Protein Kinase Cβ Modulates Ligand-induced Cell Surface Death Receptor Accumulation

Although treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) is known to protect a subset of cells from induction of apoptosis by death ligands such as Fas ligand and tumor necrosis factor-α-related apoptosis-inducing ligand, the mechanism of this protection is unknown. This study demonstrated that protection in short term apoptosis assays and long term proliferation assays was maximal when Jurkat or HL-60 human leukemia cells were treated with 2–5 nm PMA. Immunoblotting demonstrated that multiple PKC isoforms, including PKCα, PKCβ, PKCϵ, and PKCθ, translocated from the cytosol to a membrane-bound fraction at these PMA concentrations. When the ability of short hairpin RNA (shRNA) constructs that specifically down-regulated each of these isoforms was examined, PKCβ shRNA uniquely reversed PMA-induced protection against cell death. The PKCβ-selective small molecule inhibitor enzastaurin had a similar effect. Although mass spectrometry suggested that Fas is phosphorylated on a number of serines and threonines, mutation of these sites individually or collectively had no effect on Fas-mediated death signaling or PMA protection. Further experiments demonstrated that PMA diminished ligand-induced cell surface accumulation of Fas and DR5, and PKCβ shRNA or enzastaurin reversed this effect. Moreover, enzastaurin sensitized a variety of human tumor cell lines and clinical acute myelogenous leukemia isolates, which express abundant PKCβ, to tumor necrosis factor-α related apoptosis-inducing ligand-induced death in the absence of PMA. Collectively, these results identify a specific PKC isoform that modulates death receptor-mediated cytotoxicity as well as a small molecule inhibitor that mitigates the inhibitory effects of PKC activation on ligand-induced death receptor trafficking and cell death.

In addition, it has been suggested that PKC activation can affect sensitivity to death ligands. In particular, several groups have previously reported that treatment with PMA, a well characterized activator of PKC and several other enzymes, inhibits Fas-or TRAIL-mediated apoptosis in some cells (reviewed in Refs. [32][33][34]. Early studies proposed several mechanisms for the ability of PMA to protect susceptible cells from death ligand-induced apoptosis, including activation of extracellular signal-regulated kinase (ERK) (35), which phosphorylates the antiapoptotic Bcl-2 family members Bcl-2 (36) and , or activation of the transcription factor NF-B (38), which enhances expression of a number of antiapoptotic polypeptides (39,40). In contrast, subsequent reports provided evidence that PMA affects death signaling more directly by disrupting recruitment of FADD and procaspase 8 to the intracellular domains of ligated Fas (32,33), DR4, and DR5 (34). These latter studies, however, left a number of questions unanswered. First, although prior results demonstrated that PMA provides short term protection from apoptosis, it has been unclear whether PMA is merely delaying the apoptotic process or affecting the number of cells that ultimately die. Second, even though a variety of PKC isoforms, including PKC␣, PKC␤, PKC⑀, PKC, PKC␦, and PKC, have been implicated in pro-and antiapoptotic signaling (41), it remains uncertain which PKC isoforms are responsible for modulating the death receptor pathway. Third, the mechanism by which PKC activation diminishes DISC formation has remained unclear. This study was undertaken to address these issues.
Cell Culture and Transfection-HL-60 cells from Robert T. Abraham (Wyeth Pharmaceuticals, Pearl River, NY), Jurkat cells from Paul Leibson (Department of Immunology, Mayo Clinic, Rochester, MN), and the Jurkat variant I2.1 (FADDdeficient, Refs. 44,45) were maintained in log phase in RPMI 1640 medium containing 100 units/ml penicillin G, 100 g/ml streptomycin, 2 mM glutamine, and 10% heat-inactivated fetal bovine serum (medium A). For transfection, cells grown to a concentration of 3-4 ϫ 10 5 cells/ml in medium A without antibiotics (medium B) were centrifuged at 200 ϫ g for 10 min, resuspended at 25 ϫ 10 6 cells/ml in 0.4 ml of medium B, and incubated with 40 g of the indicated plasmid DNA along with 5-10 g of pEGFP-N1, which encodes EGFP, for 5 min. Samples were electroporated using a BTX 820 or 830 square wave electroporator (BTX, San Diego) at 280 V for 10 ms. After 5 min, cells were placed in fresh medium B and cultured for 24 -48 h as indicated before treatment. Control experiments using a plasmid encoding EGFP typically indicated a 60 -80% transfection efficiency as assessed by flow cytometry 24 -48 h after electroporation.
Colony Forming and Cell Outgrowth Assays-To assess the effects of treatment on clonogenic survival, aliquots containing 4 -8 ϫ 10 5 log phase HL-60 cells in medium A were treated for 6 h with the indicated concentrations of TRAIL and/or PMA, sedimented at 100 ϫ g for 5 min, washed once with medium A, diluted into molten 0.3% (w/v) agar in the medium of Pike and Robinson (46), and plated in 35-mm gridded plates as described previously. After a 10 -14-day incubation, colonies containing Ͼ50 cells were counted on an inverted microscope at ϫ25 magnification.
Because Jurkat cells do not form colonies under these conditions, long term survival and proliferation were assessed using a culture repopulation assay similar to that described by Simonian et al. (47). In brief, aliquots containing 10 6 cells in medium A were treated for 6 h with the indicated concentrations of TRAIL or CH-11 in the absence or presence of PMA, sedimented at 100 ϫ g for 5 min, washed once with medium A, and diluted into 10 ml of medium A. Flasks were examined daily to determine the point at which cell density again reached 1 ϫ 10 6 /ml. To provide a standard curve, aliquots containing 10, 100, 1000, 10 4 , 10 5 , and 10 6 untreated Jurkat cells were also diluted into 10 ml of medium A and examined for subsequent regrowth to 10 6 cells/ml. T98G cells (American Type Culture Collection, Manassas, VA) were maintained in medium A supplemented with 1 mM pyruvate (medium C). After subconfluent monolayers were trypsinized, aliquots containing 250 cells were plated in multiple 35-mm dishes containing 2 ml of medium C and allowed to attach overnight. Graded concentrations of TRAIL and enzastaurin were then added to triplicate plates. After a 4-h incubation, plates were washed twice in serum-free RPMI 1640 medium and incubated in drug-free medium C for an additional 7 days. The resulting colonies were stained with Coomas-sie Blue and counted. Diluent-treated control plates typically contained ϳ150 colonies. Colony forming assays using HeLa cells and HCT116 were performed similarly except that 300 (HeLa) or 500 (HCT116) cells were plated to have 150 -200 colonies on the diluent-treated plates.
Apoptosis Assays -After treatment with the indicated concentrations of death ligand, PMA, and/or enzastaurin, cells were washed and stained with APC-conjugated annexin V as described (48,49). 2-3 ϫ 10 4 events were collected from the FL2 (excitation 488 nm, emission 585 Ϯ 21 nm) and FL4 (excitation 635 nm, emission 661 Ϯ 8 nm) channels of a BD Biosciences FACSCalibur flow cytometer and analyzed using CellQuest software (BD Biosciences). Apoptotic and nonapoptotic untransfected Jurkat cells treated with APC-annexin V alone were utilized to identify cut points for EGFP ϩ versus EGFP Ϫ and annexin V ϩ versus annexin V Ϫ cells. Alternatively, untransfected cells were lysed at 4°C in buffer D (0.1% (w/v) Triton X-100 and 50 g/ml propidium iodide in 0.1% (w/v) sodium citrate) and subjected to flow cytometry to assess DNA fragmentation (45,50). In some experiments, cells were also fixed in 3:1 (v/v) methanol/acetic acid, stained with 1 g/ml Hoechst 33258, and examined by fluorescence microscopy for apoptotic changes in nuclear morphology (33).
Cell Fractionation-After treatment with PMA for 30 -60 min, cells were harvested for fractionation. All further steps were performed at 4°C. Cells were washed twice with PBS and then swelled for 30 min in hypotonic lysis buffer (10 mM NaCl, 3 mM MgSO 4 , 10 mM Tris-HCl (pH 7.4 at 4°C)) supplemented with the protease inhibitors leupeptin (10 g/ml), pepstatin A (10 g/ml), and ␣-phenylmethylsulfonyl fluoride (1 mM). Cells were lysed in a tight-fitting Dounce homogenizer until Ͼ95% of the cells were ruptured. The mitochondria and nuclei were removed by sedimentation at 4000 ϫ g for 15 min. Samples were then spun at 100,000 ϫ g for 1 h to separate the membrane fraction (pellet) from the cytosol (supernatant).
Plasmids-The parental pFRT-HIP and pCMS3-EGFP-HIP plasmids used for shRNA silencing were described previously (52). Isoform-specific RNA interference target sequences were selected using a custom program designed to parse the output of the GeneBee RNA secondary structure prediction program. Sequences selected for the targeted isoforms were as follows: PKC␣, 5Ј-GAACAACAAGGAAUGACUU-3Ј; PKC␤, 5Ј-GGAAGCUGUGGCCAUCUGC-3Ј; PKC⑀, 5Ј-GCCACCCU-UCAAACCACGCAUU-3Ј; and PKC, 5Ј-UUGGAUGAG-GUGGAUAAA-3Ј. . PMA provides long term protection from agonistic anti-Fas antibody and TRAIL. A, after a 5-min pretreatment with the indicated concentration of PMA or diluent (0.1% ethanol), Jurkat cells were treated for 6 h with CH-11 agonistic anti-Fas antibody at 25 ng/ml. At the completion of the incubation, samples were stained with PI and analyzed by flow microfluorimetry as described under "Experimental Procedures." B, results from samples in A and additional samples from the same experiment. Inset, results of treatment at 25 ng/ml CH-11 as a function of PMA concentration. C, after 5 ϫ 10 7 Jurkat cells were treated for 90 min with 500 ng/ml CH-11 in the absence or presence of the indicated PMA concentration, cells were lysed, and the Fas DISC was immunoprecipitated as described previously (33) using anti-mouse IgM precoupled to protein G-Sepharose. Immunoprecipitates were subjected to SDS-PAGE and immunoblotting with anti-FADD antibody. D, after a 5-min pretreatment with 5 nM PMA or diluent, cells were treated for 6 h with the indicated concentration of CH-11. At the completion of the incubation, cells were washed in drug-free medium, diluted 1:10 in medium, and examined for ability to regrow to a density of ϳ1 ϫ 10 6 cells/ml as described previously (51). Inset, standard curve from this experiment, indicating time required for various dilutions of untreated cells to proliferate to a density of 1 ϫ 10 6 cells/ml. E, after a 5-min pretreatment with the indicated concentration of PMA or diluent, Jurkat cells were treated for 6 h with the indicated concentration of recombinant human TRAIL. At the completion of the incubation, samples were stained with APC-conjugated annexin V and analyzed by flow microfluorimetry. F, after a 5-min pretreatment with 2.5 nM PMA or diluent, cells were treated for 6 h with the indicated concentration of TRAIL, washed in drug-free medium, diluted 1:10 in medium, and examined for ability to regrow to a density of ϳ1 ϫ 10 6 cells/ml as illustrated in D. G, after a 5-min pretreatment with the indicated concentration of PMA or diluent, HL-60 cells were treated for 4 h with the indicated concentration of recombinant human TRAIL, stained with PI, and analyzed by flow microfluorimetry. H, after a 5-min pretreatment with 2 nM PMA or diluent, cells were treated for 6 h with the indicated concentration of TRAIL, washed in drug-free medium, diluted into 0.3% agar, and assayed for ability to form colonies. Error bars, ϮS.D. of quadruplicate aliquots.
To generate Fas fused at its C terminus with S peptide (Fas-S peptide), cDNA encoding full-length Fas (GenBank TM accession number M67454) was isolated by reverse transcription-PCR using a 3Ј primer designed to add a linker and S peptide (GAGAGAGAGGAP)MKETAAAKFERQHMDS in-frame at the C terminus. The PCR product was cloned into pcDNA3.1 using EcoRI and XbaI.
Assay for Ligand Binding-To measure the binding of death ligands, 1 ϫ 10 6 cells were incubated with agonistic CH-11 anti-Fas antibody or 1 g/ml anti-FLAG antibody plus FLAGtagged FasL or FLAG-tagged TRAIL in the absence or presence of PMA for 90 min at 37°C. Cells were then quickly cooled, washed twice with ice-cold PBS containing 2% (v/v) heat-inactivated fetal bovine serum, and incubated with APC-conjugated anti-mouse IgM (for CH-11 binding) or Alexa Fluor647-labeled anti-mouse IgG (to detect ligand-bound anti-FLAG) for an additional 45 min on ice. Following two washes with ice-cold PBS, cells were resuspended in 4% formaldehyde in PBS and stored in the dark at 4°C for Ͻ18 h until flow microfluorimetry was performed.
Biotinylation of Cell Surface Proteins and Fas Internalization Assay-For cell surface biotinylation as well as phosphorylation analysis, we created JFSD9 cells, which stably express Fas-S peptide, by transfecting the Fas-deficient line JM14A5 (45, 53) with pcDNA3.1 encoding Fas-S peptide. After selection in medium A containing 400 g/ml G418 and cloning by limiting dilution, this line was chosen from among other clones because Fas was readily detectable on its surface by flow cytometry. Following treatment as specified in the figure legends, aliquots were quickly chilled by adding 4 volumes of ice-cold PBS containing 0.1 mM CaCl 2 and 1 mM MgCl 2 (PBS-CM) and washed three times with the same buffer. Cell surface proteins were biotinylated by incubating cells in 1.0 ml of PBS-CM containing 1 mM NHS-LC-Biotin (Pierce) on ice for 1 h. The reaction was stopped by incubating cells with ice-cold PBS-CM containing 100 mM glycine. After three washes with PBS-CM, cells were lysed in 1.0 ml DISC buffer as described above. Following clarification of the lysates, Fas was pulled down by incubating lysates for 2-3 h at 4°C with anti-S peptide antibody covalently coupled to agarose and then washing the bound proteins as described (49). After release from beads and SDS-PAGE as described below, polypeptides were transferred to nitrocellulose and visualized using horseradish peroxidase-coupled streptavidin or anti-S peptide antibody.
DISC Immunoprecipitation-To isolate the Fas DISC, aliquots containing 5 ϫ 10 7 cells were resuspended in 10 ml of medium A and incubated with diluent or 10 M enzastaurin for 30 min. Following addition of PMA and/or CH-11 (final concentrations 20 nM and 500 ng/ml, respectively), the incubation was continued for an additional 90 min at 37°C. Cells were then sedimented at 200 ϫ g for 10 min and lysed in 1 ml of DISC buffer on ice for 30 min. After the lysate was cleared by centrifugation at 18,000 ϫ g for 15 min, an aliquot of supernatant (4 mg of protein) was transferred to a new tube containing 30 l of beads with rabbit anti-mouse IgM antibody cross-linked to protein G-Sepharose. Beads were rotated at 4°C for 90 min and washed five times in DISC buffer. Bound polypeptides were released by boiling the beads for 5 min with 40 l of SDS sample buffer and loaded on SDS-polyacrylamide gels.
Sample Preparation and SDS-PAGE-After drug treatment or fluorescence-activated cell sorting, cells were sedimented at 200 ϫ g for 10 min, washed once with ice-cold RPMI 1640 medium containing 10 mM HEPES (pH 7.4 at 4°C), solubilized in buffered 6 M guanidine hydrochloride under reducing conditions, and prepared for electrophoresis (54). Aliquots containing 50 g of protein (estimated by the bicinchoninic acid method as described in Ref. 55) were separated on SDS-polyacrylamide gels, electrophoretically transferred to nitrocellulose, and probed as described (56,57). Where indicated, signals on x-ray film were quantitated using ImageJ software.
Apoptosis Assays in Primary Leukemia Isolates-After informed consent was obtained, samples of bone marrow were harvested from acute myelogenous leukemia patients prior to chemotherapy according to an IRB-approved protocol. Following sedimentation on a Ficoll-Paque (1.077 g/cm 3 ) step gradient (58), the mononuclear cells were recovered and counted on a hemocytometer. Aliquots containing 1 ϫ 10 6 cells in 1 ml of medium A were incubated for 24 h in the presence of varying concentrations of enzastaurin and/or TRAIL, sedimented, lysed in buffer D, and analyzed for DNA content by flow microfluorimetry as described above. A minimum of 20,000 events/sample was counted.
Statistics-Unless otherwise indicated, experiments were repeated at least three times with similar results. Where results of multiple experiments are summarized, data are expressed as the mean Ϯ S.D. of the indicated number of independent experiments. Groups were compared using two-sided Student's t tests (unpaired unless indicated).

Low Nanomolar PMA Concentrations Provide Short and Long Term Protection from Death Ligand-induced Toxicity-
To extend our earlier observation that PMA inhibits death ligand-induced apoptosis, we examined the effects of various PMA concentrations on death induced by the agonistic anti-Fas antibody CH-11. These studies demonstrated that PMA inhibited CH-11-induced apoptosis in Jurkat cells (Fig. 1, A and B) with half-maximal inhibition at 0.5 nM (Fig. 1B, inset). This protection occurred over the same range of PMA concentrations that inhibited DISC formation (Fig. 1C), consistent with the hypothesis (32-34) that diminished DISC formation plays a role in the PMA-induced protection from death ligand-induced death.
To rule out the possibility the PMA inhibits the development of apoptotic stigmata without affecting the number of cells that ultimately die (59), we examined long term survival of CH-11treated Jurkat cells using a culture repopulation assay. As indicated in Fig. 1D, PMA markedly enhanced the ability of Jurkat cells to grow back after Fas receptor ligation. PMA similarly inhibited TRAIL-induced apoptosis (Fig. 1E) and enhanced long term survival in these cells (Fig. 1F). Thus, low nanomolar PMA concentrations affect the number of cells killed by death ligands rather than merely affecting the kinetics of apoptosis.
Although the majority of previous studies examining the effect of PMA on death ligand-induced apoptosis were performed in Jurkat cells, the protective effect of PMA was not limited to this cell line. PMA also protected HL-60 cells (Fig.  1G) 4 and T98G glioblastoma cells (supplemental Fig. S1) from TRAIL-induced apoptosis. Clonogenic assays confirmed that PMA likewise enhanced the ability of cells to survive and multiply after TRAIL treatment (Fig. 1H). Thus, PMA provided long lasting protection against death ligand-induced cytotoxicity in multiple cell types.
Dose-dependent Activation of Specific PKC Isoforms-In an initial attempt to identify PKC isoform(s) that might contribute to PMA-induced protection from death ligand-induced apo- ptosis, cells were treated with various PMA concentrations and then fractionated into cytosol and membranes to separate inactive from active species (60 -62). Immunoblotting demonstrated that PMA causes translocation of several PKC isoforms over the range of concentrations that protect Jurkat and HL-60 cells (Fig. 2). In particular, PMA caused a dose-dependent translocation of PKC␣, PKC␤, PKC⑀, and PKC from cytosol to membranes in both cell lines. In contrast, other isoforms such as PKC␥, PKC␦, PKC, PKC, and PKC and the closely related enzyme PKD did not translocate or translocated in only one of the lines ( Fig. 2A and data not shown). These observations raised the possibility that PKC␣, PKC␤, PKC⑀, and/or PKC might be responsible for the effects of PMA on death ligandinduced killing.
Predominant Role for PKC␤ in the Protective Effects of PMA-To further assess the role(s) of these various isoforms in protecting cells from death ligand-induced apoptosis, we created isoform-selective RNA interference constructs for PKC␣, PKC␤, PKC⑀, and PKC. When Jurkat cells were transiently transfected with the indicated constructs and harvested for immunoblotting, there was extensive reduction of target protein levels with minimal alteration of levels of the nontargeted PKC isoforms (Fig. 3A). In further experiments, this down-reg-ulation was maximal at 48 h and was sustained for 24 -48 h thereafter.
In subsequent studies, cells were co-transfected with these RNA interference constructs and, to mark transfected cells, plasmid encoding EGFP. After cells were incubated for 2 days to allow target protein down-regulation, diluent or varying concentrations of PMA were added 5-10 min before the death ligand. As was the case with untransfected cells (Fig.  1), treatment with PMA protected empty vector-transfected cells from death ligand-induced apoptosis (Fig. 3, B and C, and supplemental Fig. S2). The percentage of cells undergoing CH-11-induced apoptosis was diminished from ϳ80% in the absence of PMA to ϳ30% in the presence of 10 nM PMA (Fig. 3B). Similar results were observed with TRAIL treatment (Fig. 3C). Thus, transfection per se did not alter the behavior of the cells. This ability of PMA to protect from death ligand-induced apoptosis was also not substantially altered by transfection with shRNA directed against PKC⑀ or PKC (Fig. 3, B and C, and supplemental Fig. S2).
PKC␣ shRNA induced limited protection from PMA. When cells were treated with 30 ng/ml CH-11 in the presence of 5 nM PMA, for example, the percentage of apoptotic cells increased from 33% in cells transfected with empty vector to 50% in cells transfected with PKC␣ shRNA (Fig. 3B and supplemental Fig.  S2). PKC␤ shRNA induced an even more dramatic effect (Fig. 3, B and C, and supplemental Fig. S2). Although there was little effect on apoptosis in the absence of PMA, the effect of PMA on death receptor-initiated apoptosis was severely blunted (Fig. 3, B and C, and supplemental Fig. S2), raising the possibility that PKC␤ plays a major role in mediating the effect of PMA on death ligand-induced apoptosis.
PKC␤ Inhibitor Enzastaurin Also Reverses the Effect of PMA-To further examine the role of PKC␤ in death ligand-induced apoptosis, we evaluated the effects of enzastaurin, a small molecule PKC␤ inhibitor that is currently undergoing clinical testing in diabetes and cancer (63)(64)(65)(66)(67). Initial experiments examined 10 M enzastaurin, a concentration previously shown to suppress PMA-induced substrate phosphorylation in PKC␤expressing U937 cells (68). As was the case with PKC␤ shRNA, enzastaurin by itself did not induce apoptosis under the conditions of these assays but markedly blunted the effects of PMA on CH-11-induced apoptosis in Jurkat cells (Fig. 4, A and B, and  supplemental Fig. S3). This sensitizing effect was seen at enzastaurin concentrations as low as 0.5 M and was almost maximal at 5 M (Fig. 4C), a concentration range that inhibits PKC substrate phosphorylation in cells (Fig. 4D) (68), diminishes activation-associated PKC␤ down-regulation (Fig. 4D), and is sustainable in patients (64). Consistent with our previous results, PMA inhibited FADD recruitment to Fas, and enzastaurin mitigated this inhibition (Fig. 4E). As a result, enzastaurin facilitated CH-11-induced caspase 8 activation and subsequent downstream events in the presence of PMA (Fig. 4F).
Our previous results demonstrated that PMA protects Jurkat cells from the cytotoxicity of TRAIL, CH-11, and Fas ligand but not apoptosis induced through the mitochondrial pathway by camptothecin or etoposide (33), raising the possibility that PKC plays a specific role in modulating the death receptor pathway. Consistent with this possibility, treatment with enzastaurin had no effect on the induction of apoptosis by etoposide, staurosporine, or camptothecin (Fig. 4G).
In further experiments, enzastaurin also facilitated TRAIL-induced apoptosis in Jurkat cells (Fig. 5, A and B). This sensitizing effect was again observed at concentrations as low as 0.5 M and was maximal at 5 M (Fig. 5C). Similar sensitization was observed in HL-60 cells (Fig. 5D). Consistent with these results, enzastaurin also mitigated the effects of PMA as assessed by colony forming assays in the latter system (Fig. 5E).
PMA Diminishes Cell Surface Death Receptors -Previous experiments showing that treatment of cells with PMA interrupts death ligand-induced killing by disrupting recruitment of FADD to death receptors (32-34) raised the possibility that PMA might be inducing phosphorylation of death receptors or FADD. Preliminary experiments failed to demonstrate a PMA-induced change in FADD subcellular distribution 6 or increase in FADD phosphorylation (supplemental Fig. S5A). In contrast, PMA enhanced phosphorylation of tagged Fas (supplemental Fig. S5B). Mass spectrometry tentatively identified six phosphorylation sites (supplemental Fig. S5, C-E). Although Thr 198 of Fas is a weak consensus PKC phosphorylation site, phosphorylation of Thr 198 was not detected in multiple separate mass spectrometry experiments. Additional studies demonstrated that mutation of these putative phosphorylation sites to alanine singly (supplemental Fig. S6) or in combination (supplemental Fig. S7) failed to affect the ability of PMA to diminish FADD recruitment and inhibit Fas-mediated apoptosis. Accordingly, further experiments focused on other potential mechanisms by which PMA could affect Fas signaling.
To examine cell surface Fas levels, we initially transfected JM14A5 cells, a Jurkat variant that lacks cell surface Fas (45)   is resistant to CH-11 (45,53), with Fas tagged at its C terminus with S peptide. Control experiments demonstrated that Fas-S peptide signals like endogenous Fas (Fig. 6, A and B). In particular, treatment of reconstituted cells with CH-11 induced binding of FADD to Fas-S peptide (Fig. 6A, upper panel, lane 1) and subsequent apoptosis (Fig. 6B). In further experiments, cells expressing Fas-S peptide were treated with CH-11 agonistic anti-Fas antibody for 90 min in the absence and presence of PMA and then lysed so that CH-11-bound Fas and FADD could be recovered by immunoprecipitation. 7 Results of this analysis demonstrated that less Fas was recovered with CH-11 when the incubation was performed in the presence of PMA (Fig. 6A,  lanes 3 and 4 versus lane 1). Importantly, the total amount of Fas detectable by blotting whole cell lysates with anti-S peptide antibody did not change (Fig. 6A, lower  panel).
Subsequent experiments sought to extend these findings to endogenous untagged death receptors. Because of concern that anti-Fas antibodies can yield spurious results on immunoblots, alternative techniques were applied. In one line of experiments, parental Jurkat cells were incubated for 90 min with increasing CH-11 concentrations in the absence or presence of PMA; and cell surface antibody was subsequently detected by staining with APC-conjugated anti-mouse IgM at 4°C followed by flow cytometry. Consistent with the results observed in transfected cells (Fig.  6A), less CH-11 was detectable on the cell surface when the incubation was performed in the presence of PMA (Fig. 6, C and D). Similarly, when parental Jurkat cells were treated with FasL in the absence and presence of PMA, less cell surface FasL was detected in the presence of PMA (Fig. 6E). Treatment with PMA alone at 37°C for 90 min failed to alter the amount of CH-11 subsequently bound at 4°C (Fig. 6F), suggesting that both PMA and ligand must be present in order for the effect of PMA to be detectable. When this effect of PMA and ligand on cell surface Fas was assessed in I2.1 cells, a Jurkat variant that lacks FADD and is resistant to Fas-mediated apoptosis (44,45), decreased cell surface death receptor was again observed (Fig.  6G), indicating that the effect of PMA does not require Fas-FADD signaling. Collectively, these results suggest that PMA is diminishing Fas-mediated apoptosis, at least in part, by decreasing the amount of cell surface receptor available for ligand-induced activation.
Enzastaurin or PKC␤ shRNA Increases Cell Surface Fas-In view of the ability of enzastaurin to reverse the effects of PMA on Fas-mediated apoptosis (Fig. 4), we next examined the ability of enzastaurin to reverse PMA-induced Fas downregulation. When enzastaurin was added 30 min prior to CH-11 and PMA, subsequent flow cytometry demonstrated that enzastaurin restored the levels of CH-11 on the cell surface (Fig. 7A).
Similar effects of enzastaurin were observed using two additional approaches. In the first, cells expressing Fas-S peptide were treated with CH-11 for 90 min at 37°C in the absence or presence of PMA and/or enzastaurin and then lysed so that  Fig. 6C. MFI, mean fluorescence intensity. B, aliquots containing 5 ϫ 10 7 JFSD9 cells were pretreated with diluent or 10 M enzastaurin for 30 min and then incubated with 500 ng/ml CH-11 in the absence or presence of 20 nM PMA for 90 min at 37°C. Lysates were prepared as described under "Experimental Procedures." Fas bound to CH-11 on the cell surface was recovered with anti-mouse IgM coupled to protein G-Sepharose. Immunoprecipitates were probed with antibodies to S peptide and FADD. Bottom panel, cell lysates from same cells. C, 48 h after JFSD9 cells were transfected with empty vector or PKC␤ shRNA, cells were treated with 500 ng/ml CH-11 in the absence or presence of PMA. After cells were chilled and washed, CH-11 pulldowns prepared as described for Fig. 6A (top) and cell lysates (bottom) were probed with antibodies to the indicated antigens. HSP90 served as a loading control. Numbers below lanes, relative PKC␤ signal of lysates after correction for HSP90 content. D, aliquots containing 5 ϫ 10 7 JFSD9 cells were pretreated with diluent or 10 M enzastaurin for 30 min and then incubated with 500 ng/ml CH-11 in the absence or presence of 20 nM PMA for 90 min at 37°C. After cells were rapidly chilled on ice, cell surface proteins were biotinylated for 1 h at 4°C as described under "Experimental Procedures." Following preparation of cell lysates, total cellular Fas-S peptide was recovered on beads containing anti-S peptide antibody (clone 6.2) coupled to protein G-Sepharose. After washing, bound proteins were subjected to SDS-PAGE and blotted with peroxidase-coupled streptavidin (top) for cell surface Fas, S peptide antibody (middle, for total Fas), or FADD (bottom).
CH-11-bound Fas could be immunoprecipitated and probed with anti-S peptide antibody. As shown in Fig. 7B, PMA diminished the amount of Fas-S peptide bound to CH-11; and enzastaurin reversed this effect. PKC␤ shRNA likewise restored the binding of CH-11 to Fas-S peptide in the presence of PMA (Fig. 7C).
In the second approach, cells expressing Fas-S peptide were treated with CH-11 in the absence or presence of PMA and/or enzastaurin for 90 min at 37°C and then cooled to 4°C. After cell surface proteins were biotinylated using cell-impermeable reagents (70,71), total cellular Fas-S peptide was pulled down, subjected to SDS-PAGE, and probed with peroxidase-coupled streptavidin. Results of this analysis demonstrated that the amount of Fas accessible for biotinylation on the cell surface was markedly diminished after treatment with PMA (Fig. 7D,  lanes 4 and 5); and this effect was again reversed by enzastaurin (Fig. 7D, lane 6). Collectively, the results in Fig. 7 using three independent methods suggest that enzastaurin or PKC␤ shRNA is reversing the effects of PMA by preventing a PMAinduced decrease in cell surface Fas.
PMA Diminishes Fas Trafficking to the Cell Surface-Previous studies have demonstrated that PMA stimulates clathrinmediated endocytosis of a number of cell surface receptors, including the transferrin receptor and epidermal growth factor receptor (72,73). Consistent with these results, transfection with the C-terminal domain of the adapter protein AP-180 (74) or with dominant negative Eps-15 (75), two constructs that have previously been reported to inhibit clathrin-mediated uptake, inhibited PMA-induced loss of the transferrin receptor on the surface of Jurkat cells (supplemental Fig. S8A, upper  panel). In contrast, these constructs had little effect on PMAinduced loss of CH-11 binding (supplemental Fig. S8A, lower  panel). Likewise, mutation of Fas Tyr 291 to Phe, a change reported to inhibit clathrin-mediated Fas internalization in some cells (21), had no effect on the PMA-induced decrease in CH-11 cell surface binding in Jurkat cells (supplemental Fig.  S8B). Collectively, these results suggested that PMA is modulating cell surface death receptors by a process other than clathrin-mediated endocytosis.
Further examination of Fig. 7D indicated that more Fas-S peptide was detected on the cell surface in the presence of CH-11 than in its absence (Fig. 7D, lanes 4 and 1), raising the possibility that agonistic anti-Fas antibody is trapping the death receptor as it traffics to the cell surface. In further support of this hypothesis, cells incubated with CH-11 for increasing lengths of time were observed to have more cell surface Fas-S peptide accessible for biotinylation by cell-impermeable reagents (Fig. 8A, lanes 1-5). In contrast, treatment with CH-11 ϩ PMA did not result in an increase in cell surface Fas (Fig. 8A, lanes 6 -10). Similar results were observed when cells were stained with CH-11 at 37°C for increasing lengths of time, cooled to 4°C, stained with APCconjugated secondary antibody, and analyzed by flow cytometry (Fig. 8B). Collectively, these results suggest that PMA is inhibiting ligand-induced accumulation of cell surface death receptor.

Enzastaurin Enhances TRAIL-induced Apoptosis in the
Absence of PMA Treatment-Classical PKC isoforms are activated, in part, by phospholipase C␥-mediated production of diacylglycerol after growth factor receptor ligation (61,76). Further examination of Fig. 7, A and D, indicated that enzastaurin induced a slight but reproducible increase in cell surface Fas in the absence of PMA. Likewise, enzastaurin appeared to increase CH-11-and TRAIL-induced apoptosis in the absence of PMA (Fig. 4B, Fig. 5, B and D, and supplemental Fig. S3). These observations raised the possibility that tonic signaling by PKC␤, which is known to occur in a number of neoplasms (77)(78)(79)(80), might contribute to death ligand resistance even in the absence of PMA. Consistent with this possibility, PKC␤ shRNA enhanced TRAIL-induced apoptosis in T98G cells (Fig. 9A). In further experiments (Fig. 9, B-D), enzastaurin also enhanced the amount of TRAIL bound to the cell surface and its ability to induce apoptosis in this cell line. In addition, enzastaurin enhanced the antiproliferative effects of TRAIL (Fig. 9E) or agonistic anti-DR5 antibodies (Fig. 9F) in clonogenic assays. Enhanced antiproliferative effects were likewise observed when HeLa cervical cancer and HCT116 colon cancer cells were treated with TRAIL and enzastaurin compared with TRAIL alone (supplemental Fig. S9).
To rule out the possibility that these effects were unique to tissue culture cell lines, we also examined the ability of enzastaurin to enhance TRAIL-induced apoptosis in human acute myelogenous leukemia isolates. Preliminary studies established that these cells express abundant levels of PKC␤ (Fig. 10A). Although enzastaurin by itself had little effect on these cells (Fig. 10B, inset), it nonetheless enhanced TRAIL-induced apoptosis (Fig. 10, B and C). Formal mathematical analysis using the median effect method of Chou and Talalay (81), a standard approach to assessing the effects of combined treatments (82), indicated that the effects were more than additive at the vast majority of data points in the nine acute myelogenous leukemia samples, as indicated by the combination index that was Ͻ1 (Fig. 10D).

DISCUSSION
Results of this study demonstrated that PMA enhances short term and long term survival of death ligand-treated cells and that these effects occur at low nanomolar PMA concentrations. In further experiments, these protective effects of PMA were inhibited by PKC␤ shRNA or enzastaurin. In addition, PMA diminished the amount of cell surface death receptor, whereas PKC␤ shRNA or enzastaurin reversed this effect. Collectively, these observations suggest that PMA and enzastaurin modulate the trafficking of Fas and DR5 to the cell surface, thereby affect-ing the amount of receptor available for ligation and the activation of the death receptor pathway. These observations have implications not only for current understanding of the death receptor pathway, but also potentially for the clinical development of death ligands as anticancer agents.
Although a number of previous studies reported that PMA can protect from death ligand-induced apoptosis, it was previously unclear whether cell death was delayed or abrogated. In this study, outgrowth assays (Fig. 1, D and F) as well as clonogenic assays (Fig. 1H and Fig. 5E) demonstrated that concurrent PMA treatment enhanced the number of cells that were able to survive and proliferate after TRAIL exposure. These results are consistent with scattered reports showing that proliferating cells, which often have activated PKCs, are less sensitive to TRAIL (83,84) and that selection for TRAIL resistance yields cells with accelerated proliferation (85).
During the course of the studies on PMA-induced protection from death ligands, we also observed that the protective effects are close to maximal at PMA concentrations in the 1-2 nM range ( Fig. 1, B, E, and G). Further experiments showed that a number of PKC isoforms are activated over this concentration range (Fig. 2). Although shRNA that selectively down-regulates . Because enzastaurin has no effect by itself under the conditions of these assays, its ability to enhance the effects of TRAIL meets the definition of synergy (82). Error bars, Ϯ S.D. of triplicate aliquots. MFI, mean fluorescence intensity.
PKC⑀ or PKC had little effect on PMA-induced protection, PKC␣ shRNA mitigated the effects of PMA to a limited extent, and PKC␤ shRNA markedly diminished the effects of PMA (Fig. 3). Consistent with these results, the PKC␤ inhibitor enzastaurin diminished the effects of PMA on DISC formation (Fig.  4E) and subsequent apoptosis (Fig. 4, A, B, and F).
In further experiments, three independent approaches detected fewer death receptors on the surfaces of cells treated with death ligand in the presence of PMA compared with its absence. First, in cells expressing epitope-tagged Fas, diminished receptor was pulled down with CH-11 in the presence of PMA ( Fig. 6A and Fig. 7B). Second, when cell surface proteins were biotinylated and total cellular Fas was recovered using the epitope tag, less Fas was derivatized if the incubation included PMA (Fig. 7D and Fig. 8A). Finally, when cells were treated with death ligand at 37°C, cooled to 4°C, and reacted with fluorochrome-conjugated secondary antibodies, flow cytometry detected less endogenous Fas (Fig. 6, C and D, Fig. 7A, and Fig.  8B) or FasL (Fig. 6E) on the cell surface if the incubation included PMA. During the course of these studies we noted that the PMA-induced decrease in cell surface receptor appeared to be greater using the former two assays. In view of the fact that effects of PMA on cell surface ligand binding were somewhat less dramatic than the protection against death ligand-induced death (cf. Fig. 6, B and D), we cannot rule out the possibility that PKC␤ is also involved in other changes that protect cells from death ligand-induced apoptosis. Although the reason for the quantitative difference between the assays is currently unknown, all three assays led to the conclusion that PMA is acting, at least in part, by regulating the number of cell surface death receptors.
PKC isoforms have previously been implicated in down-regulation of other types of cell surface receptors through enhanced receptor internalization (72). In contrast, results of this study failed to provide evidence for PMA-induced internalization of Fas. PMA did not stimulate clathrin-mediated Fas internalization, as indicated by (i) the inability of overexpressed c-AP180 or dominant negative Eps-15 to prevent the PMA-induced decrement in cell surface Fas (supplemental Fig. S8A) and (ii) the failure of the Fas Y291F mutation to mitigate the effects of using an approach that renders the analysis equivalent to isobologram analysis (82). Note that a combination index Ͻ1.0 indicates synergy. PMA (supplemental Fig. S8B). Moreover, the failure of PMA to cause a decrease in cell surface Fas over time (Fig. 8) argues against the possibility that PMA is accelerating a clathrin-independent internalization mechanism.
Instead, our observations suggest a model in which CH-11 is progressively trapping Fas as it spontaneously traffics to the Jurkat cell surface in a PKC␤-regulated manner. In particular, two independent lines of evidence (Fig. 8, A and B) demonstrate that cell surface Fas increases during time of exposure to agonistic antibody. Both experiments also show that PMA markedly diminishes this increase. These observations not only explain our prior observation that the amount of Fas DISC detectable in Jurkat cells increases progressively with time of death ligand incubation (33), but also provide an explanation for the ability of PMA to protect cells when added only 5 min before death ligands. Additional experiments demonstrated that the effects of PMA on cell surface Fas were reversed by PKC␤ shRNA (Fig. 7C) or enzastaurin (Fig. 7, A, B, and D). To our knowledge, these experiments provide the first evidence that PKC plays an important role in trafficking of death receptors.
Previous studies have suggested the possibility that death receptor trafficking might be a regulated process. In particular, Bennett et al. (86) reported that DNA damage increases the amount of cell surface Fas in a p53-dependent manner. Our results complement and extend those of Bennett et al. (86) by (i) showing that trafficking of TRAIL receptors is also regulated (Fig. 9B), (ii) implicating PKC in this process, and (iii) demonstrating that the PKC-mediated process can be observed in cells that lack p53 (HL-60, Fig. 5, D and E), as well as cells that contain p53 (e.g. HCT116, supplemental Fig. S9).
Although enzastaurin is often described as a PKC␤-selective inhibitor (66,87), the assignment of PKC␤ as the isoform responsible for modulating death receptor trafficking must be viewed as tentative. Even though the concentrations of enzastaurin utilized in this study are therapeutically achievable (64) and are similar to those required to inhibit PMA-induced PKC substrate phosphorylation in cells expressing PKC␤ (68), these effects are observed at concentrations where enzastaurin could conceivably inhibit other kinases (69), especially other PKC isoforms (63). On the other hand, the ability of another PKC␤selective inhibitor (supplemental Fig. S4B) and PKC␤ shRNA to mitigate the effects of PMA provides additional support for the view that PKC␤ might play a major role in modulating death receptor trafficking.
In view of the fact that PKC isoforms are often activated by growth factor signaling, we asked whether enzastaurin might also modulate TRAIL signaling in the absence of PMA. Consistent with this possibility, enzastaurin enhanced TRAIL binding as well as TRAIL-induced antiproliferative effects in a number of tissue culture cell lines ( Fig. 9 and supplemental Fig. S9). Moreover, enzastaurin synergistically enhanced TRAIL-induced apoptosis in clinical acute myelogenous leukemia isolates (Fig. 10). These results are consistent with the view that ongoing signaling might dampen the response to TRAIL even in the absence of exogenous phorbol esters. Further studies are required to determine the extent of this death ligand-inhibiting signaling in various preclinical and clinical settings as well as determine the effect of this signaling on the response to TRAIL and agonistic antibodies in the clinical setting.
Previous studies have indicated that the relationship between expression of TRAIL receptors and TRAIL sensitivity is complex. On the one hand, up-regulation of DR4 and/or DR5 after DNA damage (88,89) or proteasome inhibition (90 -92) is associated with enhanced TRAIL sensitivity. On the other hand, basal levels of expression of DR4 and DR5 levels have not always correlated with TRAIL sensitivity (93). This lack of correlation has previously been attributed to overexpression of decoy receptors, XIAP, antiapoptotic Bcl-2 family members, and/or c-FLIP (6,9,93) as well as post-translational modifications of DR4 and DR5 (19). The present results suggest that receptor trafficking also needs to be taken into account. In particular, basal measurements of cell surface DR4 or DR5 might not fully reflect the levels of receptor available at the cell surface upon prolonged ligand exposure. On the other hand, it is also important to emphasize that the present studies were all performed with cell lines that exhibit some degree of TRAIL sensitivity before treatment with PKC␤ shRNA or enzastaurin. Based on the mechanism identified through this study, it is unclear whether enzastaurin or PKC␤ shRNA would be able to overcome TRAIL resistance that results from overexpression of decoy receptors, c-FLIP, antiapoptotic Bcl-2 family members, or XIAP. Further study is required to address this issue.
In summary, the results of this study not only suggest that PKC␤ mediates the effects of PMA on death receptor signaling but also demonstrate that these effects reflect an effect on death ligand trafficking. Whether enzastaurin, which enhances cell surface death receptors, will be a useful addition to death ligand treatment requires further preclinical and clinical investigation.