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J Biol Chem, Vol. 273, Issue 47, 31471-31479, November 20, 1998
From the CBS human colon carcinoma cells are
poorly tumorigenic in athymic nude mice, whereas FET colon carcinoma
cells are non-tumorigenic. Both cell lines have well differentiated
properties in tissue culture. Transforming growth factor Transforming growth factor The contribution of TGF- We have addressed the issue of understanding the pathological
disruption of growth regulation by autocrine TGF- Previously, we contrasted the biological properties of these early
stage malignant cell models with those of highly progressed colon
carcinoma cell lines (18-21). In contrast to early stage models,
highly malignant colon carcinoma cells are completely growth
factor-independent with respect to stimulation by exogenous growth
factors, are anchorage-independent, and form tumors in 100% of athymic
mice with as little as 106 cells. Importantly, these highly
malignant cells express TGF- TGF- Cell Culture--
The human colon carcinoma cell lines were
originally isolated from primary tumors as described previously (18)
and continuously maintained in a chemically defined serum-free medium
consisting of McCoy's 5A medium (Sigma) supplemented with pyruvate,
vitamins, amino acids, antibiotics, insulin (20 µg/ml, Sigma),
transferrin (4 µg/ml, Sigma), and EGF (10 ng/ml, R & D Systems,
Minneapolis, MN) (20). Working cultures were maintained at 37 °C in
a humidified atmosphere of 5% CO2 and routinely checked
for mycoplasma contamination. Limiting dilution clones were obtained by
plating the parental cells into 96-well tissue culture plates at one
cell per well. Typical limiting dilution clones (designated as CBS4 and
FET6) were used for transfection to ensure the uniformity of the
isolated transfectants. TGF- Transfections--
A 930-bp fragment of full-length TGF- Administration of Tetracycline--
Tetracycline (Sigma) was
dissolved in 50% ethanol and stored at RNA Isolation and Analysis--
Total RNA was isolated from
confluent cell cultures by lysis in guanidine isothiocyanate and
purified by ultracentrifugation through a cesium trifluoroacetic acid
gradient as described previously (25). TGF- Conditioned Medium and TGF- Growth Assays--
Cells were plated at a clonal density of 300 cells/well into 24-well tissue culture plates in serum-free medium in
the presence or absence of EGF. Tetracycline was used at a final
concentration of 0.1 µg/ml. Cells were grown for 13 days without
changing the culture medium. The cells were stained by addition of 500 µl of 0.2% of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (Sigma) to each well for 2 h. Following removal of the
medium and staining solution, the colonies were solubilized in 800 µl of Me2SO. The plates were placed on a shaker for 5 min at
room temperature after which they were read immediately at 595 nm. The
absorbance of dissolved colonies is directly proportional to the number
of cells in culture.
Proliferation Assays--
Cells were plated at different cell
densities (30,000-360,000 cells/well) into 6-well tissue culture
plates in serum-free medium lacking EGF. Every other day the medium was
changed. The cells were pulse-labeled with
methyl[3H]thymidine (Amersham Pharmacia Biotech) for
1 h. DNA synthesis was measured by incorporation of
methyl-[3H]thymidine into trichloroacetic
acid-precipitable material.
The mitogenesis assays were performed as described previously (16). The
cells were plated at a density of either 3.0 × 104
cells per well in 24-well tissue culture plates or 1.2 × 105 cells per well in 6-well plates in medium containing
transferrin, insulin, and EGF. The cells were rendered quiescent by
growth factor and nutrient deprivation for 6 days in supplemental
McCoy's medium after reaching saturation density (7 days). Previous
work (16) showed that CBS and FET cells treated by this protocol had a
labeling index of 5-10% as determined by [3H]thymidine
incorporation followed by autoradiography. Cells were released from
quiescence with supplemental McCoy's medium (nutrients alone) or
serum-free medium (nutrients and growth factors). The cells were
pulse-labeled with methyl[3H]thymidine for 1 h. DNA
synthesis was measured by incorporation of
methyl[3H]thymidine into trichloroacetic
acid-precipitable material at the peak of DNA synthesis 18 h after
release from quiescence (16).
Immunoprecipitation and Western Immunoblotting--
Cells was
washed with cold phosphate-buffered saline and harvested in cold lysis
buffer (0.5% Nonidet P-40, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl with protease inhibitors freshly added). Crude cell
lysates were passed through a 21-gauge needle 4× to sheer DNA,
incubated on ice for 30 min, and centrifuged for 20 min at 4 °C.
Samples were incubated overnight at 4 °C with anti-EGFR antibody or
anti-activated EGFR antibody (Transduction Laboratories, Lexington,
KY). Immunoprecipitates were subsequently incubated with 50% protein
A-agarose suspension (Life Technologies, Inc.) for 30 min at 4 °C
and then were washed twice with cold lysis buffer 3 times followed by
centrifugation. Pellets were resuspended in sample buffer (0.125 M Tris-HCl (pH 6.8), 4% SDS, 10% glycerol, and 4%
Anchorage-independent Growth--
Soft agarose assays were
performed as described previously (18). Briefly, 3,000 cells suspended
in serum-free growth medium minus EGF containing 0.4% agarose (Sigma)
were plated per well into 6-well tissue culture plates containing 0.8%
agarose underlayers. Colonies were allowed to grow for 2 weeks at
37 °C in a humidified incubator at 5% CO2, stained with
1 ml of p-iodonitrotetrazolium violet (Sigma), and photographed.
Tumorigenicity--
NEO control and TGF- Flow Cytometry Analysis--
Flow cytometry was performed as
described previously (43). Briefly, trypsinized cells were washed with
cold phosphate-buffered saline and resuspended in 0.4-0.6 ml of low
salt propidium iodide stain (0.03 g/ml polyethylene glycol, 0.05 mg/ml
propidium iodide (Sigma), 0.1% Triton X-100, and 4 mM
sodium citrate). The suspension was incubated with 10 µl of RNase A
(2 mg/ml, DNase-free) at 37 °C for 20 min. An equal volume of high
salt stain (0.03 g/ml polyethylene glycol, 0.05 mg/ml propidium iodide
(Sigma), 0.1% Triton X-100, and 400 mM sodium chloride)
was then added. The mixture was vortexed gently and stored at 4 °C
overnight. The cell cycle phase distribution was performed using a
FACScan flow cytometer (Becton-Dickinson, San Jose, CA), and cell cycle
parameters were obtained using a ModFit LT program (Verity Software
House Inc.).
Expression of TGF-
TGF- Cloning Efficiency of CBS4 TGF- Mitogenesis of TGF- Activation of EGFR Family Members and SHC--
Previous work had
shown that growth arrest induced by nutrient and growth factor
deprivation was accompanied by reduced expression of endogenous TGF-
Sustained TGF- Reduction of Lag Time in TGF- Expression of TGF-
We next examined the tumorigenic properties of CBS and FET transfectant
cells in vivo. TGF- We have demonstrated that high levels of autocrine TGF- Moreover, these results address important issues regarding the
mechanism whereby autocrine TGF- The original autocrine hypothesis was formulated to account for the
growth advantage of malignant cells over their normal counterparts
(33). As with most hypotheses, the addition of new information over the
course of time suggests the need for fine tuning of the hypothesis. One
of the most important pieces of information pertinent to the autocrine
hypothesis in recent years has been the recognition that normal cells
demonstrate autocrine-positive activity (frequently in the form of
TGF- Most intriguing was the observation that high levels of autocrine
TGF- It could be speculated that TGF- The use of a tetracycline-repressible transfection system provided a
particularly powerful control for determining the alterations in
biological properties resulting from TGF- Although the levels of TGF- We thank Dr. H. Bujard, University of
Heidelberg, Germany, for kindly providing the tetracycline-controllable
expression plasmids. We also thank Dr. Gillian M. Howell for critical
reading of the manuscript and helpful discussion.
*
This work was supported by National Institutes of Health
Grants CA34432, CA54807 (to M. G. B.), CA68316, and CA43703 (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.
§
Both authors contributed equally to this work.
**
To whom correspondence should be addressed: Dept. of Surgery and
Biochemistry, the University of Texas, Health Science Center at San
Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78284-7840. Tel.:
210-567-4524; Fax: 210-567-3447.
The abbreviations used are:
TGF- 2
D. Wang and M. G. Brattain, unpublished observations.
Autocrine Transforming Growth Factor 
Provides a Growth
Advantage to Malignant Cells by Facilitating Re-entry into the Cell
Cycle from Suboptimal Growth States*
§,
,,,,,, and Departments of Biochemistry and Molecular
Biology, Medical College of Ohio, Toledo, Ohio 43699, the
¶ Department of Surgery and Biochemistry, the University of Texas
Health Science Center, San Antonio, Texas 78284-7840, and the
CWRU/Ireland Cancer Center and Department of Medicine, Case
Western Reserve University, Cleveland, Ohio 44106
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(TGF-)
was ectopically expressed by stable transfection of a TGF- cDNA
under repressible tetracycline control. The TGF--transfected cells
showed enhanced clonal initiation and shortened lag phase growth in
tissue culture without an alteration in doubling time in exponential
phase relative to untransfected cells. Furthermore, the TGF-
transfectants showed increased independence from exogenous growth
factors in clonal growth assays and induction of DNA synthesis after
release from quiescence. Growth factor independence was associated with
sustained epidermal growth factor receptor activation in quiescent
TGF--transfected cells and the requirement of exogenous insulin for
stimulation of quiescent cells to re-enter the cell cycle. Higher
cloning, reduced lag time in tissue, and the acquisition of growth
factor independence for DNA synthesis without a change in doubling time of TGF--transfected cells indicate that autocrine TGF- functions by facilitating re-entry into the cell cycle from sub-optimal growth
states rather than promoting or controlling the proliferation of
actively cycling cells. The modulation of growth regulation by
autocrine TGF- was associated with increased malignant properties as
TGF- transfectants showed increased tumorigenicity in athymic nude
mice. The administration of tetracycline reversed the effects of
TGF- expression in these cells both in vivo and in
vitro, indicating that the alterations of the biological
properties were due to the expression of TGF-. Since these cells are
continuously grown in a completely chemically defined medium without
serum supplementation, it was possible to assign the mechanism
underlying the generation of growth factor independence to the
replacement of a requirement for exogenous insulin in parental cells by
autocrine TGF-.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(TGF-)1 is a potent
mitogen structurally and functionally related to the epidermal growth
factor (EGF) family of proteins (1). It binds to the EGF receptor (EGFR) to produce a positive growth stimulus (2, 3). Elevated expression of TGF- has been associated with neoplastic
transformation (4, 5). Overexpression of TGF- by stable transfection
of a constitutive vector induced transformation of normal cultured fibroblasts (6). Co-infection with retroviruses encoding both TGF-
and EGFR produced NIH3T3 cell clones that grew efficiently in soft agar
and formed tumors in nude mice (7). Transgenic mouse experiments
revealed that the overexpression of TGF- results in the appearance
of malignant tumors in liver, mammary gland, and benign tumors in skin
(8-10). Moreover, overexpression of TGF- in vivo
enhanced oncogene-induced carcinogenesis in pancreas, liver, and
mammary gland (11-13) and accelerated chemically induced hepatocarcinogenesis (14, 15).
in the malignant progression of human
carcinomas has not been well studied. Moreover, although it is clear
that TGF- promotes tumorigenicity and malignant progression in a
variety of in vitro and in vivo assays, it is not
apparent what specific growth advantages it imparts to cells in the
various contexts of proliferative function and what specific growth
functions it replaces in generating the independence from exogenous
growth factors associated with malignant progression. More
specifically, it is not known whether autocrine TGF- functions by
driving exponential growth or whether it is more critical in other
contexts of proliferation such as re-entry into the cell cycle from
growth-arrested states. These gaps in our understanding of TGF-
function in malignancy reflect a general lack of understanding as to
how autocrine-positive growth factors contribute a growth advantage to
malignant cells, especially in light of the well documented autocrine
activity of normal cells (34). Thus, an in depth understanding of how TGF- works in malignancy will provide direction for the
investigation of other autocrine factors. Moreover, the understanding
of the pathological nature of growth regulatory disruptions associated with malignant progression may lead to new approaches for the diagnosis, prevention, and treatment of cancer.
using growth factor-dependent cell line models (CBS and FET cells) grown
in completely chemically defined medium. Consequently, specific
exogenous growth factor requirements have been defined for exponential
proliferation as well as re-entry into the cell cycle from
growth-arrested states (16, 17). These growth
factor-dependent cell lines were stably transfected with a
human TGF- cDNA under repressible control by tetracycline in
order to generate a strong autocrine TGF- loop so that specific
growth requirements assumed by autocrine TGF- could be determined
and evaluated in the context of malignant progression as well.
Repressible tetracycline control has permitted analysis of specific
growth states in tissue culture and in vivo which are
dependent upon autocrine TGF-, whereas the completely chemically
defined medium in which these cells are continuously maintained and
grown permits the determination of specific exogenous growth factor
requirements replaced by autocrine TGF- activity.
and EGFR constitutively in
growth-arrested states in contrast to CBS and FET cells in which
TGF- is down-regulated during the establishment of growth arrest.
CBS and FET cells are well differentiated in tissue culture as
indicated by retention of basolateral polarity, microvilli, and
transport function (18-21). Moreover, they are weakly
anchorage-independent and poorly tumorigenic in athymic mice (18, 20).
These cells are dependent upon exogenous growth factors for DNA
synthesis with a requirement for insulin to re-enter the cell cycle
from a growth-arrested state.
transfection using a tetracycline-repressible system permitted
the generation of CBS and FET cells in which TGF- was not
down-regulated at growth arrest since the transgene was not under
control of the TGF- promoter. This created a model that could then
be modulated to behave in the same manner as the highly malignant cells
described above in which TGF- was expressed in growth arrest or to
behave like weakly malignant cells that down-regulate TGF- in growth
arrest when tetracycline was added. Comparison of wild type cells with
TGF- transfectants shows that autocrine TGF- generates malignant
progression in a tetracycline-repressible manner by the criteria of
tumorigenicity in athymic mice and anchorage-independent growth. In
addition, several lines of evidence indicate that autocrine TGF-
mitigates the requirements for exogenous insulin and EGF in this model
system. Finally, we show that inappropriate TGF- expression in
transfected cells provides a growth advantage over wild type cells by
enhancing the ability of growth-arrested cells to re-enter the cell
cycle in association with inappropriate EGFR activation in growth
arrest. Inappropriate EGFR activation resulted in the recruitment of
other EGFR family members to form complexes with the activated EGFR in
growth-arrested TGF--transfected cells. The recruitment of other
EGFR family members expanded the potential for additional signal
transduction pathways. The advantage of growth factor independence
would be of particular importance to malignant cells under
environmental conditions that are limiting to growth. Environmental
restriction on growth appears to be common in solid tumors such as
colon carcinoma in which labeling studies have shown that fewer than
1% of the malignant cells are cycling at any given time (41).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
transfectants and control cells were
routinely maintained in serum-free medium containing 650 µg/ml active
geneticin (Life Technologies, Inc.).
cDNA (23) was inserted into the EcoRI site of the
polycloning region in the tetracycline-repressible vector pUHD10-3
(22) in the sense orientation relative to the human cytomegalovirus
(hCMV) minimal promoter to create the expression plasmid pTET-TGF-.
Orientation of the insert was confirmed by restriction digestion. The
hCMV minimal promoter requires a tetracycline-controlled transactivator
(tTA) that is constitutively produced by another vector. The latter
vector (pUHD15-1/NEO) also contains the neomycin-resistant gene which
can be used for antibiotic selection (24). Because tetracycline
prevents tTA from binding to tet operators placed upstream of the hCMV
minimal promoter, it represses the tTA-dependent expression
system. The expression vector (10 µg) and the tTA-expressing vector
(1 µg) were co-transfected into cells by electroporation (Bio-Rad) at
250 V and 960 millifarads. The control cells were transfected with the
tTA-NEO selection vector and the cloning vector without any insert.
Selection of stable transfectants was carried out by addition of
geneticin (650 µg/ml). After 3 weeks of selection,
geneticin-resistant clones were isolated and expanded to screen for
TGF- expression.
20 °C. Tetracycline was
added to the culture medium at a final concentration of 0.1 µg/ml.
Fresh tetracycline was added when changing medium (2× weekly).
Tetracycline was added to the drinking water of athymic nude mice at a
concentration of 3 mg/ml on day 6 and 2 mg/ml on days 7 through 21 of
the in vivo study.
RNase protection assays
were performed as described previously (21). Briefly, a 306-bp high
specific riboprobe was generated with the TGF- riboprobe template in
the presence of [-32P]UTP (NEN Life Science Products)
by Sp6 RNA polymerase. The riboprobe was hybridized with 20 µg of
total RNA for 12-16 h. Following RNase digestion of excess riboprobe,
the protected fragment was electrophoresed on a 6% polyacrylamide, 7 M urea sequencing gel. Loading was normalized by
simultaneous hybridization of the RNA with an actin probe that yields a
145-bp protected fragment (21). The protected endogenous TGF-
fragment was 283 bp, whereas the protected, transfected TGF-
fragment was 306 bp, since the expression vector contained an
additional 23 bp of vector sequence. This provided the ability to
distinguish the expression of the transfected TGF- mRNA from the
endogenous mRNA. Quantitative analysis of the protected mRNA
was performed with an Ambis Analysis System (Ambis, San Diego, CA).
Enzyme-linked Immunosorbent
Assay--
Cells were plated at 120,000 cells per well in 6-well
plates in serum-free medium lacking EGF in the presence or absence of tetracycline at a final concentration of 0.1 µg/ml. On day 4, 48-h
conditioned medium was collected and clarified by centrifugation. Cells
were enumerated with a hemocytometer after trypsinization. TGF-
levels were measured with a TGF- enzyme-linked immunosorbent assay
kit (Oncogene Science) following the manufacturer's instructions. The
TGF- levels were normalized to cell numbers and expressed as TGF-
protein per 106 cells.
-mercaptoethanol), heated for 5 min at 95 °C, and resolved by
7.5% SDS-polyacrylamide gel electrophoresis followed by transfer to
nitrocellulose membranes. Membranes were blocked with TTBS buffer (20 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 0.05% Tween
20) containing 5% nonfat dried milk for 2 h at 4 °C with
continuous rotation. The immunoprecipitates with anti-EGFR antibody
were then detected with phosphotyrosine monoclonal antibody (PY99) from
Santa Cruz Biotechnology. The immunocomplexes with anti-activated EGFR
antibody were then immunoblotted with anti-ErbB2 and anti-ErbB3
polyclonal antibodies (Santa Cruz Biotechnology) and anti-SHC antibody
(Upstate Biotechnology Inc.), respectively. The blots were incubated
with horseradish peroxidase-conjugated secondary antibody for 1 h
at room temperature and then washed twice with water, 5 min with TTBS
buffer, and 5 more times with water. The membrane was immersed in ECL
for 1 min and exposed to ECL film. EGFR activation was also directly
detected with an anti-activated EGFR antibody (Transduction Laboratories, Lexington, KY). This activated EGFR antibody is unique in
that it reacts only with the tyrosine-phosphorylated (activated) EGF
receptor (26). The cell lysates were subjected to SDS-polyacrylamide
electrophoresis gel followed by transfer to nitrocellulose membranes.
Then the membrane was probed with the anti-activated EGFR antibody directly.
transfectant cells
were injected subcutaneously behind the anterior forelimb of
5-6-week-old Balb/c athymic mice. Mice were maintained in a
genotobiotic environment before and during each experiment.
Tetracycline was given to mice in drinking water at a concentration of
3 mg/ml on day 6 and 2 mg/ml on day 7 through day 21 of inoculation.
Growth curves for xenografts were determined by externally measuring
tumors in two dimensions using a caliper. Volume (V) was
determined by the following equation, where L is length and
W is the width of the tumor: V = (L × W2) × 0.5.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
--
Following transfection,
geneticin-resistant clones were isolated and expanded for screening of
TGF- expression. Four clones (designated CBS4 S2, S3, S5,
and S6) expressing high TGF- mRNA and protein were
identified. The CBS4 transfectants (Fig. 1, lanes 4, 6, 8, and
10) expressed 15-25-fold higher TGF- mRNA levels
relative to CBS4-NEO (lane 2). As described under
"Experimental Procedures," the size of the protected transfected
TGF- mRNA fragment in RNase protection assays was larger than
the protected endogenous TGF- mRNA. Tetracycline (0.1 µg/ml)
repressed the expression of transfected TGF- mRNA (Fig. 1,
lanes 3, 5, 7, and 9).
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Fig. 1.
Expression of TGF- mRNA in CBS4
transfectants. The CBS4 TGF- transfectants (S2, S3,
S5, and S6) and control cells (NEO) were plated in serum-free
medium in the presence (+) or absence () of 0.1 µg/ml tetracycline.
The endogenous and the transfected TGF- mRNA levels in 20 µg
of total RNA were determined by an RNase protection assay
("Experimental Procedures"). The protected endogenous and
transfected TGF- fragments as well as internal control actin are
indicated on the right. The molecular weight markers are
shown on the left.
protein levels in conditioned medium also showed a marked
increase of 15-25-fold as compared with CBS4-NEO (Fig.
2 and data not shown). Similarly, the
TGF- protein levels were repressed by the presence of 0.1 µg/ml
tetracycline in the growth medium (Fig. 2). Therefore, the
overexpression of TGF- mRNA is reflected by increased expression
of the amount of TGF- released into culture medium. Similar results
were obtained from FET-NEO and TGF--transfectant cells.
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Fig. 2.
Expression of TGF- protein. The CBS4
parental cells (SF), NEO, and TGF-
transfectants (S2) were plated into 6-well plates at a
density of 120,000 cells/well in serum-free medium minus EGF in the
presence or absence of 0.1 µg/ml tetracycline (Tet). The
48-h conditioned media were collected on day 4, and the TGF- levels
were measured with a TGF- enzyme-linked immunosorbent assay kit.
TGF- levels were normalized to cell number and expressed as TGF-
protein per 106 cells (mean ± S.E. n = 3).
Transfectants--
The effects
of expression of TGF- on the clonal growth of CBS4 cells were
determined. Our previous work has shown that CBS cells require
exogenous growth factors for optimal growth (16, 20). We hypothesized
that the overexpression of TGF- would reduce the growth factor
requirements of CBS4 cells and result in increased clonal formation in
the absence of growth factors. As expected, the clonal initiation of
CBS4 cells overexpressing TGF- was markedly increased, as compared
with the CBS4-NEO cells (Fig. 3). The
control cells (NEO) showed an EGF-dependent phenotype in
cloning efficiency experiments. The TGF- transfectants showed an
EGF-independent phenotype in the absence of tetracycline treatment (Fig. 3A). These findings suggested that overexpression of
TGF- could move more cells from the non-dividing, quiescent stage
back into the cell cycle and drive more cells to initiate the clonal growth. However, in the presence of tetracycline which suppressed expression of TGF- in the transfected cells, the clones revealed a
growth factor-dependent phenotype similar to that of the
NEO control cells (Fig. 3B). These data provide one line of
evidence that autocrine TGF- enhances re-entry into the cell cycle
under environmental conditions that are not optimal for growth.
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Fig. 3.
Expression of TGF- modulates cloning
efficiency. CBS4 TGF- transfectants (S2, S3,
and S5) and control cells (NEO) were plated at
clonal density (300 cells per well) in 24-well plates in serum-free
medium without EGF (TI) or with 10 ng/ml EGF
(TIE). The cells were untreated (A) or treated
with tetracycline (Tet) at a final concentration of 0.1 µg/ml (B). A
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay
was performed to stain colonies 13 days after plating. The absorbance
of dissolved colonies in Me2SO was measured at 595 nm
(mean ± S.E. n = 4).
Transfectants--
Another line of evidence
of increased growth factor independence of TGF- transfectants was
demonstrated by mitogenesis studies. Quiescent growth-arrested CBS
cells require exogenous growth factors in addition to fresh nutrients
for re-entry into the cell cycle and induction of DNA synthesis as
described previously (16). CBS4 control cells required exogenous
insulin for initiation of DNA synthesis (NEO in Fig.
4A). In contrast, DNA
synthesis by quiescent TGF- transfectants stimulated with fresh
medium lacking any growth factors (nutrient replenishment alone) was
comparable to that of cells released with insulin-containing medium or
a medium containing insulin, transferrin, and EGF (Fig. 4A).
Cell cycle distribution of wild type and TGF- transfectant cells was determined by flow cytometry following the 6-day period of growth factor and nutrient deprivation to generate quiescence in order to
ensure that growth arrest in TGF- transfectants was comparable to
control cells. Flow cytometry analysis of CBS NEO and CBS TGF- transfectants indicated that 91 and 92% of cells were in
G0/G1, respectively. When tetracycline was used
to repress the expression of transfected TGF-, the TGF-
transfectants regained the insulin-dependent phenotype for
DNA synthesis (Fig. 4B). These data demonstrated that
expression of TGF- reduced the requirements for exogenous growth
factors by these cells, thus providing growth factor independence for
re-entry into the cell cycle from quiescence. These results provide a
second line of evidence that autocrine TGF- functions by enhancing
growth and survival under stringent environmental conditions. Similar
results were obtained for FET6 TGF- transfectant cells (data not
shown). Moreover, the acquisition of growth factor independence was
reflected by the ability of TGF- transfectants to proliferate in the
absence of growth factor supplementation, whereas control cells could
not sustain proliferation in the absence of exogenous growth factors
added to the medium (Fig. 4C). Addition of tetracycline to
the medium of TGF- transfectants generated a revertant growth
factor-dependent phenotype.
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Fig. 4.
Mitogenesis of TGF- transfectants.
CBS4 TGF- transfectants (S2) and control cells
(NEO) were plated at 120,000 cells/well in 6-well plates
(A) or 30,000 cells/well in 24-well plates (B) in
serum-free medium without EGF. After a 7-day cultivation period, the
confluent cells were rendered quiescent by deprivation of growth
factors for 6 days. Cells were stimulated to initiate DNA synthesis by
treatment with either serum-free medium lacking any growth factors
(SM), serum-free medium with insulin (I),
serum-free medium with transferrin and insulin (TI), or
serum-free medium with transferrin, insulin, and EGF (TIE).
Some cells received 0.1 µg/ml tetracycline (B, lower
panel). The DNA synthesis was measured by
[3H]thymidine incorporation assays. The data were plotted
as fold increase of DNA synthesis stimulated by addition of fresh
medium versus DNA synthesis with no medium change (mean ± S.E. n = 3). C, FET6 TGF-
transfectants (FET-S) and NEO cells as well as TGF- transfectants
treated with 0.1 µg/ml tetracycline were plated at 120,000 cells/well
in 6-well plates in medium without any growth factor supplements. Cell
proliferation was then determined by [3H]thymidine
incorporation as described above. Data are expressed as mean ± S.E. (n = 3).
in poorly tumorigenic cells such as CBS and FET (27). Thus, it would be
expected that autocrine TGF- activity would also be reduced.
Reduction of autocrine TGF- activity should be reflected by reduced
EGFR activation. Growth arrest of CBS4 cells is accompanied by reduced
EGFR activation (NEO, Q in Fig. 5), thus reflecting reduced autocrine
TGF- activity in these quiescent cells. In contrast, TGF- in
transfected cells should not be under endogenous cellular control, and
autocrine TGF- should remain elevated in quiescent cells. Fig. 5
shows that quiescent TGF--transfected cells retain a high level of
EGFR activation (S2, Q) relative to NEO cells (NEO, Q). Activation
of the EGFR was determined by direct immunoblotting with an antibody
directed against activated EGFR in Fig. 5A or by
immunoprecipitation with anti-EGFR antibody followed by Western
blotting with PY99 antibody directed against phosphotyrosine (Fig.
5B). Normalization of protein loading was confirmed by
reblotting with anti-EGFR antibody. Similarly, FET TGF-
transfectants also showed sustained EGFR activation (Fig.
5C). The sustained activation of EGFR in TGF--transfected cells is probably responsible for the abrogation of exogenous activation of the insulin receptor or, more likely, the insulin-like growth factor I receptor (IGFI-R) as a prerequisite for re-entry into
the cell cycle and initiation of DNA synthesis.
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Fig. 5.
Activation of EGFR family members and
SHC. A, the cell lysates from exponential
(L) and quiescent (Q) CBS4 S2 and NEO cells
were subjected to SDS-polyacrylamide gel electrophoresis followed by
transfer to nitrocellulose membranes. Membranes were probed with
anti-activated EGFR antibody that is specific for activated EGF
receptors and visualized by sequential incubation with horseradish
peroxidase-conjugated secondary antibody. B, the total
proteins (4 mg) were immunoprecipitated with anti-EGFR antibody.
Immunoprecipitates were analyzed by anti-phosphotyrosine antibody
(PY99). The same membrane was reblotted with EGFR antibody
for normalization. C, EGFR activation of FET6 TGF-
transfectants and FET NEO cells was determined as described in
A. D, the cell lysates were immunoprecipitated
(IP) with anti-(activated) EGFR antibody. Immunoprecipitates
were probed with anti-ErbB2, ErbB3, and SHC antibodies, respectively.
WB, Western blot.
binding and EGFR activation could lead to the
recruitment of other EGFR family members to form complexes with EGFR
and thus further augment the potential for enhanced signal transduction
(28-30). The activated EGFR antibody was employed to immunoprecipitate
activated EGFR followed by Western analysis to probe for ErbB2 and
ErbB3 complex formation with the EGFR. Heterodimer formation between
activated EGFR and ErbB2 or ErbB3 was observed in exponential CBS4
TGF- cells as well as exponential CBS4 NEO (Fig. 5). When cells were
rendered quiescent, the CBS4 TGF- transfectants retained high levels
of heterodimerization, whereas a significant reduction was seen in NEO
control cells (Fig. 5D). Activated EGFR should also induce
downstream components of signal transduction. The association of SHC
SH2 domain proteins with activated EGFR was therefore examined. Results
indicated that only the 52- and 46-kDa isoforms of SHC interacted with
the activated EGFR in CBS cells. Quiescent TGF--transfected cells retained a high level of association with SHC relative to NEO cells.
These results suggested that autocrine TGF- activity in CBS4 cells
not only induced higher EGFR phosphorylation and activated other EGFR
family members but also stimulated the association of activated EGFR
with downstream molecules.
Transfectants--
Lag phase of
cell culture is the period of adaptation following subculture and
reseeding during which cells demonstrate reduced proliferation. If
increased autocrine TGF- activity mitigates the effects of
environmental conditions retarding growth, it would be expected that
the time required for entering exponential growth by TGF-
transfected cells would be reduced relative to CBS4 NEO cells. CBS4
TGF- transfectant cells (S2) exhibited a shortened lag phase as
compared with CBS4-NEO cells when cultures were inoculated at low cell
density (Fig. 6, A-D). The
effect of low cell density on the time to reach exponential growth in
tissue cultures was abrogated by increasing the number of cells plated.
The growth curves in Fig. 6 indicate that increasing the number of
cells plated leads to a reduction in the difference between the TGF- transfectants and CBS NEO cells. At high cell density the exponential growth rates of the transfectant and control cells were virtually identical. Thus, TGF- expression appears to generate increased clonal initiation of cultures at a given inoculum resulting in shortened lag phase. The lack of effect on exponential growth indicates
that autocrine TGF- functions by allowing re-entry into the cell
cycle rather than affecting cell cycle transit. The results in Fig. 6,
A and E, were confirmed using flow cytometry (Table I). Flow cytometric data from day
11 at an inoculum of 104 cells (Fig. 6A) were
compared for TGF- transfectants and wild type cells. The percentages
of cells from TGF- transfectants in S or G2/M were
approximately 1.5-fold higher than wild type cells, indicating earlier
escape from lag phase by TGF- transfectants. This is contrasted by
comparison of TGF- transfectants with wild type cells at day 2 and 4 after seeding at high density (3.6 × 105 cells) in
order to abrogate lag phase where percentages of cells in S and
G2/M were essentially equal for both cell types.
View larger version (15K):
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Fig. 6.
Growth curves. The CBS4 NEO and CBS4
TGF- clone (S2) were plated at different cell
densities into 6-well tissue culture plates in serum-free medium
lacking EGF. A, 10,000 cells/well; B, 30,000 cells/well; C, 60,000 cells/well; D, 120,000 cells/well; E, 360,000 cells/well. The culture medium was
changed every other day. Starting at day 2, the DNA synthesis of the
cells of triplicate wells was measured by [3H]thymidine
incorporation assays. The mean of the three determinations and standard
errors are presented.
Flow cytometry analysis
transfectants and control cells
plated at low and high density.
Enhanced Malignant Properties--
CBS cells
have previously been shown to have low cloning efficiency in soft
agarose and to be poorly tumorigenic in athymic nude mice (18, 20).
Consequently, we compared the ability of CBS TGF- transfectants and
control cells to form colonies in soft agarose because this property is
considered to be reflective of malignancy (31, 32). TGF-
transfectants showed both increased colony number and colony size,
compared with CBS4-NEO cells (Fig. 7).
The number of colonies from TGF- transfectants was increased more
than 2-fold relative to NEO (Fig. 7A). Tetracycline
treatment reversed these effects (Fig. 7B). These in
vitro data demonstrated that expression of TGF- increased the
malignant properties of the transfectants.
View larger version (54K):
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Fig. 7.
Anchorage-independent growth. CBS4 NEO
cells and TGF- clones (S2 and S5) were
plated at 3,000 cells per well in 6-well plates in soft agarose
containing serum-free medium lacking EGF and tetracycline
(A). Some cells were treated with (+) tetracycline at 0.1 µg/ml or remained untreated () (B). Colonies were
allowed to grow for 2 weeks, stained with
p-iodonitrotetrazolium violet (Sigma), and photographed.
Colonies were counted, and bar graphs were drawn
(C and D).
transfectants and control NEO cells
(5 × 106) were injected into athymic mice to
determine whether increased expression of TGF- would affect the
tumorigenicity. Both CBS4 NEO cells and S2 clone formed xenografts
in 10/10 inoculations by day 6. However, the S2 clone showed a
marked increase in tumor growth, giving rise to a 4-fold increase in
tumor volume as compared with CBS4-NEO (Fig.
8A). Repression of TGF- by
addition of tetracycline in the drinking water of mice from day 6 to
day 21 of the inoculation resulted in reversion of the slope of the
growth curve to that of the NEO control (Fig. 8A). After
terminating tetracycline treatment, S2 tumors regained a rapid
growth rate similar to that of S2 cells without tetracycline
treatment. Thus, the overexpression of TGF- in CBS4 cells is
sufficient to enhance malignant properties of these cells in
vivo. In contrast to CBS4 cells, FET6 cells do not form
progressively growing tumors (42). FET6 cells form initial nodules of
approximately 200 mm3 at a high subcutaneous inoculum
(107 cells) which ultimately disappear. Therefore, it was
of interest to determine whether TGF- transfection could generate a
progressively growing phenotype which would not regress after initial
tumor nodule formation. Fig. 8B shows that TGF-
transfection of FET6 cells resulted in a fully tumorigenic
phenotype in 10/10 inoculations, whereas tetracycline treatment of the
animals from day 10 to day 24 resulted in complete loss of
tumorigenicity.
View larger version (20K):
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Fig. 8.
TGF- -mediated enhanced
tumorigenicity. A, CBS4 S2 TGF- transfectants and
NEO cell lines were injected at inocula of 5 × 106
cells subcutaneously behind the anterior forelimb of athymic mice. One
set of animals inoculated with TGF- transfectants (S2 + TET) was treated with tetracycline (3 mg/ml on day 6 and 2 mg/ml on days 7-21 in the drinking water), and a second set was left
untreated. Tumors were measured externally on the indicated days in two
dimensions using a caliper. B, FET6 TGF- transfectants
were characterized for tumorigenicity as described above for CBS cells
except that tetracycline treatment was continued until day 24 in this
experiment.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
contribute to the progression of malignant properties in the CBS4 and
FET6 human colon carcinoma cell lines. The CBS TGF--transfected cells formed larger tumors than control cells in athymic nude mice, and
repression of TGF- by tetracycline treatment in vivo resulted in the reacquisition of tumor growth at the same rate as
control cells in the CBS model. TGF- transfection was permissive for
progressive tumor growth in the non-tumorigenic FET6 cell line, whereas
tetracycline treatment led to the complete resolution of tumors.
Moreover, the TGF- transfectants showed decreased exogenous growth
factor requirements for clonal initiation in tissue culture and the
initiation of DNA synthesis, as well as increased cloning efficiency in
soft agarose in vitro. These results demonstrated that
autocrine TGF- plays a role in the acquisition of properties
associated with malignant progression of cells such as growth factor
independence and tumorigenicity.
activity can impart a growth advantage to malignant cells. One issue is whether autocrine TGF- provides independence from other growth factors as well as the expected
mitigation of any exogenous EGF family ligands required for EGFR
activation to enhance cell cycle entry. The utilization of model
systems in which cells are grown in completely defined medium permitted
the recognition that autocrine TGF- activation can free the cells
from an exogenous source of IGFI receptor activation for re-entry into
the cell cycle from quiescence. In addition, the present work provides
three lines of evidence showing that autocrine TGF- activity can
enhance re-entry into the cell cycle under environmental conditions
that are highly restrictive to parental CBS cells. These included
increased growth factor-independent clonality, decreased lag time in
tissue culture, and complete abrogation of exogenous growth factor
requirements for DNA synthesis from a quiescent state. These effects
were contrasted by the lack of difference of doubling times observed in
cells plated at high cell density. Thus, it appears that the primary
function of TGF- does not involve actively cycling cells. Perhaps
some other autocrine factors function in this growth state. It is
important to note that demonstration of autocrine TGF- in normal
cells and nonmalignant adenoma cells has thus far been restricted to
actively cycling cells (34-37).
) as an integral element of their growth regulation (34). The
observation of autocrine TGF- activity in normal cells indicates the
need to re-examine how TGF- imparts a growth regulatory advantage to
malignant cells. Normal cells do not, in as far as we know now, exhibit
independence from exogenous growth factors for re-entry into the cell
cycle under the various conditions used in this study. Normal
keratinocytes, for example, have an absolute requirement for exogenous
growth factor activation of the EGFR at low cell densities but do not require exogenous EGF or TGF- for clonal expansion (35, 36). Sensitivity to EGFR blockade during clonal expansion indicates the need
for autocrine-mediated EGFR activation in actively cycling cells.
Similarly, nonmalignant colon adenoma cells show autocrine TGF-
activity during exponential growth but are incapable of clonal growth
without supplementation (37). Thus, in this regard, parental CBS cells
appear to show many of the growth restrictions of non-malignant cells.
Taken together, these lines of evidence and the results reported herein
for TGF--transfected CBS cells imply that the principal advantage of
autocrine TGF- in the malignant cells may be the enhanced ability to
re-enter the cell cycle from growth arrest in environments that are not
conducive to proliferation. As such labeling studies have shown that
less than 1% of malignant cells are actively cycling in colon cancers
(41). Similar observations have been made with other solid tumors.
These observations suggest the tumor environment is not particularly
conducive to cell proliferation. Thus, an autocrine factor enabling
re-entry of cells into the cell cycle could be highly advantageous to
the malignant cells. A similar advantage could easily be envisioned for
cells involved in seeding metastases since the number of cells
initiating a metastatic deposit is generally regarded as being quite limited.
not only replaced requirements for exogenous EGF but for
insulin as well. Insulin through activation of the IGFI receptor could
enhance EGFR expression and/or activation in growth
factor-dependent cells. Alternatively, insulin could supply
a different array of signal transduction pathways from the EGFR.
Independence from insulin is intriguing because it implies that signal
transduction by inappropriate autocrine TGF- differs qualitatively
and/or quantitatively from the autocrine TGF- demonstrated by CBS4
control cells.
transfectants generate higher
levels of EGFR activation such that the increased magnitude encompasses
insulin signal transduction. However, exponentially growing TGF-
transfectants and wild type cells show similar levels of EGFR
activation. Thus, although large increases in TGF- resulted from
transfection, autocrine activation of EGFR in growth-arrested TGF-
transfectants was only about 3-fold over wild type cells. This suggests
that it is the inappropriate activation of EGFR in the growth-arrested
state by cells which normally down-regulate TGF- and lower EGFR
activation that provides independence from insulin. Inappropriate EGFR
activation in growth-arrested states may generate signal transduction
consequences that might not occur in the context of exponential cells.
For example, EGFR activation in growth-arrested TGF- transfectant
cells could involve the modification of signal transduction pathways
through the generation of new dimer interactions between EGFR and other
EGFR family members that might themselves be differentially regulated
in exponential and quiescent cells. Inappropriate expression of TGF-
and/or recruitment of other EGFR family members could similarly
interact with cell cycle control molecules such that there is overlap
with insulin function. Finally, autocrine TGF- expression could
cause autocrine activation of IGFI receptor through induction of IGFI and/or IGFII or might abrogate dependence on insulin for EGFR activation in cell cycle re-entry. Along this line we have noted that
insulin induces re-expression of down-regulated TGF- in the wild
type CBS and FET models where these cells are stimulated by exogenous
growth factor treatment to re-enter the cell cycle from
quiescence.2 Inappropriate
EGFR activation as seen in TGF- transfectants could abrogate this
type of requirement for insulin. Thus, our results raise an important
issue regarding the mechanistic advantage of autocrine TGF- in
malignancy which remains to be resolved.
transfection. We have
reported the use of this repressible vector in reversing growth
properties in tissue culture (38). Although tetracycline-repressible systems have been utilized in transgenic mice (39, 40), this is the
first example that we are aware in which this type of system has been
used to control tumor growth in vivo. When mice harboring TGF- transfectants were given tetracycline, tumor growth was minimal
and was virtually identical to that of NEO control. Removal of
tetracycline was effective in permitting a growth rate that was then
the same as that of TGF- transfectant cell inoculated mice that had
not received tetracycline. This result is significant because it
directly demonstrates that indolent tumor growth in an in
vivo setting can be stimulated to undergo significant progressive changes by a single event, which in this case is aberrant TGF- expression.
expression were increased 15-25-fold in
TGF- transfectants, the level of EGFR activation in exponential
transfected cells was only about 2-3-fold higher than NEO controls. A
similar level was observed in quiescent TGF--transfected cells.
Since autocrine TGF- functions through the EGFR exclusively, this
result implies that malignant progression could result from relatively
small increments of TGF-. This raises an important issue as to the
role of autocrine TGF- in the pathogenesis of growth regulation of
highly progressed malignant cells. Is the level of autocrine TGF-
important or is it inappropriate continued expression during growth
arrest that imparts an advantage? The results from this study cannot
directly answer this question. However, they are consistent with the
hypothesis that it is inappropriate expression since there is a
relatively small change in endogenous EGFR activation of the TGF-
transfection as described above. The hypothesis that inappropriate
expression of TGF- at growth arrest is the important determinant of
autocrine growth advantage is also consistent with a lack of effect on
doubling time in TGF--transfected cells compared with CBS NEO
controls. Along these lines, previous work has shown that highly
progressed human colon carcinoma cell lines, such as HCT116, are
independent of exogenous growth factors for re-entry into the cell
cycle and show increased TGF- expression in growth arrest similarly
to the TGF- CBS4 transfectants generated in this study (16, 27).
Moreover, blockade of autocrine TGF- in HCT116 cells results in the
loss of growth factor independence for DNA synthesis (21).
ACKNOWLEDGEMENTS
FOOTNOTES
, transforming growth factor ; EGF, epidermal growth factor; EGFR, EGF
receptor; tTA, tetracycline controlled transactivator; NEO, neomycin; hCMV, human cytomegalovirus; bp, base pair; IGFI, insulin-like growth
factor I.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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