High intensity ERK signal mediates hepatocyte growth factor-induced proliferation inhibition of the human hepatocellular carcinoma cell line HepG2

Hepatocyte growth factor (HGF) induces growth stimulation of a variety of cell types, but it also induces growth inhibition of several types of tumor cell lines. The molecular mechanism of the HGF-induced growth inhibition of tumor cells remains obscure. We have investigated the intracellular signaling pathway involved in the antiproliferative effect of HGF on the human hepatocellular carcinoma cell line HepG2. HGF induced strong activation of ERK in HepG2 cells. Although the serum-dependent proliferation of HepG2 cells was inhibited by the MEK inhibitor PD98059 in a dose-dependent manner, 10 microM PD98059 reduced the HGF-induced strong activation of ERK to a weak activation; and as a result, the proliferation inhibited by HGF was completely restored. Above or below this specific concentration, the restoration was incomplete. Expression of constitutively activated Ha-Ras, which induces strong activation of ERK, led to the proliferation inhibition of HepG2 cells, as was observed in HGF-treated HepG2 cells. This inhibition was suppressed by the MEK inhibitor. Furthermore, HGF treatment and expression of constitutively activated Ha-Ras changed the hyperphosphorylated form of the retinoblastoma tumor suppressor gene product pRb to the hypophosphorylated form. This change was inhibited by the same concentration of MEK inhibitor needed to suppress the proliferation inhibition. These results suggest that ERK activity is required for both the stimulation and inhibition of proliferation of HepG2 cells; that the level of ERK activity determines the opposing proliferation responses; and that HGF-induced proliferation inhibition is caused by cell cycle arrest, which results from pRb being maintained in its active hypophosphorylated form via a high-intensity ERK signal in HepG2 cells.     

Antiproliferative mechanism of a cannabinoid agonist by cell cycle arrest in human gastric cancer cells

For gastric cancers, the antineoplastic activity of cannabinoids has been investigated in only a few reports and knowledge regarding the mechanisms involved is limited. We have reported previously that treatment of gastric cancer cells with a cannabinoid agonist significantly decreased cell proliferation and induced apoptosis. Here, we evaluated the effects of cannabinoids on various cellular mediators involved in cell cycle arrest in gastric cancer cells. AGS and MKN-1 cell lines were used as human gastric cancer cells and WIN 55,212-2 as a cannabinoid agonist. Cell cycles were analyzed by flow cytometry and western blotting. Treatment with WIN 55,212-2 arrested the cell cycle in the G0/G1 phase. WIN 55,212-2 also upregulated phospho-ERK1/2, induced Kip1/p27 and Cip1/WAF1/p21 expression, decreased cyclin D1 and cyclin E expression, decreased Cdk 2, Cdk 4, and Cdk 6 expression levels, and decreased phospho-Rb and E2F-1 expression. ERK inhibitor decreased the proportion of G0/G1 phase which was induced by WIN 55,212-2. Inhibition of pAKT led to cell cycle arrest in gastric cancer cells. Cell cycle arrest preceded apoptotic response. Thus, this cannabinoid agonist can reduce gastric cancer cell proliferation via G1 phase cell cycle arrest, which is mediated via activation of the MAPK pathway and inhibition of pAKT.     

Hepatocyte growth factor induces redistribution of p21(CIP1) and p27(KIP1) through ERK-dependent p16(INK4a) up-regulation, leading to cell cycle arrest at G1 in HepG2 hepatoma cells

Hepatocyte growth factor (HGF) has an anti-proliferative effect on many types of tumor cell lines and tumors in vivo. We found previously that inhibition of HGF-induced proliferation in HepG2 hepatoma cells is caused by cell cycle arrest at G1 through a high intensity ERK signal, which represses Cdk2 activity. To examine further the mechanisms of G1 arrest by HGF, we analyzed the Cdk inhibitor p16(INK4a), which has an anti-proliferative function through cell cycle arrest at G1. We found that HGF treatment drastically increased endogenous p16 levels. Knockdown of p16 with small interfering RNA reversed the arrest, indicating that the induction of p16 is required for G1 arrest by HGF. Analysis of the promoter of the human p16 gene identified the proximal Ets-binding site as a responsive element for HGF, and this responded to the high intensity ERK signal. HGF treatment of the cells led to a redistribution of p21(CIP1) and p27(KIP1) from Cdk4 to Cdk2. The redistribution was blocked by the knockdown of p16 with small interfering RNA, which restored the Cdk2 activity repressed by HGF, demonstrating the requirement of p16 induction for the redistribution and eventual repression of Cdk2 activity. Our results reveal a signaling pathway for G1 arrest induced by HGF.     

Up-regulation of p21CIP1 expression mediated by ERK-dependent and -independent pathways contributes to hepatocyte growth factor-induced inhibition of HepG2 hepatoma cell proliferation

Strong activation of the ERK signal is required for hepatocyte growth factor (HGF) to inhibit proliferation of the human hepatocellular carcinoma cell line HepG2. However, it is still to be elucidated whether the activation alone is sufficient to induce the inhibitory effect. In this study, we constructed HepG2 cell clones expressing a high level of epidermal growth factor receptor (EGFR), and examined the effect of the strong activation of ERK on the proliferation of the cell clones. EGF treatment of the cell clones induced strong activation of ERK similar to HGF treatment, but did not inhibit cell proliferation. HGF treatment of the cell clones up-regulated the expression of a Cdk inhibitor p16(INK4a), which has previously been shown to be required to inhibit the proliferation of HepG2 cells, but EGF treatment did not. Furthermore, EGF treatment of the cell clones did not induce the up-regulation of another Cdk inhibitor p21(CIP1), whereas HGF treatment did. Knockdown of p21 by siRNA restored the proliferation of HepG2 cells inhibited by HGF, and restored Cdk2 activity suppressed in HGF-treated HepG2 cells. These results suggest that strong activation of ERK alone is not sufficient, and some other pathway(s), which is activated through the HGF receptor but not through EGFR, is also required to induce the up-regulation of p16 and p21 expression, and also suggest that in addition to the up-regulated expression of p16, that of p21 contributes to the suppression of Cdk2 activity leading to the inhibition of proliferation of HGF-treated HepG2 cells.     

Coupling of Grb2 to Gab1 mediates hepatocyte growth factor-induced high intensity ERK signal required for inhibition of HepG2 hepatoma cell proliferation

Activation of the extracellular signal-regulated kinase (ERK) pathway is a key factor in the regulation of cell proliferation by growth factors. Hepatocyte growth factor (HGF)-induced cell cycle arrest in the human hepatocellular carcinoma cell line HepG2 requires strong activation of the ERK pathway. In this study, we investigated the molecular mechanism of the activation. We constructed a chimeric receptor composed of the extracellular domain of the NGF receptor and the cytoplasmic domain of the HGF receptor (c-Met) and introduced a point mutation (N1358H) into the chimeric receptor, which specifically abrogates the direct binding of Grb2 to c-Met. The mutant chimeric receptor failed to mediate the strong activation of ERK, up-regulation of the expression of a Cdk inhibitor p16(INK4a) and inhibition of HepG2 cell proliferation by ligand stimulation. Moreover, the mutant receptor did not induce tyrosine phosphorylation of the docking protein Gab1. Knockdown of Gab1 using siRNA suppressed the HGF-induced strong activation of ERK and inhibition of HepG2 cell proliferation. These results suggest that coupling of Grb2 to Gab1 mediates the HGF-induced strong activation of the ERK pathway, which is required for the inhibition of HepG2 cell proliferation.     

Vanadyl bisacetylacetonate induced G1/S cell cycle arrest via high-intensity ERK phosphorylation in HepG2 cells

In recent years the anticancer properties of vanadium compounds have been noticed, but the underlying mechanisms are not well understood. In the present work, we found that vanadyl bisacetylacetonate ([VO(acac)(2)]) blocked cell cycle progression permanently at G1 phase in a dose- and time-dependent manner in HepG2 cells. This was further evidenced by the growth regulatory signals during the G1 stage. After the treatment with [VO(acac)(2)], the level of phosphorylation of retinoblastoma tumor suppressor protein (pRb) and the expressions of cyclin D1, cyclin E and cyclin A were reduced, while the expression of a cyclin-dependent kinase inhibitor p21 was increased dose-dependently. In the meantime, neither O(2)(*-) nor H(2)O(2) level was observed to increase. Interestingly, the levels of phosphorylated extracellular signal-regulated protein kinase (ERK) and Akt were highly activated. After 1-h pretreatment with a lower concentration of MEK inhibitor U0126, the level of phosphorylated pRb was restored, indicating a release of cell cycle arrest. Taken together, we suggested that [VO(acac)(2)]-induced proliferation inhibition was caused by G1/S cell cycle arrest, which resulted from the decreased level of phosphorylated pRb in its active hypophosphorylated form via a highly activated ERK signal in HepG2 cells. The results presented here provided new insight into the development of vanadium compounds as potential anticancer agents.     

p53 dephosphorylation and p21Cip1/Waf1 translocation correlate with caspase-3 activation in TGF-β1-induced apoptosis of HuH-7 cells

The p53 tumor suppressor gene product plays an important role in the regulation of apoptosis. Transforming growth factor beta1 (TGF-beta1)-induced apoptosis in hepatic cells is associated with reduced expression of the retinoblastoma protein (pRb) and subsequent E2F-1-activated expression of apoptosis-related genes. In this study, we explored the potential role of p53 in TGF-beta1-induced apoptosis. HuH-7 human hepatoma cells were either synchronized in G1, S and G2/M phases, or treated with 1 nM TGF-beta1. The results indicated that greater than 90% of the TGF-beta1-treated cells were arrested in G1 phase of the cell cycle. This was associated with enhanced p53 dephosphorylation and p21(Cip1/Waf1) expression, which coincided with decreased Cdk2, Cdk4, and cyclin E expression, compared with synchronized G1 cells. In addition, p53 dephosphorylation coincided with caspase-3 activation, and translocation of p21(Cip1/Waf1) and p27(Kip1) into the cytoplasm, all of which were suppressed by caspase inhibition of TGF-beta1-induced apoptosis. Finally, phosphatase inhibition and pRb overexpression partially inhibited p53-mediated apoptosis. In conclusion, the results demonstrated that TGF-beta1-induced p53 dephosphorylation is associated with caspase-3 activation, and cytosolic translocation of p21(Cip1/Waf1) and p27(Kip1), resulting in decreased expression of Cdks and cyclins. Further, p53 appears to mediate TGF-beta1-induced apoptosis downstream of the pRb/E2F-1 pathway.     

A pure estrogen antagonist inhibits cyclin E-Cdk2 activity in MCF-7 breast cancer cells and induces accumulation of p130-E2F4 complexes characteristic of quiescence

Estrogen antagonists inhibit cell cycle progression in estrogen-responsive cells, but the molecular mechanisms are not fully defined. Antiestrogen-mediated G(0)/G(1) arrest is associated with decreased cyclin D1 gene expression, inactivation of cyclin D1-cyclin dependent kinase (Cdk) 4 complexes, and decreased phosphorylation of the retinoblastoma protein (pRb). We now show that treatment of MCF-7 breast cancer cells with the pure estrogen antagonist ICI 182780 results in inhibition of cyclin E-Cdk2 activity prior to a decrease in the G(1) to S phase transition. This decrease was dependent on p21(WAF1/Cip1) since treatment with antisense oligonucleotides to p21 attenuated the effect. Recruitment of p21 to cyclin E-Cdk2 complexes was in turn dependent on decreased cyclin D1 expression since it was apparent following treatment with antisense cyclin D1 oligonucleotides. To define where within the G(0) to S phase continuum antiestrogen-treated cells arrested, we assessed the relative abundance and phosphorylation state of pocket protein-E2F complexes. While both pRb and p107 levels were significantly decreased, p130 was increased 4-fold and was accompanied by the formation of p130.E2F4 complexes and the accumulation of hyperphophorylated E2F4, putative markers of cellular quiescence. Thus, ICI 182780 inhibits both cyclin D1-Cdk4 and cyclin E-Cdk2 activity, resulting in the arrest of MCF-7 cells in a state with characteristics of quiescence (G(0)), as opposed to G(1) arrest.     

Hyperoxia induces S-phase cell-cycle arrest and p21(Cip1/Waf1)-independent Cdk2 inhibition in human carcinoma T47D-H3 cells

Little is known about cell-cycle checkpoint activation by oxidative stress in mammalian cells. The effects of hyperoxia on cell-cycle progression were investigated in asynchronous human T47D-H3 cells, which contain mutated p53 and fail to arrest at G1/S in response to DNA damage. Hyperoxic exposure (95% O(2), 40-64 h) induced an S-phase arrest associated with acute inhibition of Cdk2 activity and DNA synthesis. In contrast, exit from G2/M was not inhibited in these cells. After 40 h of hyperoxia, these effects were partially reversible during recovery under normoxic conditions. The inhibition of Cdk2 activity was not due to degradation of Cdk2, cyclin E or A, nor impairment of Cdk2 complex formation with cyclin A or E and p21(Cip1). The loss of Cdk2 activity occurred in the absence of induction and recruitment of cdk inhibitor p21(Cip1) or p27(Kip1) in cyclin A/Cdk2 or cyclin E/Cdk2 complexes. In contrast, Cdk2 inhibition was associated with increased Cdk2-Tyr15 phosphorylation, increased E2F-1 recruitment, and decreased PCNA contents in Cdk2 complexes. The latter results indicate a p21(Cip1)/p27(Kip1)-independent mechanism of S-phase checkpoint activation in the hyperoxic T47D cell model investigated.     

Hyperoxia Induces S-Phase Cell-Cycle Arrest and p21-Independent Cdk2 Inhibition in Human Carcinoma T47D-H3 Cells

Little is known about cell-cycle checkpoint activation by oxidative stress in mammalian cells. The effects of hyperoxia on cell-cycle progression were investigated in asynchronous human T47D-H3 cells, which contain mutated p53 and fail to arrest at G1/S in response to DNA damage. Hyperoxic exposure (95% O₂, 40–64 h) induced an S-phase arrest associated with acute inhibition of Cdk2 activity and DNA synthesis. In contrast, exit from G2/M was not inhibited in these cells. After 40 h of hyperoxia, these effects were partially reversible during recovery under normoxic conditions. The inhibition of Cdk2 activity was not due to degradation of Cdk2, cyclin E or A, nor impairment of Cdk2 complex formation with cyclin A or E and p21^Cip1. The loss of Cdk2 activity occurred in the absence of induction and recruitment of cdk inhibitor p21^Cip1 or p27^Kip1 in cyclin A/Cdk2 or cyclin E/Cdk2 complexes. In contrast, Cdk2 inhibition was associated with increased Cdk2-Tyr15 phosphorylation, increased E2F-1 recruitment, and decreased PCNA contents in Cdk2 complexes. The latter results indicate a p21^Cip1/p27^Kip1-independent mechanism of S-phase checkpoint activation in the hyperoxic T47D cell model investigated.     

Uncoupling between phenotypic senescence and cell cycle arrest in aging p21-deficient fibroblasts

Irreversible G(1) arrest in senescent human fibroblasts is mediated by two inhibitors of cyclin-dependent kinases (Cdks), p21(Cip1/SDI1/WAF1) and p16(Ink4A). To determine the physiological and molecular events that specifically require p21, we studied senescence in human diploid fibroblasts expressing the human papillomavirus type 16 E6 oncogene, which confers low p21 levels via enhanced p53 degradation. We show that in late-passage E6 cells, high Cdk activity drives the cell cycle, but population expansion is slowed down by crisis-like events, probably owing to defective cell cycle checkpoints. At the end of lifespan, terminal-passage E6 cells exhibited several aspects of the senescent phenotype and accumulated unphosphorylated pRb and p16. However, both replication and cyclin-Cdk2 kinase activity were still not blocked, demonstrating that phenotypic and replicative senescence are uncoupled in the absence of normal p21 levels. At this stage, E6 cells also failed to upregulate p27 and inactivate cyclin-Cdk complexes in response to serum deprivation. Eventually, irreversible G(1) arrest occurred coincident with inactivation of cyclin E-Cdk2 owing to association with p21. Similarly, when p21(-/-) mouse embryo fibroblasts reached the end of their lifespan, they had the appearance of senescent cells yet, in contrast to their wild-type counterparts, they were deficient in downregulating bromodeoxyuridine incorporation, cyclin E- and cyclin A-Cdk2 activity, and inhibiting pRb hyperphosphorylation. These data support the model that the critical event ensuring G(1) arrest in senescence is p21-dependent Cdk inactivation, while other aspects of senescent phenotype appear to occur independently of p21.     

Raf-Induced Cell Cycle Progression in Human TF-1 Hematopoietic Cells

Ras/Raf/MEK/ERK is a crucial pathway regulating cell cycle progression, apoptosis, and drug resistance. The Ras oncogene is frequently mutated in human cancer, which can result in the activation of the downstream Raf/MEK/ERK cascade leading to cell cycle progression in the absence of a growth stimulus. Raf-induced proliferation has been observed in hematopoietic cells. However, the mechanisms by which Raf affects cell cycle progression are not well described. To investigate the importance of Raf/MEK/ERK signaling in human hematopoietic cell growth, the effects of three different Raf genes, A-Raf, B-Raf and Raf-1, on cell cycle progression and regulatory gene expression were examined in TF-1 cells transformed to grow in response to b-estradiol-regulated DRaf:ER genes. Raf activation increased the expression of cyclin A, cyclin D, cyclin E, and p21Cip1, which are associated with G1 progression. Activated DRaf-1:ER and DA-Raf:ER but not DB-Raf:ER increased Cdk2 and Cdk4 kinase activity. The regulatory role of p16Ink4a, a potent Cdk4 kinase inhibitor, on the kinase activity of Cdk2 and Cdk4 was also examined. Raf induced p16Ink4a suppressor but this did not eliminate Cdk4 kinase activity. These results indicate that human hematopoietic cells transformed to grow in response to activated Raf can be used to elucidate the mechanisms by which various cell cycle regulatory molecules effect cell cycle progression. Furthermore, the differences that the various Raf isoforms have on Cdk4 activity and other cell cycle regulatory molecules can be determined in these cells.

Key Words:

Cell cycle, Raf, p21Cip1, p27Kip1, Cyclins, Cdks, Hematopoietic cells     

Raf-induced cell cycle progression in human TF-1 hematopoietic cells

Ras/Raf/MEK/ERK is a crucial pathway regulating cell cycle progression, apoptosis, and drug resistance. The Ras oncogene is frequently mutated in human cancer, which can result in the activation of the downstream Raf/MEK/ERK cascade leading to cell cycle progression in the absence of a growth stimulus. Raf-induced proliferation has been observed in hematopoietic cells. However, the mechanisms by which Raf affects cell cycle progression are not well described. To investigate the importance of Raf/MEK/ERK signaling in human hematopoietic cell growth, the effects of three different Raf genes, A-Raf, B-Raf and Raf-1, on cell cycle progression and regulatory gene expression were examined in TF-1 cells transformed to grow in response to beta-estradiol-regulated DeltaRaf:ER genes. Raf activation increased the expression of cyclin A, cyclin D, cyclin E, and p21(Cip1), which are associated with G(1) progression. Activated DeltaRaf-1:ER and DeltaA-Raf:ER but not DeltaB-Raf:ER increased Cdk2 and Cdk4 kinase activity. The regulatory role of p16(Ink4a), a potent Cdk4 kinase inhibitor, on the kinase activity of Cdk2 and Cdk4 was also examined. Raf induced p16(Ink4a) suppressor but this did not eliminate Cdk4 kinase activity. These results indicate that human hematopoietic cells transformed to grow in response to activated Raf can be used to elucidate the mechanisms by which various cell cycle regulatory molecules effect cell cycle progression. Furthermore, the differences that the various Raf isoforms have on Cdk4 activity and other cell cycle regulatory molecules can be determined in these cells.     

Asparanin A induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells

We recently established that asparanin A, a steroidal saponin extracted from Asparagus officinalis L., is an active cytotoxic component. The molecular mechanisms by which asparanin A exerts its cytotoxic activity are currently unknown. In this study, we show that asparanin A induces G₂/M phase arrest and apoptosis in human hepatocellular carcinoma HepG2 cells. Following treatment of HepG2 cells with asparanin A, cell cycle-related proteins such as cyclin A, Cdk1 and Cdk4 were down-regulated, while p21^WAF1/Cip1 and p-Cdk1 (Thr14/Tyr15) were up-regulated. Additionally, we observed poly (ADP-ribose) polymerase (PARP) cleavage and activation of caspase-3, caspase-8 and caspase-9. The expression ratio of Bax/Bcl-2 was increased in the treated cells, where Bax was also up-regulated. We also found that the expression of p53, a modulator of p21^WAF1/Cip1 and Bax, was not affected in asparanin A-treated cells. Collectively, our findings demonstrate that asparanin A induces cell cycle arrest and triggers apoptosis via a p53-independent manner in HepG2 cells. These data indicate that asparanin A shows promise as a preventive and/or therapeutic agent against human hepatoma.     

Multifaceted regulation of cell cycle progression by estrogen: regulation of Cdk inhibitors and Cdc25A independent of cyclin D1-Cdk4 function

Estrogens induce proliferation of estrogen receptor (ER)-positive MCF-7 breast cancer cells by stimulating G(1)/S transition associated with increased cyclin D1 expression, activation of cyclin-dependent kinases (Cdks), and phosphorylation of the retinoblastoma protein (pRb). We have utilized blockade of cyclin D1-Cdk4 complex formation through adenovirus-mediated expression of p16(INK4a) to demonstrate that estrogen regulates Cdk inhibitor expression and expression of the Cdk-activating phosphatase Cdc25A independent of cyclin D1-Cdk4 function and cell cycle progression. Expression of p16(INK4a) inhibited G(1)/S transition induced in MCF-7 cells by 17-beta-estradiol (E(2)) with associated inhibition of both Cdk4- and Cdk2-associated kinase activities. Inhibition of Cdk2 activity was associated with delayed removal of Cdk-inhibitory activity in early G(1) and decreased cyclin A expression. Cdk-inhibitory activity and expression of both p21(Cip1) and p27(Kip1) was decreased, however, in both control and p16(INK4a)-expressing cells 20 h after estrogen treatment. Expression of Cdc25A mRNA and protein was induced by E(2) in control and p16(INK4a)-expressing MCF-7 cells; however, functional activity of Cdc25A was inhibited in cells expressing p16(INK4a). Inhibition of Cdc25A activity in p16(INK4a)-expressing cells was associated with depressed Cdk2 activity and was reversed in vivo and in vitro by active Cdk2. Transfection of MCF-7 cells with a dominant-negative Cdk2 construct inhibited the E(2)-dependent activation of ectopic Cdc25A. Supporting a role for Cdc25A in estrogen action, antisense CDC25A oligonucleotides inhibited estrogen-induced Cdk2 activation and DNA synthesis. In addition, inactive cyclin E-Cdk2 complexes from p16(INK4a)-expressing, estrogen-treated cells were activated in vitro by treatment with recombinant Cdc25A and in vivo in cells overexpressing Cdc25A. The results demonstrate that functional association of cyclin D1-Cdk4 complexes is required for Cdk2 activation in MCF-7 cells and that Cdk2 activity is, in turn, required for the in vivo activation of Cdc25A. These studies establish Cdc25A as a growth-promoting target of estrogen action and further indicate that estrogens independently regulate multiple components of the cell cycle machinery, including expression of p21(Cip1) and p27(Kip1).     

IL-1beta suppresses prolonged Akt activation and expression of E2F-1 and cyclin A in breast cancer cells

Cell cycle aberrations occurring at the G(1)/S checkpoint often lead to uncontrolled cell proliferation and tumor growth. We recently demonstrated that IL-1beta inhibits insulin-like growth factor (IGF)-I-induced cell proliferation by preventing cells from entering the S phase of the cell cycle, leading to G(0)/G(1) arrest. Notably, IL-1beta suppresses the ability of the IGF-I receptor tyrosine kinase to phosphorylate its major docking protein, insulin receptor substrate-1, in MCF-7 breast carcinoma cells. In this study, we extend this juxtamembrane cross-talk between cytokine and growth factor receptors to downstream cell cycle machinery. IL-1beta reduces the ability of IGF-I to activate Cdk2 and to induce E2F-1, cyclin A, and cyclin A-dependent phosphorylation of a retinoblastoma tumor suppressor substrate. Long-term activation of the phosphatidylinositol 3-kinase/Akt signaling pathway, but not the mammalian target of rapamycin or mitogen-activated protein kinase pathways, is required for IGF-I to hyperphosphorylate retinoblastoma and to cause accumulation of E2F-1 and cyclin A. In the absence of IGF-I to induce Akt activation and cell cycle progression, IL-1beta has no effect. IL-1beta induces p21(Cip1/Waf1), which may contribute to its inhibition of IGF-I-activated Cdk2. Collectively, these data establish a novel mechanism by which prolonged Akt phosphorylation serves as a convergent target for both IGF-I and IL-1beta; stimulation by growth factors such as IGF-I promotes G(1)-S phase progression, whereas IL-1beta antagonizes IGF-I-induced Akt phosphorylation to induce cytostasis. In this manner, Akt serves as a critical bridge that links proximal receptor signaling events to more distal cell cycle machinery.