Evidence That Protein Kinase Cε Mediates Phorbol Ester Inhibition of Calphostin C- and Tumor Necrosis Factor-α-induced Apoptosis in U937 Histiocytic Lymphoma Cells
1998; Elsevier BV; Volume: 273; Issue: 37 Linguagem: Inglês
10.1074/jbc.273.37.24115
ISSN1083-351X
AutoresGeorge C. Mayne, Andrew W. Murray,
Tópico(s)Cell death mechanisms and regulation
ResumoProtein kinase C (PKC) activators, such as the tumor-promoting phorbol esters, have been reported to protect several cell lines from apoptosis induced by a variety of agents. Recent evidence suggests that PKCε is involved in protection of cardiac myocytes from hypoxia-induced cell death (Gray, M. O., Karliner, J. S., and Mochly-Rosen, D. (1997) J. Biol. Chem.272, 30945–30951). We investigated the protective effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on U937 histiocytic lymphoma cells induced to undergo apoptosis by tumor necrosis factor-α (TNF-α) or by the specific PKC inhibitor calphostin C. U937 cells were transiently permeabilized with a peptide (εV1-2) derived from the V1 region of PKCε that has been reported to specifically block translocation of PKCε. The εV1-2 peptide blocked the inhibitory effect of TPA on both TNF-α- and calphostin C-induced apoptosis. A scrambled version of εV1-2 and a peptide reported to inhibit PKCβ translocation (βC2-4) had no effect on the ability of TPA to inhibit apoptosis. These results suggest that PKCε is required for the protective effect of TPA in TNF-α- and calphostin C-induced apoptosis. Furthermore, calphostin C reduced membrane-associated PKCε activity and immunoreactivity, suggesting that PKCε may play an important role in leukemic cell survival. Protein kinase C (PKC) activators, such as the tumor-promoting phorbol esters, have been reported to protect several cell lines from apoptosis induced by a variety of agents. Recent evidence suggests that PKCε is involved in protection of cardiac myocytes from hypoxia-induced cell death (Gray, M. O., Karliner, J. S., and Mochly-Rosen, D. (1997) J. Biol. Chem.272, 30945–30951). We investigated the protective effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on U937 histiocytic lymphoma cells induced to undergo apoptosis by tumor necrosis factor-α (TNF-α) or by the specific PKC inhibitor calphostin C. U937 cells were transiently permeabilized with a peptide (εV1-2) derived from the V1 region of PKCε that has been reported to specifically block translocation of PKCε. The εV1-2 peptide blocked the inhibitory effect of TPA on both TNF-α- and calphostin C-induced apoptosis. A scrambled version of εV1-2 and a peptide reported to inhibit PKCβ translocation (βC2-4) had no effect on the ability of TPA to inhibit apoptosis. These results suggest that PKCε is required for the protective effect of TPA in TNF-α- and calphostin C-induced apoptosis. Furthermore, calphostin C reduced membrane-associated PKCε activity and immunoreactivity, suggesting that PKCε may play an important role in leukemic cell survival. protein kinase C 12-O-tetradecanoylphorbol-13-acetate tumor necrosis factor-α calphostin C interleukin-3 sphingosine 1-phosphate. The phospholipid-dependent protein kinase C (PKC)1 family of isozymes has a central role in the transduction of extracellular signals and has been implicated in tumor promotion (1Hug H. Sarre T.F. Biochem. J. 1993; 291: 329-343Crossref PubMed Scopus (1218) Google Scholar). PKCs also appear to be important in the regulation of apoptosis, and several reports have indicated that PKC activation can inhibit apoptosis. For example, the tumor-promoting phorbol ester and potent PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) prevents apoptosis induced by tumor necrosis factor-α (TNF-α), Fas, and microtubule-disrupting drugs in human lymphoma cells (2Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1618) Google Scholar, 3Takano Y. Okudaira M. Pathol. Res. Pract. 1993; 189: 197-203Crossref PubMed Scopus (55) Google Scholar); by growth factor deprivation in leukemic and normal myeloid cells (4Lotem J. Cragoe Jr., E.J. Sachs L. Blood. 1991; 78: 953-960Crossref PubMed Google Scholar); and by anticancer drug treatment of leukemic cells and a human breast cancer cell line (5Solary E. Bertrand R. Kohn K.W. Pommier Y. Blood. 1993; 81: 1359-1368Crossref PubMed Google Scholar, 6Geier A. Beery R. Haimsohn M. Hemi R. Malik Z. Karasik A. In Vitro Cell. Dev. Biol. 1994; 30A: 867-874Crossref Scopus (17) Google Scholar). TPA also inhibits apoptotic oligonucleosomal DNA degradation in isolated nuclei from thymocytes (7McConkey D.J. Hartzell P. Jondal M. Orrenius S. J. Biol. Chem. 1989; 264: 13399-13402Abstract Full Text PDF PubMed Google Scholar). Other PKC activators have also been reported to inhibit apoptosis. These include phorbol dibutyrate, which protects mouse spleen blast cells against interleukin-2 withdrawal (8Rodriguez-Tarduchy G. Lopez Rivas A. Biochem. Biophys. Res. Commun. 1989; 164: 1069Crossref PubMed Scopus (89) Google Scholar), and mezerein and bryostatin-1, which block apoptosis induced by the sphingolipid ceramide (9Jarvis W.D. Fornari Jr., F.A. Browning J.L. Gewirtz D.A. Kolesnick R.N. Grant S. J. Biol. Chem. 1994; 269: 31685-31692Abstract Full Text PDF PubMed Google Scholar). A synthetic analogue of the natural PKC activator diacylglycerol has also been reported to prevent apoptosis in human leukemic cells induced by ceramide and the isoprenoids farnesol and geranylgeraniol (9Jarvis W.D. Fornari Jr., F.A. Browning J.L. Gewirtz D.A. Kolesnick R.N. Grant S. J. Biol. Chem. 1994; 269: 31685-31692Abstract Full Text PDF PubMed Google Scholar, 10Voziyan P.A. Haug J.S. Melnykovych G. Biochem. Biophys. Res. Commun. 1995; 212: 479-486Crossref PubMed Scopus (68) Google Scholar, 11Ohizumi H. Masuda Y. Yoda M. Hashimoto S. Aiuchi T. Nakajo S. Sakai I. Ohsawa S. Nakaya K. Anticancer Res. 1997; 17: 1051-1057PubMed Google Scholar). It should be noted, however, that there are some reports that demonstrate that PKC activation can induce or increase apoptosis in some cell types (12Kizaki H. Shimada H. Ishimura Y. J. Biochem. (Tokyo). 1989; 105: 673-675Crossref PubMed Scopus (17) Google Scholar, 13Mcbain J.A. Eastman A. Simmons D.L. Pettit G.R. Mueller G.C. Int. J. Cancer. 1996; 67: 715-723Crossref PubMed Scopus (23) Google Scholar, 14Jones B.A. Rao Y.P. Stravitz R.T. Gores G.J. Am. J. Physiol. 1997; 272: G1109-G1115PubMed Google Scholar). The effects of PKC activation on apoptosis may therefore be cell type-specific and could be determined by factors such as the rate of PKC down-regulation. Evidence has recently emerged suggesting that specific PKC isozymes may be involved in the prevention of apoptosis. Mochly-Rosen and co-workers (15Gray M.O. Karliner J.S. Mochly-Rosen D. J. Biol. Chem. 1997; 272: 30945-30951Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar) have used peptides that specifically block the interaction of PKC subforms with membrane-associated binding proteins and have demonstrated that a peptide that inhibited membrane translocation of PKCε blocked the protection of cardiac myocytes from hypoxia-induced cell death. In another study, apoptosis in human leukemic cells induced by TNF-α, anti-Fas antibody, sphingomyelinase, and ceramide was associated with the redistribution of PKCδ and PKCε from the membrane to the cytosol. These redistributions were prevented by concentrations of TPA that blocked apoptosis (16Sawai H. Okazaki T. Takeda Y. Tashima M. Sawada H. Okuma M. Kishi S. Umehara H. Domae N. J. Biol. Chem. 1997; 272: 2452-2458Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Further reports suggest that PKCε is anti-apoptotic in some cells. For example, in HT58 human lymphoblastic cells, TPA causes growth inhibition and down-regulation of PKC (50Mihalik R. Farkas G. Kopper L. Benczur M. Farago A. Int. J. Biochem. Cell Biol. 1996; 28: 925-933Crossref PubMed Scopus (13) Google Scholar). These cells could be induced to undergo apoptosis by the general kinase inhibitor staurosporine, but only when the cells had been pretreated with low concentrations of TPA that down-regulated PKCε, but not PKCα and PKCβ (17Basu A. Cline J.S. Int. J. Cancer. 1995; 63: 597-603Crossref PubMed Scopus (39) Google Scholar). In oncogene-transformed rat embryo fibroblasts, susceptibility to the anticancer drug cisplatin is increased, whereas PKCε expression is decreased compared with nontransformed cells. Stable transfection of these cells with PKCε prevents cisplatin-induced apoptosis and also protects these cells against cisplatin cytotoxicity (17Basu A. Cline J.S. Int. J. Cancer. 1995; 63: 597-603Crossref PubMed Scopus (39) Google Scholar). Taken together, these reports suggest that PKCε may be of particular significance in the negative regulation of apoptosis, and this possibility has been explored further in this study. We have used U937 histiocytic lymphoma cells, in which apoptosis induced by ceramide, TNF-α, and the PKC inhibitor calphostin C is strongly inhibited by TPA. In this study, we used peptides based on unique sequences within PKC isozymes that specifically block the binding of individual isozymes to anchoring proteins, termed RACK proteins (receptors foractivated C-kinase). Recent work has demonstrated that a peptide corresponding to amino acids 14–21 in the V1 region of PKCε prevents phorbol ester-induced translocation of PKCε and inhibits contraction in cultured cardiac myocytes (18Johnson J.A. Gray M.O. Chen C.-H. Mochly-Rosen D. J. Biol. Chem. 1996; 271: 24962-24966Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). In a further study, the εV1-2 peptide inhibited hypoxic preconditioning and phorbol ester-mediated protection of cardiac myocytes from hypoxia-induced cell death. In this study, we found that this peptide inhibited the ability of TPA to prevent apoptosis induced by TNF-α and calphostin C (calC). calC also reduced putative PKCε activity and displaced PKCε from the membrane to the cytosol. ATP, benzamidine, calC, Hoechst 33258, phenylmethylsulfonyl fluoride, and TPA were obtained from Sigma. [γ-32P]ATP (10,000 Ci/mmol) was obtained from Bresatec (Adelaide, South Australia, Australia). Leupeptin was from Tokyo Kasai Kogyo Co. Aprotinin was obtained from Boehringer Mannheim. Primary antibodies for PKC isozymes were obtained from Santa Cruz Biotechnology, Inc., and secondary antibodies were from Silenius Laboratories (Hawthorn, Australia). TNF-α was kindly provided by Dr. D. Rathjen (Women's and Children's Hospital, Adelaide, South Australia). PKC SelectideTM substrate (neurogranin-(28–43)) and the PKCε translocation inhibitor peptide (EAVSLKPT; εV1-2) were from Calbiochem. PKC peptide substrate (epidermal growth factor receptor;H-Val-Arg-Lys-Arg-Thr-Leu-Arg-Arg-Leu-NH2), PKC pseudosubstrate-(19–31) inhibitor peptide, the scrambled peptide (LSETKPAV; scrambled εV1-2), and the PKCβ translocation inhibitor peptide (SLNPEWNET; βC2-4) were obtained from Auspep (Parkville, Australia). HYPERfilm-ECL was obtained from Amersham Pharmacia Biotech. Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone was obtained from Enzyme Systems Products (Dublin, CA). ECL reagent was obtained from NEN Life Science Products. The TransPortTM transient permeabilization kit was from Life Technologies, Inc. U937 human myeloid leukemic cells (American Type Culture Collection, Rockville, MD) were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 0.1 mm nonessential amino acids, 100 units/ml penicillin, and 100 mg/ml streptomycin. Cells were transferred to RPMI 1640 medium supplemented with 2.5% heat-inactivated fetal calf serum for treatment with TNF-α or calC. After addition of calC, cells were illuminated for 10 min with a 40-watt incandescent lamp at a distance of 30 cm. Cytosolic and solubilized particulate fractions were prepared by sonicating (3 × 10 s) cells in buffer A (20 mm Tris-HCl (pH 7.4), 5 mm EGTA, 2 mm EDTA, 10 mmbenzamidine, 0.01% leupeptin, 10 mm 2-mercaptoethanol, 2 mm phenylmethylsulfonyl fluoride, 0.1 unit/ml aprotinin, and 0.25 m sucrose). The sonicate was cleared of unlysed cells by centrifugation at 500 × g for 5 min, and the cleared supernatant was centrifuged at 100,000 × g for 20 min at 4 °C. The pellet was sonicated in buffer A containing Triton X-100 (as indicated), incubated on ice for 30 min, and centrifuged as described before to produce a supernatant particulate fraction. Aliquots of cytosolic and particulate fraction extracts (described above) were added to well loading buffer (20 mm Tris-HCl (pH 6.8), 40% sucrose, 6% SDS, and 10 mm 2-mercaptoethanol) (2 vol extract: 1 vol loading buffer) and heated for 5 min at 100 °C. Aliquots (5 μg of protein; determined by a modified method (19Harrington C.R. Anal. Biochem. 1990; 186: 285-287Crossref PubMed Scopus (66) Google Scholar) of Lowry et al. (49Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar)) were size-separated by SDS-polyacrylamide electrophoresis using 12% gels in a Bio-Rad minigel system (200 V, 45 min) and then electrophoretically transferred to Schleicher & Schuell nitrocellulose paper (0.2 μm; 100 V, 90 min) as described (20Jones M.J. Murray A.W. J. Biol. Chem. 1995; 270: 5007-5013Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Membranes were blocked with 5% milk powder and incubated with the primary antibodies for 1 h at 37 °C. To determine specificity, primary antibodies were preincubated for 2 h with 10-fold excess peptide against which the antibody was raised. After incubation with primary antibodies, the membranes were washed, incubated with the horseradish peroxidase-conjugated secondary antibody for 45 min, and washed again. The bound secondary antibody was detected with ECL reagent. Chemiluminescence was photographed with HYPERfilm-ECL. PKC activity in the particulate fraction was measured using the method of Durkin et al. (21Durkin J.P. Tremblay R. Buchan A. Blosser J. Chakravarthy B. Mealing G. Morley P. Song D. J. Neurochem. 1996; 66: 951-962Crossref PubMed Scopus (58) Google Scholar) with modifications. This method does not require a preliminary extraction with subsequent reconstitution and artificial activation of the enzyme with Ca2+ and phosphatidylserine. PKC activity in the particulate fraction was measured using a PKC peptide substrate based on the epidermal growth factor receptor or a highly selective peptide substrate based on neurogranin-(28–43) that allows measurement of PKC in crude extracts, and a PKC inhibitor peptide based on the pseudosubstrate-(19–31) region of PKC. The assay reaction mixture contained 4–7 μg of protein from the particulate fraction in assay buffer (50 mm Tris-HCl (pH 7.5), 5 mm MgCl2, 1 mCaCl2, 100 μm sodium vanadate, 100 μm sodium pyrophosphate, 1 mm sodium fluoride, and 100 μm phenylmethylsulfonyl fluoride) and 50 μm peptide substrate in a final volume of 90 μl. The reaction was started by the addition of 10 μl of 500 μm[γ-32P]ATP (220 cpm/pmol in Tris buffer, 0.5 μCi/assay) and stopped after 10 min by the addition of 10 μl of 75 mm orthophosphoric acid. The samples were clarified by centrifugation at 16,000 × g for 5 min, and a 90-μl sample of each supernatant was applied to Whatman P-81 paper (2 cm2). The papers were washed twice in 75 mmorthophosphoric acid with gentle agitation for 10 min. The radioactivity bound to the washed papers was determined by liquid scintillation counting. Qualitative assessment of DNA degradation was performed by agarose gel electrophoresis (22Gong J. Traganos F. Darzynkiewicz Z. Anal. Biochem. 1994; 218: 314-319Crossref PubMed Scopus (649) Google Scholar). Pelleted cells (5 × 106) were fixed by suspension in 70% ethanol at −20 °C overnight. Cells were then centrifuged at 200 × g for 5 min, and the ethanol was thoroughly removed. Cell pellets were resuspended in 40 μl of phosphate/citrate buffer (192 parts 0.2 m Na2HPO4 and 8 parts 0.1 m citric acid (pH 7.8)) at room temperature for 30 min. After centrifugation at 1000 × g for 5 min, the supernatant was transferred to new tubes, dried by vacuum in a SpeedVac (Savant Instruments, Inc., Farmingdale, NY), and reconstituted with 15 μl of sterile distilled water. The DNA extract was then incubated with the addition of 3 μl of 0.25% Nonidet P-40 and 3 μl of RNase (1 mg/ml) for 30 min at 37 °C. The extract was incubated for a further 30 min with proteinase K (3 μl, 1 mg/ml). After this digestion, 5 μl of loading buffer (0.25% bromphenol blue, 0.25% xylene cyanol, and 30% glycerol) was added, and the extract was fractionated by electrophoresis on 1.8% agarose at 5 V/cm for 3 h. DNA was visualized under UV light after staining with 0.5 g/ml ethidium bromide. U937 cells (5 × 105/ml) were treated with test compounds in RPMI 1640 medium containing 2.5% fetal calf serum. Chromatin was visualized by fluorescent microscopy after a 30-min incubation with the cell-permeable fluorochrome Hoechst 33258 (10 μg/ml). The proportion of cells undergoing apoptosis at a given time was determined by counting cells exhibiting two or more membrane blebs and brightly stained condensed and fragmented chromatin. At least 200 cells were counted for each sample. The proportion of cells undergoing apoptosis was also determined using flow cytometry (23van de Loosdrecht A.A. Ossenkoppele G.J. Beelen R.H.J. Broekhoven M.G. Drager A.M. Langenhuijsen M.M. Exp. Hematol. (N. Y.). 1993; 21: 1628-1639PubMed Google Scholar). Flow cytometry results are the percentage of cells (10,000) containing subdiploid DNA. In initial experiments, U937 cells were transiently permeabilized using the TransPortTM kit according to the manufacturer's instructions. In subsequent experiments, cells were permeabilized by electroporation. U937 cells were suspended at 10 × 106 cells/ml in serum-free RPMI 1640 medium, and 800-μl aliquots were electroporated with or without the indicated peptides at 330 microfarads and 200 V at room temperature. The electroporated cells were kept at room temperature for 10 min and then transferred to RPMI 1640 medium containing 2.5% fetal calf serum to a final concentration of 5 × 105cells/ml for treatment. In the presence of light, calC is a highly specific PKC inhibitor that interacts with the regulatory domain of the enzyme and inhibits phorbol ester binding at high concentrations (24Tamaoki T. Methods Enzymol. 1991; 201: 340-347Crossref PubMed Scopus (328) Google Scholar). calC was chosen for this study because, unlike TNF-α or ceramide, calC induced apoptosis in a large proportion of cells (Fig. 1), thus making biochemical studies more interpretable. Apoptosis induced by low concentrations of calC was acutely blocked by TPA (Figs. 1 and 2). The protective effect of TPA was less after prolonged incubations (Fig. 1), possibly because of down-regulation of PKC. calC induced apoptosis in U937 cells in a dose-dependent manner and generated characteristic oligonucleosomal DNA fragmentation (Fig. 3), membrane blebbing (data not shown), and chromatin condensation (Fig. 2). Using time-lapse microcinematography, it was observed that calC-treated U937 cells underwent fragmentation into clusters of apoptotic bodies in a similar way to cells treated with TNF-α (data not shown).Figure 2Effect of TPA on calC-induced chromatin condensation in U937 cells. U937 cells were treated with either 0.1% Me2SO (DMSO) or 125 nm calC with or without 100 nm TPA for 6 h. Chromatin in intact cells was stained with 10 μg/ml Hoechst 33258 for 30 min.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Effect of TPA on calC- and TNF-α-induced DNA fragmentation in U937 cells. U937 cells (5 × 106) were treated with 0.1% Me2SO (DMSO), with 125 nm calC with or without 100 nm TPA for 7 h, or with 1000 units/ml TNF-α with or without 100 nm TPA for 3 h. Analysis of DNA fragmentation was performed as described under “Experimental Procedures.” The results are representative of three different experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The main intracellular receptors for TPA are the PKC family of isozymes. Because U937 cells have been reported to contain different profiles of PKC isozymes, we screened our U937 cells using immunoblotting. U937 cells have been reported to express PKCα, PKCβI, PKCδ, and PKCζ, (25Pongracz J. Deacon E.M. Johnson G.D. Burnett D. Lord J.M. Leuk. Res. 1996; 20: 319-326Crossref PubMed Scopus (21) Google Scholar); PKCα, PKCβ, PKCε, and PKCζ (26Ways D.K. Messer B.R. Garris T.O. Qin W. Cook P.P. Parker P.J. Cancer Res. 1992; 52: 5604-5609PubMed Google Scholar, 27Ways D.K. Qin W. Garris T.O. Chen J. Hao E. Cooper D.R. Usala S.J. Parker P.J. Cook P.P. Cell Growth Differ. 1994; 5: 161-169PubMed Google Scholar); or PKCβI, PKCβII, PKCε, and PKCζ (28Kiley S.C. Parker P.J. J. Cell Sci. 1995; 108: 1003-1016PubMed Google Scholar). The U937 cells used in this study expressed PKCβI, PKCβII, PKCε, and PKCζ and very low levels of PKCδ, but did not contain detectable levels of PKCα or PKCθ (Fig. 4). PKCβI and PKCβII were predominantly located in the cytosol, whereas PKCε and PKCζ were distributed between the cytosolic and particulate fractions (Fig. 4). To determine whether PKCε translocation is required for the protective effect of TPA, we used a peptide derived from the V1 region of PKCε (εV1-2) that specifically inhibits the TPA-induced membrane translocation of this isoform of PKC. The εV1-2 peptide blocks the interaction between PKCε and anchoring proteins that have been termed RACK proteins. Transient permeabilization with this peptide has been demonstrated to block TPA-induced contraction and protection against hypoxia-induced cell death in cardiac myocytes (15Gray M.O. Karliner J.S. Mochly-Rosen D. J. Biol. Chem. 1997; 272: 30945-30951Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar,18Johnson J.A. Gray M.O. Chen C.-H. Mochly-Rosen D. J. Biol. Chem. 1996; 271: 24962-24966Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). Furthermore, expression of the V1 fragment of PKCε from which εV1-2 was derived blocked TPA enhancement of neurite outgrowth in PC-12 rat neuroblastoma cells (29Hundle B. McMahon T. Dadgar J. Chen C.-H. Mochly-Rosen D. Messing R.O. J. Biol. Chem. 1997; 272: 15028-15035Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Similar peptides have been identified with specificity for PKCδ (29Hundle B. McMahon T. Dadgar J. Chen C.-H. Mochly-Rosen D. Messing R.O. J. Biol. Chem. 1997; 272: 15028-15035Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) and PKCβ (βC2-4) (30Ron D. Luo J. Mochly-Rosen D. J. Biol. Chem. 1995; 270: 24180-24187Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). Clearly, such peptides provide a powerful tool to evaluate the biological roles of individual PKC subforms. Figs. 5 and 6 show the effects of the εV1-2 peptide on the TPA inhibition of calC-induced apoptosis. Apoptosis was quantified both by Hoechst chromatin staining and by fluorescence-activated cell sorter analysis. The εV1-2 peptide attenuated the protective effect of TPA against calC- or TNF-α-induced apoptosis, and the effect was dose-dependent (Fig. 5). However, the peptide had no effect on the levels of apoptosis induced by calC or TNF-α and did not induce apoptosis in untreated cells (data not shown). No significant effect was observed with a scrambled peptide containing the same amino acids as εV1-2 or with a peptide that blocks PKCβI and PKCβII binding to RACK proteins (βC2-4 (Figs. 5 and 6). These data strongly support a role for PKCε in mediating the protective effect of TPA against calC- and TNF-α-induced apoptosis. It should be noted that a low concentration of TPA (10 nm) was used in these experiments, a concentration that gave only partial protection against apoptosis (Fig. 5). The εV1-2 peptide was less effective at a higher concentration of TPA (100 nm) (data not shown), as reported previously in cardiac myocytes (18Johnson J.A. Gray M.O. Chen C.-H. Mochly-Rosen D. J. Biol. Chem. 1996; 271: 24962-24966Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar).Figure 6Effect of εV1-2 on prevention of calC-induced chromatin condensation by TPA. U937 cells were electroporated with the indicated peptides (10 nm) as described under “Experimental Procedures” and were then treated with either 0.1% Me2SO or 125 nm calC with or without 10 nm TPA for 3 h. Chromatin in intact cells was stained with 10 μg/ml Hoechst 33258 for 30 min.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A prediction from these data is that the εV1-2 peptide should specifically inhibit the TPA-induced translocation of PKCε in U937 cells. Such inhibition has been reported in cardiac myocytes (15Gray M.O. Karliner J.S. Mochly-Rosen D. J. Biol. Chem. 1997; 272: 30945-30951Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 18Johnson J.A. Gray M.O. Chen C.-H. Mochly-Rosen D. J. Biol. Chem. 1996; 271: 24962-24966Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). However, in a number of experiments including a time course (0–60 min) and several concentrations of Triton X-100, the εV1-2 peptide did not significantly inhibit 10 nm TPA-induced translocation of PKCε by Western blotting (data not shown). A similar lack of effect of the εV1-2 peptide was found in preliminary experiments with immunofluorescence localization (data not shown). We suggest two reasons for the inability to demonstrate an inhibitory effect on PKCε translocation. First, U937 cells have a high basal level of particulate PKCε (Figs. 4 and 7) (26Ways D.K. Messer B.R. Garris T.O. Qin W. Cook P.P. Parker P.J. Cancer Res. 1992; 52: 5604-5609PubMed Google Scholar), which generates a high background level of immunoreactivity. Second, the peptide blocks only the binding of PKCε to RACK proteins. Interaction of PKCε with other binding proteins will not be inhibited. For example, PKCα, PKCε, and PKCζ have been reported to bind to caveolin (31Oka N. Yamamoto M. Schwencke C. Kawabe J. Ebina T. Ohno S. Couet J. Lisanti M.P. Ishikawa Y. J. Biol. Chem. 1997; 272: 33416-33421Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). PKC also binds to AKAP79 (32Klauck T.M. Faux M.C. Labudda K. Langeberg L.K. Jaken S. Scott J.D. Science. 1996; 271: 1589-1592Crossref PubMed Scopus (483) Google Scholar) and to PICK1 (33Staudinger J. Zhou J. Burgess R. Elledge S.J. Olson E.N. J. Cell Biol. 1995; 128: 263-271Crossref PubMed Scopus (265) Google Scholar). Consequently, any effects of εV1-2 on PKCε translocation will depend on the relative proportions of RACK and other binding proteins in a particular cell type. We are currently investigating possible effects of the peptide on PKCε intracellular localization in the presence of TPA using confocal microscopy. The above results suggest that the binding of PKCε to RACK proteins mediates the protective effect of TPA against both calC- and TNF-α-induced apoptosis in U937 cells. These results further suggest that the anti-apoptotic effect of TPA requires the phosphorylation of target proteins by PKCε, as PKC only binds to RACK proteins in the presence of activating cofactors (34Mochly-Rosen D. Khaner H. Lopez J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3997-4000Crossref PubMed Scopus (442) Google Scholar). Recent reports also suggest that PKCε plays a role in the survival of cells that have not been stimulated with agents that directly activate PKC. Expression of PKCε, but not of PKCδ, extended the survival of interleukin-3 (IL-3)-dependent cells in the absence of the cytokine (35Gubina E. Rinaudo M.S. Szallasi Z. Blumberg P.M. Mufson R.A. Blood. 1998; 91: 823-829Crossref PubMed Google Scholar), and stable expression of PKCε protected transformed rat embryo fibroblasts from cisplatin-induced apoptosis (17Basu A. Cline J.S. Int. J. Cancer. 1995; 63: 597-603Crossref PubMed Scopus (39) Google Scholar). However, we found that the εV1-2 peptide had no effect on the levels of apoptosis induced by calC and TNF-α or in untreated cells, suggesting that association of PKCε with RACK proteins is not involved in U937 cell survival. PKCε does, however, appear to be inhibited in TNF-α-, Fas- and ceramide-induced apoptosis, where PKCε is displaced from the particulate fraction to the cytosol (16Sawai H. Okazaki T. Takeda Y. Tashima M. Sawada H. Okuma M. Kishi S. Umehara H. Domae N. J. Biol. Chem. 1997; 272: 2452-2458Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). We therefore determined if calC also displaced PKCε from the membrane and whether membrane-associated PKCε was active in U937 cells. PKCε immunoreactivity was divided between the particulate and cytosolic fractions of proliferating U937 cells, as has been reported by others (26Ways D.K. Messer B.R. Garris T.O. Qin W. Cook P.P. Parker P.J. Cancer Res. 1992; 52: 5604-5609PubMed Google Scholar). Particulate PKCε immunoreactivity was reduced at 10 min by calC and TNF-α (Fig. 7) and ceramide (data not shown) and was associated with increased immunoreactivity in the cytosol (Fig. 7). In calC-treated cells, the reduction in particulate-associated PKCε was sustained for at least 6 h (data not shown). This effect was reversed by TPA (Fig. 7). calC induced apoptosis and displaced particulate PKCε only when U937 cells were exposed to the inhibitor in the presence of light (Fig. 8). This suggests that the effects of calC may be due to direct inhibition of PKC, as calC must be illuminated to inhibit the enzyme (36Bruns R.F. Miller F.D. Merriman R.L. Howbert J.J. Hea
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