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: cjder@med.unc.edu (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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