Download Loss of Growth Factor Dependence and Conversion of Transforming

(CANCER RESEARCH 48. 6999-7003. December IS, 1988]
Loss of Growth Factor Dependence and Conversion of Transforming Growth
Factor-/?i Inhibition to Stimulation in Metastatic H-ras-transformed Murine
Fibroblasts1
Lois C. Schwarz, Marie-Claude Gingras,2 Grant Goldberg, Arnold H. Greenberg,3 and Jim A. Wright4
Manitoba institute of Cell Biology, and Departments of Biochemistry [L. C. S., J. A. W.] and Immunology [M. C. G., A. H. G.J, The University of Manitoba, 100 Olivia
Street, Winnipeg, Manitoba K3E OV9, Canada
ABSTRACT
Cell lines with varying tumorigenic and metastatic potentials have been
obtained by transformation of KIT1•¿
fibroblasts using radiation or transfection with 1-24 H-ras. We have observed an inverse relationship
between metastatic potential and dependence on serum for growth. The
effects of basic fibroblast growth factor, platelet-derived growth factor,
epidermal growth factor, and transforming growth factor-/?i (TGF-0i) on
these lines were then examined to determine if the changes in the serum
dependence of metastatic cells may be due to altered responsiveness to
specific growth factors (GFs). Cells were grown in monolayer culture and
DNA synthesis was measured by |CH3-3H]thymidine incorporation ex
periments. Both metastatic and nonmetastatic cells were shown to be
equivalent in their diminished responsiveness to basic fibroblast growth
factor, platelet-derived growth factor, and epidermal growth factor as
compared to their nontransformed, parental KIT1.•
cells. However, a
unique response of metastatic cells to TGF-/?i was identified. While TGFß,inhibited DNA synthesis in lOT'/i cells and a nonmetastatic tumor,
cells with intermediate to high metastatic ability were stimulated up to
5.8-fold by TGF-01. Interestingly, epidermal growth factor abrogated the
TGF-01 inhibition of the parental 10 1' 2 cells, but had no effect on the
TGF-01 response of any metastatic line. Therefore, metastatic but not
nonmetastatic cells, demonstrated a dramatically altered sensitivity to
TGF-01, a response which may be important in determining metastatic
potential.
INTRODUCTION
GFs5 have been categorized into competence and progression
families. Competence factors such as PDGF or bFGF stimulate
quiescent BALB/C-3T3 cells to enter Gì(1, 2). Progression
factors such as EGF and insulin, IGF-I or IGF-II, promote the
transit of BALB/C-3T3 cells through G, and their entry into
the S phase of the cell cycle (1, 3, 4). Competence factors act
before progression factors in BALB/C-3T3 cells.
GFs from the competence and progression families exert
their effects by stimulating the expression of a discrete set of
genes which are important for the regulation of cellular prolif
eration (5-11). GFs not only regulate the expression of genes
involved in growth, but enhanced expression of such genes can
alter the responsiveness of cells to GFs. For example, mycReceived 3/3/88; revised 7/21/88; accepted 9/16/88.
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.
1Supported by the National Cancer Institute of Canada and the Medical
Research Council of Canada.
2 Recipient of a Postdoctoral Studentship from the Manitoba Health Research
Council.
3 Terry Fox Cancer Research Scientist of the National Cancer Institute of
Canada.
4 Senior Research Scientist of the National Cancer Institute of Canada. To
whom requests for reprints should be addressed, at Manitoba Institute of Cell
Biology, 100 Olivia Street, Winnipeg, Manitoba, Canada R3E OV9.
*The abbreviations used are: GFs, growth factors; <*-MEM, ex-minimal essen
tial medium; bFGF. basic fibroblast growth factor; Cl, CIRAS-1; C2, CIRAS-2;
C3, CIRAS-3; DM. defined or serum-free medium; EGF, epidermal growth factor;
FBS, fetal bovine serum; IS. index of stimulation; IGF-I or -II, insulin-like growth
factor I or II; PDGF, platelet-derived growth factor; TGF-«,transforming growth
factor-oc; TGF-/3, transforming growth factor-/3.
transfected cells are hypersensitive to TGF-0 (12), EGF, and
bFGF ( 13). Furthermore, the effects of TGF-/3 on cells trans
fected with the myc-oncogene are dependent on the entire set
of GFs present at a given time (14). In contrast, ros-transfected
cells can become insensitive to the effects of PDGF, EGF,
TGF-0 (12, 15), and bFGF (13), but are hypersensitive to the
effects of insulin-like GFs (13). It was postulated that rastransfected cells secrete their own GFs leading to autocrine
stimulation of growth (16). Ultimately, the secretion of GFs
may lead to down regulation of receptors for GFs making such
cells insensitive to exogenous GFs. /îas-transformed cells ex
hibit enhanced metastatic potential (17-20), suggesting a direct
role for the ras-oncogene in the development of métastases(17).
In this study, we tested the hypothesis that the enhanced met
astatic potential of a series of ros-transfected cells may be
partially due to altered growth factor sensitivity.
MATERIALS
AND METHODS
Cells and Culture Media. The selection and properties of the cell
lines used in this study have been described elsewhere (17). In brief,
lOTVi mouse cells were transfected with T-24 H-ras. After transfection,
three cell lines, Cl, C2, and C3 were morphologically transformed
while the two cell lines NR3 and NR4 were morphologically nontrans
formed. NR3.1LA and NR3.1LB were derived from experimental lung
métastases,and the NR3.4 cell line was obtained from a nonregressing
S.C. tumor of NR3 cells. The MDS.R1 and MDS.R5 cell lines were
clones of radiation-transformed lOT'/z cells (21). Both MDS.R1 and
MDS.R5 cells were tumorigenic but nonmetastatic (17).
All cell lines were maintained at 37°Con 100-mm plastic tissue
culture plates (Falcon) in culture medium containing minimal essential
medium («-MEM) (Flow Laboratories) supplemented with antibiotics
and 10% (V/V) FBS (GIBCO). A serum-free medium was also used
which contained 0.4 mg transferrin (Sigma Chemical Co.) and 0.2 mg
insulin (Sigma Chemical Co.) in 100 ml of oc-MEM. In some experi
ments EGF (1 ^g/100 ml) (Collaborative Research) was added to the
serum-free medium.
Assay for DNA Synthesis. To initiate experiments, medium was
removed when the cells were subconfluent. The cells were then washed
with PBS. Medium containing 10% FBS, or serum-free medium with
or without EGF, was added to the cells for 24 h. Following this
incubation, the cells were always subconfluent and growing exponen
tially. Cells were then removed with buffered trypsin solution (Sigma
Chemical Co.) and centrifuged. The pellet was suspended in the same
medium which was added to the cells 24 h before their removal with
buffered trypsin solution. Cell number was then determined using a
model ZBI Coulter electronic particle counter (Coulter Electronics) and
was found to range between 4 and 7x10* cells/plate. Cells were then
seeded at various densities into 6-mm microtiter plates (Nunc). PDGF,
TGF-/SI (R & D Systems Inc.) or bFGF (Collaborative Research) were
added at various concentrations to triplicate cultures and the microplates were incubated at 37°Cin the presence of 5% CC«2
for 42.5 h.
Cells were pulsed with [CH3-3H]thymidine (50 i/Ci/ml) for 2.0 or 24.0
h before harvesting as indicated in the text. Growth medium was then
removed and 0.3% buffered trypsin solution was added to the wells for
15 min at 2 PC. Ice-cold trichloroacetic acid was then added to a final
concentration of 10% for at least 30 min at 4°C.Cells were harvested
6999
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TGF-tf, STIMULATION OF METASTATIC FIBROSARCOMAS
using a 12-well harvester (Skatron Inc.) attached to a source of water
for washing. Filters were dried and placed into 7.0 ml of Aquasol-2
(New England Nuclear). Radioactivity was determined by liquid scin
tillation spectroscopy using a scintillation counter (Model LS 7800,
Beckman). An IS was calculated as: dpms from cells treated with GF/
dpms from cells grown in the absence of GF as described by Schwarz
et al. (22). An IS of 2.0 or <0.5 was significant at P < 0.05 for individual
values of the IS.
Measurement of Doubling Time. All cell lines were maintained in
10% FBS. A constant number of cells (2.5 x IO4)was then transferred
to 60-mm plates (Falcon) in 2 ml Dulbecco's modified Eagle's medium
and nutrient F-12 mixture (GIBCO Laboratories) containing 0.5% FBS
(GIBCO). Proliferation of cells was followed each day by counting cells
removed with trypsin solution from duplicate plates using a /.»,Coulter
electronic particle counter (Coulter Electronics). Growth curves were
then prepared and doubling times were calculated from the linear
portion of the curves (23).
100.0
C2 C3
NR3.1LA
i NR3.4
NR3.1LB
•¿
C1
10.0
M
CO
M
CO
l NR4
1.0
HI
o>
a
x
UJ
l NR3
0.1
RESULTS
Reduced Serum Dependence of Metastatic Cells. The metastatic properties of lOT'/z cell lines which were transfected
with T-24 H-ras, or were radiation-transformed
have been
described ( 17). A summary of the metastatic properties of these
lines is presented in Table 1. It is important to note that the
10T'/2 parental cells are not tumorigenic, whereas the MDS.R1
and MDS.R5 cell lines are fully tumorigenic but nonmetastatic.
All the other cell lines demonstrated increasing potential for
metastatic behaviour as listed in Table 1.
Growth factor autonomy was initially characterized by deter
mining their ability to grow under reduced serum concentra
tions, in medium containing 0.5% FBS (Fig. 1). As cells became
more metastatic their requirements for serum declined, re
flected by their decreased doubling times. For example, the
most metastatic cells, C2 and C3, had the shortest doubling
times (23 and 26 h, respectively). In contrast, MDS.R5 and
MDS.R1 which are fully tumorigenic but nonmetastatic, re
quired 93 and 45 h, respectively, to transit one cell cycle. These
doubling times more closely resembled the 101 h of the parental
JOT1/: cells than the doubling times found for C2 and C3 cells.
This relationship between metastatic potential and doubling
times in 0.5% FBS was also maintained following standardi
zation of doubling times, accomplished by comparing the
growth of each cell line in 10% FBS to that in 0.5% FBS (data
not shown).
Responsiveness of Metastatic Cells to Growth Factors. Since
•¿
MDS.RI MDS.R5 10T1/2
20
30
40
50
90 100 110 120
Doubling Time (hours) in 0.5% FBS
Fig. 1. The relationship between metastatic potential and serum requirements.
Cells (2.5 x IO4) were plated onto 60-mm dishes in 2 ml Dulbecco's modified
Eagle's medium/nutrient F-12 mixture containing 0.5% FBS. Proliferation of
cells was followed each day by counting cells removed with trypsin solution from
duplicate plates using a Coulter counter. Doubling time was calculated from the
linear portion of growth curves.
the metastatic cells demonstrated a reduced requirement for
serum, it was next determined whether this was related to a
loss of responsiveness to specific GFs. The effects of various
concentrations of bFGF and PDGF were determined on: nontransformed parental cells (lOT'/z), tumorigenic but nonmetas
tatic cells (MDS.R5), cells of intermediate metastatic potential
(NR4), and cells of high metastatic potential (C3). The effects
of bFGF and PDGF were examined using three different media:
(a) oc-MEM containing 10% FBS; (b) serum-free medium
which contained 0.4 mg transferrin and 0.2 mg insulin in 100
ml oc-MEM; (c) serum-free medium as in (b) but in the presence
of 1 tig EGF per 100 ml oc-MEM (Table 2).
Two competence growth factors, bFGF (20 ng/ml) and
PDGF (100 ng/ml) had little effect on lOT'/z, MDS.R5, NR4,
and C3 cells in the presence of 10% FBS (Table 2). However,
DNA synthesis in lOT'/i cells was stimulated by bFGF and
Table 1 Tumorigenic and metastatic properties ofT24-H-ras-lransfected ¡OT^h PDGF in the serum-free medium (Table 2). The progression
flbroblasts
factor, EGF (10 ng/ml) further enhanced this response (Table
The data was summarized from previously reported results (17).
2). In contrast to lOT'/z cells, all three tumor lines, regardless
Experimental métastases'
of metastatic potential, exhibited relatively little response to
bFGF or PDGF, in serum-free medium with or without EGF
No. of lung
nodules
(Table 2).
(mean ±
Tumorigenicity
frequency"
The effects of TGF-/3, (10 ng/ml) on DNA synthesis were
Frequency
SE)
Cell line
determined
in initial experiments using nontransformed, paren
TWMDS.R5MDS.RINR3NR4ClNR3.4NR3.1LBNR3.1LAC2C30/125/55/56/810/1013/135/55/55/511/1111/110/120/60/31/1312/1920/274/46/66/68/814/140000.1
tal 10T'/2 cells and highly metastatic C3 cells (Fig. 2). lOT'/i
cells were inhibited by TGF-/3, at all cell densities, and were
±0.12.0
inhibited or did not respond to TGF-/S| in the presence of EGF
0.514±516±540
±
(Fig. 2). The metastatic C3 cells demonstrated a dramatically
different response to TGF-/3i, which was a stimulation of DNA
1849
±
synthesis at all cell densities, with or without EGF (Fig. 2).
±7118
±6121
Since the growth response differences between lOTVi and C3
±20
cells were similar at all cell densities, 15,000 cells/well was
" Tumorigenicity was determined following s.c. injections of 3 x 10s cells,
chosen for future experiments. To extend this study to include
* Injections were performed using 3x10* cells except for th
the MDS.R5 and
MDS.RI cells, where 10' cells were injected.
other cell lines, the effects of TGF-/3i on a large number of
7000
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TGF-01 STIMULATION OF METASTATIC FIBROSARCOMAS
Table 2 The effects ofbFGF (20.0 ng/ml) and PDGF (100.0 ng/ml) on DNA
synthesis in lOT'h fibrosarcomas cultured in fetal bovine serum (FBS) or defined
medium (DM)
Parental, nontransformed 10TV: cells, transformed, nonmetastatic MDS.RS
cells, cells of intermediate metastatic potential (NR4), or cells of high metastatic
potential (C3) were exposed for 24 h to one of three different media. They were:
10% FBS in a-MEM, DM containing a-MEM plus insulin (2.0 jig/ml) and
tmuster riii (4.0 Mg/ml) or DM plus EGF (10.0 ng/ml). Cells were then removed
with buffered trypsin solution and plated at 5000 cells/well. Either bFGF or
PDGF were then added and 42.5 h later, cells were pulsed with [3H]thymidine
for 2.0 h.
;
50
3
4.0
°
3.0
5
2.0
E
Growth
linebFGF
factor
lOT'/ibFGF
Cell
6.0
I
(mean
no.)002121002121Media10%
1.0
<5>
Ini1
i
(7) (7)
(91 TIT
191
(9)
~cr
I
(4>
'
:
i
0.75
FBSDM*DM
0.50
0.25
EGF10%
+
O
MDS.RSbFGF
IOTl/2
FBSDMDM
MDS.RS
NR3
NR4
CI
Celi
C2
C3
NR3.4
NR3.ILA
Lines
EGF10%
+
NR4bFGF
Fig. 3. Effects of TGF-/3, (10.0 ng/ml) on DNA synthesis in fibroblast cell
lines with or without EGF (10.0 ng/ml). Cells were grown to a density between 4
and 7 x 10' cells/100 mm plate in serum-free medium with (D) or without (D)
EGF for 24 h. Subconfluent lOT'/z cells in serum-free medium were not stimulated
by TGF-/SI at any cell density. However, cells which were stimulated by TGF-/3i
were most responsive to TGF-|Si within the range between 4 and 7x10* cells/
plate after the 24 h. Cells ( 15,000 cells/well) were then plated in the presence or
absence of TGF-/3, for 42.5 h. Cells were then pulsed with [3H)thymidine for 2.0
h and an index of stimulation was determined as described in "Materials and
Methods." An IS value of 1.0 represents no effect. The number of experiments
performed is indicated in parentheses. The SE was <10% for each experiment.
FBSDMDM
EGF10%
+
FBSDMDM
C3PDGF
EGF10%
+
10T'/2PDGF
FBSDMDM
EGF10%
+
MDS.RSPDGF
response to TGF-ßiin the presence or absence of EGF (Fig. 3).
In sharp contrast to lOT'/z or MDS.RS cells, lines which
demonstrated moderate to high metastatic ability were stimu
lated by TGF-/3i in all cases. This stimulation was statistically
significant in the presence or absence of EGF (Fig. 3). Similar
differences in TGF-01 response among the various cell lines
were also observed following a longer pulse of [3H]thymidine
FBSDMDM
EGF10%
+
FBSDMDM
NR4PDGF
EGF10%
+
C3Métastases
FBSDMDM
for 24 h (data not shown).
+ EGFIS"2.519.0107.11.01.30.90.62.66.90.83.86.90.952.067.81.50.80.70.81.10.70.97.02.6
"The Index of Stimulation refers to the cpm's in the presence of GF/cpm's in
the absence of GF. The effects ofbFGF were determined in 4 separate experiments
DISCUSSION
using triplicate cultures for all cell lines in each experiment. The effects of PDGF
were determined in 2 separate experiments using triplicate cultures for all cell
lines in each experiment. The SE was <10% in each experiment.
In this report we have examined the effects
* DM, defined medium, i.e., serum-free medium.
1 2 3 4 5 6
7 8 9101112131415
Cell Number
(x103)
Fig. 2. The effects of cell density on the responsiveness of fibroblast cell lines
to TGF-/SI (10.0 ng/ml). Parental, nontransformed lOT'A cells (
) or highly
metastatic C3 cells (
) were used. Cells were equilibrated for 24.0 h in medium
containing * -MEM plus insulin (2.0 ng 'ml) and transferrin (4.0 fig/ml), in the
presence or absence of EGF (10.0 ng/ml). Cells were then removed and replated
at densities of 1,000, 5,000, or 15,000 cells/well. TGF-0, was then added to cells
growing in the presence (•)or absence (•)of EGF for 42.5 h. Cells were pulsed
with [3H]thymidine during the last 2.0 h. An index of stimulation (IS) was then
calculated as described in "Materials and Methods." An IS value of 1.0 represents
no effect. Each point is the average of triplicate cultures. The SE was <10%.
lines, which varied in their metastatic properties, were deter
mined in several experiments. TGF-01 inhibited DNA synthesis
in MDS.RS cells which are tumorigenic but nonmetastatic.
This inhibition occurred in the presence or absence of EGF
(Fig. 3). Very poorly metastatic NR3 cells exhibited no obvious
of serum, bFGF,
PDGF, EGF, and TGF-/3i on the growth of metastatic fibrosarcomas in monolayer culture. This is the first demonstration
of a correlation between the metastatic properties of cells and
a reduced dependence on serum for cell proliferation. This
decreased requirement for serum was also reflected in the poor
responsiveness to specific serum growth factors such as bFGF,
PDGF, and EGF. These growth factor responses did not distin
guish between transformed metastatic and nonmetastatic tu
mors. Indeed, the responses of these cell lines were similar to
those reported for other ros-transformed cells (13, 15, 24, 25).
This suggests that altered responsiveness of cells to some GFs
reflects the tumorigenic rather than the metastatic phenotype,
since the reduced responsiveness of all transformed cells oc
curred independent of their mode of transformation and their
metastatic properties.
On the other hand, TGF-01 which was growth factor inhibi
tory for nonmetastatic cell lines, was growth stimulatory for all
metastatic tumors examined. This altered response clearly dis
tinguished cells of intermediate to high metastatic potential
from normal or very poorly metastatic cells. Normal parental
lOT'/z fibroblasts were also significantly inhibited by TGF-/3,
(Fig. 3). This latter response is similar to that previously ob
served for various normal epithelial cells such as those derived
from kidney (26), bronchus (27), skin (28), intestine (29), liver
(30), and mammary gland (31). Cells of mesenchymal origin
such as fibroblasts from late gestational stage embryos are also
inhibited by TGF-/S (32). The lack of effect of TGF-fr on
7001
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TGF-0, STIMULATION OF METASTATIC FIBROSARCOMAS
tumorigenic but poorly metastatic fibrosarcomas in our study
is similar to the response of epithelial cells following transfor
mation. For example, TGF-/3 inhibited DNA synthesis in nor
mal human bronchial epithelial cells, however no effect of TGFßwas observed in human lung carcinoma cells (27). In addition,
although isolated normal hepatocytes are very sensitive to the
antiproliferative effect of TGF-ß,transfection of these cells
with the H-ras gene also confers resistance to this growth factor
(33). Effects of TGF-0 following transformation have not been
previously determined on mesenchymal cells which are nor
mally inhibited by this growth factor. To our knowledge this is
the first demonstration that TGP-ß\can stimulate metastatic
cells while inhibiting the parental line. There has been only one
other demonstration of a similar stimulatory response of trans
formed cells in culture using lymphocytes derived from adult
T-cell leukemias (34). All other reports have demonstrated a
lack of response rather than a stimulatory effect of TGF-ßon
DNA synthesis following transformation. It should also be
noted that the inhibitory effect of TGF-0! on parental lOT'/z
cells and the nonmetastatic tumor MDS.R5 cells was partially
abrogated with EGF. EGF did not significantly alter the re
sponsiveness of any metastatic cells to TGF-01. The reasons for
the lack of effect of EGF on metastatic cells are not known.
However, cells transfected with viruses harboring the ras-on
cogene have been shown to secrete increased levels of TGF-oc
(35-38), which binds to the same receptor as EGF (39). The
continued secretion of endogenous TGF-oc and its subsequent
binding to the EGF receptor may prevent the occupancy of this
receptor by exogenous EGF and render the cells insensitive to
added EGF.
The molecular mechanisms responsible for the conversion of
an inhibitory to a stimulatory signal by TGF-ßiare unknown.
However, transformation by H-ras may affect TGF-0 respon
siveness in several ways that might explain this observation: the
type of TGF-/3 secreted, TGF-0 receptor expression, and mod
ification of TGF-/3 signals at the DNA level, could all alter the
response to exogenous TGF-ßi.TGF-/3 is produced in at least
two forms, TGF-|Si and TGF-/32 (40, 41), and the cellular
responses to these types of TGF are not identical. Indeed, there
is some evidence that tissue specific responses are unique to
each type. Xenopus mesenchymal differentiation is responsive
only to TGF-fo (42). Since an increased secretion of TGF-0 is
ubiquitous to virtually all transformed cells (38, 43), it is
possible that altered secretion rates of one type of TGF-/3 may
occur which would then modify the response to exogenous
TGF-01. That is, the combination of enhanced secretion of one
type of TGF-/3 by metastatic cells plus exogenous TGF-01 may
lead to alterations in the ratio of TGF-ßito TGF-/32 available
to the cell during the period of the assay. The second mechanism
of altered response to TGF-/3 by metastatic cells could be an
alteration in receptor expression for TGF-0] and TGF-fo- Loss
of TGF-j3i growth inhibitory responses has been suggested as a
mechanism for promoting the transformation event (33), and
retinoblastoma cell lines have been shown to lose their TGF-0
receptor (44). Since metastatic cells acquire a proliferative
response rather than simply losing a growth inhibitory re
sponse, the loss of TGF-/3 receptor is not a likely explanation.
One must postulate the appearance of a new type of receptor
which is capable of exhibiting a modified response. Since TGFß,and & likely do not have the identical receptor (45), it is
possible that either the acquisition of a new receptor type or
increased receptor numbers and affinity associated with conver
sion to the metastatic phenotype may lead to a qualitatively
altered response to exogenous TGF-/3i. Finally, a third mecha
nism might be the result of modified responses to growth factors
produced by oncogene-induced changes at the DNA level. The
metastatic ra.v transformed cells used in this study have been
shown to have significantly enhanced levels of endogenous cmyc (46). The c-myc gene is known to alter many growth factor
induced responses (15) including TGF-ß(12, 14). One interest
ing observation, which is relevant to the present discussion is
that c-myc transfection of PC 12 pheochromocytoma cells con
verts a nerve growth factor-induced differentiation to a prolif
erative response (47). TGF-0 normally has the ability to induce
differentiation in a number of cell lines (27). This differentiation
is usually accompanied by inhibition of growth and the induc
tion of a number of markers of differentiation, and in some
cases altered morphology (48,49). For example, TGF-/3 inhibits
growth of endothelial cells while inducing their differentiation
(40, 49, 51). However, the metastatic cell may now utilize the
TGF-0 as a growth factor instead of a differentiation signal as
a consequence of the transformation events leading to metas
tasis formation. This proliferative response could clearly lead
to an autocrine pathway that would promote the growth of
metastatic cells upon entry into secondary tissue sites. Since
virtually all transformed cells have increased secretion of TGFß(38, 43), this type of autocrine regulation may have general
significance for the aggressiveness of metastatic disease.
In conclusion, we have shown that moderately or highly
metastatic fibrosarcomas are growth stimulated by TGF-/S]
while nonmetastatic transformed cells of identical cell lineage
were growth inhibited, similar to the nontransformed parental
cell lines. Future experiments will be directed towards deter
mining the mechanisms responsible for the altered responsive
ness of metastatic cells to TGF-/3|.
Note Added in Proof
A recent report by D. Chadwick and A. Lagarde (JNCI 80: 318,
1988) confirms the growth factor independence of metastatic tumors.
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Loss of Growth Factor Dependence and Conversion of
Transforming Growth Factor- β1 Inhibition to Stimulation in
Metastatic H- ras-transformed Murine Fibroblasts
Lois C. Schwarz, Marie-Claude Gingras, Grant Goldberg, et al.
Cancer Res 1988;48:6999-7003.
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