast, the transcriptional activity of the TGF
deletion construct p201-CAT, which is very low, did not change during
growth from exponential phase to quiescence (Fig. 3B). In
Fig. 3C, results from several different clones are
presented. These results indicate that the major region of the TGF
promoter involved in up-regulation of TGF
expression during the
acquisition of quiescence in HCT116 cells lies between
370 and
201
of the TGF
5'-flanking DNA sequence.
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We next investigated whether this same region was involved in the
apparent down-regulation of TGF expression in FET cells rendered
quiescent. Fig. 4A shows a typical experiment in which the
CAT activity of the p370-CAT construct was measured in an FET clone
from exponential phase to quiescence. The CAT activity showed a
3-4-fold decrease as the cells became quiescent. In contrast, the
p201-CAT TGF
promoter deletion construct again had a low basal level
of transcription which did not change as the FET cells were rendered
quiescent from the confluent state (Fig. 4B). This construct
also showed no change in CAT expression during growth from exponential
phase to quiescence. Fig. 4C shows data from several
different clones represented graphically. Therefore, the same region
which mediates transcriptional activation of the TGF
promoter in
quiescent HCT116 cells shows decreased regulatory activity in quiescent
FET cells.
The finding that the region of the TGF promoter regulating TGF
expression during quiescence lies between
370 and
201 was significant because we had co-localized EGF responsiveness to the same
region of the TGF
promoter. Using the FET cell line, we examined the
EGF responsiveness of various deletion constructs of the TGF
promoter in transient transfection assays. The constructs p370-, p343-,
p247-, and p201-CAT were co-transfected with
-galactosidase into FET
cells adapted to continuous growth in serum-free medium minus EGF. At
24-48 h after plating, the cells were treated with EGF for 6 h.
The results are summarized graphically in Fig.
5A as mean ± S.E. from
several experiments. The constructs p370-, p343-, and p247-CAT all show
a significantly increased level of CAT activity after EGF treatment
(p
0.05). However, the p201-CAT construct shows no such
increase in CAT activity in response to EGF. This experiment localized
the region of the TGF
promoter responsive to EGFR activation to
between
201 and
247 of the promoter sequence.
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The sequence of this region is shown in Fig. 5B. This region
does not contain any known consensus sequences. Importantly, this
region shows no homology to known EGF-response elements which suggests
that the element represents a unique cis-element. Therefore, studies
were carried out to further define the EGF-responsive DNA sequence
within the TGF promoter. Oligo pair 1/2 spanned
246 to
220
(designated pBL-1/2-tk-CAT) and oligos 3/4 spanned
225 to
199 of
the TGF
promoter (pBL-3/4-tk-CAT). A control oligo pair, 5/6,
spanned the region
202 to
176 (pBL-5/6-tk-CAT). As shown in Fig.
5C, only the pBL-3/4-tk-CAT construct showed significantly
increased CAT activity (p
0.05) upon treatment of
FET cells with exogenous EGF. This localized the EGF-responsive region
of the TGF
promoter to the region
225 to
200.
Stable clones of FET and HCT116 were isolated containing either the
25-bp EGF-response element cloned just upstream of the heterologous
thymidine kinase promoter in a CAT vector (the vector pBL-3/4-CAT) or
the control thymidine kinase promoter-CAT vector (pBLCAT2). Typical
data of the activity of these constructs (mean ± S.E.;
n = 3) in each of the two cell lines is presented
graphically in Fig. 6. The CAT activity
of the pBL-3/4-CAT construct in HCT116 cells increases to approximately
4.5-6-fold the exponential phase activity in quiescence. In contrast,
the activity of this construct in the FET cell line exhibits
approximately a 40% decrease during the establishment of quiescence.
The activity of the control pBLCAT2 construct remains relatively
constant during the establishment of quiescence in both cell lines
(Fig. 6). Therefore, we conclude that the 25-bp region of the
TGF promoter which contains the EGF-responsive cis-element is also
involved in the autoregulation of TGF
expression during the
establishment of quiescence.
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We next examined whether nuclear protein binding to the TGF
autoregulatory cis-region was regulated in a different manner in the
two cell lines during the acquisition of quiescence. Nuclear extracts
were prepared from both exponential phase and quiescent HCT116 and FET
cells. A typical result is shown in Fig.
7A. The two cell lines show
similar low mobility, high molecular weight complexes (labeled
1 through 3 in Fig. 7A), and high
mobility, low molecular weight complexes (labeled 4 and
5), although each cell line exhibits differing amounts of
each complex. Such similarity would be expected for two cell lines
derived from the same tissue of origin. The most marked difference
between the two cell lines is in the regulation of the binding of
nuclear proteins to the DNA sequence of oligonucleotide 3/4 during the
acquisition of quiescence. Logarithmic phase HCT116 nuclear extracts
show predominantly high molecular weight complexes 1, 2, and 3, as well
as bands 4 and 5. In quiescent HCT116 nuclear extracts, the amount of
binding in bands 1, 2, and 3 is markedly increased and there is the
appearance of diffuse binding between bands 2 and 3. Nuclear protein
binding in bands 4 and 5 shows a small increase in the quiescent state compared with logarithmic growth. In contrast, logarithmic phase FET
nuclear extracts show all 3 high molecular weight complexes (bands
1 through 3), although band 1 is less intense
than bands 2 and 3. Again, there is diffuse binding between bands 2 and
3. In contrast to the situation in HCT116 cells, these high molecular weight complexes disappear in quiescent FET cell nuclear extracts. The
binding in band 4 decreases but the intensity of band 5 shows a slight
increase between logarithmic phase and quiescent extracts.
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A competition study was performed to examine the specificity of binding of the nuclear proteins to oligo 3/4. Nuclear extracts from quiescent HCT116 cells and logarithmic phase FET cells were incubated with 32P-labeled oligonucleotide sequence 3/4 in the presence of increasing amounts of unlabeled oligonucleotide 3/4 (Fig. 7B). Nuclear protein binding in the low mobility shift bands (1 through 3) are competed by the unlabeled DNA sequence equivalent to 10-100-fold excess over labeled probe and is completely abolished at 200-fold excess showing the specificity of these bands. However, bands 4 and 5 are more resistant to competition than bands 1 to 3 and remain visible even at 200-fold excess unlabeled competitor. The competition study suggests that the high molecular weight binding protein complexes in low mobility shift bands 1 to 3 are the ones of interest. It should also be noted that the bands in the quiescent HCT116 nuclear extracts are less readily competed (i.e. it takes greater amounts of cold oligonucleotide 3/4 to decrease binding) than the bands in logarithmic phase FET nuclear extracts. This may be due to the presence of higher amounts of binding protein in quiescent HCT116 cells, or the affinity of the complexes may be higher due to differential secondary modifications such as phosphorylation between the two cell lines. When the nonspecific competitor salmon sperm DNA is competed against 32P-labeled oligonucleotide 3/4, little effect on HCT116 nuclear protein binding is observed until 200 ng of nonspecific competitor (equivalent to 400-fold excess over labeled probe) is reached (data not shown).
To investigate whether the TGF autocrine loop in HCT116 cells
contributes to the up-regulation of TGF
during the establishment of
quiescence, we examined TGF
mRNA expression in a clone stably expressing TGF
antisense RNA. This clone (clone U), which
contains a disrupted TGF
autocrine loop, is dependent upon exogenous
EGF for growth much like FET cells (20). If the TGF
autocrine loop of HCT116 is the major determinant of TGF
up-regulation during the
acquisition of quiescence, disruption of this loop should abrogate the
increased TGF
expression at quiescence. In Fig. 8A, it can be seen that this
is the case. Clone U shows a markedly decreased ability to up-regulate
TGF
mRNA during the establishment of quiescence. High TGF
expressing revertants of U were isolated and designated UX (20). Like
the parental HCT116 cells, clone UX did not require exogenous EGF for
growth and were able to up-regulate TGF
mRNA during the
establishment of quiescence (Fig. 8B). The fact that EGF
specifically rescues growth of clone U (20) and the isolation of the
revertant clone UX which still expresses antisense TGF
RNA and so
contains a functional CMV promoter, provides evidence that the effects
on TGF
promoter activity in clone U are not due to squelching of
general transcription factors by the CMV promoter. Moreover, in
transient transfection studies in which the CMV expression vector was
co-transfected into HCT116 cells stably transfected with TGF
promoter CAT constructs, the activity of the CAT constructs was not
altered by the presence of the CMV promoter (data not shown).
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We next examined whether the differential regulation of TGF mRNA
in the TGF
antisense expressing clone U was mediated at the
transcriptional level. Clone U cells containing p370-CAT and clone UX
cells containing p343-CAT were obtained as described under "Materials
and Methods." These different constructs both contain the
EGF-response element and if this region is important in growth state
regulation of these clones, these constructs should show differential
regulation. These cells were grown up and harvested at various times
during the establishment of quiescence. The promoter activity of the
p370-CAT construct in clone U is not up-regulated during growth arrest
(Fig. 8C). However, the activity of the TGF
promoter
construct p343-CAT is up-regulated during the establishment of
quiescence in the revertant clone UX which behaves like the parental
HCT116 cell line (Fig. 8D). The p201-CAT construct stably transfected in U and UX cells, which does not contain the EGF-response element, showed no regulation in either cell line during the
establishment of quiescence (data not shown). This experiment provides
further evidence that autocrine TGF
function determines TGF
promoter activity in growth-arrested cells.
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DISCUSSION |
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As reported previously, the growth factor-dependent
FET cell line down-regulates endogenous TGF mRNA expression
during growth factor and nutrient deprivation leading to growth arrest
and quiescence (4, 5, 22). In contrast, the poorly differentiated,
growth factor-independent cell line HCT116 up-regulates TGF
expression upon entry into quiescence. In both cell lines, we
established that these changes were reflected by changes in the
activity of our full-length TGF
promoter construct p2813-CAT.
The HCT116 and FET cells express similar amounts of TGF mRNA
during logarithmic phase growth (22) but show different phenotypes with
respect to autocrine TGF
. The HCT116 cell line possesses a
sequestered, antibody-inaccessible TGF
autocrine loop which can only
be disrupted by constitutive expression of TGF
antisense RNA. The
FET cell line expresses a classical autocrine TGF
loop, in which
secreted TGF
interacts with its cognate EGFR on the same or adjacent
cells, and is susceptible to inhibition by EGFR blocking antibodies.
This led us to hypothesize that the changes in TGF
expression upon
the acquisition of quiescence might reflect the degree of TGF
autocrine activity in the cell, particularly as these cells are not
exposed to any exogenous growth factors and so endogenous TGF
would
be expected to be a major determinant of TGF
promoter activity.
Support for this hypothesis came when stable transfection of various
TGF
deletion promoter constructs into the FET and HCT116 cell lines
narrowed the region responsive to quiescent changes in endogenous
TGF
status to between
370 and
201. We have identified the same
region of the TGF
promoter as responsive to exogenous EGF using the
FET cell line. Moreover, this is in agreement with previous studies
which localized EGF responsiveness of the TGF
promoter to between
373 and
59 bp of the TGF
promoter but did not identify the
precise location of the element (31).
Further studies in the FET cell line led us to identify a 25-bp element
consisting of 225 to
199 of the TGF
promoter which conferred EGF
responsiveness to a heterologous thymidine kinase promoter. Studies
with stably transfected clones of HCT116 and FET containing this
construct confirmed that this region was also responsive to the altered
TGF
autocrine status in quiescent cells. Moreover, putative
transcription factors binding this element were similarly modulated in
response to growth arrest and quiescence in the two cell lines. This
25-bp element shows no homology to other described EGF-response
elements. Furthermore, there are no consensus sequences for known
transcription factors within this region. Therefore, this EGF/growth
state responsive DNA sequence probably represents a novel
cis-element.
Another important observation is the finding that deletion of the
TGF promoter construct to
201, which removes the EGF/growth state-responsive DNA sequence, results in a construct with very low
basal promoter activity. The basal activity of the p201-CAT construct
was low in several independent clones of both the HCT116 and FET cell
lines, suggesting that repression of the CAT activity was not a
positional integration effect. Moreover, the activity of the p201-CAT
construct was also very low in previous transient transfection studies
in which it was observed that the major elements controlling TGF
basal expression were localized to between
1140 and
201 (24). In
our studies localizing the EGF-response element, the p247-, p343-, and
p370-CAT constructs showed similar basal CAT activity which was lost in
the p201-CAT construct. Therefore, the EGF/growth state responsive DNA
sequence within the TGF
might also be the major determinant of basal
activity.
This autoregulatory 25-bp cis-sequence represents a significant point
in the regulation of cell growth by TGF. Previous studies in this
laboratory have shown that the degree of growth factor dependence of
the FET and HCT116 cell lines is reflected in their requirements for
release from quiescence. The growth factor-dependent FET
cell line requires both growth factors and nutrients for the subsequent
release back into the cell cycle while the growth factor-independent HCT116 cell line requires only nutrients (4, 5, 22). Therefore, it
appears that the increased production of TGF
in the HCT116 cell line
during a period of starvation allows the cells to return to a dividing
state under more stringent (growth factor deficient) conditions than
the FET cell line. This increased expression of TGF
in the growth
factor-independent cell line under adverse conditions may, therefore,
represent a deregulated transcriptional control mechanism which serves
to confer a growth advantage to this more aggressive phenotype. The
growth factor-dependent phenotype of the FET cells reflects
features of a less progressed phenotype. Like FET cells, the VACO 330 cell line, a non-malignant cell line established from a colonic,
tubular adenoma, requires EGF or TGF
for growth stimulation at low
plating density but not when plated at high density (32). Normal
keratinocytes also require exogenous EGF for clonal initiation at low
density but a high density growth proceeds in the absence of growth
factor (33). This suggests that there is very little TGF
autocrine
activity present in non-dividing, non-transformed cells.
Based on our findings in the FET and HCT116 cells, we propose that the
type of TGF autocrine activity determines whether or not TGF
production could be sustained in the absence of exogenous growth
factors and so, in circular fashion, determine the phenotype of the
cell. The growth factor-dependent FET cell line has
relatively weak TGF
autocrine activity which is insufficient to
sustain a high level of TGF
expression in the absence of exogenous
growth factors. Therefore, during the transition to the quiescent
state, TGF
expression is decreased, which in circular fashion, would necessitate the need for exogenous growth factors to aid re-entry into
the cell cycle and DNA synthesis. In contrast, the growth factor-independent HCT116 cells possess a relatively strong TGF
autocrine function, which is apparently unaffected by environmental factors such that it allows the maintenance of high levels of TGF
expression in the absence of exogenous growth factors. This ability to
maintain highly active TGF
autocrine function in the absence of
growth factors in turn re-enforces the growth factor-independent phenotype in these cells through transcriptional activation of TGF
.
In turn, this positive feedback control not only contributes to the
growth factor-independent phenotype of the cells but increases the
level of TGF
autocrine function as well. Further support for this
model is provided by the studies with the TGF
RNA antisense expressing clone U, which was derived from the HCT116 cell line. Clone
U shows dependence on exogenous growth factors for growth and no longer
up-regulates TGF
mRNA during growth arrest and quiescence.
Furthermore, the activity of the p370-CAT construct which contains the
25-bp autoregulatory element is no longer activated during the
establishment of quiescence in this growth factor-dependent clone. However, the revertant clone UX which has switched to a high
TGF
expressing, growth factor-independent phenotype despite the
presence of antisense TGF
RNA (20), again up-regulates TGF
mRNA expression during growth arrest and the establishment of
quiescence. The activity of the TGF
promoter construct p343-CAT, which contains the TGF
autoregulatory element, is also up-regulated in the revertant clone UX during the establishment of quiescence. Therefore, TGF
autocrine function which allows for inappropriate TGF
expression in the non-dividing state may confer the growth advantage and growth factor independence of the progressed tumor cell,
rather than just increased TGF
mRNA expression alone.
Although we have identified a transcriptional mechanism as the control
point for the differences in TGF regulation in the two cell types,
it remains to be determined how EGFR signaling affects the
transcription factors involved. The gel shift assay shows increased
binding of nuclear proteins during the acquisition of quiescence in
HCT116 cells and decreased binding in growth arrested FET cells.
However, it is not clear whether the binding of the transcription
factors is regulated transcriptionally or post-transcriptionally
through EGFR or growth state-regulated signaling peptides such as the
mitogen-activated protein kinases and cyclin-dependent
kinases.
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ACKNOWLEDGEMENTS |
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We thank Anna M. Chlebowski, Jenny Zak, and Suzanne Payne for preparation of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA34432 and CA54807.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.
§ These authors contributed equally to this work.
¶ Present address: Dept. of Surgery, The University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78284-7840.
To whom correspondence should be addressed. Tel.:
210-567-5706; Fax: 210-567-3447; E-mail: howellg{at}uthscso.edu.
** This work performed in partial fulfillment of the requirements for the Ph.D. degree.
1
The abbreviations used are: TGF, transforming
growth factor
; EGF, epidermal growth factor; EGFR, epidermal growth
factor receptor; CAT, chloramphenicol acetyltransferase; NEO, neomycin; tk, thymidine kinase; CMV, cytomegalovirus.
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REFERENCES |
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