FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2047899612> ?p ?o ?g. }

• W2047899612 endingPage "40162" @default.
• W2047899612 startingPage "40155" @default.
• W2047899612 abstract "The typical proliferative response of hepatocytes to tumor necrosis factor (TNF) can be converted to a cytotoxic one by transcriptional arrest. Although NF-κB activation is critical for hepatocyte resistance to TNF toxicity, the contribution of other TNF-inducible transcription factors remains unknown. To determine the function of c-Myc in hepatocyte sensitivity to TNF, stable transfectants of the rat hepatocyte cell line RALA255-10G containing sense and antisense c-myc expression vectors were isolated with increased (S-Myc cells) and decreased (AN-Myc cells) c-Myc transcriptional activity. While S-Myc cells proliferated in response to TNF treatment, AN-Myc cells underwent 32% cell death within 6 h. Fluorescent microscopic studies indicated that TNF induced apoptosis and necrosis in AN-Myc cells. Cell death was associated with DNA hypoploidy and poly(ADP-ribose) polymerase cleavage but occurred in the absence of detectable caspase-3, -7, or -8 activation. TNF-induced, AN-Myc cell death was dependent on Fas-associated protein with death domain and partially blocked by caspase inhibitors. AN-Myc cells had decreased levels of NF-κB transcriptional activity, but S-Myc cells maintained resistance to TNF despite NF-κB inactivation, suggesting that c-Myc and NF-κB independently mediate TNF resistance. Thus, in the absence of sufficient c-Myc expression, hepatocytes are sensitized to TNF-induced apoptosis and necrosis. These findings demonstrate that hepatocyte resistance to TNF is regulated by multiple transcriptional activators. The typical proliferative response of hepatocytes to tumor necrosis factor (TNF) can be converted to a cytotoxic one by transcriptional arrest. Although NF-κB activation is critical for hepatocyte resistance to TNF toxicity, the contribution of other TNF-inducible transcription factors remains unknown. To determine the function of c-Myc in hepatocyte sensitivity to TNF, stable transfectants of the rat hepatocyte cell line RALA255-10G containing sense and antisense c-myc expression vectors were isolated with increased (S-Myc cells) and decreased (AN-Myc cells) c-Myc transcriptional activity. While S-Myc cells proliferated in response to TNF treatment, AN-Myc cells underwent 32% cell death within 6 h. Fluorescent microscopic studies indicated that TNF induced apoptosis and necrosis in AN-Myc cells. Cell death was associated with DNA hypoploidy and poly(ADP-ribose) polymerase cleavage but occurred in the absence of detectable caspase-3, -7, or -8 activation. TNF-induced, AN-Myc cell death was dependent on Fas-associated protein with death domain and partially blocked by caspase inhibitors. AN-Myc cells had decreased levels of NF-κB transcriptional activity, but S-Myc cells maintained resistance to TNF despite NF-κB inactivation, suggesting that c-Myc and NF-κB independently mediate TNF resistance. Thus, in the absence of sufficient c-Myc expression, hepatocytes are sensitized to TNF-induced apoptosis and necrosis. These findings demonstrate that hepatocyte resistance to TNF is regulated by multiple transcriptional activators. tumor necrosis factor TNF receptor Fas-associated protein with death domain 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide fluorescence-activated cell sorting poly(ADP-ribose) polymerase inhibitor of apoptosis polyacrylamide gel electrophoresis Tumor necrosis factor (TNF)1 is a pleiotrophic cytokine that can induce either proliferative or cytotoxic responses in a variety of cultured cells including hepatocytes (1Tracy K.J. Remick D.G. Friedland J.S. Cytokines in Health and Disease. Marcel Dekker, New York1997: 223-240Google Scholar). The biological effects of TNF in cultured hepatocytes are relevant to the liverin vivo, since TNF also acts as a hepatic mitogen (2Akerman P. Cote Y. Yang S.Q. McClain C. Nelson S. Bagby G.J. Diehl A.M. Am. J. Physiol. 1992; 263: G579-G585Crossref PubMed Google Scholar, 3Yamada Y. Kirillova I. Peschon J.J. Fausto N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1441-1446Crossref PubMed Scopus (840) Google Scholar) or cytotoxin (4Blazka M.E. Wilmer J.L. Holladay S.D. Wilson R.E. Luster M.I. Toxicol. Appl. Pharmacol. 1995; 133: 43-52Crossref PubMed Scopus (298) Google Scholar, 5Czaja M.J. Xu J. Alt E. Gastroenterology. 1995; 108: 1849-1854Abstract Full Text PDF PubMed Scopus (191) Google Scholar, 6Hishinuma I. Nagakawa J.I. Hirota K. Miyamoto K. Tsukidate L. Yamanaka T. Katayama K.I. Yamatsu I. Hepatology. 1990; 12: 1187-1191Crossref PubMed Scopus (169) Google Scholar) in vivo, depending on the pathophysiological setting. TNF has been implicated as a mediator of hepatocyte death following injury from toxins, ischemia/reperfusion, and hepatitis virus (for a review, see Ref. 7Bradham C.A. Plümpe J. Manns M.P. Brenner D.A. Trautwein C. Am. J. Physiol. 1998; 275: G387-G392PubMed Google Scholar). In the absence of an injurious cofactor such as a toxin, hepatocytes are resistant to TNF cytotoxicity, and the mechanism by which they become sensitized to TNF-induced death in the setting of cell injury remains unknown. The pathway from TNF stimulation to cell death has been well described (for a review, see Ref. 8Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5154) Google Scholar). TNF binding to the type 1 TNF receptor (TNFR-1) causes receptor trimerization and the recruitment and binding of a series of intracellular proteins including TNFR-associated death domain protein and Fas-associated protein with death domain (FADD). FADD binding leads initially to activation of caspase-8, and subsequently to activation of caspase-3, resulting in apoptosis (8Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5154) Google Scholar). While the steps in the TNF death pathway leading to apoptosis are known, the mechanism by which cells inactivate this caspase cascade and maintain resistance to TNF toxicity is unclear. A recent advance in our understanding of cellular TNF resistance has come from the demonstration that activation of the transcription factor NF-κB is critical for the induction of cellular resistance to TNF toxicity (9Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2935) Google Scholar, 10Liu Z. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1783) Google Scholar, 11Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2449) Google Scholar, 12Wang C.-Y. Mayo M.W. Baldwin Jr., A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2512) Google Scholar). Inhibition of NF-κB activation in cultured hepatocytes (13Bradham C.A. Qian T. Streetz K. Trautwein C. Brenner D.A. LeMasters J. Mol. Cell. Biol. 1998; 18: 6353-6364Crossref PubMed Scopus (367) Google Scholar, 14Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar) or in the liver in vivo (15Iimuro Y. Nishiura T. Hellerbrand C. Behrns K. Schoonhoven R. Grisham J.W. Brenner D.A. J. Clin. Invest. 1998; 101: 802-811Crossref PubMed Scopus (424) Google Scholar) converts the hepatocellular TNF response from one of proliferation to one of apoptosis. This finding fits well with the fact that in vitro resistance to TNF-induced cytotoxicity requires RNA and protein synthesis (16Kull F.C. Cuatrecasas P. Cancer Res. 1981; 41: 4885-4890PubMed Google Scholar), suggesting that TNF signaling up-regulates a protective cellular gene(s). NF-κB inactivation may sensitize cells to TNF toxicity by preventing the transcriptional up-regulation of an NF-κB-dependent protective gene(s). However, TNF activates other transcriptional activators, including c-Myc and AP-1, and their potential contribution to the transcriptional regulation of hepatocyte resistance to TNF toxicity is unknown. c-Myc is a transcription factor that regulates cell proliferation, differentiation, and apoptosis (for a review, see Ref. 17Dang C.V. Mol. Cell. Biol. 1999; 19: 1-11Crossref PubMed Scopus (1384) Google Scholar). c-Myc expression not only promotes proliferation but also can induce or sensitize cells to apoptosis (18Hoffman B. Lieberman D.A. Oncogene. 1998; 17: 3351-3357Crossref PubMed Scopus (134) Google Scholar, 19Packham G. Cleveland J.L. Biochim. Biophys. Acta. 1995; 1242: 11-28PubMed Google Scholar). Overexpression of c-myc under circumstances in which this gene is usually down regulated such as serum deprivation, results in apoptotic cell death in nonhepatic cells (20Evan G.I. Wyllie A.H. Gilbert C.S. Littlewood T.D. Land H. Brooks M. Waters C.M. Penn L.Z. Hancock D.C. Cell. 1992; 69: 119-128Abstract Full Text PDF PubMed Scopus (2773) Google Scholar) and in a hepatoma cell line (21Xu J. Xu Y. Nguyen Q. Novikoff P.M. Czaja M.J. Am. J. Physiol. 1996; 270: G60-G70Crossref PubMed Google Scholar). c-Myc expression has been reported to be induced by TNF alone (22Manchester K.M. Heston W.D.W. Donner D.B. Biochem. J. 1993; 290: 185-190Crossref PubMed Scopus (32) Google Scholar, 23Ninomiya-Tsuji J. Torti F.M. Ringold G.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9611-9615Crossref PubMed Scopus (34) Google Scholar) or in combination with cycloheximide (24Janicke R.U. Lee F.H.H. Porter A.G. Mol. Cell. Biol. 1994; 14: 5661-5670Crossref PubMed Scopus (94) Google Scholar). Previous investigations in nonhepatic cells have consistently reported that increased c-Myc expression initiates or promotes TNF-induced apoptosis (24Janicke R.U. Lee F.H.H. Porter A.G. Mol. Cell. Biol. 1994; 14: 5661-5670Crossref PubMed Scopus (94) Google Scholar, 25Janicke R.U. Lin X.Y. Lee F.H.H. Porter A.G. Mol. Cell. Biol. 1996; 16: 5245-5253Crossref PubMed Scopus (55) Google Scholar, 26Klefstrom J. Arighi E. Littlewood T. Jäättelä M. Saksela E. Evan G.I. Alitalo K. EMBO J. 1997; 16: 7382-7392Crossref PubMed Scopus (107) Google Scholar, 27Klefstrom J. Västrik I. Saksela E. Valle J. Eilers M. Alitalo K. EMBO J. 1994; 13: 5442-5450Crossref PubMed Scopus (134) Google Scholar). However, in TNF-dependent liver injury in vivoinduced by the toxin galactosamine (6Hishinuma I. Nagakawa J.I. Hirota K. Miyamoto K. Tsukidate L. Yamanaka T. Katayama K.I. Yamatsu I. Hepatology. 1990; 12: 1187-1191Crossref PubMed Scopus (169) Google Scholar), TNF induces hepatocyte injury and death associated with a block in the up-regulation ofc-myc mRNA expression that normally occurs during a hepatic proliferative response (28Schmiedeberg P. Biempica L. Czaja M.J. J. Cell. Physiol. 1993; 154: 294-300Crossref PubMed Scopus (51) Google Scholar). These findings suggested that hepatocytes may undergo TNF-induced death in the absence of c-Myc expression or even become sensitized to TNF toxicity by a failure to up-regulate c-Myc. We therefore tested the hypothesis that c-Myc expression promotes hepatocyte resistance to TNF toxicity by examining the sensitivity of rat hepatocyte cell lines with differential c-Myc expression to TNF toxicity. Cells lines with differential c-Myc expression were derived from the wild-type RALA255–10G rat hepatocyte cell line (29Chou J.Y. Mol. Cell. Biol. 1983; 3: 1013-1020Crossref PubMed Scopus (47) Google Scholar). These cells are conditionally transformed with a temperature-sensitive T antigen. Cells were grown at the permissive temperature of 33 °C and then maintained at 37 °C to allow suppression of T antigen expression and development of a differentiated hepatocyte phenotype as described previously (29Chou J.Y. Mol. Cell. Biol. 1983; 3: 1013-1020Crossref PubMed Scopus (47) Google Scholar). All experiments were performed in cells cultured at 37 °C. The c-myc cDNA subcloned into the expression vector pMEP4 (Invitrogen, San Diego, CA) as described previously (21Xu J. Xu Y. Nguyen Q. Novikoff P.M. Czaja M.J. Am. J. Physiol. 1996; 270: G60-G70Crossref PubMed Google Scholar) was transfected into RALA hepatocytes using LipofectAMINE Plus (Life Technologies, Inc.) according to the manufacturer's instructions. Stable transfectants were selected by resistance to 200 μg/ml hygromycin (Calbiochem). The subsequent experiments employed pooled transfectants expressing sense (S-Myc cells) and antisense (AN-Myc cells) c-myc constructs. All cells were cultured in 50 μm zinc for 4 days prior to the start of experiments in order to induce transgene expression from pMEP4, which contains a zinc-inducible human MT IIa promoter. In some experiments, cells were treated with rat recombinant TNF (TNF-α, R & D Systems, Minneapolis, MN) at a concentration of 10 ng/ml, 50 μm C2 ceramide (Biomol, Plymouth Meeting, PA), or 1.25 μmol/106 cells of hydrogen peroxide (H2O2) (Sigma). To inhibit caspase activity, cells were pretreated for 1 h before the addition of TNF with the following caspase inhibitors dissolved in dimethyl sulfoxide: 100 μm Val-Ala-Asp-fluoromethylketone (BACHEM, Torrance, CA), 50 μm N-[(indole-2-carbonyl)-alaninyl]-3-amino-4-oxo-5-fluoropentanoic acid (IDN-1529), orN-[(1,3-dimethylindol-2-carbonyl)-valinyl]-3-amino-4-oxo-5-fluoropentanoic acid (IDN-1965) (IDUN Pharmaceuticals, La Jolla, CA). IDN-1529 and IDN-1965 have broad anti-caspase activity, inhibiting caspase-1, -3, -6, and -8. 2J. Wu, personal communication. RALA hepatocytes were transiently transfected with luciferase reporter genes using LipofectAMINE Plus. Cells were transfected with NF-κB-Luc (30Galang C.K. Der C.J. Hauser C.A. Oncogene. 1994; 9: 2913-2921PubMed Google Scholar), which contains three NF-κB binding sites, or pMyc 3E1b-Luc (31Gupta S. Seth A. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3216-3220Crossref PubMed Scopus (140) Google Scholar), which contains three c-Myc binding sites, driving firefly luciferase reporter genes. Cells were cotransfected with pRL-TK (Promega, Madison, WI) a Renilla luciferase vector driven by a Herpes simplex virus thymidine kinase promoter, which served as a control for transfection efficiency. To assay luciferase activity, cells were washed in phosphate-buffered saline and lysed in 1% Triton X-100, and the cell extract was assayed for firefly luciferase activity in a luminometer. Renilla luciferase was assayed in the same sample according to the manufacturer's instructions. Firefly luciferase activity was then normalized to Renillaluciferase activity. RNA was extracted from cells as described previously (32Czaja M.J. Weiner F.R. Freedman J.H. J. Cell. Physiol. 1991; 147: 434-438Crossref PubMed Scopus (25) Google Scholar). Steady-state mRNA levels were determined by Northern blot hybridizations using samples of 20 μg of total RNA (32Czaja M.J. Weiner F.R. Freedman J.H. J. Cell. Physiol. 1991; 147: 434-438Crossref PubMed Scopus (25) Google Scholar). The membranes were hybridized with [32P]dCTP (PerkinElmer Life Sciences)-labeled cDNA clones for lactate dehydrogenase A (33Shim H. Chun Y.S. Lewis B.C. Dang C.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1511-1516Crossref PubMed Scopus (254) Google Scholar) and glyceraldehyde-3-phosphate dehydrogenase (34Tso J.Y. Sun X.-H. Kao T.-H. Reece K.S. Wu R. Nucleic Acids Res. 1985; 13: 2485-2502Crossref PubMed Scopus (1760) Google Scholar). The hybridized filters were washed under stringent conditions (32Czaja M.J. Weiner F.R. Freedman J.H. J. Cell. Physiol. 1991; 147: 434-438Crossref PubMed Scopus (25) Google Scholar). Relative cell number was determined by the MTT assay, as described previously (14Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar). Cell survival was calculated as a percentage of control cells by taking the optical density reading of cells given a particular treatment, dividing that number by the optical density reading for the untreated, control cells, and then multiplying by 100. The numbers of apoptotic and necrotic cells were determined by examining cells under fluorescence microscopy following costaining with acridine orange and ethidium bromide (14Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar). The percentage of cells with apoptotic morphology (nuclear and cytoplasmic condensation, nuclear fragmentation, membrane blebbing, and apoptotic body formation) under acridine orange staining was determined by examining >400 cells/dish. Necrosis was determined by the presence of ethidium bromide staining in the same cell population. The identification of hypoploid cells by FACS detection of DNA loss after controlled extraction of low molecular weight DNA was performed as described previously (35Jones B.E. Lo C.R. Liu H. Srinivasan A. Streetz K. Valentino K.L. Czaja M.J. J. Biol. Chem. 2000; 275: 705-712Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Cells were trypsinized and centrifuged, and the cell pellets were fixed in 70% ethanol and placed at −20 °C for a minimum of 17 h. The cells were washed and resuspended in Hanks' buffered saline solution and incubated in phosphate-citric acid buffer (0.2 m Na2HPO4, 0.1 mcitric acid, pH 7.8) for 5 min. The cells were then centrifuged, and the pellet was resuspended in Hanks' buffered saline solution containing propidium iodide (20 μg/μl) and RNase (100 μg/ml). Following a 30-min incubation at room temperature, the cells were analyzed on a FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA) at an excitation of 488 nm. DNA fluorescence pulse processing was used to discriminate between single cells and aggregates of cells (Doublet Discrimination) by evaluating the FL2-Width versusFL2-Area scatter plot. Light scatter gating was used to eliminate smaller debris from analysis. An analysis gate was set to limit the measurement of hypoploidy to an area of 10-fold loss of DNA content. For protein isolation for Western immunoblots of c-Myc and protein-disulfide isomerase, cells were washed in phosphate-buffered saline, centrifuged, and resuspended in lysis buffer composed of 50 mm Tris, pH 7.5, 150 mm sodium chloride, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mm dithiothreitol, 1 mm EDTA, 1 mm EGTA, 1 mmphenylmethylsulfonyl fluoride, and 2 μg/ml of pepstatin A, leupeptin, and aprotinin. Cells were then mixed at 4 °C for 30 min. After centrifugation, the supernatant was collected, and the protein concentration was determined by the Bio-Rad protein assay. Fifty micrograms of protein were resolved on 10% SDS-PAGE as described previously (14Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar). Membranes were stained with Ponceau red to ensure equivalent amounts of protein loading and electrophoretic transfer among samples. Membranes were exposed to a rabbit anti-c-Myc polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or protein-disulfide isomerase rabbit antiserum (36Terada K. Manchikalapudi P. Noiva R. Jauregui H.O. Stockert R.J. Schilsky M.L. J. Biol. Chem. 1995; 270: 20410-20416Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), at 1:1000 dilutions followed by a goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (Life Technologies) at a 1:20,000 dilution. Proteins were visualized by chemiluminescence (SuperSignal West Dura Extended; Pierce). For poly(ADP-ribose) polymerase (PARP) immunoblots, cells were washed in phosphate-buffered saline, centrifuged, and resuspended in lysis buffer containing 20 mm Tris, pH 7.5, 1% SDS, 2 mm EDTA, 2 mm EGTA, 6 mmβ-mercaptoethanol, and the protease inhibitors as above. After a 10-min incubation on ice, the cell suspension was sonicated. Fifty micrograms of protein were resolved on 8% SDS-PAGE and immunoblotted with a rabbit anti-PARP polyclonal antibody (Santa Cruz Biotechnology) at a 1:1000 dilution followed by a goat anti-rabbit antibody at a 1:20,000 dilution. For caspase immunoblots, cells were scraped in medium; centrifuged; resuspended in lysis buffer containing 10 mm HEPES, pH 7.4, 42 mm MgCl2, 1% Triton X-100, and the protease inhibitors listed previously; and mixed at 4 °C for 30 min. Fifty micrograms of protein were resolved on 10% SDS-PAGE and immunoblotted with rabbit polyclonal anti-caspase-3, -7, and -8 antibodies (IDUN Pharmaceuticals) at 1:2000, 1:1000, and 1:4000 dilutions, respectively, followed by a goat anti-rabbit secondary antibody at a 1:10,000 dilution. To examine mitochondrial cytochrome c release, mitochondrial fractions were prepared by differential centrifugation in sucrose as described previously (35Jones B.E. Lo C.R. Liu H. Srinivasan A. Streetz K. Valentino K.L. Czaja M.J. J. Biol. Chem. 2000; 275: 705-712Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Fifty micrograms of mitochondrial protein were subjected to 15% SDS-PAGE as described above. A mouse anti-cytochrome c monoclonal IgG (Pharmingen, San Diego, CA) and a mouse anti-cytochrome oxidase subunit IV monoclonal IgG (Molecular Probes, Inc., Eugene, OR) were used at 1:1000 dilutions together with a goat anti-mouse IgG conjugated to horseradish peroxidase (Life Technologies). The following adenoviruses were employed: a control virus Ad5LacZ that expresses theEscherichia coli β-galactosidase gene; NFD-4 containing a dominant negative FADD; a CrmA-expressing adenovirus; and Ad5IκB, which expresses a mutated IκB that irreversibly binds NF-κB, preventing its activation (13Bradham C.A. Qian T. Streetz K. Trautwein C. Brenner D.A. LeMasters J. Mol. Cell. Biol. 1998; 18: 6353-6364Crossref PubMed Scopus (367) Google Scholar). Viruses were grown in 293 cells; purified by banding twice on CsCl gradients; dialyzed against 5 mm Tris, pH 8.0, 50 mm MgCl2, 3% glycerol, and 0.05% bovine serum albumin; and stored at −80 °C. Cells were infected with 5 × 109 particles of the appropriate virus per 35-mm culture dish (∼1.5 × 103 particles/cell or 5–15 plaque-forming units/cell) as described previously (14Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar). Nuclear proteins were isolated by the method of Schreiber et al. (37Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3917) Google Scholar), modified as described previously (21Xu J. Xu Y. Nguyen Q. Novikoff P.M. Czaja M.J. Am. J. Physiol. 1996; 270: G60-G70Crossref PubMed Google Scholar). Electrophoretic mobility shift assays were performed on 5 μg of protein with a 32P-end-labeled oligonucleotide for the NF-κB consensus sequence (Santa Cruz Biotechnology). The DNA binding reaction was performed as described previously (21Xu J. Xu Y. Nguyen Q. Novikoff P.M. Czaja M.J. Am. J. Physiol. 1996; 270: G60-G70Crossref PubMed Google Scholar); the samples were resolved on a 4% polyacrylamide gel, dried, and subjected to autoradiography. All numerical results are reported as mean ± S.E. and represent data from a minimum of three independent experiments performed in duplicate. RALA hepatocytes were transfected with the pMEP4 expression vector containing the c-myc cDNA in either a sense or antisense orientation. Stable transfectants were selected in hygromycin and initially screened for c-myc expression by Northern blot analysis. Two polyclonal cell lines were selected in which expression of sense c-myc (S-Myc cells) and antisense c-myc(AN-Myc cells) constructs resulted in maximally increased and decreasedc-myc levels, respectively. Western immunoblotting confirmed that S-Myc cells had increased c-Myc levels compared with AN-Myc cells, while the two cell lines had equivalent levels of the constitutively expressed protein-disulfide isomerase (Fig. 1 A). The relative amounts of c-Myc transcriptional activity in the two cells lines were measured with a transiently transfected c-Myc firefly luciferase reporter, and the results were normalized to a cotransfected Renillaluciferase reporter under the control of a minimal reporter. c-Myc transcriptional activity in untreated cells was increased over 14-fold in S-Myc cells as compared with AN-Myc cells (Fig. 1 B). Although c-Myc-dependent transcriptional activity increased in both cell lines following TNF treatment, the activity in AN-Myc cells was still less than 10% of the activity in S-Myc cells (Fig. 1 B). As additional evidence of differential c-Myc transcriptional activity in the two cell lines, mRNA levels for the c-Myc-dependent lactate dehydrogenase A gene (33Shim H. Chun Y.S. Lewis B.C. Dang C.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1511-1516Crossref PubMed Scopus (254) Google Scholar), were determined by Northern blot analysis. S-Myc cells had significantly increased expression of lactate dehydrogenase A relative to AN-Myc cells, while RNA levels of the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase gene were equivalent in the two cell lines (Fig. 1 C). Thus, as assessed by protein levels, transcriptional activity, and c-Myc-dependent gene expression, c-Myc levels were increased in S-Myc cells relative to AN-Myc cells. TNF treatment of RALA hepatocytes results in a proliferative response (14Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar), similar to the known mitogenic effect of TNF on the liver in vivo following partial hepatectomy (2Akerman P. Cote Y. Yang S.Q. McClain C. Nelson S. Bagby G.J. Diehl A.M. Am. J. Physiol. 1992; 263: G579-G585Crossref PubMed Google Scholar, 3Yamada Y. Kirillova I. Peschon J.J. Fausto N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1441-1446Crossref PubMed Scopus (840) Google Scholar). To convert the TNF response from proliferation to apoptosis requires either cotreatment with the RNA synthesis inhibitor actinomycin D or inhibition of activation of the transcription factor NF-κB (14Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar, 35Jones B.E. Lo C.R. Liu H. Srinivasan A. Streetz K. Valentino K.L. Czaja M.J. J. Biol. Chem. 2000; 275: 705-712Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Similar to wild-type RALA hepatocytes, S-Myc cells were resistant to TNF toxicity as determined by MTT assays 6 and 24 h after TNF treatment (Fig. 2 A). Despite the use of highly confluent cultures, S-Myc cell number increased 11% at 24 h, indicating that these cells underwent a proliferative response to TNF, a result identical to previous findings in wild-type cells (14Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar). In contrast, TNF treatment of AN-Myc cells resulted in a 32% decrease in cell number within only 6 h and only a slight further decrease in cell number by 24 h (Fig. 2 A). In keeping with previously published results (14Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar), TNF at 10 ng/ml resulted in a maximal death response because no further decrease in cell number occurred when AN-Myc cells were treated with a higher TNF concentration of 30 ng/ml (data not shown). To examine whether AN-Myc cell sensitivity to TNF toxicity represented a nonspecific sensitization to any cell death stimulus, cell survival was determined following treatment with C2 ceramide and H2O2. Ceramide is a known apoptotic stimulus that has been implicated as a downstream mediator of TNF-induced cell death (38Kolesnick R.N. Krönke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (730) Google Scholar). The oxidant H2O2 triggers apoptosis in many cell types, including RALA cells (39Jones B.E. Lo C.R. Liu H. Pradhan Z. Garcia L. Srinivasan A. Valentino K.L. Czaja M.J. Am. J. Physiol. 2000; 278: G693-G699Crossref PubMed Google Scholar), and oxidative stress has been implicated as a mechanism of TNF toxicity (1Tracy K.J. Remick D.G. Friedland J.S. Cytokines in Health and Disease. Marcel Dekker, New York1997: 223-240Google Scholar). Identical to previous reports in wild-type RALA hepatocytes (40Jones B.E. Lo C.R. Srinivasan A. Valentino K.L. Czaja M.J. Hepatology. 1999; 30: 215-222Crossref PubMed Scopus (45) Google Scholar), both S-Myc and AN-Myc cells were resistant to ceramide toxicity at the 6-h time point, at which sensitization to TNF toxicity had occurred (Fig. 2 A). Both S-Myc and AN-Myc cells underwent significant cell death 24 h after H2O2 treatment (Fig. 2 A), indicating no significant alteration in sensitivity to this toxin between the two cell lines. Inhibition of c-Myc expression did not sensitize RALA hepatocytes indiscriminately to any form of cell death but specifically modulated resistance to TNF-induced cell death. Cell death from TNF may result from apoptosis or necrosis depending on the cell type. To determine which type of cell death occurred in TNF-treated AN-Myc cells, cells were examined for morphological and biochemical evidence of apoptosis. AN-Myc cells were examined under fluorescence microscopy following costaining with acridine orange and ethidium bromide to quantitate the numbers of apoptotic and necrotic cells. Over the 6 h after TNF treatment, AN-Myc cells had marked increases in both the numbers of apoptotic and necrotic cells (Fig. 2 B). As additional evidence that inhibition of c-Myc expression sensitized cells to death at least in part from apoptosis, FACS analysis was performed to quantitate the numbers of hypoploid cells as a measure of the presence of DNA fragmentation. Despite 24 h of culture in serum-free medium, S-Myc cells had a low level of hypoploidy that decreased slightly with TNF t" @default.
• W2047899612 created "2016-06-24" @default.
• W2047899612 creator A5008681401 @default.
• W2047899612 creator A5010440823 @default.
• W2047899612 creator A5013520965 @default.
• W2047899612 creator A5013770831 @default.
• W2047899612 creator A5030051266 @default.
• W2047899612 creator A5031699197 @default.
• W2047899612 creator A5039706801 @default.
• W2047899612 creator A5051851067 @default.
• W2047899612 date "2000-12-01" @default.
• W2047899612 modified "2023-09-26" @default.
• W2047899612 title "Inhibition of c-Myc Expression Sensitizes Hepatocytes to Tumor Necrosis Factor-induced Apoptosis and Necrosis" @default.
• W2047899612 cites W1492606093 @default.
• W2047899612 cites W1751616175 @default.
• W2047899612 cites W1888783223 @default.
• W2047899612 cites W1981691673 @default.
• W2047899612 cites W1997261857 @default.
• W2047899612 cites W1998822013 @default.
• W2047899612 cites W2016730049 @default.
• W2047899612 cites W2021180042 @default.
• W2047899612 cites W2025128734 @default.
• W2047899612 cites W2025713422 @default.
• W2047899612 cites W2043493252 @default.
• W2047899612 cites W2053736456 @default.
• W2047899612 cites W2063229165 @default.
• W2047899612 cites W2065257477 @default.
• W2047899612 cites W2071198735 @default.
• W2047899612 cites W2072858020 @default.
• W2047899612 cites W2077548707 @default.
• W2047899612 cites W2080187994 @default.
• W2047899612 cites W2081948388 @default.
• W2047899612 cites W2082575834 @default.
• W2047899612 cites W2085718426 @default.
• W2047899612 cites W2088333883 @default.
• W2047899612 cites W2090685205 @default.
• W2047899612 cites W2094892715 @default.
• W2047899612 cites W2096012158 @default.
• W2047899612 cites W2097774235 @default.
• W2047899612 cites W2098650898 @default.
• W2047899612 cites W2110029891 @default.
• W2047899612 cites W2113999599 @default.
• W2047899612 cites W2117217908 @default.
• W2047899612 cites W2126909895 @default.
• W2047899612 cites W2140075835 @default.
• W2047899612 cites W2148582464 @default.
• W2047899612 cites W2167490100 @default.
• W2047899612 cites W2185702474 @default.
• W2047899612 cites W2344494361 @default.
• W2047899612 cites W2410968240 @default.
• W2047899612 cites W4255313435 @default.
• W2047899612 cites W9096134 @default.
• W2047899612 doi "https://doi.org/10.1074/jbc.m001565200" @default.
• W2047899612 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11016920" @default.
• W2047899612 hasPublicationYear "2000" @default.
• W2047899612 type Work @default.
• W2047899612 sameAs 2047899612 @default.
• W2047899612 citedByCount "35" @default.
• W2047899612 countsByYear W20478996122014 @default.
• W2047899612 countsByYear W20478996122015 @default.
• W2047899612 countsByYear W20478996122017 @default.
• W2047899612 countsByYear W20478996122021 @default.
• W2047899612 crossrefType "journal-article" @default.
• W2047899612 hasAuthorship W2047899612A5008681401 @default.
• W2047899612 hasAuthorship W2047899612A5010440823 @default.
• W2047899612 hasAuthorship W2047899612A5013520965 @default.
• W2047899612 hasAuthorship W2047899612A5013770831 @default.
• W2047899612 hasAuthorship W2047899612A5030051266 @default.
• W2047899612 hasAuthorship W2047899612A5031699197 @default.
• W2047899612 hasAuthorship W2047899612A5039706801 @default.
• W2047899612 hasAuthorship W2047899612A5051851067 @default.
• W2047899612 hasBestOaLocation W20478996121 @default.
• W2047899612 hasConcept C118776929 @default.
• W2047899612 hasConcept C126322002 @default.
• W2047899612 hasConcept C134018914 @default.
• W2047899612 hasConcept C17991360 @default.
• W2047899612 hasConcept C185592680 @default.
• W2047899612 hasConcept C190283241 @default.
• W2047899612 hasConcept C3020084786 @default.
• W2047899612 hasConcept C502942594 @default.
• W2047899612 hasConcept C503630168 @default.
• W2047899612 hasConcept C55493867 @default.
• W2047899612 hasConcept C71924100 @default.
• W2047899612 hasConcept C86803240 @default.
• W2047899612 hasConceptScore W2047899612C118776929 @default.
• W2047899612 hasConceptScore W2047899612C126322002 @default.
• W2047899612 hasConceptScore W2047899612C134018914 @default.
• W2047899612 hasConceptScore W2047899612C17991360 @default.
• W2047899612 hasConceptScore W2047899612C185592680 @default.
• W2047899612 hasConceptScore W2047899612C190283241 @default.
• W2047899612 hasConceptScore W2047899612C3020084786 @default.
• W2047899612 hasConceptScore W2047899612C502942594 @default.
• W2047899612 hasConceptScore W2047899612C503630168 @default.
• W2047899612 hasConceptScore W2047899612C55493867 @default.
• W2047899612 hasConceptScore W2047899612C71924100 @default.
• W2047899612 hasConceptScore W2047899612C86803240 @default.
• W2047899612 hasIssue "51" @default.
• W2047899612 hasLocation W20478996121 @default.

• next