Key words: Raf, Rac, Rho, cyclin D1, p21CIP1, p27KIP1

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.

1. Introduction
The involvement of Ras proteins in cell signaling and in regulation of cell proliferation is well-established. Our knowledge on the signaling pathways that are regulated by Ras is considerable. Ras functions as a nodal point, where it is activated by diverse extracellular stimuli. Once activated, Ras in turn interacts with a diverse spectrum of effectors and initiates a multitude of cytoplasmic signaling cascades. Similarly, 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 regulatory 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 connect them with the cell cycle regulatory machinery.

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 function as GDP/GTP-regulated switches that cycle between an active GTP-bound state and an inactive GDP-bound state. Mitogenic signals stimulate a transient formation of active GTP-bound Ras and activated 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 activating proteins (GAPs; p120 GAP, NF1-GAP, etc.) facilitate 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 extracellular 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 specificity kinases, which then activate the p42/p44 ERK mitogen-activated protein kinases (MAPKs). The phosphoinositide 3-phosphate lipid kinases (PI3Ks) represent the second best characterized effectors of Ras [13]. Activated PI3K, a lipid kinase, facilitates the conversion of phosphatidylinositol 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 requirement for Ras
Mitogenic stimuli promote the entry of quiescent cells into the first gap phase (G1) and initiation of DNA synthesis (S phase) of the cell cycle [4]. Exit from or entry into the G0 quiescent state are 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 activities, 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-responsive 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 modifications including phosphorylation by CDK-activating kinase (CAK) to yield active holoenzymes. Further into G1, cyclin E complexes with CDK2 and causes additional phosphorylation and inactivation of Rb. With sufficient phosphorylation of Rb, E2F is released and transactivates 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 classified into two distinct families on the basis of their structural and functional characteristics. The members of the INK4 family of CKIs (p16Ink4a, p15Ink4b, p18Iik4c, and p19Ink4d) contain multiple ankyrin repeats and act as negative regulators of CDK4/6 by binding to the catalytic subunit 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 established the importance and requirement for endogenous Ras for cell cycle progression and the ability of oncogenic Ras to promote growth factor-independent cell cycle entry. First, studies in NIH 3T3 fibroblasts 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 throughout most of G1 for normal cell cycle progression [17-20]. Second, the ability of activated Ras alone to stimulate quiescent NIH 3T3 fibroblasts 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 activation (Fig. 1) [22-24]. The first phase of Ras activity was shown to occur rapidly following serum stimulation of quiescent cells. The second phase of Ras activation 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. Interestingly, whereas an activation of the Raf/ERK pathway is associated with the first peak of Ras activation, the later peak of activation did not correlate with ERK activation, and instead, PI3K/Akt activation [24].

One of the first links to be established between Ras-dependent signaling and Rb function was demonstrated using Ras neutralizing antibodies or dominant negative Ras and asynchronously growing primary Rb+/+ or Rb-/- mouse embryo fibroblasts [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 fibroblasts 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 specific 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 also apparent that 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 versus continuously proliferating cells [32]. Finally, cell type differences in how Ras regulates the cell cycle machinery further complicate our ability to define 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 accumulation 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 upregulation of cyclin D1 expression is dependent on Ras function and constitutive expression of cyclin D1 can overcome the requirement for Ras for proliferation 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 fibroblasts or IEC-18 and RIE-1 rat intestinal epithelial cells is accompanied by upregulation of cyclin D1 transcription and protein expression [35-37]Oncogenic Ras transformation of NIH 3T3 and Rat-1 fibroblasts, 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 oligonucleotides 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 sufficient 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 pathway. For example, transient [42-45] or sustained [38](6438}[29] activation of Raf or MEK in rodent fibroblasts caused increased levels of cyclin D1. In contrast, whereas activated Ras increased cyclin D1 expression in RIE-1 rat intestinal epithelial cells, activated 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 sufficient 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 identified to facilitate both Raf-dependent and Raf-independent stimulation of transcription. Albanese et al. identified ERK-dependent stimulation of Ets-2 and the cyclin D1 promoter as well as an AP-1 site activated by Raf-independent activation of the Jun/JNK pathway in JEG-3 human trophoblasts [46]. Interestingly, this AP-1 site was found to be dispensable for Ras-mediated stimulation of the cyclin D1 promoter in NIH 3T3 fibroblasts 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 transcription 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 particular 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 threonine 286 (T286) which initiates its degradation. Interestingly, 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 phosphorylation of GSK-3?b negatively regulates its activity, thus promoting increased cyclin D1 protein levels [49]. A role for this signaling mechanism in oncogenic Ras upregulation of cyclin D1 protein expression was indicated by the increased half-life of cyclin D1 in Ras-transformed NIH 3T3 cells, but not in cells expressing a PI3K-deficient 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 pathway 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 antagonizes Ras growth stimulation. For example, three groups found that expression of low levels of activated 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 deficient fibroblasts demonstrated the importance of p21Cip1 in mediating this inhibitory response [53,54]. These results reflect the fact that the degree of Ras activation can influence the biological actions of Ras. Ras upregulation of p21Cip1 is mediated, in part, by upregulation of transcription [55]. Finally, keratinocytes 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 fibroblasts 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 fibroblasts, 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 upregulation of p21Cip1. This upregulation is mediated, in part, at the level of transcription [53,55]. However, although p53-mediated stimulation of p21Cip1 expression 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 artificially 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]. Furthermore, 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 activation 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 determine 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 translational controls [64] and by ubiquitin-mediated proteolysis [65]. Cyclin E-CDK2 phosphorylates p27Kip1 at threonine 187 (T167) and causes its degradation. Studies have shown that the F-box protein p45Skp2 recognizes p27Kip1 phosphorylated at T187 and initiates the ubiquitin-dependent proteolysis [66,66]. Mitogen 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 downregulation 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 p27Kip`. For example, 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 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 fibroblasts or REF52 rat fibroblasts, and instead, required additional signals to cause p27Kip1 reduction [36,70]. These different observations may reflect the different consequences of transient versus 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 identified, 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 activated, each Rho GTPase interacts with a wide spectrum 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 transformation [3,30], (Aznar S & Lacal JC 2001 Cancer Letters 165:1). 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 first demonstrated by observations that C3 exoenzyme inhibition of RhoA, or dominant negative inhibition of Rac or Cdc42 blocked serum-induced DNA synthesis in rodent fibroblasts [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 activation 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 fibroblasts, activated Rho GTPases alone were not sufficient to stimulate DNA synthesis in quiescent NIH 3T3 cells [81].

Rho GTPases can also regulate the activities of CKIs. Marshall and colleagues reported that microinjected oncogenic Ras is mitogenic in Swiss 3T3 cells grown in the presence of serum, but is growth inhibitory when the cells are serum-starved [81]. Upregulation 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-transformed 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 fibroblasts 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 in FRTL-5 rat thyroid cells [84]. In contrast, it was concluded that RhoA activity did not influence significantly p27Kip1 expression in Ras-transformed Swiss 3T3 cells or in ras mutation-positive BE and HCT15 human colon carcinoma cell lines [57]. These different observations may reflect cell type differences RhoA regulation of CKIs.

8. Concluding Remarks
The mechanism by which aberrant Ras and Rho GTPase activation promotes oncogenesis clearly 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 incomplete and complex. One important issue that has complicated the delineation of a simple relationship between Ras and cell cycle regulation is that this relationship may exhibit significant cell type differences. Another complication is that a majority of studies have evaluated the consequences of transient overexpression of activated Ras. While such approaches are advantageous in defining 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 define 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 Merck.

Figure Legends

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 first 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 hyperphosphorylation 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 downregulated 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.

Fig. 3. The consequences of Ras show cell type and duration of activation differences. Two major issues have made it difficult to define a simple relationship between Ras activation and changes in the function of G1 regulators. First, the consequences of oncogenic Ras expression can vary significantly in different cell types. Second, the consequences of transient Ras activation versus sustained Ras activation can also be strikingly different. Shown in this figure are the consequences of sustained Ras activation in NIH 3T3 mouse fibroblasts and RIE-1 rat intestinal epithelial cells {Pruitt, Pestell, et al. 2000 9514 /id}. Ras activation of Raf alone is sufficient 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.

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