From the ** Departments of Surgery and Biochemistry, University of
Texas Health Science Center, San Antonio, Texas 78284, the
¶ Department of Pharmacology, University of Kentucky,
Lexington, Kentucky 40536, the Department of Medicine, Case
Western Reserve University Ireland Cancer Center and School of
Medicine, Cleveland, Ohio 44106, and the
Department of
Biochemistry, Medical College of Ohio, Toledo, Ohio 43699
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ABSTRACT |
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Ectopic expression of the
As mediators of cell matrix-cell interaction, integrins impart
diverse biological properties to the cells that express them. A number
of studies have demonstrated that integrin expression affects tumor
cell proliferation and progression (1-3). Immunohistochemical analysis
showed that the expression of integrins is altered in human tumors
compared with corresponding normal cells. Many neoplastic cells show
reduced expression of integrins (4-9). Overexpression of certain
integrins, such as Loss of negative growth factors can also lead to abnormal growth in
transformed cells. Transforming growth factor- Two lines of evidence indicate that autocrine negative TGF- Previously, we showed that vitamin D3 was inhibitory to
wild type MCF-7 clones that expressed RII and to RII-null clones
expressing ectopic RII (29). In contrast, RII-null clones were
refractory to inhibition (29). Response to vitamin D3 was
associated with induction of higher RII levels and enhanced TGF- Cell Culture--
MCF-7 cells were originally obtained from
American Type Culture Collection and adapted to McCoy's 5A medium
supplemented with 10% fetal bovine serum (FBS), pyruvate, vitamins,
amino acids, and antibiotics (30). Working cultures were maintained at
37 °C in a humidified atmosphere of 5% CO2 and
routinely checked for mycoplasma contamination as described previously
(21). MCF-7 limiting dilution clones were obtained by diluting the
parental cells in 96-well tissue culture plates at 0.5 cell/well as
described previously (21). The strain of MCF-7 cells utilized in this study is insensitive to TGF- Integrin RNA Analysis--
Total RNA was isolated from cultured cells by
the guanidine isothiocyanate method (31). For detection of FN mRNA
levels, cells (106) were plated in 100-mm culture dishes
coated with poly-L-lysine (10 µg/ml; Sigma) or FN (10 µg/ml; Collaborative Biomedical Products) for 4 days in 10% FBS
medium, and total RNA was then isolated. For detection of RII mRNA
levels, cells (106) were plated in 100-mm culture dishes
coated with poly-L-lysine (10 µg/ml) or FN (10 µg/ml)
for 1 and 2 h in McCoy's 5A medium supplemented with 2% bovine
serum albumin (Sigma) and 24 and 96 h in McCoy's 5A medium
supplemented with 10% FBS.
The construction of the integrin Immunoprecipitation--
To determine cell surface integrin
Cell Adhesion and 3-(4,5-Dimethyl Thiazol-2-yl)-2,5-diphenyl
Tetrazolium Bromide Assays--
A 96-well Corning tissue culture plate
was coated overnight at room temperature with FN at concentrations of
0, 1.0, 2.5, 5.0, and 10.0 µg/ml, blocked with 3% bovine serum
albumin for 3 h, and then rinsed once with phosphate-buffered
saline. Confluent cells were trypsinized, plated at 4 × 104 cells/well on FN-coated plates in serum-free medium,
and incubated for 15 min. Unattached cells were gently washed away by
three rinses with serum-free medium. The relative number of attached cells was determined by the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay as described previously (33).
The specificity of cell adhesion to FN was determined using a
monoclonal anti-human integrin Tumorigenicity--
Exponentially growing cells of MCF-7 Neo
pool and Mitogenesis Assay--
Inhibition of cell proliferation by
exogenous TGF- Plating Efficiency Assay--
The effect of TGF- Transient Transfection and Luciferase Assay--
The
TGF- Receptor Cross-linking--
Human TGF- Expression of
Ectopic Expression of RII in Induction of Autocrine Negative TGF-
To confirm the enhancement of autocrine TGF-
We previously showed that autocrine TGF- Growth Inhibitory Effects of TGF- Effect of Integrin Expression on Tumorigenicity--
To assess the
effect of A number of studies have indicated that loss or reduced expression
of integrin receptors results in abnormal cell growth (10, 11, 12). To
test the hypothesis that Previously, we showed that blockade of
FN/ Our results suggest that An interesting aspect of this study was the enhancement of RII
expression and TGF- In a previous study, we reported that disruption of
There is evidence that integrins transduce signals cooperatively with
other growth factor systems in the regulation of cell proliferation.
The proliferative response of murine mammary carcinoma cells to
platelet-derived growth factor-BB and basic fibroblast growth factor is
dependent on the extracellular matrix environment, indicating that
modification of extracellular matrix and/or surface integrin receptors
may regulate responsiveness to these growth factors (41). Reciprocal
enhancement of 5 integrin subunit in cancer cells with little or
no endogenous expression of this integrin often results in reduced
proliferation as well as reduced malignancy. We now show that
inhibition resulting from ectopic expression of
5
integrin is due to induction of autocrine negative transforming growth
factor-
(TGF-
) activity. MCF-7 breast cancer cells do not express
either
5 integrin or type II TGF-
receptor and hence are unable to generate TGF-
signal transduction. Ectopic expression of
5integrin expression enhanced cell adhesion to
fibronectin, reduced proliferation, and increased the expression of
type II TGF-
receptor mRNA and cell surface protein. Receptor
expression was increased to a higher level in
5
transfectants by growth on fibronectin-coated plates. Induction of type
II TGF-
receptor expression also resulted in the generation of
autocrine negative TGF-
activity because colony formation was
increased after TGF-
neutralizing antibody treatment. Transient
transfection with a TGF-
promoter response element in tandem with a
luciferase cDNA into cells stably transfected with
5
integrin resulted in basal promoter activities 5-10-fold higher than
those of control cells. Moreover, when
5 transfectants
were treated with a neutralizing antibody to either TGF-
or integrin
5, this increased basal promoter activity was blocked.
Autocrine TGF-
activity also induced 3-fold higher endogenous
fibronectin expression in
5 transfectants relative to
that of control cells. Re-expression of type II receptor by
5 transfection also restored the ability of the cells to
respond to exogenous TGF-
and led to reduced tumor growth in athymic nude mice. Taken together, these results show for the first time that
TGF-
type II receptor expression can be controlled by
5
1 ligation and integrin signal
transduction. Moreover, TGF-
and integrin signal transduction appear
to cooperate in their tumor-suppressive functions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
5
1, can reverse
tumorigenicity and anchorage-independent growth in some transformed
cells (10, 11). Perturbation of integrin
5
1binding to its ligand stimulates the
growth of a variant of the K562 cells (12). Recently, we showed
expression of integrin
5subunit selectively blocks DNA synthesis (13) and disruption of
5
1ligation enhanced DNA synthesis in a
mitogen-activated protein kinase-dependent but epidermal growth factor receptor-independent manner (14). Extracellular matrix
recognition by integrin
5
1 may,
therefore, play a role in the negative control of cell growth, which
may be lost in some cancer cells.
(TGF-
)1 has been
identified as a potent growth inhibitor in various normal as well as
transformed cell types (15, 16). Escape from the negative growth
control by TGF-
is an important step during oncogenic transformation
(15) because the growth of normal mammary epithelial cells is inhibited
by TGF-
, whereas their transformed counterparts are often resistant
to its inhibitory effects (17-19). Loss of TGF-
sensitivity in
estrogen receptor + breast cancer cells has frequently been associated
with loss of the TGF-
type II receptor (RII), which, along with the
TGF-
type I receptor (RI), is necessary for TGF-
signal
transduction (20, 21). The importance of loss of TGF-
signaling in
breast cancer cells was demonstrated by differences in tumorigenicity
of MCF-7 breast cancer clones with and without RII expression (22) and
the ability of re-expression of RII in MCF-7 cells to inhibit
tumorigenicity in athymic mice (21). RII has been shown to be a tumor
suppressor gene by the criteria that its mutational inactivation is
associated with a hereditary form of colon cancer and that RII ectopic
expression in cancer cells from individuals with this form of cancer
reverses malignancy in athymic mice (24, 25). Re-expression of RI in cancer cells that are deficient in this receptor also reverses tumorigenicity (26).
may be
more important than response to exogenous TGF-
in controlling tumor
growth. The first of these was the demonstration that removal of
autocrine TGF-
activity by stable transfection of a TGF-
antisense expression vector leads to malignant progression of cancer
cells in athymic mice (27, 28). This approach blocked autocrine TGF-
activity because endogenous TGF-
was removed from these cells but
did not affect the expression of TGF-
receptors and, therefore,
permitted the retention of response to exogenously produced TGF-
in
the tumor environment from nonmalignant cells. The occurrence of tumor
formation indicated that exogenous TGF-
produced by nonmalignant
cells was insufficient to achieve tumor suppression. The second line of
evidence involved the re-expression of RII in a cell line that was
homozygous for mutational inactivation of the gene. Re-expression of
RII regenerated autocrine negative TGF-
but did not regenerate an
inhibitory response to exogenous TGF-
(25). However, reversion of
tumorigenicity did occur, indicating that autocrine TGF-
was
critical. Thus, the available evidence indicates that mechanisms for
regeneration of RII expression and autocrine negative TGF-
will be
of importance in our understanding of controls of TGF-
signal
transduction in malignancy and may lead to novel treatment or
prevention approaches for cancer.
autocrine negative activity, suggesting that other growth modulators
may cause inhibition by inducing TGF-
autocrine negative activity.
Given the ability of
5 integrin to affect growth in a
negative fashion (13, 14), we hypothesized that re-expression of cell
surface
5
1 integrin in cancer cells
deficient in the expression of the
5 subunit would lead
to regeneration of RII expression and autocrine negative TGF-
activity. This hypothesis was tested by stable transfection of a MCF-7
breast cancer clone lacking both RII and
5 integrin expression with an
5 integrin cDNA. Transfection
resulted in re-expression of RII and regeneration of autocrine TGF-
activity, as well as response to exogenous TGF-
. RII expression and
TGF-
responses were dependent upon
5
1
ligation to endogenous MCF-7 fibronectin (FN) and were enhanced when
exogenous FN was used to coat culture plates, indicating that growth
inhibition by
5
1 ligation involves
induction of autocrine TGF-
activity.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
because it lacks RII (21).
5 Stable Transfection--
An integrin
5 expression vector for mammalian cells was used for
stable transfection as described previously (13). The plasmid was
linearized and transfected into a typical MCF-7 limiting dilution clone
(designated MCF-7 LDC4) by electroporation with a Bio-Rad Gene Pulser
at 250 V and 960 microfarads. Control cells were transfected with a
Neo-containing plasmid. The transfected cells were plated in 100-mm
culture dishes in 10% FBS medium for 2 days. Selection of stable
transfectants was carried out by adding Geneticin (600 µg/ml; Life
Technologies, Inc.) into the medium. After three weeks,
Geneticin-resistant clones were ring-cloned and expanded for screening
of
5 expression. The control clones were pooled and
designated the MCF-7 LDC4 Neo pool.
5 subunit and RII
antisense probes has been described (21, 32). The FN riboprobe plasmid was constructed by subcloning a 232-base pair
BamHI-PvuII fragment of the human FN cDNA
into a pBSK(
) vector (Stratagene Cloning System). T7 RNA
polymerase was used to synthesize the FN antisense probe (13, 32). The
RNase protection assay was performed by hybridization of the
radioactive riboprobes with total RNA (20 µg) isolated from the
control or
5-transfected cells as described previously
(32).
5
1 expression, cell surface proteins were
labeled with biotin, immunoprecipitated with an anti-
5
subunit monoclonal antibody (Life Technologies, Inc.), and analyzed by
SDS-polyacrylamide gel electrophoresis as described previously
(32).
5 subunit antibody (Life
Technologies, Inc.). The cells were incubated in the absence or
presence of the antibody (1:100 dilution) for 30 min at 4 °C and
then plated at 4 × 104 cells/well in 96-well plates
coated with FN (10 µg/ml). Determination of cell adhesion and the
3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay
were performed as described above.
5 sense clone 15 were inoculated subcutaneously
as described previously (21). Growth curves for xenografts were
determined by measuring the volume (V) of tumors.
V is expressed as V = (L × W2) × 0.5, where L is the length and
W is the width of the xenograft.
1 in Neo and
5-transfected
cells was determined by measuring [3H]thymidine
incorporation as described previously (21). Briefly, cells were plated
in 24-well plates at 1.5 × 104 cells/well in the
presence of various concentrations of TGF-
1 (0.2-10
ng/ml). After 4 days of incubation, cells received a 1-h pulse with
[3H]thymidine (25 µCi) and were washed with
supplemental McCoy's 5A medium three times, and DNA was precipitated
with 10% trichloroacetic acid and then solubilized with 0.2 M sodium hydroxide. The amount of
[3H]thymidine incorporated was analyzed by liquid
scintillation counting in a Beckman LS7500 scintillation counter.
Growth inhibition by TGF-
1 is represented as the
percentage of [3H]thymidine incorporation of
TGF-
1-treated cells relative to untreated cells.
1
neutralizing antibody on the clonogenic potential of control and
5-transfected cells was compared to determine autocrine
TGF-
activity as described previously (25, 26). Cells were seeded at
low density (400 cells/well) in 24-well plates in the presence of
control IgG (10 µg/ml; R & D Systems, Inc.) or TGF-
1
neutralizing antibody (10 µg/ml; R & D Systems, Inc.). After 8 days
of incubation without medium change, cell colonies were fixed with 1%
glutaraldehyde, stained with 0.1% crystal violet, and dissolved in 1%
Triton X-100 as described by Westergren-Thorsson et al.
(34).
-responsive luciferase expression plasmid (p3TP-Lux) was used
for transient transfections, and luciferase assays were performed as
described previously (35). MCF-7 Neo control and
5
sense-transfected cells were transfected with 30 µg of p3TP-Lux and
10 µg of
-galactosidase plasmid by electroporation with a Bio-Rad
Gene Pulser at 250 V and 960 microfarads. The electroporated cells were
plated onto 6-well tissue culture plates. After the cells attached,
control IgG (10 µg/ml) and TGF-
1 neutralizing antibody
(10 µg/ml) were added. The cells were harvested with 200 µl of
lysis buffer (luciferase assay system; Promega). Luciferase activity
was measured in the first 10 s after substrate addition using a
luminometer (Berthold Lumat LB 9501) and expressed as arbitrary
units after normalization with
-galactosidase activity.
1 was
purified and iodinated by the chloramine T method as described
previously (36). Equal numbers of the cells (105) were
plated in 6-well plates, and after 5 days, binding and cross-linking of
200 mM 125I-TGF-
1 to the cell
monolayer were performed as described by Segarini et al.
(37). Labeled cells were solubilized in 200 µl of 1% Triton X-100.
Equal amounts of cell lysate protein were electrophoresed by 4-10%
gradient SDS-polyacrylamide gel electrophoresis under reducing
conditions and exposed for autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
5
1 Integrin in MCF-7
Cells--
We transfected the
5 subunit into a typical
limiting dilution clone, MCF-7 LDC4. The
5-positive
transfectants were initially screened for increased expression of
5 mRNA by RNase protection assays (Fig.
1). Two positive clones (designated cl.5
and cl.15) were isolated that expressed higher levels of
5 subunit mRNA than the untransfected MCF-7 LDC4 and
Neo-transfected (Neo pool) control cells. Increased
5
mRNA expression was accompanied by increased cell surface
expression (Fig. 2). The levels of cell surface integrin
1 subunit dimerized by
5
subunit were also increased, indicating that the expression of
1 subunit might be up-regulated by increased
5 subunit expression or that excess
1
subunit already present could complex with the increased levels of
5 subunit. In contrast to
5
1, the levels of
2
1 and
3
1 were unchanged in
5 transfectants (data not shown),
ruling out the possibility that increased
5 expression
might compete with other
subunits for dimerizing with the
1 subunit. We next determined whether the increased
5
1 enhanced adhesion to FN (Fig.
3A). Both
5
transfectants showed 7-8-fold increased adhesion to FN-coated plates
at FN concentrations ranging from 5 to 10 µg/ml, whereas Neo control
cells showed only 3-4-fold enhancement. In addition, both
5 transfectants showed ~5-fold increases of binding at
FN concentrations ranging from 0 to 2.5 µg/ml, relative to Neo
control cells. The specificity of cell adhesion was shown by blocking the attachment to FN with an anti-
5 subunit antibody
(Fig. 3B), thus indicating that enhanced cell attachment to
FN was due to increased cell surface
5
1expression.
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Fig. 1.
Integrin
5 mRNA levels in
5 sense-transfected MCF-7 LDC4.
MCF-7 limiting dilution clone 4 was stably transfected with the
integrin
5 sense expression vector
(
5S.) and selected with Geneticin.
Total RNA from two positive clones (cl.5 and
cl.15) was isolated and compared with untransfected (MCF-7
LDC4) and Neo-transfected (Neo pool) control cells for
5
mRNA levels by RNase protection assay. Actin mRNA levels were
used to normalize loading of the samples. M.W. std.,
molecular weight standard.
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Fig. 2.
Cell surface integrin
5 and
1 protein levels in
5 sense-transfected MCF-7 LDC4.
Cell surface proteins from MCF-7 LDC4 Neo pool,
5 sense
clone 5, and
5 sense clone 15 were labeled with biotin.
The biotinylated integrin from equal cell numbers of each cell type was
immunoprecipitated with an anti-human
5 subunit antibody
and analyzed by SDS-polyacrylamide gel electrophoresis and Western
blotting.
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Fig. 3.
Adhesion of MCF-7 LDC4 integrin
5 sense transfectants to FN.
A, MCF-7 LDC4, Neo,
5S.5, and
5S.15 cells were incubated in 96-well plates coated with
different concentrations of FN for 15 min. Unattached cells were washed
away, and the number of adherent cells determined by 3-(4,5-dimethyl
thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. B,
specificity of adhesion to FN by
5
1was
ascertained by preincubating cells either with (+) or without (
)
5
1monocloned antibody (1:100) dilution
for 30 min at 4 °C prior to adhesion on FN (10 µg/ml).
C, DNA synthesis was measured by [3H]thymidine
incorporation in cells plated for growth curves. Cells were seeded into
24-well plates (1.5 × 104 cells/well), and DNA
incorporation determined at the indicated times as described under
"Materials and Methods." Each point is the mean ± S.E. of
nine determinations from three separate experiments. Ab,
antibody.
5 expression leading to enhanced
5
1 ligation blocked DNA synthesis,
whereas disruption of ligation led to increased DNA synthesis in other
model systems (13, 14). If our hypothesis that enhanced
5
1 function leads to induction of
autocrine negative TGF-
activity was correct, ectopic expression of
5 should result in reduced cell proliferation. Growth
curves of
5-transfected clones showed more than 50%
inhibition of proliferation relative to wild type cells (Fig.
3C).
5
Transfectants--
Previously, we showed that MCF-7 cells were
insensitive to TGF-
1 because they expressed nearly
undetectable levels of RII (21). Therefore, if
5
1-mediated growth inhibition was
associated with autocrine negative TGF-
activity, RII expression
must be restored. To test this hypothesis, we examined RII mRNA
levels in
5 transfectants by RNase protection assay
(Fig. 4). High steady state RII mRNA
levels were induced in
5-transfected cells compared with
Neo control cells when the cells were cultured on
poly-L-lysine. This was probably due to enhanced production
of endogenous FN, as described below. Growth on exogenous FN further
increased RII mRNA levels to 2.5-fold (as determined by
densitometry) in
5-transfected cells, whereas it had no
effect in Neo control cells. RI mRNA levels remained the same in
5-transfected and Neo-transfected cells (data not
shown). TGF-
receptor cross-linking with
125I-TGF-
1 showed little binding to RII of
control cells, whereas substantially higher binding of
TGF-
1 was observed in the
5 transfectants
(Fig. 5). Interestingly, binding of
TGF-
1 to RI was fairly prominent in control cells, as
was binding to RIII. This has been observed in previous studies of this
cell line (21, 22), as well as in other cell lines (38). Transfection
of
5 subunit resulted in a substantial increase in RI
binding. This is in accordance with the increased RII expression
because this receptor is thought to be responsible for RI recruitment
to the cell surface (35).
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Fig. 4.
TGF- RII mRNA
levels in MCF-7 LDC4
5 sense
transfectants. Total RNA (20µg) harvested from MCF-7 LDC4 Neo
pool,
5S.5 and
5S.15 cells, plated either
on poly-L-lysine or FN at the indicated time points was
used in RNase protection assays with an antisense RII probe. Actin was
used to normalize RNA loading.
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Fig. 5.
Cell surface expression of
TGF- receptors in MCF-7 LDC4
5 sense transfectants.
125I-Labeled TGF-
was cross-linked to its cell surface
receptor on the control MCF7 LDC4 Neo pool,
5S.5, and
5S.15 cells as described under "Materials and
Methods."
Activity by
5
1 Transfection--
To determine
whether autocrine negative TGF-
activity was induced as a result of
5
1 expression, TGF-
1
neutralizing antibody blockade of endogenously produced
TGF-
1 was employed, utilizing a previously described
clonal assay (25, 26). Cells expressing autocrine negative TGF-
activity will show enhanced colony formation and growth as a result of
the antibody-mediated neutralization of TGF-
1, whereas
those cells that do not express autocrine negative activity will be
unaffected by antibody treatment. Cell growth and colony formation are
determined by crystal violet staining. Standard curves were performed
measuring crystal violet levels with known numbers of MCF-7 cells to
ensure that the assay was performed over a linear range of MCF-7 cells.
As shown in Fig. 6, TGF-
1
neutralizing antibody stimulated colony formation in
5
transfectants, whereas it had no effect on Neo control cells (Fig.
6A). The percentage of stimulation by the antibody was
calculated and plotted in Fig. 6B. TGF-
1
neutralizing antibody treatment resulted in 25% stimulation for clone
5 and 55% for clone 15.
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Fig. 6.
Effect of
TGF- 1 neutralizing antibody on the
plating efficiency of MCF-7 LDC4 integrin
5 sense transfectants.
A, cells (400) were plated on FN-coated (10 µg/ml) 24-well
plates either in the presence or absence of neutralizing TGF-
antibody (1:100). Cell growth was quantitated by crystal violet
staining 8 days later. B, after staining, cell colonies were
dissolved in 1% Triton X-100, and absorbance measurements at 595 nm
and cell number are expressed as the percentage of stimulation. Each
value is the mean ± S.E. of four replicates.
activity after
5 expression, we compared the activity of a
TGF-
-responsive promoter in control cells with that in
5-transfected cells. The p3TP-Lux promoter contains a
TGF-
response element from the plasminogen activator inhibitor gene
inserted upstream of the luciferase reporter gene and has been
extensively utilized as a marker for TGF-
responsiveness (23, 35).
Therefore, it would be expected that induction of autocrine TGF-
activity would result in enhanced expression of the p3TP-Lux construct
in
5 transfectants relative to Neo control cells. Fig.
7 shows that both
5
transfectants expressed 5-10-fold higher levels of luciferase activity
than Neo control. If increased luciferase activity of p3TP-Lux
construct was due to autocrine TGF-
, neutralizing antibody treatment
would reduce expression of the reporter construct. As shown in Fig.
7A, TGF-
1 neutralizing antibody treatment
resulted in a substantial decrease in luciferase reporter activity in
both
5-transfected clones, whereas it had no effect on
Neo control cells. This experiment was repeated four times, and similar
results were obtained. Similarly, an
5 neutralizing antibody was used to show that disruption of
5
1 ligation to FN resulted in
approximately a 60% reduction in the enhanced luciferase activity
associated with the
5 clone 15 transfectant (Fig.
7B), thus confirming that the enhanced endogenous TGF-
activity was dependent on the ectopic
5 expression.
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Fig. 7.
Effect of
TGF- 1 neutralizing antibody on the
transcription of a TGF-
-responsive
promoter. A, 3TP-Lux and pSV
-galactosidase plasmids
were transiently transfected into Neo,
5S.5, and
5S5.15 cells by electroporation, and luciferase activity
was measured 48 h after transfection in cells treated with IgG or
TGF-
neutralizing antibody (10µg/ml). Values are means of
duplicate samples. B, 3TP-Lux transfectants were treated
with either
5 antibody or TGF-
neutralizing antibody
(10 µg/ml).
controlled steady state
levels of FN in both native and RII-transfected cells (25, 32).
Consequently, induction of autocrine TGF-
should be associated with
increased endogenous FN expression by
5 transfectants.
The
5-transfected cells showed a 3-fold increase (as
determined by densitometry) in FN mRNA levels compared with Neo
control cells (Fig. 8A). FN
mRNA levels were further increased in
5-transfected cells when the cells were plated on FN, whereas the levels in Neo
control cells still remained the same. The enhanced FN expression was
due to autocrine TGF-
as shown by the ability of TGF-
neutralizing antibody treatment to repress FN expression in
5 transfectant cells to a level comparable to that of
NEO controls (Fig. 8B).
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Fig. 8.
Fibronectin mRNA levels in MCF-7
LDC4 5 sense transfectants.
A, RNase protection assay was performed to determine FN
mRNA levels in Neo and
5S.15 cells grown either on
poly-L-lysine or FN-coated plates for 2 days. Actin
mRNA was used to normalize samples. B, Cells were plated
on poly-L-lysine and treated with IgG (
) or TGF-
neutralizing antibody (Ab) (+), and FN mRNA levels were
measured, as described above.
1 on
5 Transfectants--
Induction of autocrine TGF-
activity suggested that response to exogenous TGF-
effects should
also result from
5 transfection. The MCF-7 LDC4 Neo pool
was insensitive to TGF-
1 in the absence or presence of
exogenous FN (Fig. 9). The
5 transfectants showed reduced basal proliferation
relative to NEO controls as indicated above in Fig. 3C. DNA
synthesis in the
5 transfectants was further inhibited
by TGF-
1 in a dose-dependent manner (Fig.
9). When the two
5 transfectants were plated in 24-well
culture plates coated with exogenous FN (10 µg/ml), increased
sensitivity to TGF-
1 was demonstrated. Increased
sensitivity on FN was likely due to the increased RII expression when
cells were grown on FN as demonstrated in Fig. 4, above.
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Fig. 9.
Growth inhibition of integrin
5 sense transfectants by
TGF-
1. MCF-7 LDC4 Neo pool
and the two
5 sense transfectants were plated in 24-well
plates coated with or without FN (10 µg/ml) at 1.5 × 104 cells/well in the presence of various concentrations of
TGF-
1. DNA synthesis was assayed 4 days later by
measuring [3H]thymidine incorporation. Thymidine
incorporation in the presence of TGF-
1 was calculated as
percentage of the thymidine incorporation in the absence of
TGF-
1 for each clone. Each point is the mean ± S.E. of nine determinations from three separate experiments.
5 expression on the malignant properties of
MCF-7 cells, we inoculated Neo control and
5-transfected clone 15 into ovariectomized, estrogen-supplemented nude mice as
described previously (21). The size of xenografts formed was monitored
with time (Fig. 10). Initially, MCF-7
LD 4 Neo pool and
5 clone 15 formed similar sized
xenografts (< 200 mm3) until day 8 after inoculation.
After day 8, growth was delayed in
5 clone 15 compared
with Neo control. At day 28, Neo controls formed ~2.2-fold larger
xenografts than
5 clone 15. This result indicates that
integrin
5
1 expression in MCF-7 cells can
partially reverse the malignant properties of the cell line.
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Fig. 10.
Xenograft growth of MCF-7 LDC4 Neo and
integrin 5 sense clone 15. Exponentially growing (1 × 107 cells) MCF-7 LDC4 Neo
pool and
5 sense clone 15 (cl15) were
subcutaneously inoculated into ovariectomized athymic nude mice
supplemented with 17
-estradiol. Tumors were measured externally on
the indicated days in two dimensions using a caliper. Xenograft volume
was determined from the equation V = (L × W2) × 0.5, where L is the length
and W is the width of the tumor. Each point represents the
mean ± S.E. of 10 xenografts.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
5
1 ligation has
tumor suppressor effects mediated through autocrine TGF-
, we
restored integrin
5 expression in MCF-7 cells. The MCF-7
cell line provided a good model system for this study in that it
expressed low amounts of
5 integrin and was insensitive
to the growth inhibitory effects of TGF-
1 due to
repression of RII expression (21-23). Integrin
5
transfectants expressed similar levels of cell surface integrin as
Hs578T cells, another breast cancer cell line that was sensitive to
growth inhibitory effects of
TGF-
1.2 The
5 transfection resulted in an increase in expression of RII, which was accompanied by increased autocrine TGF-
activity as
assessed by 1) enhanced clonality following TGF-
neutralizing antibody treatment; 2) decreased endogenous activity of a
TGF-
-sensitive reporter system in response to TGF-
neutralizing
antibody treatment with either TGF-
1 or
5
antibodies; and 3) stimulation of FN expression, which was reversed by
TGF-
neutralizing antibody treatment. Up-regulation of RII
expression was also reflected by increased sensitivity to inhibition by
treatment with exogenous TGF-
.
5
1 ligation by antibodies against FN
or integrin
5 subunit stimulated DNA synthesis in cancer
cell lines with moderate to high
5
1 cell
surface expression (13, 14). These results are consistent with a
previously described model that suggests that moderate adhesion to a
loosely organized extracellular matrix facilitated both migration and
growth, but strong adhesion to a fully organized extracellular matrix
suppressed proliferation and contributed to inhibition of growth (4). Thus, the low level of expression of integrin
5
1 on the cell surface of wild type MCF-7
cells could contribute to weak adhesion and hence abnormal growth in
MCF-7 cells. Ectopic
5 expression leads to results
consistent with a model suggesting that higher
5
1 surface expression allows for greater
adhesion due not only to
5, but to greater endogenous FN
expression as well. Inhibition of proliferation is either due to or
augmented by the generation of autocrine TGF-
activity. Exogenous FN
coating allows for even stronger adhesion and further enhancement of
autocrine TGF-
activity. Most importantly, the results indicate that
5
1 ligation and autocrine TGF-
interact in a reciprocal manner that is self-sustaining for both
autocrine negative activity and cell-extracellular matrix interactions.
Moreover, this interaction is tumor-suppressive. This model may well
apply to other systems given that autocrine TGF-
signaling and
5
1 ligation have both been individually associated with tumor suppression in various model systems. Autocrine TGF-
has been shown to control steady state
5
expression (32) in model systems that also show tumor-suppressive
TGF-
function (28).
5
1 may have a
negative growth regulatory role in some cancer cells and normal cells
through modulation of TGF-
sensitivity. Apparently, signal
transduction mechanisms for activating the TGF-
pathway are
essentially intact in MCF-7 cells because restoration of
5
1 expression leads to autocrine as well
as exogneous TGF-
inhibitory responses. However, other types of
cancer cells may be resistant to this mode of regulation despite
5
1 expression because of perturbations of
the TGF-
pathway resulting from malignant transformation. For
example, HCT116 colon carcinoma cells express high levels of integrins that mediate adhesion to FN, but this cell line still exhibits a highly
malignant phenotype due to a mutated RII gene (25). It is also possible
that downstream signaling messengers encoded by oncogenes or tumor
suppressor genes that participate in either a primary or secondary
manner in signal transduction are abnormally modulated in some cell types.
function when
5 transfectants
were plated on FN-coated plates. These results, along with the
demonstration that
5 antibody treatment blocks autocrine
TGF-
activity, show that
5
1 ligation
to FN is critical to the generation of RII expression and hence
TGF-
-mediated signal transduction. Enhanced basal expression of FN
in MCF-7
5 transfectants that were not grown on
FN-coated plates was also observed. We postulate that increased steady
state expression of FN after
5 transfection allowed for
5
1 ligation, which was critical to the
basal RII expression and TGF-
sensitivity associated with
transfectant cells that were not exposed to FN-coated plates.
5
1/FN ligation resulted in stimulation of
DNA synthesis (14). DNA synthesis was associated with up-regulated CDK2
activity without alterations of CDK inhibitors. DNA synthesis was also
found to be dependent upon extracellular receptor kinase 1 and 2 activation. Thus, this previous study indicated
5
1 ligation to FN had a repressive effect
on cell cycle progression through repression of cyclin A and CDK2
expression. Exogenous treatment with TGF-
has been shown to
down-regulate cyclin A (39). Interestingly, TGF-
has also been
reported to repress ERK1/2 activation in some types of cells (40). This
suggests that DNA synthesis resulting from disruption of
5
1 ligation may be a reflection of the
disruption of integrin related autocrine TGF-
activity resulting in
up-regulation of ERK activation and subsequent promotion of cell cycle transit.
5
1-mediated adhesion by
insulin and of insulin-mediated signal transduction by
5
1 have recently been reported (42).
However,
5
1 ligation does not appear to
modulate expression of insulin receptor in this system. Our results
indicate that the TGF-
signaling pathway can be rescued by
re-expression of integrin in MCF-7 cells. Ligation of integrins with
their extracellular matrix ligands has been shown to regulate gene
expression in a number of studies (43). However, because studies on the
regulation of RII mRNA transcription and stability are limited, it
is not yet clear how ligation of
5
1
integrin to FN increases RII mRNA level. Nevertheless, the induction of RII and autocrine TGF-
activity by
5
1 ligation suggests that
integrin-mediated signal transduction plays a cooperative role with
TGF-
signal transduction in tumor suppression. Moreover, the results
indicating reciprocal positive control of autocrine TGF-
and
5
1 ligation suggest that TGF-
signal
transduction and
5
1 integrin signal
transduction participate in a mutually self-sustaining
tumor-suppressive autocrine loop.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. Massague of Memorial
Sloan-Kettering Cancer Center for the TGF--responsive construct and
Jenny Zak and Lorraine Gilmore for the skillful preparation of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA 38173, CA 50457, and CA 72001 (to M. G. B.); CA 63480, CA 75253, and CA 79683 (to L. Z. S.); and CA 68316 and CA 43703 (to J. K. V. W.).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.
§ Performed in partial fulfillment of the requirements for the Ph.D. degree Department of Biochemistry, Medical College of Ohio, Toledo, Ohio 43699
To whom correspondence should be addressed: Depts. of Surgery
and Biochemistry, University of Texas Health Science Center at San
Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78284. Tel.: 210-567-4524; Fax: 210-567-4664.
2 D. Wang and M. Brattain, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
TGF-, transforming growth factor-
;
RI, RII, and RIII, TGF-
type I, type
II, and type III receptor, respectively;
5, integrin
5;
5S.5 and
5S.15,
5 sense clones 5 and 15;
cl., clone;
FN, fibronectin;
FBS, fetal bovine serum.
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REFERENCES |
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