(Received for publication, February 4, 1997, and in revised form, April 30, 1997)
From the Department of Cell and Molecular Biology and
the ¶ Department of Pharmacological and Physiological Sciences,
St. Louis University School of Medicine, St. Louis, Missouri 63104 and
the § Department of Physiology, The Johns Hopkins
University School of Medicine, Baltimore, Maryland 21205
We have generated stable IIC9 cell lines, Goa1
and Goa2, that overexpress full-length antisense
Go RNA. As shown previously, expression of
antisense Go
RNA ablated the
subunit of the
heterotrimeric G protein, Go, resulting in growth in the
absence of mitogen. To better understand this change in IIC9 phenotype,
we have characterized the signaling pathway and cell cycle events
previously shown to be important in control of IIC9 G1/S
phase progression. In this paper we clearly demonstrate that ablation
of Go
results in growth, constitutively active Ras/ERK,
elevated expression of cyclin D1, and constitutively active cyclin
D1-CDK complexes, all in the absence of mitogen. Furthermore, these
characteristics were abolished by the transient overexpression of the
transducin heterotrimeric G protein
subunit strongly suggesting the
transformation of Go
-ablated cells involves
Go
subunits. This is the first study to implicate a
heterotrimeric G protein in tumor suppression.
In IIC9 cells, a subclone of Chinese hamster embryo fibroblasts, platelet-derived growth factor (PDGF)1 is a potent mitogen (1). PDGF stimulates an increase in cyclin D1 expression concomitant with an increase in cyclin D1-CDK activity (1). Cyclin D1 is an important G1 protein in that its delayed early induction in response to mitogen is required for G1 progression (2). Microinjection of cyclin D1 antibodies or antisense cyclin D1 plasmids into normal fibroblasts arrests them in G1 but has no effect on cells already beyond the G1/S boundary (3, 4). Thus, accumulation of cyclin D1 in G1 in response to mitogen is required for progression through the restriction point and entrance into S phase (2, 5, 6). Cyclin D1 preferentially binds to its catalytic partner, cyclin-dependent kinase 4 (CDK4), to form a holoenzyme (7-10). The activated cyclin D1-CDK4 complex preferentially binds to and phosphorylates the retinoblastoma gene product (pRb) (11-16). Hence, the mitogen-induced activation of cyclin D1-CDK4 complexes allows for progression through the restriction point in vivo presumably through the hyperphorylation and inactivation of pRb in concert with other G1 cyclin-CDK complexes.
We and others previously have shown that PDGF stimulates ERK1 activity in IIC9 and CCL39 cells and that inhibition of this activity is sufficient to cause the loss of PDGF-induced cyclin D1 expression, as well as a loss of cyclin D1-CDK activity (1). The loss of PDGF-induced cyclin D1-CDK activity was correlated with G1 growth arrest. Attention has focused on the mitogen-induced signals involved in cell growth and more recently those signals regulating mitogen-dependent induction cyclin D1 (1, 6). Expression of constitutively active mutant Ras has been shown to transform several cell types and elevate cyclin D1 expression (3, 17-19). The role of Ras proteins in the mitogenesis and transformation of cells is mediated, in part, by a downstream cascade of serine-threonine kinases that terminates with the activation of p42 and p44 MAPKs (ERK1, ERK2). Activated ERKs phosphorylate several nuclear factors that control gene expression. Evidence for the role of the Ras-mediated MAPK cascade is well documented with kinase-deficient mutants of Raf-1, MEK and MAPKs inhibiting Ras transformation.
Recent evidence has shown that Go activates ERK (20).
Go
activation of ERK is mediated by a novel protein
kinase C-dependent mitogenic signaling pathway which is
independent of Ras activation. The studies reported here examine the
effects of ablation of Go
by overexpression of antisense
Go
RNA in IIC9 cells on the Ras/ERK pathway and cell
cycle activities known to be important in mitogen-induced growth.
Currently, there is evidence suggesting that
subunits from
pertussis toxin-sensitive heterotrimeric G proteins are capable of ERK
activation through a Ras-dependent signaling pathway
(20-23). The
-mediated ERK activation is blocked by the
expression of dominant negative Ras (22). Our data demonstrate that
loss of Go
expression in IIC9 cells results in
unregulated growth by constitutively activating the Ras/ERK pathway, a
pathway we and others have shown positively regulates cyclin D1-CDK
activity through the increased expression of cyclin D1.
IIC9 cells, a subclone of Chinese
hamster embryo fibroblasts (46), were grown and maintained in DMEM
(Life Technologies, Inc., Grand Island, NY) containing 10% fetal calf
serum and 2 mM L-glutamine (all chemicals were
obtained from Sigma, unless specified otherwise). Subconfluent cultures
were growth-arrested by washing once with serum-free DMEM and cultured
for 48-60 h in serum-free media. PDGF was obtained from Calbiochem (La
Jolla, CA). Stable Go antisense transfectants were
produced and maintained as described previously (48). Briefly,
pcDNAI containing Go
cDNA in an antisense
orientation to the cytomegalovirus promoter was transfected into IIC9
cells using LipofectAmineTM protocol (Life Technologies,
Inc.). Following an 18-h transfection period, cells were cultured for
48 h in DMEM containing 10% fetal calf serum to allow for the
expression of neomycin-resistant gene products. Transfected IIC9 cells
were grown for several weeks in selection media containing G418 (500 µg/ml). G418-resistant clones were isolated and subcultured in DMEM
containing 10% fetal calf serum and 250 µg/ml G418. Several
Go
-ablated clones were isolated of which two, Goa1 and
Goa2 (described in the previous paper (48)), were used for the studies
in this paper. The experiments in this paper utilized both clones even
though the data presented is only that of the Goa1 clone.
Transient transfection of IIC9 cells using LipofectAmineTM
resulted in 85-90% expression efficiency as visualized by
-galactosidase staining. IIC9 cells were also transiently
transfected with Go
antisense cDNA (3 µg/ml) for
18 h. Transfected cells were stimulated with serum for 20-24 h
and cultured in serum-free media for 48 h for subsequent
experiments. Similarly, Goa1 cells were transiently transfected with
Gt
cDNA (3 µg/ml) to achieve the phenotype of the
Goa1/Gt
cell type. However, Goa1/Gt
cells
were cultured in serum-free media for only 24 h to ensure the
continued expression of Gt
protein in the absence of
mitogen.
PDGF (10 ng/ml) was added directly to
serum-deprived IIC9 and Goa1 cells. Cells (1-2 × 106) were then harvested at 24 h after addition of
PDGF by scraping in cold 1 × PBS. Harvested cells were pelleted
at 10,000 × g for 5 min and lysed with 30 µl of
solubilization buffer (25 mM Hepes, 300 mM
NaCl, 0.2 mM EDTA, 1.5 mM MgCl2,
0.1% Triton X-100, 20 mM -glycerophosphate, 0.1 mM sodium vanadate, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, and 0.5 mM phenylmethylsulfonyl fluoride).
IIC9 and Goa1 cells were incubated with aphidicolin (5 µg/ml) for 12-16 h to achieve growth arrest. Arrested cells were released by washing twice with DMEM and incubating in serum-free DMEM. Cells were then harvested and lysed 8 h after release from aphidicolin arrest as described above. Protein concentrations of lysates were determined by Bio-Rad Protein Assay (Bio-Rad) as recommended by the manufacturer. Lysates/proteins (15 µg) were electrophoresed on 10% SDS-polyacrylamide gels. Separated proteins were then transferred to polyvinylidene difluoride membranes (Millipore, Boston, MA). Membranes were probed with a cyclin D1 polyclonal antibody (Santa Cruz Biotechnology). Goat anti-rabbit IgG (H+L) horseradish peroxidase conjugate (Bio-Rad) was added as the secondary antibody and specific protein bands were visualized using ECL (Amersham) as recommended by the manufacturer.
Northern BlotsTotal RNA was isolated from IIC9 and Goa1
cells (4-8 × 106) cultured on 100-mm dishes with
Trizol Reagent (Life Technologies, Inc.) using the manufacturer's
protocol. RNA was electrophoresed on 2% agarose/formaldehyde gels.
Formaldehyde was removed by washing gels in 0.5 M ammonium
acetate. RNA was transferred onto Hybond N+ nylon membranes
(Amersham) using the TurboblotterTM system (Schleicher & Schuell, Keene, NH). RNA was cross-linked onto membranes with an
Ultraviolet Crosslinker (Amersham) using the manufacturers protocol.
Transferred RNA was visualized using a methylene blue/sodium acetate
stain. Randomly-labeled [-32P]dCTP cDNA probes
(murine cDNA for cyclin D1 was a generous gift from Dr. Charles
Sherr) were made using the Random Primed DNA Labeling Kit (Boehringer
Mannheim, Germany). Blots were probed simultaneously with cyclin D1 and
glyceraldehyde-3-phosphate dehydrogenase probes for 2 h at
65 °C using Rapid-hyb buffer (Amersham) and washed once at room
temperature with 5 × SSPE (20 mM EDTA, 1 M NaCl, 50 mM
NaH2PO4-H2O), 0.1% SDS and
subjected to either autoradiography or direct quantitation with a
PhosphorImagerTM (Molecular Dynamics). More stringent
washes were done at 65 °C with 0.5% SDS when necessary.
Thymidine incorporation was performed as described previously (1) with minor modifications. Briefly, growth-arrested IIC9 cells were stimulated with PDGF (10 ng/ml) for approximately 20 h. Goa1 cells were serum-starved by washing twice with DMEM and incubated for 48 h in serum-free DMEM supplemented with 2 mM L-glutamine. To growth-arrest Goa1 cells, aphidicolin (5 µg/ml) was added to serum-deprived cells as described above. Following a 24-h incubation, aphidicolin-arrested cells were released from arrest by washing twice with DMEM. Following primary incubation, cells were incubated for 3 h with 1 µCi of [3H]thymidine/ml (NEN Life Sciences Products). 3H-Labeled cells were washed twice with cold 1 × PBS and the DNA was precipitated by incubating the cells for 30 min with cold 5% trichloroacetic acid. The trichloroacetic acid-precipitated DNA was washed twice with cold 5% trichloroacetic acid and solubilized with 2% sodium bicarbonate, 0.1 N NaOH. The solution was neutralized by addition of one-fifth volume of 5% trichloroacetic acid and the trichloroacetic acid-precipitated [3H]DNA was quantitated by scintillation counting.
ERK AssayGrowth-arrested IIC9 cells were stimulated with
mitogen at 37 °C. After 15 min the media was removed and the cells
were washed with 1 × PBS. Goa1 cells were either grown in serum
(10%), serum-starved for 48 h, arrested with aphidicolin (5 µg/ml), or released from aphidicolin arrest for 8 h as described
above and washed with 1 × PBS. After washing, cells were lysed by
scraping in 300 µl of solubilization buffer (20 mM
Tris-HCl, pH 8, 1 mM sodium vanadate, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 2 mM EDTA,
1% Triton X-100, 50 mM -glycerophosphate, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin). The lysates
were assayed for ERK activity as described previously (1).
Cyclin D1/CDK activity was
assayed as described previously (8) with modifications. Briefly,
growth-arrested IIC9 cells were stimulated with PDGF (10 ng/ml) and
harvested at 0 and 24 h after stimulation by scraping in cold
1 × PBS and lysed in 50 µl of IP buffer (50 mM
Hepes, 150 mM NaCl, 0.1 mM sodium vanadate, 1 mM EDTA, 2.5 mM EGTA, 1 mM
dithiothreitol, 10 mM -glycerophosphate, 1 mM sodium fluoride, 0.1% Tween 20, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin). Goa1 cells were grown in
serum, serum-deprived for 48 h, aphidicolin-arrested, or released
from aphidicolin arrest for 8 h and harvested by scraping in cold
1 × PBS. Lysates were sonicated briefly and insoluble material
was pelleted by centrifugation at 10,000 × g for 10 min. Cyclin D1 monoclonal antibody (2 µg) was added to supernatants
and incubated at 4 °C. After 1-2 h cyclin D1 complexes were
precipitated for 2-3 h with protein G-Sepharose. Cyclin D1 immune
complexes were washed 4 times with 1 ml of cold IP buffer and 2 times
with 1 ml of cold wash buffer (50 mM Hepes, 10 mM MgCl2, and 1 mM dithiothreitol).
Cyclin D1 immune complexes were pelleted and resuspended in 30 µl of
reaction buffer (50 mM Hepes, 10 mM
MgCl2, 1 mM dithiothreitol, 2.5 mM
EGTA, 10 mM
-glycerophosphate, 0.1 mM sodium
vanadate, and 20 µM ATP). The immune complexes were
incubated for 30 min at 30 °C with 2 µg/ml soluble GST-Rb fusion
protein (GST-Rb cDNA was a generous gift from Dr. Mark Ewen) and 5 µCi of [
-32P]ATP. The reaction was stopped by
addition of 15 µl of 2 × Laemmli sample buffer. Samples were
boiled for 5 min and subjected to SDS-polyacrylamide electrophoresis.
Gels were dried and subjected to autoradiography.
The assay is a modification of the protocol by Downward et al. (47). Serum-deprived IIC9 cells or Goa1 cells were labeled for 18 h with [32P]Pi at 0.2 mCi/100 mM dish in phosphate-free DMEM. Cells were washed twice with phosphate-free media and with a saline buffer (50 mM Tris-HCl, pH 7.5, and 150 mM NaCl). Cells were incubated with PDGF (10 ng/ml) for 15 min and washed twice with cold 1 × PBS. Cells were harvested and lysed by scraping in 500 µl of IP buffer (50 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 150 mM NaCl, 1% Triton X-100, 2 mM p-nitrophenyl phosphate, 10 µg/ml pepstatin, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). After a 10-min incubation the homogenate was centrifuged at 750 × g for 5 min. The supernatant was treated with 100 µl of bovine serum albumin-coated charcoal for 5 min at 4 °C and then centrifuged at 750 × g to remove charcoal. A monoclonal p21ras antibody (Oncogene) was added to supernatants and Ras immune complexes were precipitated with protein G plus agarose (Oncogene) overnight at 4 °C. The precipitate was washed twice with IP buffer and 3 times with wash buffer (Tris-HCl, pH 7.5, 20 mM MgCl2, and 150 mM NaCl). Final pellets were drained and bound nucleotides were eluted in 20 µl of elution buffer (20 mM Tris-HCl, pH 7.5, 20 mM EDTA, 2% SDS, 0.5 mM GDP, and 0.5 mM GTP). Eluants were heated at 65 °C for 5 min and centrifuged. The supernatant was spotted onto a polyethyleneimine-cellulose thin layer plate (Merck) and developed with 0.75 M KH2PO4 (pH 3.4). GDP and GTP 32P-labeled fractions were quantified by scintillation counting.
We previously generated a panel of stable IIC9 clones
(including Goa1 and Goa2 clones) overexpressing full-length
Go RNA (48). We examined the phenotype of two of these
clones, Goa1 and Goa2. In contrast to IIC9 cells, Goa1 and Goa2 cells
do not express Go
protein. Goa1 and Goa2 cells formed
multiple foci in monolayer cultures and anchorage independent colonies
in soft agar (48).
To understand the mechanism for the transformed phenotype of these
cells, we examined whether Goa1 cells growth arrested upon removal of
mitogen. Goa1 cells did not growth arrest upon removal of serum (Fig.
1A) and flow cytometry showed Goa1 cells
randomly distributed throughout the cell cycle with a slight majority
of cells in S phase (data not shown). To arrest and synchronize
populations of mitogen-independent Goa1 cells, aphidicolin, a novel DNA
polymerase inhibitor, was utilized. Addition of aphidicolin in the
absence of serum resulted in growth arrest within 12-16 h after
treatment (Fig. 1B). Previous studies have shown that
removal of aphidicolin by washing with serum-free media is sufficient
to allow cells to enter S phase in the absence of mitogen within 4-8 h
(5). However, aphidicolin-released cells arrest when they reach the subsequent G1/S restriction point of the next cycle. Goa1
cells resumed cell cycle progression after release from aphidicolin in
serum-free media and continued through subsequent cycles in the absence
of mitogen while IIC9 cells arrested at the proceeding G1/S
restriction point (Fig. 1B) further demonstrating the
mitogen-independent growth of Goa1 cells. Transient overexpression of
Go
antisense RNA also resulted in mitogen-independent
growth suggesting that the transformed phenotype of the stable Goa1
cell type is a result of Go
ablation and not a result of
a deletion of another gene due to homologous recombinantion (data not
shown).
Ablation of Go
IIC9 cells overexpressing constitutively
activated Ras form multiple foci when grown in soft agar and do not
growth arrest when serum-depleted (data not shown). To examine whether
the Ras pathway was involved in the transformation of Goa1 cells we
first examined Ras activation. In growth-arrested IIC9 cells, levels of
activated Ras as determined by the ratio GTP/(GTP + GDP) associated with Ras were quite low (Fig. 2) and increased 6-fold
within 5 min after the addition of PDGF (Fig. 2) or several other
growth factors (data not shown). In contrast, Goa1 cells exhibited high levels of activated Ras in the absence of mitogen similar to levels found in IIC9 cells treated with PDGF (Fig. 2). Addition of PDGF did
not increase the level of activated Ras above the levels seen in
serum-depleted Goa1 cells. Similar results were observed with a second
clonal Go-ablated cell line (Goa2) (data not shown) indicating that ablation of Go
in IIC9 cells results in
significant activation of Ras. These results are consistent with the
observed inability of these cells to growth arrest with the removal of mitogen and suggest that in certain cell types loss of
Go
could result in neoplastic transformation.
Goa1 Cells Exhibit Constitutively Active ERK
Recent data from
several laboratories has suggested the importance of ERK activation in
Ras-dependent growth. We have previously demonstrated that
suppression of PDGF-induced ERK activation blocked G1
progression in IIC9 cells (1). To determine whether ablation of
Go resulted in constitutive activation of the ERK
pathway, we next examined the endogenous activity of ERK in Goa1 cells. Addition of PDGF (Fig. 3) and several other growth
factors (data not shown) to growth-arrested IIC9 cells increased ERK
activity approximately 7-8-fold within 15 min. As previously found in
CCL39 cells (24) and IIC9 cells (1) addition of thrombin or PDGF to
growth-arrested cells induced a biphasic increase in ERK activity. A
rapid 8-12-fold increase of ERK activity within 5-10 min is followed
by a sustained 4-6-fold increase in ERK activity. Asynchronous Goa1
cells grown in serum-free media express levels of ERK activity (7-9-fold) (Fig. 3) similar to the levels of ERK activity of IIC9 cells stimulated with PDGF (Fig. 3).
Elevated Expression of Cyclin D1 in the Absence of Mitogen
An
ERK-responsive region has recently been identified in the cyclin D1
promoter (25) and we have previously shown that addition of PDGF to
IIC9 cells induces cyclin D1 mRNA and protein expression (1). In
addition, we and others have shown that inhibition of mitogen-induced
ERK activity blocks expression of cyclin D1 and progression of IIC9
cells through G1 (1, 6). Many tumor cell lines express
elevated levels of oncogenic Ras and cyclin D1 (26-28). To identify a
possible downstream target of the Goa1 constitutively active Ras/ERK
pathway, we measured levels of cyclin D1. Go ablation
conferred increased cyclin D1 protein expression by approximately
3-4-fold (Fig. 4A). The levels of cyclin D1
in Goa1 cells remained constitutively elevated in the absence of mitogen.
We next investigated the effect of aphidicolin arrest and release on cyclin D1 expression. Treatment of Goa1 cells with aphidicolin for 12 h resulted in sustained levels of cyclin D1 protein in the absence of mitogen (Fig. 4A). Matsushime et al. (5) previously showed that Bac1.2F5A macrophages released from aphidicolin arrest required the presence of growth factor (CSF-1) to sustain the expression of cyclin D1. Release of IIC9 cells from aphidicolin arrest in the absence of PDGF resulted in the rapid (within 5 h) decrease in the levels of cyclin D1 protein (Fig. 4A). However, cyclin D1 protein levels in Goa1 cells did not decrease significantly when released from aphidicolin in the absence of PDGF suggesting a significant difference in the requirement of sustained presence of growth factor for cyclin D1 expression. It is clear that the constitutive activation of the Ras/ERK pathway (Figs. 2 and 3) provides the sustained mitogenic signals required for the continued expression of cyclin D1. Cyclin D1 protein expression remained high through the next round of replication (approximately 24 h after aphidicolin release) (Fig. 4A). In the absence of mitogen, cyclin D1 mRNA levels in Goa1 cells were similar to the levels found in IIC9 cells treated with PDGF (Fig. 4B). Aphidicolin-treated and released Goa1 cells exhibited a 1.6-fold decrease in cyclin D1 mRNA expression although these levels were still 3-fold higher than serum-deprived IIC9 cells (Fig. 4B) providing further evidence for the positive role of the Ras/ERK pathway in sustaining cyclin D1 expression in Goa1 cells in the absence of mitogen.
Cyclin D1-CDK Complexes Are Constitutively ActivePhosphorylation of Rb by active cyclin D1-CDK complexes has been demonstrated to be required for progression through G1 in several cell types (12-16). Although several transformed tumor cells express abnormally high levels of cyclin D1, the role of cyclin D1 in tumor formation is unclear. However, cyclin D1-CDK activity is thought to play an important role in mitogen-induced progression of cells through G1. In the absence of mitogen, IIC9 cells contain low levels of cyclin D1-CDK activity (Fig. 4C). PDGF treatment induced a 6-fold increase in cyclin D1-CDK activity within 4 h (data not shown) and sustained this level of activity through 24 h (Fig. 4C). In contrast to IIC9 cells, Goa1 cells display significant cyclin D1-CDK activity in the absence of mitogen (Fig. 4C). In addition, treatment of Goa1 cells with PDGF did not increase further cyclin D1-CDK activity (data not shown). Treatment of Goa1 cells with aphidicolin as well as release from aphidicolin arrest in serum-free media did not result in a decrease in cyclin D1-CDK activity (Fig. 4C) further correlating the mitogen independence of Goa1 cells with the activation of the cyclin D1-CDK complexes in the absence of mitogen. Preliminary results from our laboratory also suggest that high levels of cyclin D1-CDK activity are required to retain the transformed phenotype of Goa1 cells. These data further suggest that in certain cell types Ras transformation involves the up-regulation of cyclin D1-CDK activity.
A Possible Role for GoThe ablation of Go from IIC9 cells
provides interesting insights into the mechanisms of G1
progression as well as cellular transformation. Ablation of
Go
from IIC9 cells results in growth and high levels of
Ras activity in the absence of mitogen (Fig. 2). In addition, Goa1
cells contain constitutive levels of ERK activity (Fig. 3) as well as
cyclin D1-CDK activity (Fig. 4C). We and others have shown
that ERK activity is essential for mitogen-induced expression of cyclin
D1 and subsequent cyclin D1-CDK activity. Inhibition of ERK activity
not only resulted in a concomitant loss of cyclin D1-CDK activity but
also resulted in growth arrest (1). These data show that Ras
transformation through the ablation of Go
results in
sustained cyclin D1-CDK activity and suggest that in certain cell types
Ras transformation may involve up-regulation of cyclin D1-CDK
activity.
Recent evidence demonstrating that the Go subunit
stimulates ERK activity through a Ras-independent mechanism suggested a role for Go
in ERK activation (20). Ablation of
Go
from IIC9 cells resulted in constitutive elevation of
Ras and ERK activation in the absence of mitogenic stimulation. Several
studies, however, have provided new insights into G protein-coupled
signaling (29). Overexpression of
subunits also activates the
Ras/ERK pathway (21, 22). In addition, several studies present findings
suggesting that G
activates the Ras/ERK pathway through the
recruitment of cytosolic or membrane-associated factors containing
pleckstrin homology domains or through direct activation of G
with Ras (29). Transfection of a bARKct G
antagonist which
contains a
-binding region significantly reduces Ras activation
(23). Furthermore, transfection of dominant negative Ras eliminates ERK
activation suggesting that G
-mediated ERK activation involves Ras
(22).
To determine whether the transformation of Goa1 cells was mediated by
G activation of the Ras/ERK pathway in the absence of
Go
, we transiently transfected Goa1 cells with
Gt
(Goa1/Gt
) which has previously been
used to sequester G
subunits (21, 22). Transient overexpression
of Gt
did not result in the expression of
Go
protein, demonstrating continued ablation of
Go
in the presence of Gt
(data not
shown). As observed in IIC9 cells, Goa1/Gt
cells growth
arrested when cultured in the absence of mitogen (Fig.
5A). Stimulation with PDGF resulted in
progression through S phase and this progression was inhibited by
addition of aphidicolin as described previously (Fig. 1). In contrast
to Goa1 cells, stimulation of Goa1/Gt
cells with PDGF
resulted in a significant increase in [3H]thymidine
incorporation as compared with mitogen-deprived cells (Fig.
5A). Goa1/Gt
cells also required the presence
of PDGF after release from aphidicolin arrest for entrance into S phase
24 h after release (data not shown).
Goa1/Gt cells displayed a 60-70% decrease in ERK
activity compared with unstimulated Goa1 cells (Fig. 5B).
Addition of PDGF to Goa1/Gt
cells resulted in a marked
stimulation of ERK activity demonstrating that these cells were able to
respond to PDGF (Fig. 5B). These data demonstrate that
transfection of Gt
significantly reduces ERK activity in
unstimulated Goa1/Gt
cells but does not inhibit the
ability of PDGF to stimulate ERK activity in Goa1/Gt
cells. Cyclin D1 expression in IIC9 cells as well as macrophages requires mitogen after release from aphidicolin arrest (5). Absence of
mitogen results in a rapid decrease in cyclin D1 (Fig. 4A).
However, withdrawal of PDGF from Goa1 cells did not decrease cyclin D1
levels (Fig. 4, A and B). This regulation is
restored in Goa1/Gt
cells. Release of
Goa1/Gt
cells from aphidicolin arrest results in a
2.5-fold decrease of cyclin D1 mRNA expression (Fig.
5C). Concomittant with a decrease in cyclin D1 protein and mRNA expression, cyclin D1-CDK activity was significantly reduced in Goa1/Gt
cells in the absence of PDGF (Fig.
5D). It is important to note that the decrease in cyclin
D1-CDK activity is due to cyclin D1 down-regulation since the levels of
inhibitors of this complex (p27KIP1 and
p16INK4) are relatively high in cycling Goa1
cells.2
These data suggest that sequestration of Go by
Gt
results in a reversal of the Goa1 phenotype rendering
the cells sensitive to the machinery involved in the regulation of cell
cycle progression. This results in the restoration of mitogen-induced
regulation of cyclin D1 expression and its role in G1
progression. Previous results from our laboratory (1) and others (6)
show that mitogen-induced ERK activation is required for growth and the positive regulation of cyclin D1 expression. Preliminary results from
our laboratory now suggest that ERK activation absolutely is required
to maintain the transformed phenotype of Goa1 cells independent of
Go
-mediated signaling.2
This is the first study to implicate a heterotrimeric G protein,
Go, in tumor suppression. We have shown that ablation of Go
from IIC9 cells results in cellular transformation
(48) and that this involves the constitutive activation of the Ras/ERK pathway and the mitogen-independent activation of cyclin D1-CDK complexes. Transient ablation of Gq, Gi2, and
Gi3 in IIC9 cells did not result in an increase in basal
ERK activity in the absence of mitogenic stimulation suggesting that
only the specific
associated with Go
activate the
Ras/ERK pathway.3 The possibility that
Go
is a tumor suppressor becomes more intriguing by its
genomic location. The Go
gene (GNAO1) is located on
chromosome 16 at position 16q13 and is the only heterotrimeric G
protein gene on chromosome 16 (30-33). The instability of chromosome
16 has been well documented and has led to the discovery of several break points in its lower (q) arm (34-38). These chromosomal
abnormalities (translocations and deletions) are now thought to be
associated with a wide array of human cancers including breast,
prostate, colorectal, pituitary, alveolar, Ewing sarcomas, Wilms'
tumors, and turban tumors all of which map near or at the 16q13 region (34, 36-38, 39-45). Our data is the first to implicate
Go
in tumor suppression and warrants further
investigation into the apparent tumor suppressive properties of
Go
.
We thank Dr. Charles Sherr for murine cyclin D1 cDNA and Dr. Mark Ewen for GST-Rb cDNA.