(Received for publication, August 24, 1995; and in revised form, March 1, 1996)
From the
Using a gastric derived tumor line, we investigated the
involvement of 1 integrin and Rho in cell growth regulation in
response to collagen. The addition of C3 exoenzyme from Clostridium
botulinum to specifically ribosylate and inhibit the function of the
rho gene products inhibited cellular proliferation in a
dose-dependent fashion. C3 exoenzyme exhibited broad cytostatic activity
toward a number of tumor lines and induced G
/G
accumulation, cyclin A inhibition, and pronounced alterations in cell
morphology. Integrin-mediated adhesion to collagen led to the expression
of the cyclin A gene whose expression could be blocked using anti-
1
integrin monoclonal antibodies. Phospholipid levels were induced upon
1 integrin-mediated adhesion to collagen, and the phospholipid
induction was inhibited by either antibodies to
1 integrin or
pretreatment of cells with C3 exoenzyme. Significant reduction in
phospholipid levels correlated with proliferation for a panel of tumor
lines deprived of adhesion to substrate. These results implicate a novel
role for integrins and Rho in the regulation of tumor growth in response
to matrix.
The integrins are a family of adhesion receptors that play a role in the interaction of cells with the extracellular matrix(1, 2) . Cellular interactions with the extracellular matrix play a role in diverse processes such as differentiation(3, 4) , lymphocyte activation (5) , and tumor cell dissemination and metastasis(6) . Collagen is a major component of the extracellular matrix that serves as a scaffold for cell binding but also plays a role in cell differentiation and growth(7) .
In vitro, interaction of cells
with a collagen matrix can induce arachidonic acid production in HeLa
cells (8) and glandular differentiation in a human colon
carcinoma line(9) . Clustering of the 2
1 integrin, a
receptor for collagen, induces stimulation of tyrosine phosphorylation and
accumulation of GTP bound Ras in a human lymphoblastic cell line(10) . The interaction of cells with the extracellular matrix can
also regulate growth by creating a permissive effect for the action of
mitogens(11, 12) . Therefore, specific interaction
between integrins and matrix components provide another level of growth
regulation. A potential mediator of integrin signaling is the small GTP
binding protein Rho. It is a member of the Ras family of GTPases that
regulates the formation of actin stress fibers in response to growth
factors(13) . Integrins can also regulate the formation of actin
stress fibers(14) , which suggests a convergence of integrin and
Rho signaling pathways.
A role in mitogen signaling has been demonstrated for Rho using Val-14 mutations, analogous to Val-12 oncogenic mutations in Ras, which induced transformation of fibroblasts(15) . Amplification of Rho by transfection resulted in cells that exhibited increased tumorigenicity, higher saturation density, and reduced serum dependence(16) . Mutations in Dbl, a guanine exchange factor for Rho, also induced cellular transformation(17, 18) . Recent reports have demonstrated the induction of phospholipid levels in untransformed fibroblasts upon adhesion to fibronectin by a mechanism involving Rho. This pathway may provide a mechanism for integrating adhesion with soluble growth factors that generate phospholipid derived second messenger signals(19, 20) . These findings suggest a role for Rho in the regulation of both cell growth and cell architecture. A means for elucidating the function of Rho has employed the use of the ADP-ribosylation exoenzyme C3 from Clostridium botulinum. C3 could efficiently modify Rho A, B, and C by ribosylation to alter their function, presumably by inhibiting interaction with downstream effectors(21, 22) . The other Rho family proteins such as Rac-1 and CDC42 are poor substrates in vitro(23, 24) , and therefore C3 provides a sensitive means for elucidating Rho function.
In these studies,
we investigated a role for integrins and Rho in growth regulation of a
tumor line. Cyclin A and cyclin D are regulators of S phase
progression(25, 26) . They have been implicated in
tumorigenesis(25, 27) , and cyclin A and has also
been shown to serve as a link between adhesion and cell cycle
progression(28) . Using a gastric tumor line ST2 whose
proliferation was previously shown to be regulated by adhesion(29) , we have demonstrated specific 1 integrin-dependent
regulation of cyclin A expression, cell cycle progression, and
phospholipid synthesis on collagen matrix using monoclonal antibodies that
block these events. We also demonstrated the involvement of Rho in this
integrin-mediated process by inactivating Rho in tumor cells using C3
exoenzyme. These studies address novel mechanisms relating an integrin and
Rho in mediating tumor growth in response to matrix.
The lysate was added to 1 ml of
washed glutathione-agarose beads (Pharmacia) and mixed gently at 4 °C.
The beads were centrifuged at 500 g for 5 min, washed
five times with 14 ml of PBS containing 1% Triton X-100, and washed three
times with 14 ml of 50 mM Tris-HCl (pH 7.5), 150 mM
NaCl, 2.5 mM CaCl
, and the beads were resuspended in
0.5 ml of the Tris buffer. 30 NIH units of purified bovine
-thrombin
was added and incubated for 16 h at 4 °C to cleave the fusion protein.
The beads were centrifuged at 1000
g for 2 min, washed
twice with 1 ml of the Tris solution, and concentrated using a Centricon-3
(Amicon, Beverly, MA). The purified product ran as a single band at
approximately 25 kDa on a 11.25% SDS-polyacrylamide gel (33) .
Figure 1:
Inhibition of
proliferation by C3. A, dose-dependent inhibition by C3
exoenzyme. Cells (4000 cells/well) were plated in triplicate in 96-well
plates coated with collagen (see ``Materials and Methods'') with
the indicated concentrations of C3. After 5 days in culture, cell number
was determined as described under ``Materials and Methods,'' and
relative cell number was calculated by dividing the number of cells
treated with C3 by the number of cells that were left untreated.
B, C3 inhibition of proliferation. Cells (4000 cells/well) were
plated in 96-well tissue culture plates in the absence or the presence of
C3 exoenzyme (50 µg/ml). After 5 days in culture, the number of cells
in each well was determined as described under ``Materials and
Methods,'' and the percentage of inhibition was determined as 100%
(1 - cell number cultured with C3/cell number cultured
without C3). The values are expressed as means of triplicate ±
S.E.
Figure 2:
C3 induced morphological changes. The indicated cell
lines were plated on collagen-coated wells either in the presence or the
absence of 50 µg/ml C3 and incubated for 24 h at 37 °C in a 5%
CO humidified incubator.
Figure 3: Culturing cells in the presence of C3 induces ADP-ribosylation of Rho. ST7 (lanes 1 and 2), ST2 (lanes 3 and 4), A375 (lanes 5 and 6), HMEC-1 (lanes 7 and 8), 131-INI (lanes 9 and 10), and FOGERTY (lanes 11 and 12) were left either untreated (lanes 1, 3, 5, 7, 9, and 11) or treated with 50 µg/ml C3 (lanes 2, 4, 6, 8, 10, and 12) for 3 days, harvested by brief trypsinization, and transferred to microcentrifuge tubes. The cells were then ADP-ribosylated in vitro and separated by SDS-polyacrylamide gel electrophoresis as described under ``Materials and Methods.''
Figure 4: Cell cycle analysis of C3-treated cells. ST2 cells were plated on collagen either in the presence (A) or the absence (B) of C3, incubated for 48 h, and then analyzed by propidium iodide staining and flow cytometry as described under ``Materials and Methods.''
We next investigated the effect of adhesion and C3 on
cell cycle progression at the transcriptional level by analyzing the
expression of cyclin A and cyclin D, two regulators of cell cycle
progression that have been implicated in tumorigenesis(25, 38) . We first investigated the effect of integrin-mediated adhesion
to collagen on the expression of cyclin A message. ST2 cells were
initially deprived of adhesion for 30 h by transferring the cells to
culture dishes coated with polyHEMA, a substance used to inhibit cell
attachment (11) (Fig. 5A). By culturing the
cells in the absence of adhesion(11, 39) , the effect
of adhesion to specific substrates can then be analyzed. Upon transfer
from polyHEMA to collagen-coated dishes, the cells expressed low levels of
cyclin A (Fig. 5A, lane 1). However, by 18 h on
collagen-coated dishes (lane 2), the expected 2.7- and
1.8-kilobase alternatively polyadenylated forms of cyclin A mRNA (25) were induced significantly, and their levels continued to rise
24 (lane 3) and 36 h (lane 4) after plating. To
demonstrate that integrin-mediated attachment to collagen was required for
induction, in Fig. 5B, the cells were initially deprived
of adhesion for 30 h to induce low levels of cyclin A message (lane
1). The cells were then plated on collagen (lane 2 and
3) or maintained on polyHEMA (lane 4 and 5)
either in the absence (lane 2 and 4) or the presence of
an anti-1 integrin monoclonal antibody (lane 3 and
5). After 24 h, cyclin A expression was induced when the cells
were plated on collagen (lane 2) but was blocked in the presence
of anti-
1 integrin monoclonal antibody (lane 3). Cells
maintained on polyHEMA did not express cyclin A either in the presence or
the absence of monoclonal antibody (lanes 4 and 5). The
results demonstrated that adhesion to collagen could induce the expression
of cyclin A message, and its expression was dependent on
1
integrin-mediated adhesion.
Figure 5:
Integrin-mediated induction of cyclin A
mRNA. A, kinetics of cyclin A induction. After 30 h on
polyHEMA, the cells were replated on collagen-coated plates. RNA from 2
10
cells were extracted at 0 (lane 1), 18 (lane 2), 24 (lane 3), and 36 (lane 4) h and
then analyzed by Northern blot analysis. B, integrin-dependent
induction of cyclin A. Cells were transferred to polyHEMA for 30 h (lane 1) and then transferred to collagen-coated dishes (lanes 2 and 3) or maintained on polyHEMA (lanes
4 and 5) in the absence (lanes 2 and 4)
or the presence (lanes 3 and 5) of the anti-
1
integrin monoclonal antibody 33B6 for an additional 36 h in a
humidified incubator. Migration of the 28S and 18S ribosomal RNA band
is indicated to the left of each autorad. Below each
panel shows the corresponding ethidum bromide-stained 28S and 18S
ribosomal RNA bands immobilized to nylon
membrane.
The expression of cyclin A, which was
regulated by 1 integrin-mediated adhesion, is also known to be
regulated by growth factors (serum)(40) . Also, because C3
induced G
/G
accumulation ( Fig. 4and
Table 1), we compared the effect of C3 treatment, serum
withdrawal, and deprivation of adhesion on the expression of cyclins A and
D1(26, 38) . In Fig. 6A, ST2 cells
grown on collagen-coated dishes (lane 1) expressed 2.7- and
1.8-kilobase alternatively polyadenylated forms of cyclin A mRNA. When the
cells were deprived of adhesion by plating them for 30 h in polyHEMA
coated dishes (lane 2) or deprived of serum (lane 3),
cyclin A mRNA decreased significantly. The addition of C3 exoenzyme to ST2
plated on collagen also showed reduced cyclin A expression after 30 h
(lane 4). The decrease in cyclin A expression in response to
deprivation of adhesion, serum starvation, and Rho inactivation by C3
treatment is consistent with the effect of integrins, Rho, and mitogenic
factors to regulate proliferation at similar points in the cell cycle(19, 20, 28, 38) . Although C3
inactivation of Rho is also known to induce cell rounding and detachment
from substrate(41) , which could potentially inhibit cyclin A
expression, in this system, C3 induced morphological alterations but did
not inhibit cell attachment. These results demonstrate a distinct but
cooperative role for integrins, Rho, and serum factors leading to cell
cycle progression.
Figure 6:
Inhibition of cyclin A expression by C3
treatment. ST2 cells were plated on collagen in 10% FBS (lane
1), on polyHEMA with 10% FBS (lane 2), on collagen under
reduced (0.5%) serum (lane 3), or on collagen in the presence
of 10% FBS plus 50 µg/ml C3 (lane 4). After 30 h, total
RNA from 2 10
cells were isolated and analyzed by
Northern blot analysis using probes specific for either cyclin A (A) or cyclin D1 (B). Migration of the 28S and 18S
ribosomal RNA band is indicated to the left of each autorad. Below each panel shows the corresponding ethidum
bromide-stained 28S and 18S ribosomal RNA bands immobilized to nylon
membrane.
In a similar experiment, the blots were probed
with cyclin D1 (Fig. 6B), and the expected alternatively
polyadenylated 4.8 and 1.7 cyclin D1 message was detected(26) .
In contrast to cyclin A, cyclin D1 expression was not affected by any of
the treatments, which demonstrates that not all cyclin mRNA expression was
affected by these different modes of treatment. Also, the lack of
inhibitory effect of reduced serum on cyclin D1 was of interest because
growth factors are known to regulate the expression of cyclin D1(26) . However, disregulated expression of cyclin D, by relieving
requirements for G to G
cell cycle transition, has
been proposed as a mechanism contributing to tumorigenesis(38, 42) . These results indicate that tumor proliferation
may also require integrin signaling and Rho to achieve progression into S
phase.
Figure 7:
1 integrin-mediated induction of
endogenous phospholipid synthesis is blocked by C3 pretreatment. ST2
cells were initially plated on polyHEMA for 24 h either without (lanes 1-4) or with (lanes 5 and 6) 50
µg/ml C3. The cells were then maintained on polyHEMA (PH) (lanes 1, 3, and 5) or transferred to a
collagen (CL) (lanes 2, 4, and 6)
substrate for 3 h. The cells were then incubated in phosphate-free
medium containing [
P]orthophosphoric acid for 5
h, and the lipids were extracted and analyzed by TLC as described under
``Materials and Methods.''
Figure 8:
Decreased phospholipid synthesis is
accompanied by reduced proliferation upon deprivation of substrate. A, reduced phospholipid synthesis on polyHEMA. The indicated
cells were plated on substrate or transferred to polyHEMA-coated dishes
for 24 h. The cells were then incubated in phosphate-free medium
containing [P]orthophosphoric acid for 5 h, and
the lipids were extracted and analyzed by TLC as described under
``Materials and Methods.'' B, reduced proliferation
of tumor cells on polyHEMA. The indicated cell lines were plated on
either polyHEMA or tissue culture plastic. After 5 days in culture, the
number of cells in each well was determined by crystal violet staining
as described under ``Materials and Methods,'' and the
percentage of inhibition was expressed as a ratio of cell number
deprived of adhesion or maintained as attached
cells.
Numerous examples have demonstrated integrin signaling, including clustering of the fibronectin receptor leading to protein tyrosine phosphorylation of focal adhesion tyrosine kinase (43) and cytoplasmic alkalinization(44) . In peripheral blood T cells, antibodies to integrins or matrix components have been shown to possess growth stimulatory activities(5, 45) .
In these studies we have shown that
cyclin A and phospholipids were regulated by adhesion to collagen, and the
induction was inhibited by an anti-1 integrin monoclonal antibody.
ADP-ribosylation of Rho by C3 exoenzyme inhibited proliferation, induced
G
/G
accumulation, and also inhibited
integrin-mediated induction of phospholipid synthesis as well as cell
cycle progression as measured by the expression of cyclin A. The ability
of ADP-ribosylation of Rho to inhibit integrin-mediated cell cycle
progression demonstrates a convergence of integrin and Rho signaling
pathways leading to positive regulation of tumor cell growth as a
consequence of cell attachment to substratum. Cyclin A expression was also
inhibited by culturing the cells under low serum (0.5%) conditions (Fig. 6A), and this correlated with G
/G
cell cycle arrest (not shown). These results demonstrate an additional
level of cooperativity between integrins, Rho, and soluble serum factors
leading to cell cycle progression. The generation of second messengers
leading to proliferation induced by many growth factors relies on a pool
of phospholipids (46) and therefore, the synthesis of
phospholipids induced by integrins in tumor cells may play a pivotal role
in the integration of growth factors and adhesion leading to growth
stimulation.
The profound morphological alterations accompanying
ADP-ribosylation of Rho is consistent with alterations in the level of
phospholipids. Phospholipid can alter the function of cytoskeletal
components including -actinin(47) , gelsolin(48) , profilactin(49) , and talin (50) , and
therefore, changes in morphology induced by C3 ribosylation of Rho may in
part be explained by alterations in phospholipid levels induced by C3
treatment. Cells maintained in suspension overnight show reduced
phospholipid synthesis and correspondingly showed similar alterations in
morphology, which was evident upon reattachment to collagen coated
plastic. This alteration was transient, and the cells resumed normal
morphology after several hours on collagen (not shown).
These
results are in agreement with those of other investigators who
demonstrated that enhanced inositol lipid synthesis and platelet-derived
growth factor-induced inositol lipid synthesis breakdown in response to
fibronectin involved the regulation of phosphatidylinositol 4-phosphate
5-kinase by Rho(19, 20) . Our studies using intact
cells demonstrated that integrin-mediated adhesion could regulate not only
PIP but PIP as well. Treatment of cells with C3 also inhibited
the synthesis of PIP
and PIP. The observed differences may be
due to more complex molecular interactions in intact cells in which, for
example integrin-mediated induction of phospholipids may influence
downstream activation of protein kinase C, which in turn, may indirectly
alter phospholipid metabolism.
Various studies have implicated a role for integrins in cancer. The mechanism by which adhesion molecules promote metastasis is not clear but may involve adhesion to platelets to escape immunologic detection (51) . Integrins can mediate extravasation of cells from circulation into tissue to sequester them from immunologic detection or physical damage caused by shear forces under conditions of flow(6) . An additional role for integrins in promoting metastasis may involve generation of growth promoting signals. Anchorage-independent growth is a function of the suppression of the apoptotic pathway (52) and or the activation of growth promoting pathways. The growth-promoting effect on tumors through integrins may therefore occur as a separate event in the context of mutations leading to suppression of apoptosis.
A number of tumors have been shown to
overexpress growth factor receptors. Karyotype analysis of ST2 has
revealed the amplification of chromosome 7p, which carries the gene for
the epidermal growth factor receptor(29) . Therefore, the
epidermal growth factor receptor may play a relevant role in contributing
to tumor proliferation of ST2, and its overexpression may facilitate rapid
tumor growth under conditions of limiting soluble factors(53, 54) . The binding of growth factors can lead to
phospholipid turnover and the generation of second messengers, which
ultimately act on components that regulate cell cycle progression and cell
division. Several components that respond to growth factor stimulation and
that have also been implicated to play a role in tumorigenesis include
cyclin A and cyclin D. Results using ST2 demonstrated that cyclin D
expression is disregulated and may contribute to its tumorigenicity.
Cyclin D is required for exit from G into G
, and
therefore, constitutive expression of cyclin D may result in the inability
of ST2 to remain in G
such that the cells will, in the absence
of physiologic stimulation, progress to G
. Full progression
into S phase not only requires cyclin D but also the expression of cyclin
A. In contrast to cyclin D1, however, cyclin A expression in ST2 cells
required not only soluble factors (serum) but also integrin-mediated
adhesion. The ability of cyclin A to mediate adhesive signals has been
shown in untransformed fibroblast lines(28) , and therefore,
integrin-mediated adhesion contributing to tumor proliferation may be a
feature that may be retained upon cell transformation in some cases.