Protein Kinase Cδ Is Required for Survival of Cells Expressing Activated p21RAS*

Inhibition of protein kinase C (PKC) activity in transformed cells and tumor cells containing activated p21RAS results in apoptosis. To investigate the pro-apoptotic pathway induced by the p21RAS oncoprotein, we first identified the specific PKC isozyme necessary to prevent apoptosis in the presence of activated p21RAS. Dominant-negative mutants of PKC, short interfering RNA vectors, and PKC isozyme-specific chemical inhibitors directed against the PKCδ isozyme demonstrated that PKCδ plays a critical role in p21RAS-mediated apoptosis. An activating p21RAS mutation, or activation of the phosphatidylinositol 3-kinase (PI3K) Ras effector pathway, increased the levels of PKCδ protein and activity in cells, whereas inhibition of p21RAS activity decreased the expression of the PKCδ protein. Activation of the Akt survival pathway by oncogenic Ras required PKCδ activity. Akt activity was dramatically decreased after PKCδ suppression in cells containing activated p21RAS. Conversely, constitutively activated Akt rescued cells from apoptosis induced by PKCδ inhibition. Collectively, these findings demonstrate that p21RAS, through its downstream effector PI3K, induces PKCδ expression and that this increase in PKCδ activity, acting through Akt, is required for cell survival. The p21RAS effector molecule responsible for the initiation of the apoptotic signal after suppression of PKCδ activity was also determined to be PI3K. PI3K (p110CAAX, where AA is aliphatic amino acid) was sufficient for induction of apoptosis after PKCδ inhibition. Thus, the same p21RAS effector, PI3K, is responsible for delivering both a pro-apoptotic signal and a survival signal, the latter being mediated by PKCδ and Akt. Selective suppression of PKCδ activity and consequent induction of apoptosis is a potential strategy for targeting of tumor cells containing an activated p21RAS.


Inhibition of protein kinase C (PKC) activity in transformed cells and tumor cells containing activated p21
RAS results in apoptosis. To investigate the pro-apoptotic pathway induced by the p21 RAS oncoprotein, we first identified the specific PKC isozyme necessary to prevent apoptosis in the presence of activated p21 RAS . Dominant-negative mutants of PKC, short interfering RNA vectors, and PKC isozyme-specific chemical inhibitors directed against the PKC␦ isozyme demonstrated that PKC␦ plays a critical role in p21 RAS -mediated apoptosis. An activating p21 RAS mutation, or activation of the phosphatidylinositol 3-kinase (PI3K) Ras effector pathway, increased the levels of PKC␦ protein and activity in cells, whereas inhibition of p21 RAS activity decreased the expression of the PKC␦ protein. Activation of the Akt survival pathway by oncogenic Ras required PKC␦ activity. Akt activity was dramatically decreased after PKC␦ suppression in cells containing activated p21 RAS . Conversely, constitutively activated Akt rescued cells from apoptosis induced by PKC␦ inhibition. Collectively, these findings demonstrate that p21 RAS , through its downstream effector PI3K, induces PKC␦ expression and that this increase in PKC␦ activity, acting through Akt, is required for cell survival. The p21 RAS effector molecule responsible for the initiation of the apoptotic signal after suppression of PKC␦ activity was also determined to be PI3K. PI3K (p110 CAAX , where AA is aliphatic amino acid) was sufficient for induction of apoptosis after PKC␦ inhibition. Thus, the same p21 RAS effector, PI3K, is responsible for delivering both a pro-apoptotic signal and a survival signal, the latter being mediated by PKC␦ and Akt. Selective suppression of PKC␦ activity and consequent induction of apoptosis is a potential strategy for targeting of tumor cells containing an activated p21 RAS .
The ras oncogene family is among the most commonly mutated group of genes in human cancer. Its protein products code for three closely related p21 RAS proteins, including H-Ras, K-Ras, and N-Ras. p21 RAS proteins are localized in the inner plasma membrane, bind GDP and GTP, and possess an intrinsic GTPase activity. p21 RAS proteins function as plasma mem-brane-bound guanine nucleotide-binding proteins and act as molecular switches, thereby regulating signal transduction pathways for hormones, growth factors, and cytokine receptors (1). Several downstream effector proteins of p21 RAS have been identified that bind preferentially to p21 RAS in the GTP-bound state, including Raf, phosphatidylinositol 3-kinase (PI3K), 2 and a family of GDP-GTP exchange factors for the Ral small GTPases (Ral-GDS). Raf proteins, which are proto-oncogeneencoded serine/threonine kinases, activate the MEK-ERK signaling pathway. PI3K activation results in the activation of the anti-apoptotic serine/threonine kinase Akt, among other molecules. Other p21 RAS targets include the GTPase-activating proteins, p120 GAP and neurofibromin (2). p21 RAS proteins were shown to influence proliferation, differentiation, transformation, and apoptosis by relaying mitogenic and growth signals from the membrane into the cytoplasm and the nucleus.
Specific point mutations localized in codons 12,13,59,61,63,116,117, and 146 can lock the p21 RAS protein in the active GTP-bound state and permit stimulation of downstream signaling cascades in the absence of extrinsic p21 RAS activation. Ras mutations can be found in human malignancies with an overall frequency of 20%. A particularly high incidence of ras gene mutations has been reported in malignant tumors of the pancreas (80 -90%, K-ras), in colorectal carcinomas (30 -60%, K-ras), in non-melanoma skin cancer (30 -50%, H-ras), and in hematopoietic neoplasia of myeloid origin (18 -30%, K-and N-ras) (3).
In addition to its central involvement in cell proliferation, recent studies indicate that the presence of an activated p21 RAS protein sensitizes transformed or malignant cells to apoptotic stimuli (4 -9). Various signaling pathways have been proposed for this pro-apoptotic activity. Chou et al. (10) reported activated p21 RAS can cause apoptosis in transformed murine fibroblast cells through activation of the transcription factor NFB. Another study suggested that the p21 RAS /MAPK pathway is involved in Ras-specific apoptosis (11). The latter study also found that activating p21 RAS mutations increased colon cancer cell sensitivity to 5-fluorouracil-induced apoptosis through the negative regulation of gelsolin expression (12). Our previous studies demonstrated that suppression of protein kinase C (PKC) activity in cells expressing activated p21 RAS rapidly * This work was supported by the Philip Morris External Research Program, NCI Grants CA108100 and CA112102 from the National Institutes of Health, a Littlefield-AACR Grant in Metastatic Colon Cancer Research, and the Karin Grunebaum Cancer Research Foundation (to D. V. F.). 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. 1  induces apoptosis via FADD/caspase-8 signaling (9). We also found that reactive oxygen species are necessary as downstream effectors of the Ras-mediated apoptotic response to PKC inhibition (7). There are at least 12 PKC isoforms that are classified into three subfamilies according to the structure of the N-terminal regulatory domain, which determines their sensitivity to the second messengers Ca 2ϩ and diacylglycerol (13). Despite the high degree of homology, however, there is a surprising degree of nonredundancy. Thus, individual PKC isoforms mediate different and unique cellular functions in different cell types and different tissues (14). PKC␦ belongs to the subfamily of novel isoforms (PKC␦, PKC⑀, PKC, and PKC), which are insensitive to Ca 2ϩ . PKC␦ is widely regarded as having pro-apoptotic properties (15)(16)(17). Caspase activation mediates cleavage of PKC␦, which results in release of the active catalytic domain (18,19). In addition, PKC␦ activity is known to initiate a number of pro-apoptotic signals, such as increased expression and stability of p53 (20,21), mitochondrial cytochrome c release (22,23), and c-Ab1 activation (24). But recent studies have also shown that PKC␦ can protect cells against apoptotic stimuli under certain conditions (25). PKC␦ has been reported to regulate B-lymphocyte survival (26). Knock-out experiments have shown that PKC␦-deficient mice have a severely deregulated immune system and develop autoimmune disease (27,28). Thus, PKC␦ activation can serve as a pro-apoptotic signal, or as a survival signal, to determine cell fate.
This study examines the mechanism of apoptotic signaling induced by the p21 RAS oncoprotein. We found that PKC␦ plays a critical role in suppressing p21 RAS -mediated apoptosis, and selective inhibition of this isozyme initiates apoptosis in cells containing activated p21 RAS . Our data further demonstrate that unregulated Ras activity, through activation of the downstream effector PI3K, up-regulates PKC␦ expression and subsequently activates Akt, generating an anti-apoptotic effect and protecting against Ras-mediated apoptosis.
The chemical inhibitors used in this study specific to PKC isozymes, PI3K, p21 RAS , and MAPK are listed in Table 1. All inhibitors were dissolved in dimethyl sulfoxide for use, and their effects were measured relative to dimethyl sulfoxide (vehicle)-treated controls. The concentration of all inhibitors was optimized to produce greater than 90% inhibition of target molecule activity.
Cell Proliferation Assay-Cell proliferation was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Roche Applied Science). The number of viable cells growing in a single well on a 96-well microtiter plate was estimated by adding 10 l of MTT solution (5 mg/ml in phosphate-buffered saline (PBS)). After 4 h of incubation at 37°C, the stain was diluted with 100 l of dimethyl sulfoxide. The optical densities were quantified at a test wavelength of 550 nm and a reference wavelength of 630 nm on a multiwell spectrophotometer.
Assay of PKC␣ and PKC␦ Kinase Activities-PKC␣ and PKC␦ activities were measured with an assay kit (Upstate Cell Signaling). After 2 days of treatment with inhibitors, cells were lysed in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 20 mM MgCl 2 , 5 mM EGTA, 1 mM orthovanadate, 50 g/ml phenylmethylsulfonyl fluoride, and 3 g/ml aprotinin. PKC␣ and PKC␦ were immunoprecipitated from 200 g of protein extracts as described above. Immunocomplexes were washed three times with the kinase buffer (20 mM Tris-HCl, 10 mM MgCl 2 , pH 7.5) and then incubated with a PKC-specific peptide substrate, [␥-32 P]ATP, and inhibitors of cAMPdependent kinase and calmodulin kinase for 10 min at 30°C. 32 P incorporated into the substrate was separated from residual 32 P using p81 filters and subsequently quantified by scintillation counting. ␤-Actin antibody was used as negative control in immunoprecipitations.
p21 Ras Activity Assays-Cells were cultured in DMEM containing 0.5% serum for 48 h. Then cells were lysed in a buffer containing 25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 0.25% sodium deoxycholate, 10% glycerol, and 25 mM NaF. Protein was normalized to 1 g/l, and activated Ras was affinity-precipitated by mixing 1 mg of cell lysate with 10 g of Raf-RBD-agarose bead conjugate (Upstate Biotechnology, Inc.) for 60 min at 4°C. The conjugates were washed three times in lysate buffer and then separated on a 10% SDS-polyacrylamide gel. Proteins were transferred onto a polyvinylidene difluoride membrane and immunoblotted with a monoclonal p21 RAS antibody (BD Transduction Laboratories).
DNA Profile Analysis-1 ϫ 10 5 cells were plated in a 60-mm dish and grown until confluent. Cells were harvested and resuspended with 1 ml of a 35% ethanol/DMEM solution for 5 min at room temperature. Cells were collected and stained with solution containing 50 g of propidium iodide/ml and 25 units of RNase/ml in PBS and incubated in the dark for 30 min at room temperature for flow cytometric analysis.
Cell Apoptosis Assay-Terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay was used for apoptosis assay. Briefly, cells were fixed with 4% paraformaldehyde in PBS overnight at 4°C. The samples were washed three times in PBS and permeabilized with 0.2% Triton X-100 in PBS for 15 min on ice. After rinsing twice with PBS, the samples were incubated with the TMR red TUNEL reagent (Roche Applied Science) at 37°C in the dark, according to the manufacturer's instructions. Apoptotic cells were identified by fluorescent microscopy.
Statistical Analyses-Results are expressed as mean Ϯ S.D. Statistical analysis was performed using Student's t test, and p values Ͻ0.05 were considered significant.

Selective Down-regulation of PKC␦ Induces Apoptosis in Cells
with Activated p21 Ras -To begin determination of whether a specific PKC isozyme is responsible for suppression of Rasmediated apoptosis, six isozyme-specific or nonspecific PKC inhibitors were used to suppress PKC␣, PKC␣/␤, PKC␤1/2, PKC␦, or general PKC activity (Table 1). Optimal maximal effective concentrations of each inhibitor (Ͼ90% inhibition of relevant enzyme activity) were determined in pilot experiments ( Fig. 1A or data not shown). The elevated activity of p21 RAS in KBalb, MIA PaCa-2, and NIH/3T3-Ras cell lines compared with their counterpart cell lines (Balb, BxPc-3, and NIH/3T3, respectively) was confirmed by an activated p21 RAS pulldown assay (Fig. 1B). Cells were treated for 48 h, and cell proliferation was quantitated by an MTT assay. Among the isozyme-specific inhibitors, rottlerin dramatically and specifically decreased the proliferation of both MIA PaCa-2 and NIH/3T3-Ras cells (both of which express a mutant, activated p21 RAS protein), compared with the corresponding Hs766T cells and NIH/3T3 cells (which contain wild-type p21 RAS ) ( Fig. 1,C and D). The pan-PKC inhibitor bisindolylmaleimide I and the diacylglycerol antagonist 1-O-hexadecyl-2-O-methyl-rac-glycerol also strongly suppressed the growth of the cells containing activated p21 RAS , consistent with our previous studies, while having no significant effect on most of the cell lines that contained a wildtype p21 RAS (7,8). In contrast, rottlerin modestly but consistently stimulated proliferation of those cell lines expressing wild-type p21 RAS (Balb and Hs766T cells). Cytofluorometric analysis of propidium iodide-stained nuclei showed that rottlerin caused 24 -35% apoptosis (manifested as hypodiploid nuclei) in activated Ras-containing cell lines after 60 h of treatment, whereas the matched cells containing wild-type p21 RAS displayed only 12-14% apoptosis (Fig. 1E). PKC␦ expression and activity were analyzed in the rottlerin-treated cells to confirm suppression of activity.
To confirm inhibition of PKC␦ activity by rottlerin, both in vivo and in vitro kinase assays were performed. For the in vivo assays, cells were treated with rottlerin at 20 M for 48 h and then lysed, and PKC␦ was immunoprecipitated by a specific anti-PKC␦ antibody. The incorporation of [␥-32 P]ATP into a PKC substrate peptide (QKRPSQRSKYL) by the immunoprecipitates was quantitated. Exposure to rottlerin, at the concentrations used in the apoptosis studies described above, blocked greater than 85% of the PKC␦ activity in all of the cell lines tested, except Balb ( Fig. 2A). In contrast, rottlerin produced only a slight, statistically insignificant decrease in PKC␣ activity (Fig. 2B). For the in vitro kinase assay, rottlerin was added to immunopurified PKC␦ protein and incubated for 4 h, and PKC␦ kinase activities were assayed using the artificial substrate. Rottlerin directly inhibited 55-90% of PKC␦ activity (Fig. 2C).
Cell lines expressing activated p21 RAS consistently demonstrated elevated levels of PKC␦ activity relative to the corresponding lines containing wild-type p21 RAS (Fig. 2, A and B).
Similarly, total levels of PKC␦ protein were elevated in the cell lines containing activated p21 RAS relative to the corresponding lines containing wild-type p21 RAS (Fig. 2D). The data obtained with the chemical inhibitors of PKC isozymes are consistent with PKC␦ being the relevant PKC target for Ras-mediated apoptosis.
Unexpectedly, exposure to rottlerin for 48 h suppressed PKC␦ protein levels in all cell lines tested (Fig. 2D), whereas the levels of other PKC isozymes, including PKC␣, -␤, -, and -, were not changed by rottlerin (Fig. 2E). The magnitude of suppression of PKC␦ levels by rottlerin in the cells containing activated p21 RAS (3-5-fold) approached the magnitude of the suppression of PKC␦ activity in these cells by rottlerin (6 -10-fold). Thus, although we can demonstrate direct inhibition of PKC␦ activity by rottlerin in in vitro assays, the marked suppression in PKC␦ activity observed after 48 h in vivo may be because of suppression of isozyme protein levels as well as direct inhibition of enzyme activity.
As the specificity of chemical kinase inhibitors is never absolute, we employed two specific genetic techniques to suppress PKC␦ activity. At 48 h after transfection of each of two PKC␦specific hairpin vectors targeted at different PKC␦ sequences into matched pairs of cell lines, BxPc-3/MIA PaCa-2 and NIH/ 3T3/NIH/3T3-Ras, immunoblot analysis demonstrated that expression of PKC␦ protein was significantly diminished by transfection with the PKC␦-siRNA-2 vector. In contrast, the PKC␦-siRNA-1 vector did not produce significant knockdown of PKC␦ protein (Fig. 3A). Because the efficiency of transient transfection in these cells was less than 50%, these analyses likely underestimate the activity of the siRNAs in an individual cell. Control experiments showed no changes of PKC␦ by vehicle alone or scrambled siRNA. PKC␦ siRNA also had no effect on PKC␣ expression. Seventy two hours after transfection with pRNA-U6.1-GFP-control siRNA (scrambled hairpin sequence) or pRNA-U6.1-GFP-PKC␦-siRNA-2, all cell lines were harvested to determine the apoptotic fraction by TUNEL assay.  Table 1. The corresponding solvents at equivalent volumes were used as vehicle controls. After 48 h of treatment, cell growth was evaluated by MTT assay or fluorescence-activated cell sorter. Results are presented as the means Ϯ S.D. Statistical significance was determined using a paired Student's t test, and p values Ͻ0.05 were considered significant (*, p Ͻ 0.01). A, p21 RAS -specific suppression of growth by PKC␦ inhibition. Human pancreatic carcinoma cell cultures Hs766T (wild-type p21 RAS ) and MIA PaCa-2 (activated p21 RAS ) were treated with different doses of rottlerin, and cell growth was assayed by MTT assay. B, assay of cellular p21 RAS activity. Nuclear free lysates containing a total of 400 g of protein from each indicated cell type were used for analysis of Ras activity by Raf-RBD pulldown. Equal loading was demonstrated by re-probing the blot with anti-␤-actin antibody. Pan-Ras protein expression levels were also analyzed. Lanes 1-6 represent Balb, KBalb, NIH/3T3, NIH/3T3-Ras, BxPc-3, and MIA PaCa-2 cells, respectively. C, growth of NIH/3T3 and NIH/3T3-Ras cells treated with isozyme-specific and nonspecific PKC inhibitors. D, growth of Hs766T and MIA PaCa-2 cells treated with isozyme-specific and nonspecific PKC inhibitors. E, nuclear DNA content of cells after inhibition of PKC␦. After 60 h of treatment with rottlerin, cells were fixed and stained with propidium iodide, and the apoptotic (hypodiploid) fractions (M1 fractions) were evaluated by flow cytometry. Counts refers to cell numbers; FL2-H is a log scale of fluorescence channels. Similar results were observed in three independent experiments. Bis-1, bisindolylmaleimide I; HMG, 1-O-hexadecyl-2-O-methyl-rac-glycerol.
Cells that took up the vector DNA were identified by green fluorescence. TUNEL-positive cells stained red. Superimposition displayed transfected, apoptotic cells as yellow (Fig. 3, C  and D). For cells transfected with the PKC␦ hairpin vector, 40 -50% cells were undergoing apoptosis at the 48-h time point, whereas cells transfected with the control scrambled hairpin vector displayed a frequency of apoptosis of less than 10% (Fig. 3H).
Similar results were obtained when a dominant-negative, kinase-dead PKC␦ mutant protein was used as an alternative method of blocking specifically PKC␦ activity in cells expressing an activated p21 RAS or wild-type p21 RAS (Fig. 3, E and F). Transfection of NIH/3T3-Ras cells with the dominant-negative PKC␦ vector produced a 30 -40% fraction of cells with a hypodiploid (apoptotic) DNA content, compared with a less than 5-10% apoptotic fraction in cells expressing a wild-type p21 RAS (Fig. 3H). Transfection of the empty vector as a control generated no significant apoptosis above background levels. The induction of apoptosis by the competitive expression of dominant-negative PKC␦ with a single-base mutation, rendering it catalytically inactive, also demonstrates that it is the kinase activity of PKC␦ that is required for the survival of cells expressing activated p21 RAS , rather than a noncatalytic function of the molecule. We studied the effects of knockdown of PKC␣ (via expression of a PKC␣-siRNA) as a specificity control. Forty eight hours after transfection of PKD-PKC␣V6, the levels of PKC␣ protein were significantly decreased in both NIH/3T3 and NIH/3T3-Ras cells (Fig. 3B). Analysis of apoptosis demonstrated that PKC␣ inhibition by PKC␣-siRNA induced apoptosis in ϳ20% of both NIH/3T3 and NIH/3T3-Ras cells, with no selective toxicity for cells containing an activated p21 RAS (Fig. 3,  G and H). This finding was consistent with results of the MTT  (20 M) or with the same volume of vehicle after they reached 80% confluence. After 48 h, cells were harvested, lysed in 50 l lysis buffer, and the lysates separated and subjected to immunoblot analysis using anti-PKC isoform-specific antibodies. Lanes 1-4 represent Balb, KBalb, NIH/3T3, and NIH/3T3-Ras cells, respectively. The immunoblots shown in the left and right panels were separated on the same gel and immunoblotted at the same time. Because the samples in the left panel came from untreated cells, whereas the samples in the right panel came from rottlerin-treated cells, and because rottlerin-treatment inhibited cell growth/proliferation, fewer numbers of cells were obtained in these groups, and therefore less protein/lysate was available for assay from the treated group compared with the vehicle control group. The mean activities Ϯ S.D. were obtained from at least three independent experiments, and assays from immunoprecipitations using an anti-␤-actin antibody were used to determine the background activity, which was subtracted. Student's t test was used for statistical analysis. (*, p Ͻ 0.01; ࡗ, p Ͻ 0.001.) assay (Fig. 1, C and D). Collectively, the data demonstrate that the PKC␦ isozyme plays a critical survival role in p21 RAS -induced apoptosis.
PKC␦ Inhibition Induces Mitochondrial Apoptotic Pathways in Cells Expressing an Activated p21 Ras -To further characterize the molecular mechanisms of p21 RAS -mediated apoptotic pathways, we investigated the influence of mitochondrial apoptotic signaling when PKC␦ activity is suppressed by assay of caspase-3 and caspase-9 activation. Immunoblot analysis demonstrated that exposure to rottlerin activated both procaspase-3 and procaspase-9 exclusively in the cells expressing activated p21 RAS (Fig. 4, A and B). For caspase-3, the full-length protein (35 kDa) and the large cleavage fragment (17 kDa) were detected (Fig. 4A); three activation fragments from caspase-9 zymogen (35, 17, and 10 kDa) were detected (Fig. 4B).
The PI3K Ras Effector Pathway Is Sufficient to Sensitize Cells to Apoptosis by PKC␦ Inhibition-Although previous studies have demonstrated that either constitutive expression of activated p21 RAS or acute increases in endogenous p21 RAS activity stimulate apoptosis following inhibition of PKC activity in multiple types and lineages of cells, the roles of specific p21 RAS downstream effectors in the process have never been determined. In general, three major effector pathways activated by Ras have been defined as follows: Raf1/MAPK, Ral-GDS, and PI3K. To begin to address this question, p21 RAS effector loop mutants, consisting of the activating Ras mutation (V12) and a second mutation (Ser-35, Gly-37, or Cys-40) were employed. The three RasV12 mutants (Ser-35, Gly-37, or Cys-40) differ in their ability to bind to p21 RAS effectors (Raf, Ral-GEFs, and the p110 subunit of PI3K, respectively) (29). Cells were treated with 20 M rottlerin for 60 h and subjected to flow cytometric analysis. All three p21 RAS downstream effector-loop mutants stimulated apoptosis to some extent after inhibition of PKC␦, although expression of the C40 mutant consistently generated the greatest amount of apoptosis (data not shown).
The Ras effector loop mutants are not completely specific in their activation of a single Ras effector pathway, and each activates all three pathways to some degree. To more clearly identify the downstream effector of p21 RAS relevant to Ras-mediated apoptosis, we activated single effector pathways using expression vectors for activated PI3K (p110 CAAX ), Raf (Raf-22W), and Ral-GEF (RIF-CAAX) into NIH/3T3 cells. The dominant-negative RalA-22N vector was used as control. Constitutive activation of the PI3K pathway (transfection of p110 CAAX ) was capable of inducing apoptosis after PKC␦ inhibition (apoptotic frequency 38.22%). In contrast, the other Ras effectors Raf and Ral-GEF induced little apoptosis in response to PKC␦ suppression (Fig. 5A).
To independently confirm that the downstream p21 RAS effector PI3K plays a critical role in Ras-mediated apoptosis, a PI3K-specific inhibitor (LY294002) and a MAPK inhibitor (PD98095) were employed. In dose-ranging studies, we evaluated concentrations of LY294002 and PD98095 from 1.0 to 60 M, assaying for efficiency of target enzyme inhibition and for toxicity. Even at 60 M concentrations of LY294002 or PD98095, only modest and nonstatistically significant levels of apoptosis were observed. At 10 M concentrations, there were no signs of toxicity or apoptosis (data not shown). Their effectiveness in suppressing the relevant target pathways at 10 M concentrations was confirmed by immunoblot (Fig. 5, B and C). An NIH/3T3 cell line stably expressing p110 CAAX was pretreated with LY294002 (10 M) for 30 min, and then rottlerin  (20 M) was added for 60 h. In the presence of the PI3K inhibitor, the apoptosis induced by rottlerin decreased nearly 50% (Fig. 5D). Similarly, in KBalb cells, LY294002 blocked 50% of the apoptosis induced by PKC␦ inhibition (Fig. 5E). In cells that contained an activated p21 RAS , but not those with activation of the single PI3K effector pathway, the MAPK inhibitor PD98095 also suppressed p21 RAS -dependent apoptosis to some extent, but to a substantially lesser degree than did PI3K inhibition. Collectively, these data demonstrate that the pro-apoptotic signal manifested during Ras-mediated apoptosis following PKC␦ inhibition is mediated mainly through the downstream effector PI3K, although the MAPK effector pathway may contribute to some extent in this process.
The Anti-apoptotic Akt Ras Effector Pathway Is Regulated by PKC␦-One direct downstream target of the PI3K pathway are the members of the Akt kinase family, which have well characterized pro-survival and anti-apoptotic activities, mediated through regulation of the mitochondrial apoptotic machinery and the expression of genes involved in this process. To deter-mine the role of Akt in p21 RAS -mediated apoptosis, we first examined Akt levels and activity in three paired cell lines with wild-type or mutant Ras alleles. These cells were treated with rottlerin for 48 h or left untreated. Akt activity, as assessed by quantitation of serine 473-phosphorylated Akt, was down-regulated ϳ50% by rottlerin in all three cell lines expressing activated Ras (Fig. 6A). In contrast, no significant changes in levels of p-Akt were induced in the cell lines expressing wild-type Ras. Exposure to rottlerin also suppressed total Akt levels to a modest extent in some cell lines, but this effect was independent of the presence of activated p21 RAS . If the suppression of Akt activation by PKC␦ suppression in activated Ras-containing cells was responsible for the resulting apoptosis, enforced expression of activated Akt should be able to effect rescue from apoptosis. MIA Paca-2 and NIH/3T3-Ras were co-transfected with a PKC␦ hairpin vector (which efficiently suppressed PKC␦ levels in these experiments by at least 85%, see Fig. 3A for an example) and a constitutively activated Akt (vAkt) expression vector, or a cAkt expression vector as a control. Akt activity in these cells was confirmed by immunoblotting for phosphorylated (activated) Akt (Fig. 6B). In both cell lines containing activated Ras, expression of vAkt reversed ϳ70 -80% of the apoptosis induced by knockdown of PKC␦. Expression of a nonactivated, wild-type Akt, however, did not similarly rescue the cells (Fig. 6,  C-E). To further support a role for Akt in Ras-mediated apoptosis induced by PKC␦ inhibition, Akt activity was analyzed after PKC␦ knockdown. Balb and KBalb cells were treated with a PKC␦ siRNA or a scrambled siRNA with a fluorescein tag (serving as control and to quantitate transfection efficiency). After 48 h, cells were harvested, and p-Akt expression was analyzed. p-Akt expression was dramatically decreased when PKC␦ expression was knocked down by PKC␦ siRNA in KBalb cells (Fig. 6F). For reasons not yet elucidated, we found PKC␦-siRNA was consistently not as effective in down-regulating PKC␦ in Balb cells as it was in Balb cells containing a mutated, activated p21 RAS (KBalb cells) (e.g. 62% suppression in Balb compared with 83% suppression in KBalb). Accordingly, the effects of PKC␦ down-regulation on p-AKT levels cannot be directly compared between Balb and K-Balb cells in these studies. Collectively, these studies indicate that activation of Akt is required for survival of cells expressing activated Ras, that PKC␦ is required for the activation of Akt by Ras, and that induction of Ras-mediated apoptosis by suppression of PKC␦ is effected through interference with this anti-apoptotic Ras effector pathway.
A pro-apoptotic p21 RAS effector, NORE1 (33), which binds to Ras-GTP and is a member of the RASSF family of tumor suppressors, has been described by Eckfeld et al. (34).
To determine whether activation of this Ras effector pathway plays any role in Ras-mediated apoptosis induced by suppression of PKC␦, we employed vectors expressing the fragment of NORE1, which binds to its apoptotic effector MST1 (amino acids 358 -413), or the C-terminal noncatalytic segment of MST1 (amino acids 307-487) as glutathione S-transferase fusion peptides, each of which has been demonstrated to block formation of the NOR-MST1 apoptotic complex (33). Neither peptide attenuated the apoptosis induced by suppression of PKC␦ (data not shown).
p21 Ras Up-regulates PKC␦ Protein Levels Post-transcriptionally-We observed that cell lines expressing an activated p21 RAS invariably expressed substantially more PKC␦ protein than did their wild-type p21 RAS -expressing counterparts (Fig. 7A), suggesting that p21 RAS activity may up-regulate PKC␦ protein expression as well as activity (Fig. 2, D and E). To test this hypothesis, NIH/3T3 cells were transfected with pSG5-H-(V12)Ras, and PKC␦ protein levels were assayed over time. PKC␦ protein levels increased rapidly after transfection, reaching peak levels at 18 h (Fig. 7B). In contrast, transfection with the empty pSG5 vector had no effect on PKC␦ protein levels. Conversely, when p21 RAS activity, or PI3K activity, in KBalb and NIH/3T3-Ras cells was inhibited by the Ras inhibitor (E,E)-[2-oxo-2-[[(3,7,11-trimethyl-2,6,10-dodecatrienyl)oxy]amino]ethyl] phosphonic acid, (2,2-dimethyl-1-oxopropoxy)methyl ester, Na or the PI3K inhibitor LY294002, respectively, PKC␦ protein  ). B, pAkt protein levels in MIA PaCa-2 and NIH/3T3-Ras cells, assayed by immunoblotting with an antibody specific for phosphorylated serine 473 Akt, or for total Akt, or for ␤-actin. Both cell lines were transfected with pEGFP and with pEF1␣ empty vector, or pEF1␣-cAKT, or pEF1␣-vAkt. GFP expression was used to normalize for transfection efficiency. C and D, activated Akt rescues cells from apoptosis induced by PKC␦ knockdown. MIA PaCa-2 and NIH/3T3-Ras cells were cultured on 4-well chamber slides and were co-transfected with pRNA-U6.1-GFP-PKC␦ hairpin vector and with pEF1␣-vAkt or pEF1␣-cAkt vectors. The PKC␦-hairpin vector efficiently suppressed PKC␦ levels in these experiments by at least 85% (e.g. see Fig. 3A). After 72 h, cells were fixed with 4% paraformaldehyde for 1 h at room temperature and then subjected to TUNEL assay. Shown are ϫ200 magnifications. E, quantitation of apoptotic cells in the transfected populations (*, p Ͻ 0.001). F, immunoblot of lysates from Balb or KBalb cells treated with PKC␦-specific siRNA or scrambled, control siRNA. Antibodies against PKC␦, total Akt, phosphoserine 473 Akt, and ␤-actin were used. The results are representative of at least two independent experiments. levels fell (Fig. 7C). This regulation of PKC␦ by p21 RAS is not at the level of transcription, as PKC␦ transcript levels, assessed by quantitative RT-PCR, did not vary as a function of p21 RAS activity (data not shown).
To determine which p21 RAS effector pathway mediated the up-regulation of PKC␦ protein, p110 CAAX , Raf-22w, RIF CAAX, and RalA-28N expression vectors were each stably transfected into two different cell lines, and PKC␦ protein levels were quantitated (Fig. 8A). The p110 CAAX expression vector was consistently the most potent inducer of PKC␦ expression in both Balb and NIH/3T3 cells, although activation of each of the other two effector pathways could also induce PKC␦ protein expression to a variable extent. In agreement, the C40 p21 RAS effector loop mutant, which activates predominantly the PI3K pathway, was the most potent of the effector mutants for induction of PKC␦ protein levels (Fig. 8B). The expression of other isozymes of PKC (-␣ and -), ␤-actin, and tubulin were not changed by expression of p21 RAS or PI3K (Fig. 8, C and D). Similarly, levels of p53, cyclin D1, and p16 were not changed by expression of the p21 RAS or PI3K (data not shown).

DISCUSSION
The p21 RAS family members include critical molecular switches transducing signals to diverse downstream pathways, ultimately controlling such processes as proliferation, cytoskeletal integrity, apoptosis, cell adhesion, and cell migration (35)(36)(37)(38). p21 RAS and Ras-related proteins are frequently deregulated in cancers, leading to invasion and metastasis and enhancement of survival by activation of anti-apoptotic pathways. Paradoxically, several studies have demonstrated that enforced, high level expression of oncogenic p21 RAS can induce a permanent growth arrest in normal cells, mimicking natural senescence (39,40). Activation of the Raf-1/MEK/p38MAPK pathway is thought to be essential for oncogenic p21 RAS -induced senescence (40,41). Additionally, inactivation of the ARF/p53 tumor suppressor pathway in mouse fibroblasts and skin keratinocytes, or inactivation of the p16/Rb tumor suppressor pathway in human fibroblasts, can bypass p21 RAS -induced senescence, suggesting that cellular fate resulting from oncogenic p21 RAS signaling is dependent upon the cellular context and the integration of tumor suppressor signals (39,42). Our laboratory and others have established p21 RAS as a modulator of apoptosis in transformed cells and malignant cells and also in normal cells. Through FADD, caspase 8, and downstream effector reactive oxygen species, p21 RAS sensitizes cells to apoptosis induced by PKC inhibition (7,43,44). However, the specific molecular mechanism by which oncogenic p21 RAS mediates apoptosis in  the setting of inhibition of PKC activity remains incompletely understood. As different PKC isozymes may have opposing functions with respect to cellular proliferation, differentiation, and apoptosis, a more complete understanding of this process required identification of the specific PKC isozyme required for survival of cells expressing activated p21 RAS , along with elucidation of the specific p21 RAS downstream effectors evoking the apoptotic outcome.
In this study, we demonstrate that PKC␦ activity is required to prevent the induction of apoptosis in cells expressing activated p21 RAS . It is noteworthy that p21 RAS activity, and in particular activation of the PI3K pathway, up-regulates PKC␦ protein levels, thus positively reinforcing an anti-apoptotic, protective response to p21 RAS dysregulation in the cell. Conversely, when this induction is prevented by siRNA knockdown of PKC␦, programmed cell death is initiated. Pro-apoptotic activity engendered by activated Ras has been described in a number of other systems (45), and isoform differences in the proapoptotic effects of Ras have been reported, with K-Ras sensitizing cells to ␥-irradiation-induced apoptosis but H-Ras exerting a protective effect (46). Activated K-Ras has been reported to sensitize cells to the pro-apoptotic effects of PKC agonists through phosphorylation of serine residue 181 of p21 RAS and its translocation to intracellular membranes (47). K-Ras, but not H-Ras, was reported to bind to and potentially inactivate or sequester the anti-apoptotic proteins Bcl-2 and Bcl-XL (48). In contrast to these observations of isotype-specific differences in p21 RAS functions, we have previously reported, and confirm herein, that both activated K-Ras and H-Ras sensitize cells to apoptosis induced by inhibition of PKC␦ activity, which prevents their obligate activation of the Akt survival pathway.
PKC␦ has been reported to both positively and negatively regulate apoptotic programs (49 -52). These findings have generated conflicting hypotheses as to the role of PKC␦ in the control of cell proliferation and survival. The normal phenotype of PKC␦-null mice demonstrates that PKC␦ is not required for appropriate control of cell proliferation during normal development (28,53). In contrast, PKC␦ may be recruited during cellular transformation and become necessary for one or more components of the malignant phenotype. Inhibition of PKC␦ was reported to suppress the metastatic potential of breast cancer cells (54) and to reduced their survival (55). Similar results were reported using non-small cell lung cancer cells (56). Our findings support this hypothesis. We find that PKC␦ functions as a survival signal in a variety of cells with dysregulated activation of p21 RAS . PKC␦ expression is up-regulated in response to p21 RAS activity, primarily through PI3K activation, and is required for the survival of these cell lines. However, PKC␦ is not required for the survival or proliferation of the counterparts of these cell lines containing wild-type p21 RAS , whether or not they are transformed, and indeed suppression of PKC␦ actually leads to a small but reproducible increase in the proliferation or normal cells.
Activated Akt is a well established survival factor, exerting anti-apoptotic activity by suppressing the release of cytochrome c from the mitochondria (57). Activated Akt also inactivates the pro-apoptotic factors BAD and procaspase-9 by direct phosphorylation (58). A number of reports have suggested relationships between PKC activity and Akt activation. One recent study showed that PKC␦ contributes to the phosphorylation of Ebp1 in PC12 cells (59). Phosphorylation of Ebp1 is required for its association/interaction with active nuclear Akt, and the Akt-Ebp1 complex mediates an anti-apoptotic effect in intact cells. A very recent study suggested that both PKC␦ and PKC⑀ can negatively regulate Akt activity in mouse keratinocytes (60), although this study utilized only chemical inhibitors lacking absolute specificity. Whereas PKC␣ has been shown to promote the dephosphorylation and inactivation of Akt in prostate cancer cells (61), another study demonstrated that phosphorylation of PKC␦ protects glioma cells from TRAIL-induced apoptosis by activation of Akt (62). In our study, we prove that Akt activity can be regulated by PKC␦ (Fig.  6F) and that cells in which PKC␦ has been selectively suppressed by treatment with PKC␦-siRNA can be rescued from apoptosis by enforced expression of a constitutively activated Akt (Fig. 6C). These data suggest that oncogenic p21 RAS protein, through PI3K and PKC␦, induces Akt activity, initiating an anti-apoptotic signaling cascade that is required for their survival. However, whether PKC␦ alone is the major regulator of Akt activity under all conditions remains to be elucidated, as the levels of p-Akt were only slightly changed after knocking down PKC␦ in cells containing wild-type (normal) Ras (Fig. 6F). There are two possible explanations for the differences observed on PKC␦ actions of Akt between normal cells and those containing activated p21 RAS as follows: the first is the relative differences in efficiency of transient transfection, and the second is that the robust regulation of Akt activity by PKC␦ requires the involvement of an activated p21 RAS protein.
It is noteworthy that the PI3K effector pathway, in addition to generating the anti-apoptotic signal mediated through PKC␦ and activation of Akt (and up-regulation of PKC␦ levels and activity), is also responsible for the pro-apoptotic signal delivered in Ras-transformed cells, which is uncovered and made manifest by inhibition of PKC␦. We show that isolated activation of the PI3K pathway is sufficient to render cells susceptible Ras-mediated apoptosis and, conversely, that inhibition of the PI3K pathway is sufficient to protect cells from apoptosis initiated by suppression of PKC␦.
In conclusion, this study significantly extends our understanding of the mechanism underlying p21 RAS -mediated apoptosis by identifying the molecules required to redirect p21 RAS signaling from a proliferative/transforming outcome toward instead an apoptotic fate. Furthermore, elucidation of the particular PKC isozyme necessary for survival of cells transformed by p21 RAS suggests that selective suppression of PKC␦ activity, and the consequent induction of apoptosis, is a potential strategy for targeting of tumor cells containing an activated p21 RAS GTPase.