Download Autocrine Transforming Growth Factor Provides a Growth Advantage

THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 273, No. 47, Issue of November 20, pp. 31471–31479, 1998
Printed in U.S.A.
Autocrine Transforming Growth Factor a Provides a Growth
Advantage to Malignant Cells by Facilitating Re-entry into the Cell
Cycle from Suboptimal Growth States*
(Received for publication, August 31, 1998)
Dianhua Jiang‡§, Haisu Yang§¶, James K. V. Willsoni, Jiurong Liang‡, Lisa E. Humphrey¶,
Elizabeth Zborowskai, Degeng Wang‡, Jason Fosteri, Robert Fan‡, and Michael G. Brattain¶**
From the ‡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 iCWRU/Ireland Cancer Center and Department of Medicine, Case Western Reserve University,
Cleveland, Ohio 44106
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 a (TGF-a) was ectopically expressed by stable transfection of a TGF-a cDNA under
repressible tetracycline control. The TGF-a-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-a 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-a-transfected cells and the requirement of exogenous insulin for stimulation of quiescent cells to reenter 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-a-transfected cells indicate that autocrine
TGF-a 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-a was associated with increased malignant
properties as TGF-a transfectants showed increased tumorigenicity in athymic nude mice. The administration
of tetracycline reversed the effects of TGF-a 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-a. 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-a.
* 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. This 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.
This paper is available on line at http://www.jbc.org
Transforming growth factor a (TGF-a)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-a has been associated with neoplastic transformation (4, 5). Overexpression of TGF-a by stable transfection of a constitutive vector induced transformation of normal
cultured fibroblasts (6). Co-infection with retroviruses encoding both TGF-a 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-a results in the appearance of malignant tumors in liver, mammary gland, and benign tumors in skin
(8 –10). Moreover, overexpression of TGF-a in vivo enhanced
oncogene-induced carcinogenesis in pancreas, liver, and mammary gland (11–13) and accelerated chemically induced hepatocarcinogenesis (14, 15).
The contribution of TGF-a in the malignant progression of
human carcinomas has not been well studied. Moreover, although it is clear that TGF-a 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-a 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 growtharrested states. These gaps in our understanding of TGF-a
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-a 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.
We have addressed the issue of understanding the pathological disruption of growth regulation by autocrine TGF-a using
growth factor-dependent cell line models (CBS and FET cells)
1
The abbreviations used are: TGF-a, transforming growth factor a;
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.
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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-a cDNA under repressible control by tetracycline in order to generate a strong autocrine TGF-a loop so that
specific growth requirements assumed by autocrine TGF-a
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-a, 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-a activity.
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-a and
EGFR constitutively in growth-arrested states in contrast to
CBS and FET cells in which TGF-a 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 growtharrested state.
TGF-a transfection using a tetracycline-repressible system
permitted the generation of CBS and FET cells in which TGF-a
was not down-regulated at growth arrest since the transgene
was not under control of the TGF-a 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-a was expressed in growth arrest or to behave like weakly
malignant cells that down-regulate TGF-a in growth arrest
when tetracycline was added. Comparison of wild type cells
with TGF-a transfectants shows that autocrine TGF-a 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-a mitigates the requirements for exogenous insulin and EGF in this model system.
Finally, we show that inappropriate TGF-a expression in
transfected cells provides a growth advantage over wild type
cells by enhancing the ability of growth-arrested cells to reenter 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-a-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
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 mg/ml, Sigma), transferrin (4
mg/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-a transfectants and control cells were routinely maintained in serum-free medium containing 650 mg/ml active geneticin (Life Technologies, Inc.).
Transfections—A 930-bp fragment of full-length TGF-a 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-a. 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 mg) and the tTA-expressing vector (1 mg) 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 mg/ml). After 3 weeks of
selection, geneticin-resistant clones were isolated and expanded to
screen for TGF-a expression.
Administration of Tetracycline—Tetracycline (Sigma) was dissolved
in 50% ethanol and stored at 220 °C. Tetracycline was added to the
culture medium at a final concentration of 0.1 mg/ml. Fresh tetracycline
was added when changing medium (23 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.
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-a RNase protection assays were performed
as described previously (21). Briefly, a 306-bp high specific riboprobe
was generated with the TGF-a riboprobe template in the presence of
[a-32P]UTP (NEN Life Science Products) by Sp6 RNA polymerase. The
riboprobe was hybridized with 20 mg 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-a fragment was 283 bp, whereas the protected, transfected TGF-a 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-a
mRNA from the endogenous mRNA. Quantitative analysis of the protected mRNA was performed with an Ambis Analysis System (Ambis,
San Diego, CA).
Conditioned Medium and TGF-a 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 mg/ml. On day 4, 48-h conditioned
medium was collected and clarified by centrifugation. Cells were enumerated with a hemocytometer after trypsinization. TGF-a levels were
measured with a TGF-a enzyme-linked immunosorbent assay kit (Oncogene Science) following the manufacturer’s instructions. The TGF-a
levels were normalized to cell numbers and expressed as TGF-a protein
per 106 cells.
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
mg/ml. Cells were grown for 13 days without changing the culture
medium. The cells were stained by addition of 500 ml of 0.2% of 3-[4,5dimethylthiazol-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 ml of Me2SO. The plates were placed on
TGF-a Enhances Malignancy
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 serumfree 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 3 104 cells per well in
24-well tissue culture plates or 1.2 3 105 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 43 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% b-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.
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-a 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 5 (L 3
W2) 3 0.5.
Flow Cytometry Analysis—Flow cytometry was performed as described previously (43). Briefly, trypsinized cells were washed with cold
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FIG. 1. Expression of TGF-a mRNA in CBS4 transfectants. The
CBS4 TGF-a transfectants (aS2, aS3, aS5, and aS6) and control cells
(NEO) were plated in serum-free medium in the presence (1) or absence
(2) of 0.1 mg/ml tetracycline. The endogenous and the transfected
TGF-a mRNA levels in 20 mg of total RNA were determined by an
RNase protection assay (“Experimental Procedures”). The protected
endogenous and transfected TGF-a fragments as well as internal control actin are indicated on the right. The molecular weight markers are
shown on the left.
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 ml of RNase A (2 mg/ml, DNasefree) 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 (BectonDickinson, San Jose, CA), and cell cycle parameters were obtained
using a ModFit LT program (Verity Software House Inc.).
RESULTS
Expression of TGF-a—Following transfection, geneticin-resistant clones were isolated and expanded for screening of
TGF-a expression. Four clones (designated CBS4 aS2, aS3,
aS5, and aS6) expressing high TGF-a mRNA and protein were
identified. The CBS4 transfectants (Fig. 1, lanes 4, 6, 8, and 10)
expressed 15–25-fold higher TGF-a mRNA levels relative to
CBS4-NEO (lane 2). As described under “Experimental Procedures,” the size of the protected transfected TGF-a mRNA
fragment in RNase protection assays was larger than the protected endogenous TGF-a mRNA. Tetracycline (0.1 mg/ml) repressed the expression of transfected TGF-a mRNA (Fig. 1,
lanes 3, 5, 7, and 9).
TGF-a 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-a protein levels
were repressed by the presence of 0.1 mg/ml tetracycline in the
growth medium (Fig. 2). Therefore, the overexpression of
TGF-a mRNA is reflected by increased expression of the
amount of TGF-a released into culture medium. Similar results
were obtained from FET-NEO and TGF-a-transfectant cells.
Cloning Efficiency of CBS4 TGF-a Transfectants—The effects of expression of TGF-a on the clonal growth of CBS4 cells
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TGF-a Enhances Malignancy
FIG. 2. Expression of TGF-a protein. The CBS4 parental cells
(SF), NEO, and TGF-a transfectants (aS2) 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 mg/ml tetracycline (Tet). The 48-h
conditioned media were collected on day 4, and the TGF-a levels were
measured with a TGF-a enzyme-linked immunosorbent assay kit.
TGF-a levels were normalized to cell number and expressed as TGF-a
protein per 106 cells (mean 6 S.E. n 5 3).
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-a 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-a was markedly increased, as compared with the CBS4NEO cells (Fig. 3). The control cells (NEO) showed an EGF-dependent phenotype in cloning efficiency experiments. The
TGF-a transfectants showed an EGF-independent phenotype
in the absence of tetracycline treatment (Fig. 3A). These findings suggested that overexpression of TGF-a 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-a 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-a enhances re-entry into the cell cycle
under environmental conditions that are not optimal for
growth.
Mitogenesis of TGF-a Transfectants—Another line of evidence of increased growth factor independence of TGF-a 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-a 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-a 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-a
transfectants was comparable to control cells. Flow cytometry
analysis of CBS NEO and CBS TGF-a transfectants indicated
that 91 and 92% of cells were in G0/G1, respectively. When
FIG. 3. Expression of TGF-a modulates cloning efficiency.
CBS4 TGF-a transfectants (aS2, aS3, and aS5) 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 mg/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 6 S.E. n 5 4).
tetracycline was used to repress the expression of transfected
TGF-a, the TGF-a transfectants regained the insulin-dependent phenotype for DNA synthesis (Fig. 4B). These data demonstrated that expression of TGF-a 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-a functions by enhancing growth and survival
under stringent environmental conditions. Similar results
were obtained for FET6 TGF-a transfectant cells (data not
shown). Moreover, the acquisition of growth factor independence was reflected by the ability of TGF-a 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-a transfec-
TGF-a Enhances Malignancy
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FIG. 4. Mitogenesis of TGF-a transfectants. CBS4 TGF-a transfectants
(aS2) 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 mg/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 6 S.E. n 5 3). C, FET6 TGF-a
transfectants (FET-aS) and NEO cells as
well as TGF-a transfectants treated with
0.1 mg/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 6 S.E. (n 5 3).
tants generated a revertant growth factor-dependent phenotype.
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-a in poorly tumorigenic cells such as
CBS and FET (27). Thus, it would be expected that autocrine
TGF-a activity would also be reduced. Reduction of autocrine
TGF-a 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-a activity in these quiescent cells. In contrast, TGF-a in
transfected cells should not be under endogenous cellular control, and autocrine TGF-a should remain elevated in quiescent
cells. Fig. 5 shows that quiescent TGF-a-transfected cells retain a high level of EGFR activation (aS2, 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 antiEGFR 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-a transfectants also showed sustained EGFR activation (Fig. 5C). The sustained activation of
EGFR in TGF-a-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.
Sustained TGF-a 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
FIG. 5. Activation of EGFR family members and SHC. A, the cell
lysates from exponential (L) and quiescent (Q) CBS4aS2 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-a 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.
exponential CBS4 TGF-a cells as well as exponential CBS4
NEO (Fig. 5). When cells were rendered quiescent, the CBS4
TGF-a 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 iso-
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TGF-a Enhances Malignancy
forms of SHC interacted with the activated EGFR in CBS cells.
Quiescent TGF-a-transfected cells retained a high level of association with SHC relative to NEO cells. These results suggested that autocrine TGF-a 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.
Reduction of Lag Time in TGF-a 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-a activity mitigates the
effects of environmental conditions retarding growth, it would
be expected that the time required for entering exponential
growth by TGF-a transfected cells would be reduced relative to
CBS4 NEO cells. CBS4 TGF-a transfectant cells (aS2) 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-a transfectants and CBS
NEO cells. At high cell density the exponential growth rates of
the transfectant and control cells were virtually identical.
Thus, TGF-a 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-a 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-a transfectants and wild type
cells. The percentages of cells from TGF-a transfectants in S or
G2/M were approximately 1.5-fold higher than wild type cells,
indicating earlier escape from lag phase by TGF-a transfectants. This is contrasted by comparison of TGF-a transfectants
with wild type cells at day 2 and 4 after seeding at high density
(3.6 3 105 cells) in order to abrogate lag phase where percentages of cells in S and G2/M were essentially equal for both cell
types.
Expression of TGF-a 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-a transfectants and control cells to form colonies in soft
agarose because this property is considered to be reflective of
malignancy (31, 32). TGF-a transfectants showed both increased colony number and colony size, compared with CBS4NEO cells (Fig. 7). The number of colonies from TGF-a 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-a
increased the malignant properties of the transfectants.
We next examined the tumorigenic properties of CBS and
FET transfectant cells in vivo. TGF-a transfectants and control
NEO cells (5 3 106) were injected into athymic mice to determine whether increased expression of TGF-a would affect the
tumorigenicity. Both CBS4 NEO cells and aS2 clone formed
xenografts in 10/10 inoculations by day 6. However, the aS2
clone showed a marked increase in tumor growth, giving rise to
a 4-fold increase in tumor volume as compared with CBS4NEO (Fig. 8A). Repression of TGF-a 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, aS2 tumors regained a rapid growth
FIG. 6. Growth curves. The CBS4 NEO and CBS4 TGF-a clone
(aS2) 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.
rate similar to that of aS2 cells without tetracycline treatment.
Thus, the overexpression of TGF-a 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-a transfection could generate a progressively
growing phenotype which would not regress after initial tumor
nodule formation. Fig. 8B shows that TGF-a transfection of
FET6 cells resulted in a fully tumorigenic phenotype in 10/10
TGF-a Enhances Malignancy
inoculations, whereas tetracycline treatment of the animals from
day 10 to day 24 resulted in complete loss of tumorigenicity.
DISCUSSION
We have demonstrated that high levels of autocrine TGF-a
contribute to the progression of malignant properties in the
CBS4 and FET6 human colon carcinoma cell lines. The CBS
TGF-a-transfected cells formed larger tumors than control cells
in athymic nude mice, and repression of TGF-a by tetracycline
treatment in vivo resulted in the reacquisition of tumor growth
at the same rate as control cells in the CBS model. TGF-a
transfection was permissive for progressive tumor growth in
TABLE I
Flow cytometry analysis
Cell cycle distribution of CBS4 TGF-a transfectants and control cells
plated at low and high density.
Cells in
CBS NEO
CBS4 aS2
75
21
4
67
25
8
66
26
8
61
30
9
69
23
8
64
28
8
%
Day 11a
Day 2b
Day 4b
G0/G1
S
G2/M
G0/G1
S
G2/M
G0/G1
S
G2/M
a
Cells were plated at low density (1 3 104/60-mm well) as described
in Fig. 6A. Flow cytometry analysis was determined at day 11.
b
Cells were plated at high density (3.6 3 105/60-mm well) as described in Fig. 6E and cell cycle analysis was determined at day 2 and
day 4 as described under “Experimental Procedures.”
FIG. 7.
Anchorage-independent
growth. CBS4 NEO cells and TGF-a
clones (aS2 and aS5) 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 (1) tetracycline at 0.1
mg/ml or remained untreated (2) (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).
31477
the non-tumorigenic FET6 cell line, whereas tetracycline treatment led to the complete resolution of tumors. Moreover, the
TGF-a 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-a plays a role in the acquisition of properties
associated with malignant progression of cells such as growth
factor independence and tumorigenicity.
Moreover, these results address important issues regarding
the mechanism whereby autocrine TGF-a activity can impart a
growth advantage to malignant cells. One issue is whether
autocrine TGF-a 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-a 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-a
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-a does not involve actively cycling cells. Perhaps some other autocrine factors function in this growth state.
31478
TGF-a Enhances Malignancy
FIG. 8. TGF-a-mediated enhanced tumorigenicity. A, CBS4 aS2
TGF-a transfectants and NEO cell lines were injected at inocula of 5 3
106 cells subcutaneously behind the anterior forelimb of athymic mice.
One set of animals inoculated with TGF-a transfectants (aS2 1 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-a transfectants were characterized
for tumorigenicity as described above for CBS cells except that tetracycline treatment was continued until day 24 in this experiment.
It is important to note that demonstration of autocrine TGF-a
in normal cells and nonmalignant adenoma cells has thus far
been restricted to actively cycling cells (34 –37).
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-a) as
an integral element of their growth regulation (34). The observation of autocrine TGF-a activity in normal cells indicates the
need to re-examine how TGF-a 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-a 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-a 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-a-transfected CBS cells imply that the principal advantage of autocrine TGF-a 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.
Most intriguing was the observation that high levels of autocrine TGF-a 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-a differs qualitatively and/or quantitatively from
the autocrine TGF-a demonstrated by CBS4 control cells.
It could be speculated that TGF-a transfectants generate
higher levels of EGFR activation such that the increased magnitude encompasses insulin signal transduction. However, exponentially growing TGF-a transfectants and wild type cells
show similar levels of EGFR activation. Thus, although large
increases in TGF-a resulted from transfection, autocrine activation of EGFR in growth-arrested TGF-a 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-a 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-a 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-a and/or recruitment of other
EGFR family members could similarly interact with cell cycle
control molecules such that there is overlap with insulin func-
TGF-a Enhances Malignancy
tion. Finally, autocrine TGF-a 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-a 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-a transfectants could abrogate this type of
requirement for insulin. Thus, our results raise an important
issue regarding the mechanistic advantage of autocrine TGF-a
in malignancy which remains to be resolved.
The use of a tetracycline-repressible transfection system provided a particularly powerful control for determining the alterations in biological properties resulting from TGF-a 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-a 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-a 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-a expression.
Although the levels of TGF-a expression were increased 15–
25-fold in TGF-a 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-a-transfected cells. Since autocrine TGF-a functions
through the EGFR exclusively, this result implies that malignant progression could result from relatively small increments
of TGF-a. This raises an important issue as to the role of
autocrine TGF-a in the pathogenesis of growth regulation of
highly progressed malignant cells. Is the level of autocrine
TGF-a 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-a transfection as described
above. The hypothesis that inappropriate expression of TGF-a
at growth arrest is the important determinant of autocrine
growth advantage is also consistent with a lack of effect on
doubling time in TGF-a-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 reentry into the cell cycle and show increased TGF-a expression
in growth arrest similarly to the TGF-a CBS4 transfectants
generated in this study (16, 27). Moreover, blockade of autocrine TGF-a in HCT116 cells results in the loss of growth factor
independence for DNA synthesis (21).
2
Acknowledgments—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.
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