ras and rho regulation of the cell cycle and oncogenesis
TRANSCRIPT
Mini-review
Ras and Rho regulation of the cell cycle and oncogenesis
Kevin Pruitt, Channing J. Der*
University of North Carolina at Chapel Hill, Lineberger Comprehensive Cancer Center, Department of Pharmacology, Chapel Hill,
NC 27599-7295, USA
Accepted 5 April 2001
Abstract
The important contribution of aberrant Ras activation in oncogenesis is well established. Our knowledge of the signaling
pathways that are regulated by Ras is considerable. However, the number of downstream effectors of Ras continues to increase
and our understanding of the role of these effector signaling pathways in mediating oncogenesis is far from complete and
continues to evolve. Similarly, our understanding of the components that control mitogen-stimulated cell cycle progression is
also very advanced. Where our understanding has lagged has been the delineation of the mechanism by which Ras causes a
deregulation of cell cycle progression to promote the uncontrolled proliferation of the cancer cell. In this review, we summarize
our current knowledge of how deregulated Ras activation alters the function of cyclin D1, p21Cip1, and p27Kip1. The two themes
that we have emphasized are the involvement of Rho small GTPases in cell cycle regulation and the cell-type differences in how
Ras signaling interfaces with the cell cycle machinery. q 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Raf; Rac; Rho; cyclin D1; p21CIP1; p27KIP1
1. Introduction
The involvement of Ras proteins in cell signaling
and in regulation of cell proliferation is well-estab-
lished. Our knowledge of the signaling pathways that
are regulated by Ras is considerable. Ras functions as
a nodal point, where it is activated by diverse extra-
cellular stimuli. Once activated, Ras in turn interacts
with a diverse spectrum of effectors and initiates a
multitude of cytoplasmic signaling cascades. Simi-
larly, our understanding of the components that
control mitogen-stimulated passage through G1 and
entry into S phase of the cell cycle is also very
advanced. A regulation of the activity of positive
and negative regulatory proteins that control the
activity of the Rb tumor suppressor protein dictates
G1 progression. Where our understanding has lagged
has been the delineation of the mechanism by which
Ras causes a deregulation of cell cycle progression to
promote the uncontrolled proliferation of the cancer
cell. Recent studies have begun to establish the links
between Ras signaling pathways and cell cycle regu-
latory proteins. One important theme that has
emerged is that the Ras-related Rho GTPases may
facilitate this regulation. A second theme involves
cell-type differences in how Ras signaling interfaces
with the cell cycle machinery. Recent excellent
reviews summarize our current understanding of
Ras signaling [1±3], cell cycle regulation [4,5], or
both [6±8]. The focus of this review will be on the
recent advances made from the study of Ras and Rho
small GTPases and the signaling mechanisms that
Cancer Letters 171 (2001) 1±10
0304-3835/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0304-3835(01)00528-6
www.elsevier.com/locate/canlet
* Corresponding author. Tel.: 11-919-966-5634; fax: 11-919-
966-0162.
E-mail address: [email protected] (C.J. Der).
connect them with the cell cycle regulatory machin-
ery.
2. Ras and signal transduction
Ras proteins are positioned at the inner face of the
plasma membrane where they serve as relay switches
to transmit extracellular signal-mediated stimuli to
cytoplasmic signaling cascades [9]. Ras proteins func-
tion as GDP/GTP-regulated switches that cycle
between an active GTP-bound state and an inactive
GDP-bound state. Mitogenic signals stimulate a tran-
sient formation of active GTP-bound Ras and acti-
vated Ras in turn interacts with downstream effector
targets. This activation is facilitated by guanine
nucleotide exchange factors (GEFs; Sos1/2,
RasGRF1/2, RasGRP, CNRasGEF). GTPase activat-
ing proteins (GAPs; p120 GAP, NF1-GAP, etc.) facil-
itate the return of Ras back to the inactive GDP-bound
state. Tumor-associated mutant Ras proteins harbor
single amino acid substitutions, primarily at residues
12, 13, and 61, that render Ras insensitive to GAP-
stimulated GTP hydrolysis [10,11]. Hence, these
oncogenic mutants of Ras are chronically-activated
proteins that continue to signal in the absence of extra-
cellular signals
The best characterized effector of Ras function are
the Raf serine/threonine kinases (A-Raf, B-Raf, c-
Raf-1) [1,2,12]. Activated Raf activates the MEK1/2
dual speci®city kinases, which then activate the p42/
p44 ERK mitogen-activated protein kinases
(MAPKs). The phosphoinositide 3-phosphate lipid
kinases (PI3Ks) represent the second best character-
ized effectors of Ras [13]. Activated PI3K, a lipid
kinase, facilitates the conversion of phosphatidylino-
sitol 4,5-phosphate (PIP2) to phosphatidylinositol
3,4,5-phosphate (PIP3). PIP3 levels are elevated in
Ras-transformed cells and promote the activation of
the Akt/PKB serine/threonine kinase. PIP3 may also
activate GEFs for the Rac small GTPase. A third class
of Ras effectors is a family of GEFs (RalGDS, RGL,
and Rlf/RGL2) that serve as activators of the Ral
small GTPases [14]. While a contribution of these
three classes of effectors to Ras transformation has
been established, the role of other candidate effectors
(e.g. AF6, Rin1, Nore1, RASSF1, PLC episilon)
remains to be elucidated [2,15,16].
3. Regulation of the Rb pathway: a requirementfor Ras
Mitogenic stimuli promote the entry of quiescent
cells into the ®rst gap phase (G1) and initiation of
DNA synthesis (S phase) of the cell cycle [4]. Exit
from or entry into the G0 quiescent state is controlled
by positive and negative regulatory proteins. G1
cyclin-dependent kinases (CDKs) serve as positive
regulators. D-type cyclins (D1, D2, D3) complex
with CDK4 and CDK6 to stimulate their kinase activ-
ities, which in turn cause the phosphorylation and
inactivation of the retinoblastoma (Rb) tumor
suppressor protein. By binding to E2F, Rb recruits
histone deacetylases to the promoters of E2F-respon-
sive genes and represses their transcription [5].
Cyclin D1, in part, regulates the kinase activities of
both CDK4 and CDK6. These complexes are formed
in the cytoplasm and are transported into the nucleus
and undergo stimulatory modi®cations including
phosphorylation by CDK-activating kinase (CAK) to
yield active holoenzymes. Further into G1, cyclin E
complexes with CDK2 and causes additional phos-
phorylation and inactivation of Rb. With suf®cient
phosphorylation of Rb, E2F is released and transacti-
vates genes required for S phase entry, including
cyclins E and A [5].
CDK inhibitors (CKIs) serve as negative regulators
of the Rb pathway [4]. CKIs are classi®ed into two
distinct families on the basis of their structural and
functional characteristics. The members of the INK4
family of CKIs (p16Ink4a, p15Ink4b, p18Ink4c, and p19Ink4d)
contain multiple ankyrin repeats and act as negative
regulators of CDK4/6 by binding to the catalytic subu-
nit and preventing formation of the active cyclin-CDK
complex. The Cip/Kip family of CKIs (p21Cip1, p27Kip1,
and p57Kip2) is more broadly acting and regulates both
CDK4/6 and CDK2 activity. Each member of the
family contains a characteristic motif within the
amino-terminal region that enables them to bind to
both cyclin and CDK subunits. The stoichiometry
between CDKs and CKIs is important and determines
the activity of Rb and the proliferative state of cells.
A number of experimental approaches have estab-
lished the importance and requirement for endogen-
ous Ras for cell cycle progression and the ability of
oncogenic Ras to promote growth factor-independent
cell cycle entry. First, studies in NIH 3T3 ®broblasts
K. Pruitt, C.J. Der / Cancer Letters 171 (2001) 1±102
with anti-Ras neutralizing antibodies or dominant
negative Ras have been shown to cause growth arrest
in the presence of serum stimulation, demonstrating
the requirement of endogenous Ras function through-
out most of G1 for normal cell cycle progression [17±
20]. Second, the ability of activated Ras alone to
stimulate quiescent NIH 3T3 ®broblasts to S phase
entry showed that Ras function could promote cellular
proliferation [21]. Third, mitogen stimulation of
quiescent cells causes a biphasic pattern of Ras acti-
vation (Fig. 1) [22±24]. The ®rst phase of Ras activity
was shown to occur rapidly following serum stimula-
tion of quiescent cells. The second phase of Ras acti-
vation was more robust and was achieved at a later
time point corresponding to mid-G1 phase and may
account for the requirement for Ras in late G1. Inter-
estingly, whereas an activation of the Raf/ERK path-
way is associated with the ®rst peak of Ras activation,
the later peak of activation did not correlate with ERK
activation, and instead, PI3K/Akt activation [24].
One of the ®rst links to be established between Ras-
dependent signaling and Rb function was demon-
strated using Ras neutralizing antibodies or dominant
negative Ras and asynchronously growing primary
Rb 1 /1 or Rb2/2 mouse embryo ®broblasts
[25,26]. Two studies showed that the inhibition of
Ras function caused formation of hypophosphorylated
and active Rb and G1 arrest of wild type cells. In
contrast, Rb null mouse ®broblasts failed to undergo
growth arrest when Ras function was blocked. Thus,
Ras-mediated growth stimulation is dependent, in
part, on causing an inactivation of Rb. Consistent
with this possibility, Rb is hyperphosphorylated and
inactivated in Ras-transformed cells [27±29].
Recent studies have begun to link speci®c Ras
signaling events with the regulation of Rb and cell
cycle progression (Fig. 2). In particular, a relationship
between Ras signaling activity and the regulation of
cyclin D1 and the CDK inhibitors, in particular p27
and p21, has been established. Additionally, in light of
previous studies that demonstrated the requirement
for Rho GTPases in Ras transformation [30,31] it is
not surprising that Rho GTPases may facilitate Ras
regulation of these components. It is also apparent that
K. Pruitt, C.J. Der / Cancer Letters 171 (2001) 1±10 3
Fig. 1. Ras is required for G1 entry and progression. Upon mitogen
stimulation of quiescent cells (G0), two peaks of Ras activation are
seen {Taylor & Shalloway 1996 8666 /id}{Gille & Downward 1999
8462 /id}. The ®rst occurs immediately on entry into G1 and is
associated with activation of the Raf/MEK/ERK protein kinase
cascade. The second occurs at mid-G1 and corresponds to activation
of the PI3K/Akt effector pathway. Ras activation is essential for
mitogen-stimulated upregulation of cyclin D1 and p21Cip1, and
downregulation of p27Kip1, protein expression.
Fig. 2. Ras and Rho GTPase regulation of G1 entry and progression.
Activated Ras and Rho GTPases promote exit from G0, passage
through G1, and entry into S phase by controlling the expression
and function of cyclin D1, p21Cip1, and p27Kip1. The activation of
cyclin D-CDK4/6 and cyclin E-CDK2 in turn promotes hyperpho-
sphorylation of Rb, leading to the release of histone deacytylase
(HDAC) and activation of E2F. Ras and Rho upregulation of cyclin
D1 expression is due primarily to stimulation of gene expression.
p27Kip1 protein function is downregulated primarily by cyclin E-
CDK2-mediated protein degradation. p27Kip1 function is also down-
regulated by association with cyclin D1-CDK4/6 complexes, thus
relieving p27Kip1 inhibition of cyclin E-CDK2, which in turn
promotes p27Kip1 degradation. Rho promotes p27Kip1 degradation
by activation of cyclinE-CDK2. Ras upregulation of p21Cip1 is
controlled, in part, by stimulation of gene expression. Rho activity
can antagonize p21Cip1 upregulation.
how signal-activated endogenous Ras and mutated
oncogenic Ras signals to regulate the cell cycle is
likely to be distinct. Furthermore, the role of Ras in
promoting cell cycle progression is distinct when
assessed in cells exiting from G0 vs. continuously
proliferating cells [32]. Finally, cell type differences
in how Ras regulates the cell cycle machinery further
complicate our ability to de®ne a simple relationship
between Ras and the cell cycle (Fig. 3).
4. Ras and Cyclin D1
Perhaps the best-characterized component of the
cell cycle machinery targeted by Ras is cyclin D1
[6±8]. Cyclin D1 is induced transcriptionally in
response to growth factor stimulation [33]. Cyclin
D1 transcription and protein expression is typically
elevated by mid-G1, associated with the second
peak of Ras activation [24], with maximal accumula-
tion occurring closer to the G1/S boundary. Cyclin D1
is rapidly degraded, so its expression is dependent on
continued growth factor stimulation until cells pass
the G1 restriction point. Serum-stimulated upregula-
tion of cyclin D1 expression is dependent on Ras
function and constitutive expression of cyclin D1
can overcome the requirement for Ras for prolifera-
tion of NIH 3T3 cells [34].
Oncogenic Ras causes upregulation of cyclin D1
gene and protein expression in a wide variety of cell
types. Transient induction of activated Ras expression
in Balb 3T3 ®broblasts or IEC-18 and RIE-1 rat intest-
inal epithelial cells is accompanied by upregulation of
cyclin D1 transcription and protein expression [35±37]
oncogenic Ras transformation of NIH 3T3 and Rat-1
®broblasts, IEC-18, NMUMG mouse mammary
epithelial cells, and RIE-1 cells is associated with
sustained upregulation of cyclin D1 protein [29,37±
40]. The treatment of Ras-transformed NIH 3T3 or
IEC-18 cells with anti-sense cyclin D1 oligonucleo-
tides caused an impairment in proliferation, indicating
a contribution of cyclin D1 upregulation to Ras-
mediated growth transformation [38,41]. However,
overexpression of cyclin D1 alone is clearly not suf®-
cient to promote Ras-mediated growth transformation
[29,38].
Ras upregulation of cyclin D1 has been attributed
mainly to Ras activation of the Raf/MEK/ERK path-
way. For example, transient [42±45] or sustained [38]
(6438) [29] activation of Raf or MEK in rodent ®bro-
blasts caused increased levels of cyclin D1. In
contrast, whereas activated Ras increased cyclin D1
K. Pruitt, C.J. Der / Cancer Letters 171 (2001) 1±104
Fig. 3. The consequences of Ras show cell type and duration of activation differences. Two major issues have made it dif®cult to de®ne a simple
relationship between Ras activation and changes in the function of G1 regulators. First, the consequences of oncogenic Ras expression can vary
signi®cantly in different cell types. Second, the consequences of transient Ras activation vs. sustained Ras activation can also be strikingly
different. Shown in this ®gure are the consequences of sustained Ras activation in NIH 3T3 mouse ®broblasts and RIE-1 rat intestinal epithelial
cells [29]. Ras activation of Raf alone is suf®cient for these changes in NIH 3T3 cells. In contrast, Ras activation of Raf-independent signaling
pathways are critical to cause the changes seen in RIE-1 cells.
expression in RIE-1 rat intestinal epithelial cells, acti-
vated Raf did not [29]. Nevertheless, inhibition of
ERK activation did block cyclin D1 upregulation in
Ras-transformed RIE-1 cells. Thus, in some cell
types, Ras activation of the Raf effector is necessary
but not suf®cient to promote the upregulation of cyclin
D1, supporting the contribution of non-Raf effector
function in cyclin D1 regulation.
The prominence of the Raf/MEK/ERK pathway in
the regulation of cyclin D1 is undisputed, but recent
studies highlight the contribution or requirement of
other Ras effector pathways for the induction of
cyclin D1. Gille and Downward found that the
second peak of serum-stimulated activation of Ras
corresponded to activation of Akt, rather than ERK
[24]. Cyclin D1 expression also corresponded to the
second peak of Ras activation and was dependent on
PI3K activity. This observation, together with the
ability of the PI3K target, Akt, to cause upregulation
of cyclin D1, indicated that Ras activation of PI3K
also contributes to the upregulation of cyclin D1 in
NIH 3T3 cells. Similarly, both Raf and PI3K effector
pathways were found to be important for oncogenic
Ras upregulation of cyclin D1 protein in RIE-1
epithelial cells [29].
Ras-mediated upregulation of cyclin D1 occurs, in
part, through stimulation of cyclin D1 transcription.
Activated versions of Raf, PI3K, or a Ral GEF alone
were able to stimulate cyclin D1 promoter activity,
possibly via distinct mechanisms [24]. Multiple
elements in the cyclin D1 promoter have been identi-
®ed to facilitate both Raf-dependent and Raf-indepen-
dent stimulation of transcription. Albanese et al.
identi®ed ERK-dependent stimulation of Ets-2 and
the cyclin D1 promoter as well as an AP-1 site acti-
vated by Raf-independent activation of the Jun/JNK
pathway in JEG-3 human trophoblasts [46]. Interest-
ingly, this AP-1 site was found to be dispensable for
Ras-mediated stimulation of the cyclin D1 promoter
in NIH 3T3 ®broblasts but essential for Ras-mediated
stimulation in RIE-1 epithelial cells [29]. Tetsu and
McCormick demonstrated that deletion of the EtsB
and CREB binding sites in the cyclin D1 promoter
strongly inhibited Ras-mediated stimulation of tran-
scription of cyclin D1 in HeLa cells [47]. Ral GEF-
mediated activation of Ral may stimulate the cyclin
D1 promoter through activation of NF-kB [48]. Thus,
multiple Ras effector pathways appear to play an
important role in the regulation of cyclin D1, in parti-
cular at the level of transcription.
A second level of regulation of cyclin D1 occurs
post-transcriptionally. The PI 3-kinase pathway
appears to post-transcriptionally regulate cyclin D1.
Cyclin D1 is known to be phosphorylated on threo-
nine 286 (T286) which initiates its degradation. Inter-
estingly, glycogen synthase kinase-3b (GSK-3b) has
been shown to phosphorylate T286 reducing its half-
life of about 10 min. It has been demonstrated that
activation of the PI3K and Akt-mediated phosphory-
lation of GSK-3b negatively regulates its activity,
thus promoting increased cyclin D1 protein levels
[49]. A role for this signaling mechanism in onco-
genic Ras upregulation of cyclin D1 protein expres-
sion was indicated by the increased half-life of cyclin
D1 in Ras-transformed NIH 3T3 cells, but not in cells
expressing a PI3K-de®cient Ras effector domain
mutant (12V/35S) or constitutively activated MEK1.
This could potentially engage another Ras effector
pathway in the regulation of cyclin D1 and allow
the stabilization of the protein in addition to increased
transcription of the gene. Finally, the PI3K/Akt path-
way may also promote increased cyclin D1 protein
expression by enhanced translation of cyclin D1
mRNA [50].
5. Ras and p21Cip1
The levels of p21 are low in serum-starved or
density-arrested quiescent cells and mitogenic stimuli
that activate the Ras/ERK pathway induce expression
of p21Cip1 protein [51,52] (Fig. 1). However, the
majority of observations suggest that p21Cip1 antago-
nizes Ras growth stimulation. For example, three
groups found that expression of low levels of acti-
vated Ras or Raf to be mitogenic for NIH 3T3 or
schwann cells, but high levels of activated Ras or
Raf caused cell cycle arrest that was associated with
a strong induction of p21Cip1 expression [43,52±54].
The failure of Raf to cause cell cycle arrest of p21Cip1
de®cient ®broblasts demonstrated the importance of
p21Cip1 in mediating this inhibitory response [53,54].
These results re¯ect the fact that the degree of Ras
activation can in¯uence the biological actions of Ras.
Ras upregulation of p21Cip1 is mediated, in part, by
upregulation of transcription [55]. Finally, keratino-
K. Pruitt, C.J. Der / Cancer Letters 171 (2001) 1±10 5
cytes lacking p21Cip1, but not p27Kip1, were shown to
be more susceptible to Ras-mediated tumorigenesis
[56]. Thus, loss of p21Kip1 function may promote
Ras transformation of both ®broblasts and epithelial
cells.
In contrast to the transient expression analyses,
p21Cip1 levels have been found to be elevated in
Ras-transformed NIH 3T3 and Swiss 3T3 mouse
®broblasts, and in RIE-1 epithelial cells [29,40,57].
Paradoxically, this suggests that stable upregulation
of p21Cip1 may be important for maintenance of the
Ras-transformed state. Thus, it is unclear whether up-
or downregulation of p21Cip1 is required to promote
Ras transformation. Furthermore, the induction of
p21Cip1 caused by serum stimulation does not appear
to require Ras function [34]. Finally, it is not clear
how high intensity Ras/Raf signaling causes the upre-
gulation of p21Cip1. This upregulation is mediated, in
part, at the level of transcription [53,55]. However,
although p53-mediated stimulation of p21Cip1 expres-
sion in response to cellular stress is well-established,
p21Cip1 induction by high Raf does not require p53
function. Thus, it has been suggested that the arti®-
cially high Ras and Raf signals may induce p21Cip1
due to the induction of cellular stress [7].
Although Cip/Kip CKIs have been considered as
negative regulators, recent evidence also supports
their positive roles in promoting G1 progression [4].
For example, Cip/Kip CKIs can be found in
complexes with active cyclin-CDKs [58±61]. Further-
more, it is believed that p21Cip1 may promote the
assembly of active cyclin D1-CDK4 complexes in
vivo, providing a means of nuclear import because
its localization signal, and increases the stability of
the complex [61]. Additionally, cyclin D-CDK
complexes may also play a role in the sequestering
Cip/Kip proteins, thereby contributing to the activa-
tion of cyclin E-CDK2 complexes. Thus the Cip/Kip
family of inhibitors appears to play a more diverse
role where their stoichiometry with respect to other
components of the cell cycle machinery may deter-
mine their overall effect.
6. Ras and p27Kip1
A link between Ras and a second CDK inhibitor
p27Kip1, where Ras causes downregulation of p27
expression, has also been observed in a variety of
cell types. p27Kip1 protein levels exhibit a pattern of
expression that is opposite that of p21Cip1 [62]. p27Kip1
levels are elevated in quiescent cells, increased by
stimuli that cause growth arrest, and downregulated
in response to mitogenic stimuli via a Ras-dependent
mechanism [34,63]. In contrast to p21Cip1, p27Kip1
mRNA levels are constant throughout the cell cycle
and p27Kip1 protein levels are regulated by transla-
tional controls [64] and by ubiquitin-mediated proteo-
lysis [65]. Cyclin E-CDK2 phosphorylates p27Kip1 at
threonine 187 (T187) and causes its degradation.
Studies have shown that the F-box protein p45Skp2
recognizes p27Kip1 phosphorylated at T187 and initi-
ates the ubiquitin-dependent proteolysis [66]. Mito-
gen activation of Ras and Ras-mediated
downregulation of p27Kip1 in late G1 involves both
suppression of protein synthesis and enhancement of
protein degradation in NIH 3T3 cells [63]. Inhibition
of PI3K, but not ERK, was found to block growth
factor-induced downregulation of p27Kip1, supporting
a role for this effector in Ras-mediated downregula-
tion of p27Kip1 levels.
Ras also regulates p27Kip1 function by modulating
its association with different CDK-cyclin complexes.
Both CDK2 and cyclin E are expressed at constant
amounts in quiescent and growing cells. Therefore,
cyclin E-CDK2 activity is controlled primarily by
the level of p27Kip1. Ras-mediated upregulation of
cyclin D1 promotes increased formation of cyclin
D1-CDK4 complexes, which then bind and sequester
p27Kip1 away from cyclin E-CDK2, thus leading to
CDK2 activation.
The Raf/MEK/ERK pathway is perhaps the best
characterized effector pathway by which oncogenic
Ras caused the downregulation of p27Kip1. For exam-
ple, the inducible activation of estrogen receptor
fusion proteins of Raf-1 [43] or MEK1 [67] caused
downregulation of p27Kip1 protein levels in NIH 3T3
cells. ERK can phosphorylate p27Kip1 in vitro and
phosphorylated p27Kip1 is impaired in binding to
CDK2 [68] (Kawada et al., 1997).
In contrast to these studies, induction of activated
MEK did not cause downregulation of p27Kip1 in NIH
3T3 cells, but did promote the sequestration of p27Kip1
by cyclin D1 [69]. When assessed in Ras-transformed
NIH 3T3 cells, one study found no change in p27Kip1
levels [40], whereas a second study found that the
K. Pruitt, C.J. Der / Cancer Letters 171 (2001) 1±106
stable expression of constitutively active Ras and Raf
in NIH 3T3 cells caused persistent upregulation of
p27Kip1 protein levels [29]. In contrast, in RIE-1 rat
intestinal epithelial cells, stable expression of Ras, but
not Raf, was shown to stably decrease p27Kip1 protein
levels. Finally, oncogenic Ras alone failed to cause a
downregulation of p27Kip1 in Balb 3T3 mouse ®bro-
blasts or REF52 rat ®broblasts, and instead, required
additional signals to cause p27Kip1 reduction [36,70].
These different observations may re¯ect the different
consequences of transient vs. sustained expression of
activated Ras, the intensity of Ras signaling, positive
and negative roles of p27Kip1 in G1 progression, as
well as cell type variations in the contribution of
p27Kip1 function in growth transformation.
7. Rho GTPases and cell cycle regulation
Rho GTPases constitute a major branch of the Ras
superfamily of small GTPases [30,71,72]. To date, at
least 18 mammalian Rho GTPases have been identi-
®ed, with RhoA, Rac1, and Cdc42 being the most
intensely studied. Like Ras, Rho GTPases function
as regulated GDP/GTP switches that are activated
by diverse extracellular stimuli that stimulate G
protein-coupled receptors, receptor tyrosine kinases,
integrins, and other cell surface receptors. Once acti-
vated, each Rho GTPase interacts with a wide spec-
trum of functionally diverse downstream effectors to
initiate cytoplasmic signaling pathways that regulate
both cytoplasmic and nuclear events.
The aberrant activation of Rho GTPases can
promote uncontrolled proliferation and growth trans-
formation [3,30,85]. Additionally, Ras and other
oncoproteins require Rho GTPase function to cause
cellular transformation. Consequently, it is not
surprising that Rho GTPases are also regulators of
cell cycle progression. This link was ®rst demon-
strated by observations that C3 exoenzyme inhibition
of RhoA, or dominant negative inhibition of Rac or
Cdc42 blocked serum-induced DNA synthesis in
rodent ®broblasts [73,74]. Conversely, microinjection
of constitutively activated mutants of RhoA, Rac1, or
Cdc42 into quiescent Swiss 3T3 cells stimulated G1
progression and DNA synthesis. Finally, loss of Rho
GTPase function may contribute, in part, to the cell
cycle arrest caused by geranylgeranyltransferase I
inhibitors of the prenylation of Rho GTPases [75,76].
Like Ras, Rho GTPases also stimulate the cyclin
D1 promoter and cause upregulation of cyclin D1
protein[77,78]. For Rac1, this occurs through activa-
tion of NF-kB [79]. Activated Rac and Cdc42, but not
RhoA, was found to promote the inactivation of Rb
and stimulate E2F-mediated transcription in NIH 3T3
cells [80]. However, in contrast to the observations
with Swiss 3T3 cells or Rat-1 rat ®broblasts, activated
Rho GTPases alone were not suf®cient to stimulate
DNA synthesis in quiescent NIH 3T3 cells [81].
Rho GTPases can also regulate the activities of
CKIs. Marshall and colleagues reported that microin-
jected oncogenic Ras is mitogenic in Swiss 3T3 cells
grown in the presence of serum, but is growth inhibi-
tory when the cells are serum-starved [81]. Upregula-
tion of p21Cip1 was observed only in the serum-starved
cells. They concluded that the ability of serum to
allow Ras growth stimulation was due to serum-
induced activation of RhoA, which in turn blocked
Ras-induced upregulation of p21Cip1. RhoA activity
also downregulated p21Cip1 expression in Ras-trans-
formed Swiss 3T3 cells as well as in colon carcinoma
cell lines [57].
Ras activation of RhoA may also facilitate the
downregulation of p27Kip1 expression. Baldassare
and colleagues found that platelet-derived growth
factor-induced degradation and downregulation of
p27Kip1 in IIC9 hamster embryo ®broblasts was Ras-
and Rho-dependent [82]. Activated RhoA alone
promoted p27Kip1 downregulation by causing an
increase in cyclin E-CDK2 activity [83]. A similar
requirement for RhoA-mediated degradation of
p27Kip1 for growth factor-stimulated DNA synthesis
was shown in FRTL-5 rat thyroid cells [84]. In
contrast, it was concluded that RhoA activity did not
in¯uence signi®cantly p27Kip1 expression in Ras-
transformed Swiss 3T3 cells or in ras mutation-posi-
tive BE and HCT15 human colon carcinoma cell lines
[57]. These different observations may re¯ect cell
type differences in RhoA regulation of CKIs.
8. Concluding remarks
The mechanism by which aberrant Ras and Rho
GTPase activation promotes oncogenesis clearly
K. Pruitt, C.J. Der / Cancer Letters 171 (2001) 1±10 7
involves a deregulation of cell cycle progression.
Much is now known regarding how Ras and Rho
signaling can control both positive (cyclin D1) and
negative (p21Cip1 and p27Kip1) regulators to facilitate
exit from G0, progression through G1, and initiation
of DNA synthesis. However, despite being a topic of
intense research study, the precise consequences of
oncogenic Ras and Rho activation on these regulators,
and their contribution to oncogenesis, remains incom-
plete and complex. One important issue that has
complicated the delineation of a simple relationship
between Ras and cell cycle regulation is that this rela-
tionship may exhibit signi®cant cell type differences.
Another complication is that a majority of studies
have evaluated the consequences of transient overex-
pression of activated Ras. While such approaches are
advantageous in de®ning the direct consequences of
Ras activation, they may not accurately convey the
cell cycle changes that support oncogenic Ras in the
cancer cell, where sustained Ras activation will lead
to both primary and secondary adaptive changes in
cell cycling. Clearly, future studies with the epithelial
cell types from which ras mutation positive cancers
arise will be required to better de®ne what aspects of
aberrant cell cycle control may be targeted to reverse
the oncogenic actions of Ras and Rho GTPases.
Acknowledgements
We thank Misha Rand for assistance in manuscript
preparation. Our studies were supported by from the
National Institutes of Health to C.J.D. (CA42978,
CA55008 and CA63071). K.P. was supported by
fellowships from the National Science Foundation
and UNCF-Merck.
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