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K-Ras and H-Ras Activation Promote Distinct Consequences on Endometrial Cell Survival

Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Oita, Japan

	ABSTRACT

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A considerable amount of evidence indicates that Ras signaling contributes to the development of endometrial cancer. We previously demonstrated that endometrial cancer cells carrying oncogenic [¹²Val]K-ras were susceptible to apoptosis. The present study examined the role of K-and H-Ras in the induction of apoptosis using rat endometrial cells (RENT4 cells). We found that constitutively activated K-Ras promoted apoptotic cell death, whereas the H-Ras mutant rescued rat endometrial cells from apoptosis. Expression of a constitutively active form of Raf-1 (Raf-CAAX) promoted apoptosis, whereas expression of a constitutively active catalytic subunit of phosphoinositide 3-kinase, p110K227E, allowed cells to escape from apoptosis. Moreover, inhibition of the MEK-MAPK pathway by the specific inhibitor, UO126, rescued the cells from apoptosis, whereas the inhibition of phosphoinositide 3-kinase by its specific inhibitor, LY294002, promoted apoptosis in RENT4 cells expressing activated K-Ras. However, both inhibitors promoted apoptosis in RENT4 cells expressing activated H-Ras. This difference in the regulation of apoptosis by the MEK inhibitor between K-Ras- and H-Ras-expressing cells depended on the interaction of effector proteins downstream of each Ras isoform. Finally, to elucidate the role of downstream K-Ras signal pathways, we generated K-Ras effector domain mutants (K12V35S, K12V40C). We examined the incidence of apoptotic cell death induced by the K-Ras effector domain mutants (K12V35S, K12V40C). The relative ratio of phospho-MAPK to phospho-Akt compared with that of mock cells was higher in K12V35S cells than in K12V40C cells. Ectopic expression of K12V35S protein increased the proportion of apoptotic cells, and in turn, the expression of K12V40C protein decreased compared with the expression of K12V protein without the effector domain mutant. These results demonstrate that K- and H-Ras-mediated signaling pathways exert distinct effects on apoptosis and that K-Ras downstream Raf/MEK/MAPK pathway is required for the induction of apoptosis in endometrial cells. Coordination of the two pathways contributes to endometrial cell survival.

	INTRODUCTION

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Ras proteins act as critical relay switches, which control signaling pathways connecting the cell surface with the nucleus and thus play a role in integrating and transmitting signals elicited by membrane receptors to downstream effector pathways. Ras controls these pathways by the binding of its effector loop region to the so-called Ras effector proteins, which in turn activates specific signaling cascades. Ras proteins in the activated GTP bound state interacts with the same set of effectors: Raf kinases; phosphoinositide 3-kinase (PI3K); Ral GDS; and AF6 (1, 2, 3, 4, 5, 6, 7) . Raf-MEK-MAPK and PI3K/Akt signaling are often simultaneously activated in response to activated Ras proteins and mediate several apparently conflicting cellular responses such as proliferation, apoptosis, growth arrest, differentiation, and senescence, depending on the duration and strength of the external stimuli as well as cell type (8, 9, 10) .

The NH₂-terminal 165 amino acids of Ras proteins contain the critical domains required for GTPase function. The NH₂-terminal 85 residues of all Ras isoforms are identical, and the subsequent 80 amino acids are 95% conserved (11) . The Ras effector domain, a critical binding site for all of the Ras effectors, is found between residues 32–40. No marked differences exist in the binding affinities of H-, N-, and K-Ras for their effector proteins (12) . Despite the similar structure and binding affinity of the effectors of Ras isoforms, several lines of evidence suggest that Ras isoforms have distinct functions. K-Ras but not H- or N-Ras functions in mouse development (13 , 14) . Specific Ras isoforms are mutated in different tumors: K-ras mutations occur in 20% of endometrial cancers, 50% of colon cancers, and 90% of pancreatic cancers, whereas N- and H-ras mutations are extremely rare in these cancers (15) . Recent evidence showed that in vivo there are marked quantitative differences in the activation of Raf-1 and PI3K by H- and K-Ras. K-Ras recruits Raf-1 to the plasma membrane more efficiently than H-Ras and is a more potent activator of membrane-recruited Raf-1 than H-Ras. In contrast, H-Ras is a more potent activator of PI3K than K-Ras (16) . Distinct hierarchies for Raf-1 activation have also been reported among the four Ras homologies (K-Ras4B>K-Ras4A[tmt]N-Ras>H-Ras; Ref. 17 ). Thus, it is probable that the activation of each Ras isoform has distinct phenotypic cell consequences via extensions of the activation of each effector pathway.

Considerable evidence indicates that Ras signaling contributes to the development of endometrial cancer. We previously demonstrated that endometrial cancer cells carrying [¹²Val]K-ras were more susceptible to apoptosis (18) . We used two human endometrial cancer cell lines: Ishikawa cells and HHUA cells. No mutations were detected at codons 12, 13, or 61 of the K-, H-, and N-rasgenes in Ishikawa cells, whereas HHUA cells harbored a single base substitution at codon 12 that resulted in the substitution of a valine in the K-rasgene. We found that the incidence of apoptotic cell death (ACD) in the absence of serum was significantly higher in HHUA cells than in Ishikawa cells (0.6% in Ishikawa cells, 7.0% in HHUA cells). To additionally confirm the effect of K-Ras gene mutations on apoptosis, expression vectors encoding wt or mutated [¹²Val]K-Ras were introduced into Ishikawa cells, and IKWT and IKK12V cells were established, respectively. We examined the incidence of ACD in IKWT and IKK12V cells by incubating the cells in the absence of serum for 3 days. Ten-fold increase in the number of positively staining cells detected by an in situ apoptosis detection assay was found for IKK12V cells (0.5% in IKWT cells versus 6.0% in IKK12V cells; Ref. 18 ). Thus, to test the role of the K- and H-Ras proteins in the development of this cancer, we manipulated the Ras-Raf-MEK-MAPK and Ras-PI3K-Akt pathways using RENT4 cells, which are immortalized rat endometrial cells (19) . The results demonstrate that the K- and H-Ras-mediated signaling pathways exert distinct effects on apoptosis and that the K-Ras downstream Raf/MEK/MAPK pathway is required for the induction of apoptosis in endometrial cells. Coordination of the two pathways contributes to endometrial cell survival.

	MATERIALS AND METHODS

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Plasmids.
pZIP-Neo SV (X)1 vector and constructs containing cDNA sequences encoding either [¹²Val]K-Ras4B or [⁶¹Leu]H-Ras, pBABE vector, and constructs [pBABE-H-ras(12V), Raf-CAAX, and p110K227E] were received as gifts from Dr. Channing Der (University of North Carolina). K-Ras 4B effector domain mutants were generated using a Transformer site-directed mutagenesis kit (Clontech) according to the manufacturer’s instructions. We used pZeo-[¹²Val]K-ras 4B cDNA as a template. The mutations were confirmed by sequencing.

Cells and Cell Cultures.
A rat endometrial cell line (RENT4 cells) was used in the present study. RENT4 cells were obtained from the European Collection of Cell Cultures. RENT4 cells harboring mutant (12V) versions of K-ras4B and mutant (61L) versions of H-Ras were established by transfecting RENT4 cells with pZIP-NeoSV (x)1 retroviral vector constructs containing cDNA sequences encoding K-Ras (12V) or H-Ras (61L) using Lipofectin (Life Technologies, Inc., Rockville, MD). Stably transfected cells were selected and isolated in growth medium containing 400 µg/ml G418 (Geneticin; Life Technologies, Inc.) to establish cell lines expressing each Ras protein. Pooled populations of each cell line were used for the assay.

Ras protein expression was confirmed by immunoprecipitation as described below. pBABE-Hras12V effector domain mutant constructs, pZeo-Kras12V constructs, pBABE-p110 K227E (activated catalytic subunit of PI3K by the amino acid substitution), and pBABE-Raf-CAAX (activated Raf-1 by the addition of the COOH-terminal plasma membrane sequence from Ras) constructs (20) were introduced into RENT4 cells by transfection and each protein was transiently expressed. Cells were then used for apoptosis assay. Parent RENT4 cells and the RENT4-derived cell lines were maintained in DMEM (Nissui, Seika, Japan) supplemented with 20 mg of Gly-His-Lys, 2 mM glutamine, 80 IU insulin (Sigma), and 10% FCS (Hyclone).

Immunoprecipitation Analysis of Ras.
Cells were labeled overnight in a growth medium supplemented with[³⁵S]methionine/cystein (Tran³⁵S-label, ICN; Costa Mesa, CA; 400 µCi/ml Met/Cys). Ras proteins were immunoprecipitated using the Y13-259 rat anti-ras monoclonal antibody (Oncogene Science, Uniondale, NY) and were resolved by SDS-PAGE. ³⁵S-labeled bands were quantitated using a Bioimage analyzer (BAS 2000, Fujix, Tokyo, Japan).

Effect of U0126 and Ly294002.
We used UO126 (Promega) and LY294002 (Calbiochem) as specific inhibitors of mitogen-activated protein kinase kinase (MEK) and PI3K, respectively. Cells were plated in DMEM supplemented with 10% FCS and incubated overnight. Subconfluent cells were treated with 10 µM U0126 or 50 µM LY294002 dissolved in DMSO for 2 or 3 days before each assay was performed.

Analysis of Phosphorylated Mitogen-Activated Protein Kinase (MAPK), Akt, Inhibitor of Nuclear Factor-B (IB), and bcl-2.
Cells were treated with each inhibitor as described above. Cells were lysed with lysis buffer [20 mM Tris HCl (pH 8.0), 1% Triton X-100, 10% glycerol, 137 mM NaCl, 1.5 mM NgCl₂, 1 µg/ml leupeptin, and 10 µg/ml aprotinin]. After centrifugation at 13,000 x g for 10 min to remove cell debris, 100 µg of the protein were subjected to SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were incubated with MAPK monoclonal antibody (Ab-1; Calbiochem), Akt polyclonal antibody (Santa Cruz Biotechnology, Inc.), phospho-specific MAPK antibody (pTEpY; Promega), phospho-Akt antibody (T308; New England Biolabs, Inc), phospho-IB antibody (Ser³²; New England Biolabs, Inc.), phospho-bcl-2 antibody (Santa Cruz Biotechnology, Inc.), and bcl-2 antibody (Santa Cruz Biotechnology, Inc.), followed by incubation with horseradish peroxidase-linked antirabbit antibodies. They were then analyzed using an enhanced chemiluminescence system (Amersham). The amount of each protein was quantitated using NIH image software.

Detection of Apoptosis.
Cells were plated at 2 x 10⁵ cell/60-mm plate in DMEM supplemented with 10% FCS and incubated in serum-free medium for 2 or 3 days.

For the in situ apoptosis detection assay, cells were fixed in 4% formalin. Endogenous peroxidase activity was quenched with 2% H₂O₂ in PBS for 5 min. The assay was carried out according to the manufacturer’s instructions (ApoTag Plus; Oncor, Inc., Gaithersburg, MD). Quantification of apoptosis was performed by counting five random fields of view at a magnification of x400/section assayed.

For fluorescence-activated cell sorting analysis, attached cells were washed twice with ice-cold PBS and prepared in NP40 lysis buffer (3.4 mM sodium citrate, 10 mM NaCl, and 0.1% NP40) containing 0.5% propidium iodide. The DNA contents of the cells was measured using the propidium iodide staining method and a FACScan (Becton Dickinson). Each fraction of cells in the G₁, S, and G₂-M was analyzed using DNA content software.

Data represent the average and SD of three independent experiments. Statistical analyses of all values were performed using the Student’s t test. Significant differences were taken for P < 0.05.

	RESULTS

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ACD Is Induced by Constitutively Active K-Ras but Is Suppressed by Active H-Ras in Rat Endometrial Cells.
To define the functions of the two Ras isoforms, K-Ras4B and H-Ras, we established four cell lines, RK12V, RH61L, RH12V, and mock cells (RV), by transfecting parent RENT4 cells with expression vectors encoding[¹²Val]K-ras, [⁶¹Leu]H-ras, [²Val]H-ras, and an empty vector, respectively. Pooled populations of each cell line were used for the assays. Western blot analyses of cell lysates from RK12V, RH61L, and RH12V cells indicated that they expressed exogenous [¹²Val]K-Ras, [⁶¹Leu]H-Ras, and [¹²Val]H-Ras proteins, respectively (Fig. 1A) . RENT4 cells, established from rat endometrial cells with the SV40T antigen, are immortalized and do not generate visible tumors in nude mice (19) . RV cells showed an appearance similar to the parent cells and were not transformed. In contrast, RK12V and RH61L cells were heterogeneous in size and shape, and some cells grew in domes. They also showed an increased saturation density compared with the mock cells (Fig. 1B) . RK12V and RH61L cells showed transformed phenotypes and thus acquired the ability to form foci in soft agar (data not shown). We incubated these cells in the absence of serum for 3 days and evaluated the incidence of ACD using an in situ apoptosis detection assay. The incidence of ACD was 1.5-fold higher in RK12V cells than in RV cells (14% in RK12V cells versus 9% in RV cells). In contrast, the expression of [⁶¹Leu]H-Ras significantly suppressed the level of ACD (0.6-fold; 9% in RV versus 5% in RH61L cells; Fig. 2A ). To exclude the possibility that this difference was because of mutation rather than the isoforms, we also analyzed the level of ACD in RH12V cells. The expression of [¹²Val]H-Ras also suppressed the level of ACD (9% in RV versus 2% in RH12V cells; data not shown). The relative ratio of positively stained cells from the in situ apoptosis detection assay for each cell line correlated with that of the sub-G1 fraction obtained from fluorescence-activated cell sorting analyses (1.6-fold for RK12V cells and 0.5-fold for RH61L cells compared with that for mock cells; Fig. 2B ). The cell population in the sub-G1 fraction was greater than that of the positively stained cells in the in situ apoptosis detection assay. This may reflect a difference in sensitivity between the two assays.

View larger version (38K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Cell growth of RK12V, RH61L and mock cells (RV). A, RK12V, RH61L, and mock cells (RV) were established by transfecting parent RENT4 cells with expression vectors encoding [12Val]K-ras4B, [61Leu]H-ras, and [12Val]H-ras and with an empty vector, respectively. Pooled populations of each cell line were used for the assays. The proteins (1000 µg) obtained by the lysis of each cell line were analyzed for immunoprecipitation of Ras proteins using Y-13 259 rat anti-Ras monoclonal antibody. Exogenous [12Val]K-Ras or H-Ras proteins migrated more slowly, and exogenous [61Leu]H-Ras proteins migrated faster than endogenous Ras proteins on SDS-PAGE. The arrows indicate each type of Ras protein. B, cells were plated at 2.5 x 104 cells/2.0 mm2 in 24-well plates in DMEM supplemented with 10% FCS and incubated overnight. The next day, the medium was replaced with DMEM supplemented with 1% FCS, and the cells were incubated for 5 days. Viable cells were counted on days 1, 3, and 5. RK12V and RH61 cells exhibited a greater increase in saturation density than did mock cells. These results were reproduced three times.

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Induction of apoptotic cell death (ACD) by activated K-Ras and protection from ACD by activated H-Ras in rat endometrial cells. A, cells (RV, RK12V, RH61L, and RH12V) were incubated with serum-free medium for 3 days and fixed in 4% formalin. The extent of ACD was determined using an in situ apoptosis detection assay. Each bar shows the mean of three independent experiments. The effects of expression of activated K-Ras or activated H-Ras were statistically significant (P < 0.005). B, cells (RV, RK12V, and RH61L) were incubated in serum-free medium for 3 days and stained with 5 µg/ml propidium iodide. The population of cells in the sub-G1 fraction was obtained by FACScan analysis. These results were reproduced three times.

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. The Ras effector pathway contributes to the induction and suppression of apoptosis. A, pBabe-Raf-CAAX and p110K227E constructs were transiently introduced into parent RENT4 cells. Subconfluent cells were incubated in DMEM supplemented with 1% FCS for 3 days. The protein (100 µg) obtained from each cell lysate was analyzed by Western blotting. Phospho-specific mitogen-activated protein kinase (MAPK) and Akt antibodies were used to detect the phosphorylated form of each protein. The relative phosphorylation levels of each protein are shown. The same results were obtained for two independent experiments. B, pBABE-Raf-CAAX and p110K227E constructs were transiently introduced into parent RENT4 cells. Subconfluent cells were incubated with serum-free medium for 3 days and stained with 5 µg/ml propidium iodide. The population of cells in the sub-G1 fraction was obtained by FACScan analysis. The proportion of sub-G1 cells is shown. These results were reproduced three times.

K-Ras-Induced ACD through the MEK/MAPK Pathway and the PI3-K/Akt Pathway Contributed to H-Ras-Mediated Suppression of ACD.
Although the K- and H-Ras isoforms qualitatively activated the same effector pathways, the present results suggested that the former promoted apoptosis, whereas the latter rescued RENT4 cells from apoptosis. Thus, we investigated how ACD is modulated by the signal transmitted through the MEK/MAPK pathway or the PI3K/Akt pathway.

We used a specific MEK inhibitor, UO126 (10 µM), and a specific PI3K inhibitor, LY294002 (50 µM), to abrogate each pathway.

First, we examined the effects of each MEK inhibitor and PI3K inhibitor on MAPK and Akt phosphorylation and compared them with those of DMSO-treated cells (Fig. 4A) . The MEK inhibitor, UO126, completely suppressed the phosphorylation of MAPK. In turn, LY294002 sharply reduced the level of phosphorylated Akt.

View larger version (50K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Apoptosis of rat endometrial cells regulated by the mitogen-activated protein kinase kinase (MEK) or phosphoinositide 3-kinase (PI3K) inhibitor. A, subconfluent cells were incubated in DMEM supplemented with 1% FCS in the presence of 10 µM U0126 or 50 µM Ly294002 for 2 days. Cells were treated with the same amount of carrier (DMSO) as the control. The protein (100 µg) obtained from each cell lysate was analyzed by Western blotting as shown in Fig. 4 . Phospho-specific mitogen-activated protein kinase (MAPK), Akt, inhibitor of nuclear factor-B, and Bcl-2 antibodies were used to detect the phosphorylated form of each protein. B, subconfluent rat endometrial cells (RV, RK12V, RH61L, and RH12V cells) were incubated in serum-free medium for 3 days in the presence of 10 µM U0126 as a MEK inhibitor or 50 µM Ly294002 as a PI3K inhibitor. Cells were treated with the same amount of carrier (DMSO) as the control. The incidence of ACD was obtained by in situ apoptosis detection. Data represent the average and SD of three independent experiments.

Several effector proteins downstream of the Raf/MAPK or PI3K/Akt pathway may be involved in executing or terminating the apoptosis program through mutual interactions. The PI3K/Akt pathway controls part of the Ras-dependent protection against apoptosis via NF-B (21) . NF-B activation involves the signal-inducible degradation of IB- phosphorylated by a cascade of specific kinases. To determine whether NF-B was activated, we tested for the phosphorylation of IB- using a phospho-specific IB- antibody. Reduced phosphorylation of Akt by the PI3K inhibitor was consistently followed by a significantly reduction in the phosphorylation of IB- (12% in RV cells, 29% in RK12V cells, and 14% in RH61L cells), contributing to the promotion of ACD. However, although the MEK inhibitor reduced the level of phosphorylated IB- in RV, RK12V, and RH61L cells, it rescued RV and RK12V cells from ACD in contrast to the promotion of ACD in RH61L cells (Fig. 4B) . These results suggested that the phosphorylation status of IB- was not associated with the induction of apoptosis mediated by the Raf/MEK/MAPK pathway in RENT4 cells.

In addition to IB-, we also investigated Bcl-2, an antiapoptotic protein. Bcl-2 protein is phosphorylated by multiple kinases, including MAPK (22) , allowing for the possibility that PI3K/Akt signaling also phosphorylates Bcl-2. Although evidence is emerging that phosphorylation regulates the function of Bcl-2, whether Bcl-2 is activated or inactivated by phosphorylation remains unclear. For example, the apoptosis induced by Taxol is accompanied by the phosphorylation of Bcl-2 (23) . In contrast, cytokine or glucocorticoid-mediated phosphorylation of Bcl-2 prevents apoptosis (24 , 25) . Thus, we investigated the change in the phosphorylation status of Bcl-2 in response to the MEK- or PI3K-specific inhibitor. The PI3K inhibitor almost abrogated the Bcl-2 phosphorylation in RV, RK12V, and RH61L cells, indicating the presence of signaling, resulting in the phosphorylation of Bcl-2 via the PI3K/Akt pathway. In turn, the MEK inhibitor failed to reduce phosphorylation levels in RV and RK12V cells. However, it significantly suppressed the phosphorylation of Bcl-2 in RH61L cells. These results demonstrated that phosphorylation of Bcl-2 prevented Ras-induced apoptosis, and the change in the level of phosphorylated Bcl-2 correlated with the incidence of ACD in RK12V and RH61L cells in response to the MEK- or PI3K inhibitor.

The K-Ras Downstream Raf/MEK/MAPK Pathway Is Important for the Induction of Apoptosis.
H-ras effector domain mutants are widely used for determining which signaling pathways are sufficient or necessary for specific Ras activity (26, 27, 28, 29) . The Ras effector domain mutant H-Ras (12V, 35S) signals through Raf but is prevented from signaling via the RalGDS and PI3K pathways. The effector domain mutant H-Ras (12V, 40C) signals through PI3K but is prevented from signaling to Raf and RalGDS. We generated K-Ras effector domain mutants (K12V35S, K12V40C) by site directed mutagenesis. RENT4 cells were transfected with pZeo-Kras effector domain mutants (K12V, K12V35S, and K12V40C) or empty vector as a control. Each protein was then transiently expressed.

First, we examined the change in the level of phosphorylated MAPK and Akt in response to the effector domain mutants, K12V 35S and K12V40C, to address whether these mutants behaved in the same way as the analogous mutants of H-Ras (Fig. 5A) . As expected, the expression of activated K12V resulted in higher levels of phosphorylated MAPK and Akt (9.6- and 2.0-fold, respectively). Expression of K12V35S protein in RENT4 cells increased the phosphorylation of MAPK compared with the expression of K12V protein. However, this mutant also increased the phosphorylation of Akt (4.4-fold), unlike the analogous mutant of H-Ras. In turn, expression of K12V40C protein suppressed the phosphorylation of MAPK but increased that of Akt, compared with the expression of K12V protein. These results suggested that K12V35S and 40C function, although their activity is less notable than that of the analogous mutant of H-Ras.

View larger version (31K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Contribution of Ras effector mutants to apoptosis in rat endometrial cells. RENT4 cells were transfected with pZeo-Kras effector domain mutants (K12V, K12V35S, and K12V40C) or each empty vector as a control. Each protein was transiently expressed. A, the level of phosphorylated mitogen-activated protein kinase (MAPK) or Akt in cells expressing each effector domain mutant was examined by Western blotting as seen in Fig. 4 . The relative ratio of phospho-MAPK to phospho-Akt compared with that in mock cells was calculated. [12Val]K-Ras protein preferentially phosphorylated MAPK over Akt. The relative ratio of phospho-MAPK to phospho-Akt was higher in K12V35S cells than in K12V40C cells. B, subconfluent cells were incubated in serum-free medium for 3 days and stained with 5 µg/ml propidium iodide. The population of cells in the sub-G1 fraction was obtained by FACScan analysis. The proportion of sub-G1 cells is shown. In K12V and K12V35S cells, the cell population in the sub-G1 fraction was significantly higher than in mock cells. Similar results were obtained from three independent experiments.

	DISCUSSION

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We demonstrated here the promotion of cell growth in immortalized rat endometrial cells by constitutively activated K-Ras and H-Ras mutants. The MEK/MAPK and PI3K/Akt signaling pathways may be involved in promoting cell growth in response to Ras mutants. In the presence of activated MEK/MAPK and PI3K/Akt signaling, the K-Ras mutant promoted apoptosis, whereas the H-Ras mutant protected against apoptosis. These results suggest that K- and H-Ras isoforms serve distinct functions, although they interact with the same set of effector proteins; the implications are that K-Ras-mediated signaling is involved in the promotion of apoptosis and H-Ras-mediated signaling to protect against apoptosis. The two Ras isoforms share extensive sequence homology and are identical in their effector binding domains. However, the hypervariable region between residues 166 and 185 differs between them (29 , 30) . One role of the hypervariable region is to cooperate with the processed COOH-terminal CAAX motif and provide a second signal for Ras plasma membrane localization. The difference in the COOH-terminal hypervariable region may influence the recruitment of Ras effectors to the plasma membrane. In fact, a recent study showed marked quantitative differences in the ability of K-and H-Ras to activate their downstream effectors. Previous studies showed that K-Ras was a significantly more potent activator of Raf-1 than H-Ras (16 , 17) , and in turn, H-Ras was a more potent activator of PI3K than K-Ras (16) . This suggests that the greater dependence of K-Ras-mediated signals on the pathway comprising Raf/MEK/MAPK, and H-Ras-mediated signals on the pathway that includes PI3K/Akt can account for the different phenotypic cell consequences.

Therefore, we examined the involvement of MEK/MAPK in the induction of, apoptosis and PI3K/Akt in preventing it. The specific PI3K inhibitor, LY294002, which inhibited phosphorylation of Akt, markedly promoted apoptosis in all types of cells that we examined. This proapoptotic effect accompanied a reduction in the phosphorylation of IB-. Although we only found that the putative pathway via Ras/PI3K/Akt modulated antiapoptotic signaling, a role for PI3K/Akt in cell survival has been suggested in cell type, including cerebellar neurons (31) , Rat1 cells, and COS7 cells (32) . Thus, the pathway comprising Ras/PI3K/Akt may be of general significance in preventing of apoptosis.

In turn, the specific MEK inhibitor had the potential to reduce cell death in cells harboring activated K-Ras (Fig. 4) . Furthermore, ectopic expression of the K-Ras effector domain mutant K12V35S promoted ACD (Fig. 5) . We found that the constitutively activated K-Ras mutant renders endometrial cells susceptible to apoptosis (Fig. 2) , suggesting the dependence of K-Ras-mediated signaling in promoting ACD on the pathway comprising Raf/MEK/MAPK. However, the apoptosis-promoting effect of the putative K-Ras/Raf/MEK/MAPK pathway is at odds with the significant increase in the basal level of cell death caused by the specific MEK inhibitor in rat endometrial cells expressing the activated [⁶¹Leu]H-Ras mutant. It seems that several effector proteins downstream of the Raf/MAPK or PI3K/Akt pathway are involved in the execution or termination of apoptosis. These effector proteins may functionally interact and determine cell fate. Bcl-2 protein is a candidate for one of these proteins because it is phosphorylated by multiple kinases, including MAPK (22) . Thus, we examined changes in the level of phosphorylated Bcl-2 in response to the specific MEK or PI3K inhibitor in RK12V and RH61L cells. The PI3K inhibitor suppressed phosphorylation of Bcl-2 in RK12V and RH61L cells, indicating that the PI3K/Akt signaling results in the phosphorylation of Bcl-2 protein in RENT4 cells. Additionally, we showed that the MEK inhibitor reduced the level of phosphorylated Bcl-2 protein in RH61L cells but not in RK12V cells. These changes are compatible with the incidence of ACD in RK12V and RH61L cells in response to the MEK- or PI3K inhibitors. The results indicates that unknown signaling downstream of each Ras isoform dilutes the effect of the MEK inhibitor in the presence of activated H-Ras mutant. However, complete understanding of the regulation of the apoptotic process downstream of each Ras isoform requires additional investigation. The effect of H-Ras-mediated signaling on cell survival lies in its ability to block the proapoptotic action of K-Ras-mediated signaling, which is absolutely required for the promotion of cell growth and contributes to the development of endometrial cancer.

	ACKNOWLEDGMENTS

 
We thank Dr. Channing Der (University of North Carolina) for generously donating pZIP neo SV(X) 1-K-Ras4B(12V), -H-Ras(61L), pBABE-Raf-CAAX, p-110K227E, and H-Ras(12V). We also thank Sawako Adachi and Yuko Hachisu for technical assistance and Aiko Kerakawauchi for assistance in preparing of the manuscript.

	FOOTNOTES

 
Grant support: Grants-in-Aid 12557138 and 11671629 from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Y. Ninomiya and K. Kato contributed equally to this work.

Requests for reprints: Kiyoko Kato, Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumihara, Beppu, Oita 874-0838, Japan. Phone: 81-977-27-1660; Fax: 81-977-27-1661; E-mail: kkatoh{at}tsurumi.beppu.kyushu-u.ac.jp

Received 12/10/02. Revised 2/ 6/04. Accepted 2/12/04.

	REFERENCES

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