Oncogenic Ras Mediates Apoptosis in Response to Protein Kinase C Inhibition through the Generation of Reactive Oxygen Species
2000; Elsevier BV; Volume: 275; Issue: 50 Linguagem: Inglês
10.1074/jbc.m007154200
ISSN1083-351X
AutoresJames S. Liou, Changyan Chen, James S. Chen, Douglas V. Faller,
Tópico(s)Cancer-related Molecular Pathways
ResumoRas is a well established modulator of apoptosis. Suppression of protein kinase C (PKC) activity can selectively induce apoptosis in cells expressing a constitutively activated Ras protein. We wished to determine whether reactive oxygen species serve as an effector of Ras-mediated apoptosis. Ras-transformed NIH/3T3 cells contained higher basal levels of intracellular H2O2 compared with normal NIH/3T3 cells, and PKC inhibition up-regulated ROS to 5-fold greater levels in Ras-transformed cells than in normal cells. Treatment withN-acetyl-l-cysteine reduced both the basal and inducible levels of intracellular H2O2 in NIH/3T3-Ras cells and antagonized the induction of apoptosis by PKC inhibition. Culturing NIH/3T3-Ras cells in low oxygen conditions, which prevents ROS generation, also inhibited the apoptotic response to PKC inhibition. These results suggest that reactive oxygen species are necessary as downstream effectors of the Ras-mediated apoptotic response to PKC inhibition. However, the generation of ROS alone is not sufficient to induce apoptosis in Ras-transformed cells because inhibition of cell cycle progression prevented the induction of apoptosis in NIH/3T3-Ras cells without inhibiting the generation of intracellular H2O2 observed after PKC inhibition. These findings suggest that continued cell cycle progression of Ras-transformed cells during PKC inhibition is also necessary for the induction of apoptosis. Ras is a well established modulator of apoptosis. Suppression of protein kinase C (PKC) activity can selectively induce apoptosis in cells expressing a constitutively activated Ras protein. We wished to determine whether reactive oxygen species serve as an effector of Ras-mediated apoptosis. Ras-transformed NIH/3T3 cells contained higher basal levels of intracellular H2O2 compared with normal NIH/3T3 cells, and PKC inhibition up-regulated ROS to 5-fold greater levels in Ras-transformed cells than in normal cells. Treatment withN-acetyl-l-cysteine reduced both the basal and inducible levels of intracellular H2O2 in NIH/3T3-Ras cells and antagonized the induction of apoptosis by PKC inhibition. Culturing NIH/3T3-Ras cells in low oxygen conditions, which prevents ROS generation, also inhibited the apoptotic response to PKC inhibition. These results suggest that reactive oxygen species are necessary as downstream effectors of the Ras-mediated apoptotic response to PKC inhibition. However, the generation of ROS alone is not sufficient to induce apoptosis in Ras-transformed cells because inhibition of cell cycle progression prevented the induction of apoptosis in NIH/3T3-Ras cells without inhibiting the generation of intracellular H2O2 observed after PKC inhibition. These findings suggest that continued cell cycle progression of Ras-transformed cells during PKC inhibition is also necessary for the induction of apoptosis. protein kinase B mitogen-activated protein reactive oxygen species protein kinase C phorbol 12-myristate 13-acetate 1-O-hexadecyl-2-O-methyl-rac-glycerol dichlorodihydrofluorescein diacetate fluorescent-activated cell sorter trichostatin A N-acetylcysteine platelet-derived growth factor cyclosporin A Ras-binding domain The ras proto-oncogene serves as a molecular switch controlling a variety of cellular processes including proliferation (1Mulcahy L.S. Smith M.R. Stacey D.W. Nature. 1985; 313: 241-243Crossref PubMed Scopus (592) Google Scholar), differentiation (2Hagag N. Halegoua S. Viola M. Nature. 1986; 319: 680-682Crossref PubMed Scopus (302) Google Scholar), and senescence (3Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3942) Google Scholar). Point mutations that cause single amino acid substitutions in the normal cellular Ras protein lead to its constitutive activation (4Barbacid M. Annu. Rev. Biochem. 1987; 56: 779-827Crossref PubMed Scopus (3772) Google Scholar). This dominant mutant form of Ras plays a major role in the multistep progression of tumorigenesis in many human tumors, with oncogenicras mutations occurring in approximately 30% of all human tumors (5Bos J.L. Cancer Res. 1989; 49: 4682-4689PubMed Google Scholar). Strikingly, over 90% of pancreatic tumors and 50% of colorectal tumors analyzed contain ras mutations. Our laboratory and others (6Downward J. Curr. Opin. Genet. & Dev. 1998; 8: 49-54Crossref PubMed Scopus (507) Google Scholar, 7Chen C.Y. Liou J. Forman L.W. Faller D.V. J. Biol. Chem. 1998; 273: 16700-16709Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 8Chen C.Y. Faller D.V. Oncogene. 1995; 11: 1487-1498PubMed Google Scholar) have established Ras as a modulator of apoptosis. Paradoxically, Ras can either inhibit or promote apoptosis, with the outcome probably dependent upon cell type and the presence of other pro-apoptotic or anti-apoptotic signals (6Downward J. Curr. Opin. Genet. & Dev. 1998; 8: 49-54Crossref PubMed Scopus (507) Google Scholar). Ras can inhibit apoptosis induced by a variety of stimuli, and protection against these apoptotic stimuli by Ras is thought to contribute to tumorigenesis. Studies have shown that phosphoinositide-3-OH kinase, a direct downstream effector of Ras, plays a crucial role in mediating Ras protection against apoptosis by activating PKB1/AKT (9Kauffmann-Zeh A. Rodriguez-Viciana P. Ulrich E. Gilbert C. Coffer P. Downward J. Evan G. Nature. 1997; 385: 544-548Crossref PubMed Scopus (1071) Google Scholar, 10Khwaja A. Rodriguez-Viciana P. Wennstrom S. Warne P.H. Downward J. EMBO J. 1997; 16: 2783-2793Crossref PubMed Scopus (935) Google Scholar). PKB/AKT activation may prevent apoptosis by phosphorylating the pro-apoptotic protein BAD (11Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4936) Google Scholar), allowing it to be sequestered by 14-3-3 proteins (12Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2253) Google Scholar). Alternatively, PKB/AKT may also activate NF-κB as a protective mechanism against apoptosis (13Romashkova J.A. Makarov S.S. Nature. 1999; 401: 86-90Crossref PubMed Scopus (1666) Google Scholar), as NF-κB activity is known to be protective against various forms of apoptosis (14Yin Foo S. Nolan G.P. Trends Genet. 1999; 15: 229-235Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar), including Ras-mediated apoptosis. Inhibition of NF-κB activity results in apoptosis of Ras-transformed cells (15Mayo M.W. Wang C.Y. Cogswell P.C. Rogers-Graham K.S. Lowe S.W. Der C.J. Baldwin Jr., A.S. Science. 1997; 278: 1812-1815Crossref PubMed Scopus (506) Google Scholar). Similarly, expression of the transcriptional repressor protein Par-4, which can inhibit NF-κB transcriptional activity, also induces apoptosis in Ras-transformed cells (16Nalca A. Qiu S.G. El-Guendy N. Krishnan S. Rangnekar V.M. J. Biol. Chem. 1999; 274: 29976-29983Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In addition to activation of phosphoinositide-3-OH kinase by Ras, activation of the Raf-MAP kinase pathway may also be necessary for Ras-mediated protection against apoptosis, downstream of growth factor signaling (17Kinoshita T. Shirouzu M. Kamiya A. Hashimoto K. Yokoyama S. Miyajima A. Oncogene. 1997; 15: 619-627Crossref PubMed Scopus (103) Google Scholar). Curiously, Ras activation of the Raf-MAP kinase pathway may actually enhance apoptosis induced by c-Myc (9Kauffmann-Zeh A. Rodriguez-Viciana P. Ulrich E. Gilbert C. Coffer P. Downward J. Evan G. Nature. 1997; 385: 544-548Crossref PubMed Scopus (1071) Google Scholar). Ras can sensitize cells to apoptosis induced by a variety of stimuli (6Downward J. Curr. Opin. Genet. & Dev. 1998; 8: 49-54Crossref PubMed Scopus (507) Google Scholar), and high level expression of oncogenic Ras alone can induce apoptosis as well (18Joneson T. Bar-Sagi D. Mol. Cell. Biol. 1999; 19: 5892-5901Crossref PubMed Scopus (153) Google Scholar). Our laboratory has demonstrated that the inhibition of PKC activity can also induce apoptosis in cells expressing activated Ras (7Chen C.Y. Liou J. Forman L.W. Faller D.V. J. Biol. Chem. 1998; 273: 16700-16709Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 8Chen C.Y. Faller D.V. Oncogene. 1995; 11: 1487-1498PubMed Google Scholar). Like NF-κB, PKC can also protect against many forms of apoptosis (19Lucas M. Sanchez-Margalet V. Gen. Pharmacol. 1995; 26: 881-887Crossref PubMed Scopus (100) Google Scholar), and basal levels of PKC activity may protect against Ras-induced apoptosis. Although the mechanisms by which Ras provides protection against apoptosis are being elucidated, less is understood about how Ras can induce apoptosis or sensitize cells to different apoptotic stimuli. Some studies (18Joneson T. Bar-Sagi D. Mol. Cell. Biol. 1999; 19: 5892-5901Crossref PubMed Scopus (153) Google Scholar, 20Goillot E. Raingeaud J. Ranger A. Tepper R.I. Davis R.J. Harlow E. Sanchez I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3302-3307Crossref PubMed Scopus (246) Google Scholar) have reported that the activation of extracellular signal-regulated kinase and/or c-Jun NH2-terminal kinase is important for Ras-mediated apoptosis, whereas others (21Parrizas M. Blakesley V.A. Beitner-Johnson D. Le Roith D. Biochem. Cell Biol. 1997; 234: 616-620Google Scholar, 22Ohmori M. Shirasawa S. Furuse M. Okumura K. Sasazuki T. Cancer Res. 1997; 57: 4714-4717PubMed Google Scholar) show no role for extracellular signal-regulated kinase or c-Jun NH2-terminal kinase activation. Elucidation of the role of extracellular signal-regulated kinase activation in Ras-mediated apoptosis is complicated by the fact that activation of the Raf-MAP kinase pathway can also inhibit apoptosis (17Kinoshita T. Shirouzu M. Kamiya A. Hashimoto K. Yokoyama S. Miyajima A. Oncogene. 1997; 15: 619-627Crossref PubMed Scopus (103) Google Scholar, 23Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5032) Google Scholar, 24Parrizas M. Saltiel A.R. Le Roith D. J. Biol. Chem. 1997; 272: 154-161Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar). One downstream effector of Ras that could potentially mediate or initiate an apoptotic process is reactive oxygen species (ROS). ROS can influence numerous intracellular pathways, including those leading to programmed cell death (25Adler V. Yin Z. Tew K.D. Ronai Z. Oncogene. 1999; 18: 6104-6111Crossref PubMed Scopus (591) Google Scholar, 26Anderson K.M. Seed T. Ou D. Harris J.E. Med. Hypotheses. 1999; 52: 451-463Crossref PubMed Scopus (55) Google Scholar). As intracellular second messengers, ROS also control a variety of Ras-mediated cellular effects (27Irani K. Goldschmidt-Clermont P.J. Biochem. Pharmacol. 1998; 55: 1339-1346Crossref PubMed Scopus (153) Google Scholar). We wished to determine whether ROS mediated apoptosis of Ras-transformed cells in which PKC was inhibited. We report here that the generation of ROS is necessary for Ras to initiate apoptosis when PKC is inhibited. However, up-regulation of ROS alone is not sufficient for the induction of apoptosis by PKC. Our studies indicate that cell cycle progression is also necessary for Ras-mediated apoptosis to occur, independent of ROS generation. The NIH/3T3 mouse embryo fibroblast line and Balb/3T3 clone A31 mouse embryo fibroblast line were obtained from the ATCC (Manassas, VA). NIH/3T3-Ras cells were produced by stable transfection of NIH/3T3 cells with v-Ha-ras and selected and maintained in 0.5 μg of geneticin/ml (Life Technologies, Inc.). KBalb cells were produced by stable transfection of Balb cells with v-Ki-ras and have been described previously (28Zullo J.N. Faller D.V. Mol. Cell. Biol. 1988; 8: 5080-5085Crossref PubMed Scopus (36) Google Scholar). KBalb-Bcl2 cells were produced by stable transfection of KBalb cells with a retroviral Bcl-2 expression vector (29Wagner A.J. Small M.B. Hay N. Mol. Cell. Biol. 1993; 13: 2432-2440Crossref PubMed Scopus (207) Google Scholar). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% donor calf serum, 2 mml-glutamine, 100 units of penicillin/ml, and 100 μg of streptomycin/ml (Life Technologies, Inc.). Cells were cultured in 24-well plates at 2 × 104 cells/well and treated in triplicate with phorbol 12-myristate 13-acetate (PMA) (Sigma) or 1-O-hexadecyl-2-O-methyl-rac-glycerol (HMG) (Calbiochem) for the times indicated. Background control wells were treated with 0.19% saponin for 10 min prior to assay. Wells were washed once with phosphate-buffered saline (PBS), and 500 μl of 1 μm calcein-AM (Molecular Probes) in PBS was added to each well. Plates were incubated at room temperature for 30 min. Fluorescence was quantified on a Cytofluor 2300 fluorescent plate reader (Applied Biosystems) at excitation/emission wavelengths of 485/530 nm. Cells were plated at 1 × 105/plate in 60-mm dishes and treated for the indicated times. Cells were harvested with trypsin/EDTA and fixed in 35% ethanol, 65% Dulbecco's modified Eagle's medium. Cells were washed once in ice-cold PBS and resuspended in 25 μg of propidium iodide/ml and 50 μg of RNase A/ml in PBS. Samples were incubated at 37 °C for 2 h, and DNA profiles were analyzed by FACS using a FACScan flow cytometer (Becton Dickinson). Cells were plated at 1 × 105/plate in 60-mm dishes and treated for the indicated times. Cells were harvested with trypsin/EDTA, washed once in PBS, and resuspended in 5 μg of 2′,7′-dichlorodihydrofluorescein diacetate (DCF) (Molecular Probes)/ml in Hanks' balanced salt solution. Samples were incubated for 10 min at room temperature and analyzed immediately by FACS. For manipulation of ROS levels, cells were either treated with 20 mm NAC (Sigma) or placed in sealed chambers (Billups-Rothenberg), which had been flushed for 30 min with 95% N2, 5% CO2 as described previously (30Kourembanas S. Hannan R.L. Faller D.V. J. Clin. Invest. 1990; 86: 670-674Crossref PubMed Scopus (338) Google Scholar) for 24 h before treatment with HMG. Cellular PKC activity was measured using a commercial assay, following the manufacturer's protocol (Upstate Biotechnology) and as described previously (7Chen C.Y. Liou J. Forman L.W. Faller D.V. J. Biol. Chem. 1998; 273: 16700-16709Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Briefly, cells were treated with 150 μm HMG for 24 h or left untreated in normal culture conditions before harvesting with trypsin/EDTA. Cells were lysed in 25 mm Tris-HCl, pH 7.5, 1% Triton X-100, 20 mm MgCl2, and 150 mm NaCl, and extracts were normalized for protein concentration. Subsequently, 100 μg of extract was incubated with a PKC-specific peptide substrate, [32P]ATP, and inhibitors of cAMP-dependent protein kinase and calmodulin kinase for 10 min at 30 °C.32P incorporated into the substrate was separated from residual 32P using p81 phosphocellulose paper and quantified by scintillation counting. NIH/3T3-Ras cells were treated as indicated for 24 h, either in low oxygen culture conditions or with sodium butyrate (5 mm) or TsA (100 ng/ml). For a control, NIH/3T3 cells were serum-starved in medium containing 0.5% donor calf serum for 24 h, and endogenous Ras was activated by PDGF (30 ng/ml) stimulation for 15 min. Ras activity was measured using the technique developed by de Rooij and Bos (31de Rooij J. Bos J.L. Oncogene. 1997; 14: 623-625Crossref PubMed Scopus (420) Google Scholar). Briefly, 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 μg/μl, and activated Ras was affinity precipitated by mixing 1 mg of cell lysate with 10 μg of Raf-1 RBD agarose bead conjugate (Upstate Biotechnology) end over end for 30 min at 4 °C. Agarose bead conjugates were washed 3 times in lysate buffer, resuspended in Laemmli sample buffer, and boiled for 5 min. Beads were pelleted, and supernatant was loaded onto a 10% SDS-polyacrylamide gel electrophoreses gel. Proteins were transferred onto a polyvinylidene difluoride membrane and immunoblotted with a pan-Ras antibody (Oncogene Research Products). Ras protein was detected using ECL-Plus chemiluminescent reagent (Amersham Pharmacia Biotech). Our laboratory has previously shown that down-regulation of PKC by chronic, high dose PMA treatment (32Young S. Parker P.J. Ullrich A. Stabel S. Biochem. J. 1987; 244: 775-779Crossref PubMed Scopus (356) Google Scholar) could selectively induce apoptosis in Jurkat human T lymphoblastoid cells stably expressing v-Ha-Ras (PH1 cells) when compared with normal Jurkat cells (7Chen C.Y. Liou J. Forman L.W. Faller D.V. J. Biol. Chem. 1998; 273: 16700-16709Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 8Chen C.Y. Faller D.V. Oncogene. 1995; 11: 1487-1498PubMed Google Scholar). Down-regulation of PKC by high dose PMA treatment also selectively induced cell death in both v-Ki-ras-transformed Balb fibroblasts (KBalb) and v-Ha-ras-transformed NIH/3T3 fibroblasts (NIH/3T3-Ras) when compared with normal Balb and NIH/3T3 cells (Fig. 1 a). Treatment with 100 nm PMA, a concentration that activates PKC but is insufficient to cause down-regulation of PKC, consistently caused less than 10% loss in cell viability of the Ras-transformed fibroblasts. In contrast, chronic high dose treatment of Ras-transformed fibroblasts with 500 nm PMA, a concentration known to cause down-regulation of PKC after prolonged exposure (32Young S. Parker P.J. Ullrich A. Stabel S. Biochem. J. 1987; 244: 775-779Crossref PubMed Scopus (356) Google Scholar), caused between 85 and 95% cell death after 72 h. No loss in cell viability was seen in normal Balb or NIH/3T3 fibroblasts with either 100 or 500 nm PMA treatment. These findings demonstrate that the suppression of PKC activity, but not activation of PKC, can trigger cell death in the presence of oncogenic Ras activity. To determine whether a pharmacological inhibitor of PKC could also selectively induce cell death in Ras-transformed cells, the diacylglycerol antagonist HMG was used to treat both normal and Ras-transformed fibroblasts. Treatment of KBalb and NIH/3T3-Ras fibroblasts with 150 μm HMG caused Ras-specific cell death, similar to that achieved using chronic, high dose PMA down-regulation of PKC. Ras-transformed cells treated with 150 μm HMG underwent 80–90% loss in cell viability after 48 h, whereas less than 10% loss of viability was observed after treatment of normal Balb and NIH/3T3 cells (Fig. 1 b). Other selective PKC inhibitors, such as Ro-31-8220 and GF109203X, were also able to induce differentially cell death in Ras-transformed cells. 2J. S. Liou and D. V. Faller, unpublished observation. Overexpression of Bcl-2 in KBalb cells by transfection with a retroviral expression vector inhibited the apoptosis induced by HMG treatment, consistent with our previous findings that Bcl-2 overexpression effectively inhibited death induced by chronic, high dose PMA treatment in cells expressing activated Ras (8Chen C.Y. Faller D.V. Oncogene. 1995; 11: 1487-1498PubMed Google Scholar). To verify that the dose of HMG that selectively induced cell death was also inhibiting PKC activity, anin vitro PKC activity assay was performed. Treatment with 150 μm HMG suppressed the cellular PKC activity of cells growing logarithmically in 10% serum-containing medium by greater than 4-fold (Fig. 1 c). Photomicroscopy of NIH/3T3-Ras cells treated with HMG showed changes in morphology consistent with apoptosis (Fig. 1 d). Quantification of apoptosis by propidium iodide staining and FACS analysis showed that NIH/3T3-Ras cells underwent increasing amounts of DNA fragmentation over a 72-h period, with over 70% of the population containing a hypodiploid DNA content at 72 h (Fig. 2 a). In contrast, no increase in DNA fragmentation was observed in parental (normal) NIH/3T3 after 72 h of HMG treatment when compared with basal levels (Fig. 2 b). Concurrent analysis of cell cycle indicated that over 98% of parental NIH/3T3 cells were arrested in the G0/G1 phase after PKC inhibition (Fig. 2 c). This cell cycle arrest was reversible, and NIH/3T3 cells resumed cell cycle progression once HMG was washed out of culture.2 To determine if reactive oxygen species served as effectors of Ras-mediated apoptosis, intracellular ROS levels were measured by FACS using the peroxide-sensitive fluorescent indicator, DCF. NIH/3T3-Ras cells showed higher basal levels of ROS compared with parental NIH/3T3 cells (Fig. 3 a). KBalb cells also showed higher basal ROS levels compared with parental Balb cells.2 HMG treatment for 24 h caused an up-regulation of ROS levels in both NIH/3T3 and NIH/3T3-Ras (Fig. 3 b). However, whereas ROS levels were up-regulated by only 2-fold in NIH/3T3 cells in response to HMG, ROS levels increased by more than 7-fold in NIH/3T3-Ras cells. Treatment with the structurally unrelated PKC inhibitors Ro-31-8220 and GF109203X also resulted in similar up-regulation of ROS levels,2 suggesting that ROS generation by HMG correlated with PKC inhibition. To establish whether the up-regulation of ROS levels is necessary for Ras-mediated apoptosis, the antioxidant N-acetylcysteine (NAC) was used to pre-treat Ras-transformed cells. Treatment of NIH/3T3-Ras cells with 20 mm NAC for 24 h reduced the basal ROS to levels similar to those in parental NIH/3T3 (Fig. 4 a). Moreover, pre-treatment of NIH/3T3-Ras cells with NAC completely inhibited the up-regulation of ROS levels induced by HMG, reducing ROS to levels comparable to those found in untreated NIH/3T3-Ras cells (Fig. 4 b). Pre-treatment of NIH/3T3-Ras cells with NAC also effectively inhibited the DNA fragmentation induced by HMG. DNA fragmentation was reduced from a 13-fold increase in cells unprotected by NAC treatment to 2-fold in cells pre-treated with NAC (Fig. 4 c). Culturing cells under a low oxygen environment is another method for inhibiting intracellular ROS generation (33Chance B. Sies H. Boveris A. Physiol. Rev. 1979; 59: 527-605Crossref PubMed Scopus (4819) Google Scholar). NIH/3T3-Ras cells were cultured in either normoxic or hypoxic conditions for 24 h before treatment with HMG. After PKC inhibition, cells were maintained in normoxic or hypoxic conditions for another 48 h before analysis of DNA fragmentation by FACS. Whereas NIH/3T3-Ras cells maintained in normoxic conditions underwent a 10-fold increase in DNA fragmentation upon HMG treatment, DNA fragmentation was reduced to only 2-fold in NIH/3T3-Ras cells cultured in hypoxia (Fig. 5 a). In contrast, serum deprivation, which can induce apoptosis independently of ROS generation (34Jacobson M.D. Raff M.C. Nature. 1995; 374: 814-816Crossref PubMed Scopus (635) Google Scholar), was able to induce DNA fragmentation in NIH/3T3 cells, regardless of normoxic or hypoxic culture conditions (Fig. 5 b).Figure 5Low oxygen inhibits DNA fragmentation caused by PKC inhibition. Cells were cultured in a low oxygen environment in sealed chambers for 24 h or left in normoxic conditions. Subsequently, NIH/3T3-Ras cells were treated with 150 μmHMG (a) or NIH/3T3 cells were serum-starved as control (b). Cells cultured in low oxygen were returned to sealed chambers for another 48 h before analysis of DNA fragmentation. The percentage of the population of cells containing less than 2n of DNA in each condition is indicated. Results shown are representative of experiments performed two times.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine the mechanism by which PKC inhibition generates ROS, studies were performed using diphenylene iodonium, an inhibitor of NAD(P)H oxidase (35Meier B. Cross A.R. Hancock J.T. Kaup F.J. Jones O.T. Biochem. J. 1991; 275: 241-245Crossref PubMed Scopus (194) Google Scholar). Pre-treatment of NIH/3T3-Ras cells with diphenylene iodonium did not inhibit the up-regulation of ROS by HMG, suggesting no role for NAD(P)H oxidase in generating ROS downstream of PKC inhibition (data not shown). Since mitochondria play an important role in many forms of apoptosis and are also a major source for ROS production (36Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar), we investigated the involvement of mitochondria as the source of ROS downstream of PKC inhibition. To explore the possibility that the inhibition of PKC generates ROS as a consequence of the opening of the mitochondrial permeability transition pore, studies were performed using cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition pore (37Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1383) Google Scholar). Pretreatment of NIH/3T3-Ras cells with CsA did not affect the basal level of ROS in the cells but effectively blocked the up-regulation of ROS by HMG treatment (Fig. 6 a). Moreover, the inhibition of HMG-induced ROS generation by CsA correlated with inhibition of apoptosis. NIH/3T3-Ras cells pretreated with CsA exhibited nearly a 10-fold reduction in DNA fragmentation induced by HMG (Fig. 6 b). Previous results from our laboratory suggested that cell cycle progression may also be necessary for Ras-mediated apoptosis (7Chen C.Y. Liou J. Forman L.W. Faller D.V. J. Biol. Chem. 1998; 273: 16700-16709Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Attempts to use the cell cycle-arresting agents hydroxyurea or aphidicolin to inhibit the cell cycle progression of Ras-transformed cells resulted in high levels of cytotoxicity. However, the cell cycle-arresting agents sodium butyrate and trichostatin A (TsA) were relatively nontoxic to NIH/3T3-Ras cells and were therefore tested for their ability to inhibit Ras-mediated apoptosis induced by PKC inhibition. Pre-treatment of NIH/3T3-Ras cells with either sodium butyrate (5 mm) (Fig. 7 a) or TsA (100 ng/ml) (Fig. 7 b) reduced the induction of DNA fragmentation in cells by HMG treatment from over 13-fold to less than 2-fold. Concurrent cell cycle analysis showed inhibition of cell cycle progression and accumulation of cells in G1 induced by both sodium butyrate and TsA treatments (Fig. 8 a). Treatment with either sodium butyrate or TsA reduced the percentage of cells in S phase by more than 5-fold. In contrast, treatment with isobutyramide, an analog of sodium butyrate (38Perrine S.P. Dover G.H. Daftari P. Walsh C.T. Jin Y. Mays A. Faller D.V. Br. J. Haematol. 1994; 88: 555-561Crossref PubMed Scopus (49) Google Scholar), had no effect on the cell cycle distribution of NIH/3T3-Ras cells (Fig. 8 a) and also did not prevent induction of apoptosis by HMG.2 Thus, inhibition of apoptosis by these compounds correlated with inhibition of cell cycle progression. Our laboratory has shown that continued Ras activity is necessary to mediate apoptosis induced by PKC inhibition, as inhibition of Ras activity with a farnesyltransferase inhibitor can inhibit apoptosis. 3J. S. Liou and D. V. Faller, manuscript in preparation. To rule out the possibility that the inhibition of apoptosis by hypoxic culture conditions, sodium butyrate treatment, or trichostatin A treatment were due to effects on Ras activity, Ras activity was analyzed by affinity binding to a Raf Ras-binding domain (RBD) peptide that only binds the activated (GTP-bound) form of Ras. Stimulation of serum-starved NIH/3T3 cells with PDGF (30 ng/ml) resulted in an increase in Ras activity as measured by the Raf-RBD-GST pulldown assay (Fig. 8 b1). NIH/3T3-Ras cells constitutively expressed a relatively high level of activated Ras, and culturing NIH/3T3-Ras cells under hypoxic conditions, or treatment with either sodium butyrate or TsA, had no inhibitory effect on the levels of Ras activity (Fig. 8 b2).Figure 8Sodium butyrate and trichostatin A inhibit cell cycle progression but do not affect Ras activity or intracellular ROS levels. a, NIH/3T3-Ras cells were treated with 5 mm isobutyramide, 5 mm sodium butyrate, or TsA (100 ng/ml) for 24 h before staining with propidium iodide and cell cycle analysis by FACS. The percentage of the population of cells in S phase for each condition is indicated. b1, NIH/3T3 cells were serum-starved in media containing 0.5% donor calf serum for 24 h and left untreated (lane 1) or stimulated with PDGF (30 ng/ml) for 15 min (lane 2). Cells were subsequently analyzed for Ras activity by GST-Raf-RBD pulldown of activated Ras and immunoblot analysis. b2, NIH/3T3-Ras cells were grown in normal culture conditions (lane 1), cultured in low oxygen (lane 2), treated with 5 mm sodium butyrate (lane 3), or treated with TsA (100 ng/ml) (lane 4). After 24 h, cells were analyzed for Ras activity by GST-Raf-RBD pulldown of activated Ras and immunoblot analysis. c, NIH/3T3-Ras cells were treated with 5 mm sodium butyrate for 24 h or left untreated before inhibition of PKC by HMG for 24 h and analysis of ROS levels by FACS. Results shown are representative of experiments performed two times.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Interestingly, despite the strong protection against apoptosis, pre-treatment of NIH/3T3-Ras cells with sodium butyrate also had no significant effect on either basal ROS levels or the up-regulation of ROS levels by HMG (Fig. 8 c). These results, together with the protective effects of antioxidants agents like NAC, suggest that the observed increase in ROS levels after PKC inhibition are necessary for Ras-mediated
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