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• W2078841413 abstract "Previously, using primary hepatocytes residing in early G1 phase, we demonstrated that expression of the cyclin-dependent kinase (CDK) inhibitor protein p21Cip-1/WAF1/mda6 (p21) enhanced the toxicity of deoxycholic acid (DCA) + MEK1/2 inhibitor. This study examined the mechanisms regulating this apoptotic process. Overexpression of p21 or p27Kip-1 (p27) enhanced DCA + MEK1/2 inhibitor toxicity in primary hepatocytes that was dependent on expression of acidic sphingomyelinase and CD95. Overexpression of p21 suppressed MDM2, elevated p53 levels, and enhanced CD95, BAX, NOXA, and PUMA expression; knockdown of BAX/NOXA/PUMA reduced CDK inhibitor-stimulated cell killing. Parallel to cell death processes, overexpression of p21 or p27 profoundly enhanced DCA + MEK1/2 inhibitor-induced expression of ATG5 and GRP78/BiP and phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) and eIF2α, and it increased the numbers of vesicles containing a transfected LC3-GFP construct. Incubation of cells with 3-methyladenine or knockdown of ATG5 suppressed DCA + MEK1/2 inhibitor-induced LC3-GFP vesicularization and enhanced DCA + MEK1/2 inhibitor-induced toxicity. Expression of dominant negative PERK blocked DCA + MEK1/2 inhibitor-induced expression of ATG5, GRP78/BiP, and eIF2α phosphorylation and prevented LC3-GFP vesicularization. Knock-out or knockdown of p53 or CD95 abolished DCA + MEK1/2 inhibitor-induced PERK phosphorylation and prevented LC3-GFP vesicularization. Thus, CDK inhibitors suppress MDM2 levels and enhance p53 expression that facilitates bile acid-induced, ceramide-dependent CD95 activation to induce both apoptosis and autophagy in primary hepatocytes. Previously, using primary hepatocytes residing in early G1 phase, we demonstrated that expression of the cyclin-dependent kinase (CDK) inhibitor protein p21Cip-1/WAF1/mda6 (p21) enhanced the toxicity of deoxycholic acid (DCA) + MEK1/2 inhibitor. This study examined the mechanisms regulating this apoptotic process. Overexpression of p21 or p27Kip-1 (p27) enhanced DCA + MEK1/2 inhibitor toxicity in primary hepatocytes that was dependent on expression of acidic sphingomyelinase and CD95. Overexpression of p21 suppressed MDM2, elevated p53 levels, and enhanced CD95, BAX, NOXA, and PUMA expression; knockdown of BAX/NOXA/PUMA reduced CDK inhibitor-stimulated cell killing. Parallel to cell death processes, overexpression of p21 or p27 profoundly enhanced DCA + MEK1/2 inhibitor-induced expression of ATG5 and GRP78/BiP and phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) and eIF2α, and it increased the numbers of vesicles containing a transfected LC3-GFP construct. Incubation of cells with 3-methyladenine or knockdown of ATG5 suppressed DCA + MEK1/2 inhibitor-induced LC3-GFP vesicularization and enhanced DCA + MEK1/2 inhibitor-induced toxicity. Expression of dominant negative PERK blocked DCA + MEK1/2 inhibitor-induced expression of ATG5, GRP78/BiP, and eIF2α phosphorylation and prevented LC3-GFP vesicularization. Knock-out or knockdown of p53 or CD95 abolished DCA + MEK1/2 inhibitor-induced PERK phosphorylation and prevented LC3-GFP vesicularization. Thus, CDK inhibitors suppress MDM2 levels and enhance p53 expression that facilitates bile acid-induced, ceramide-dependent CD95 activation to induce both apoptosis and autophagy in primary hepatocytes. Bile acids are detergent molecules, synthesized from cholesterol in the liver, that are released into the gut upon feeding and are essential for digestion (1Benage D. O'Connor K.W. J. Clin. Gastroenterol. 1990; 12: 192-194Crossref PubMed Scopus (10) Google Scholar). In the intestine, bile acids function in the solubilization and absorption of fats, certain vitamins, and cholesterol (2Holt P.R. Arch. Intern. Med. 1972; 130: 574-583Crossref PubMed Scopus (40) Google Scholar). Bile acids, post-feeding, re-enter the liver via the portal vein together with digested nutrients and are re-circulated back into the gallbladder for use during the next feeding cycle (3Roberts M.S. Magnusson B.M. Burczynski F.J. Weiss M. Clin. Pharmacokinet. 2002; 41: 751-790Crossref PubMed Scopus (450) Google Scholar). Individually, when retained within the liver because of impaired secretion into the bile canaliculi, bile acids are also known to cause hepatocellular toxicity both in vivo and in vitro (4Faubion W.A. Guicciardi M.E. Miyoshi H. Bronk S.F. Roberts P.J. Svingen P.A. Kaufmann S.H. Gores G.J. J. Clin. Investig. 1999; 103: 137-145Crossref PubMed Scopus (463) Google Scholar, 5Noto H. Matsushita M. Koike M. Takahashi M. Matsue H. Kimura J. Todo S. Artif. Organs. 1998; 22: 300-307Crossref PubMed Scopus (14) Google Scholar, 6Bloomer J.R. Allen R.M. Klatskin G. Arch. Intern. Med. 1976; 136: 57-61Crossref PubMed Scopus (40) Google Scholar, 7Koeppel T.A. Trauner M. Baas J.C. Thies J.C. Schlosser S.F. Post S. Gebhard M.M. Herfarth C. Boyer J.L. Otto G. Hepatology. 1997; 26: 1085-1091PubMed Google Scholar, 8Poupon R. Chazouilleres O. Poupon R.E. J. Hepatol. 2000; 32: 129-140Abstract Full Text PDF PubMed Google Scholar). Treatment of primary rodent and human hepatocytes as well as hepatoma cells with a physiologic concentration of the bile acid deoxycholic acid (DCA) 2The abbreviations used are:DCAdeoxycholic acidDMSOdimethyl sulfoxideCMVcytomegalovirussiRNAshort interfering RNAPBSphosphate-buffered salinesiSCRscrambled siRNAASMaseacidic sphingomyelinaseCDKcyclin-dependent kinaseERendoplasmic reticulumPERKPKR-like endoplasmic reticulum kinaseE3ubiquitin-protein isopeptide ligase. 2The abbreviations used are:DCAdeoxycholic acidDMSOdimethyl sulfoxideCMVcytomegalovirussiRNAshort interfering RNAPBSphosphate-buffered salinesiSCRscrambled siRNAASMaseacidic sphingomyelinaseCDKcyclin-dependent kinaseERendoplasmic reticulumPERKPKR-like endoplasmic reticulum kinaseE3ubiquitin-protein isopeptide ligase. has been shown to cause activation of the ERK1/2 pathway (9Qiao L. McKinstry R. Gupta S. Gilfor D. Windle J.J. Hylemon P.B. Grant S. Fisher P.B. Dent P. Hepatology. 2002; 36: 39-48Crossref PubMed Scopus (44) Google Scholar, 10Qiao L. Yacoub A. Studer E. Gupta S. Pei X.Y. Grant S. Hylemon P.B. Dent P. Hepatology. 2002; 35: 779-789Crossref PubMed Scopus (130) Google Scholar, 11Qiao L. Studer E. Leach K. McKinstry R. Gupta S. Decker R. Kukreja R. Valerie K. Nagarkatti P. El Deiry W. Molkentin J. Schmidt-Ullrich R. Fisher P.B. Grant S. Hylemon P.B. Dent P. Mol. Biol. Cell. 2001; 12: 2629-2645Crossref PubMed Scopus (203) Google Scholar, 12Werneburg N.W. Yoon J.H. Higuchi H. Gores G.J. Am. J. Physiol. 2003; 285: G31-G36Crossref PubMed Scopus (21) Google Scholar). Blockade of DCA-induced ERK1/2 and AKT activation, with inhibitors of RAS, phosphatidylinositol 3-kinase, or MEK1/2, increased apoptosis ∼10-fold within 6 h of exposure. Apoptosis was dependent on bile acid-induced, ligand-independent, and ceramide-dependent activation of the CD95 death receptor. Other studies demonstrated that overexpression of the cyclin-dependent kinase inhibitor p21Cip-1/WAF1/mda6 (p21) enhanced DCA toxicity in hepatocytes that was due to enhanced expression of the tumor suppressor p53 (9Qiao L. McKinstry R. Gupta S. Gilfor D. Windle J.J. Hylemon P.B. Grant S. Fisher P.B. Dent P. Hepatology. 2002; 36: 39-48Crossref PubMed Scopus (44) Google Scholar, 13Qiao D. Stratagouleas E.D. Martinez J.D. Carcinogenesis. 2001; 22: 35-41Crossref PubMed Scopus (102) Google Scholar). Elevated expression of p53 correlated with a p21-dependent reduction in the expression of MDM2, the E3 ligase known to regulate p53 protein levels. MDM2 is also known to be a negative regulator of p21 expression, independently of p53 function (14Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3629) Google Scholar, 15Honda R. Tanaka H. Yasuda H. FEBS Lett. 1997; 420: 25-27Crossref PubMed Scopus (1576) Google Scholar, 16Kubbutat M.H. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2798) Google Scholar, 17Tao W. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3077-3080Crossref PubMed Scopus (296) Google Scholar, 18Zhang Z. Wang H. Prasad G. Li M. Yu D. Bonner J.A. Agarwal S. Zhang R. Clin. Cancer Res. 2004; 10: 1263-1273Crossref PubMed Scopus (56) Google Scholar, 19Shieh S.Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1718) Google Scholar, 20Maki C.G. J. Biol. Chem. 1999; 274: 16531-16535Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 21Chehab N.H. Malikzay A. Stavridi E.S. Halazonetis T.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13777-13782Crossref PubMed Scopus (452) Google Scholar, 22Maya R. Balass M. Kim S.T. Shkedy D. Leal J.F. Shifman O. Moas M. Buschmann T. Ronai Z. Shiloh Y. Kastan M.B. Katzir E. Oren M. Genes Dev. 2001; 15: 1067-1077Crossref PubMed Scopus (523) Google Scholar). These findings suggested that under endogenous promoter control p21 and MDM2 may potentially titrate the expression of each other to maintain a steady state amount of p53 within the cell. deoxycholic acid dimethyl sulfoxide cytomegalovirus short interfering RNA phosphate-buffered saline scrambled siRNA acidic sphingomyelinase cyclin-dependent kinase endoplasmic reticulum PKR-like endoplasmic reticulum kinase ubiquitin-protein isopeptide ligase. deoxycholic acid dimethyl sulfoxide cytomegalovirus short interfering RNA phosphate-buffered saline scrambled siRNA acidic sphingomyelinase cyclin-dependent kinase endoplasmic reticulum PKR-like endoplasmic reticulum kinase ubiquitin-protein isopeptide ligase. This study was designed, initially, to determine the mechanisms by which the CDK inhibitor stimulated expression of p53, via reduction of MDM2 levels, and promoted bile acid toxicity in primary hepatocytes. However, based on our recent discovery using the novel cancer therapeutics sorafenib and vorinostat, activation of CD95 can promote endoplasmic reticulum (ER) stress as well as PERK- and ATG5-dependent autophagy, and reduced expression of an E3 ligase, such as MDM2, will also be predicted to increase the levels of unfolded proteins in cells. Therefore, we subsequently examined whether CDK inhibitors promoted bile acid-induced ER stress and autophagy in primary hepatocytes (23Yacoub A. Park M.A. Gupta P. Rahmani M. Zhang G. Hamed H. Hanna D. Sarkar D. Lebedeva I.V. Emdad L. Sauane M. Vozhilla N. Spiegel S. Koumenis C. Graf M. Curiel D.T. Grant S. Fisher P.B. Dent P. Mol. Cancer Ther. 2008; 7: 297-313Crossref PubMed Scopus (69) Google Scholar, 24Park M. Yacoub A. Rahmani M. Zhang G. Hart L. Hagan M. Calderwood S. Sherman M. Koumenis C. Spiegel S. Chen C.S. Graf M. Curiel D. Fisher P. Grant S. Dent P. Mol. Pharmacol. 2008; 73: 1168-1184Crossref PubMed Scopus (61) Google Scholar, 25Zhang G. Park M.A. Mitchell C. Hamed H. Rahmani M. Martin A.P. Curiel D.T. Yacoub A. Graf M. Lee R. Roberts J.D. Fisher P.B. Grant S. Dent P. Clin. Cancer Res. 2008; (in press)Google Scholar, 26Park M.A. Zhang Z. Mitchell C. Hamed H. Rahmani M. Martin A.P. Yacoub A. Koumenis C. Spiegel S. Norris J. Hylemon P.B. Graf M. Fisher P.B. Grant S. Dent P. Cancer Biol. Ther. 2008; (in press)Google Scholar). Autophagy is a ubiquitous process in which cells degrade cytosolic materials such as proteins and organelles, and this process continuously occurs at a basal level in eukaryotic cells. In this process, cytoplasmic constituents are sequestered into forming membrane vesicles referred to as autophagosomes, which then fuse with lysosomes to form an autolysosome. In the autolysosome the contents of the vesicle are degraded and recycled. Autophagy has been primarily researched in yeast as a response to nutrient depletion, and there are at least 25 yeast genes specifically involved in the autophagic process, and the levels of their gene products are directly elevated when autophagy is up-regulated. Recent studies have shown that yeast ATGs have very similar mammalian homologues arguing that autophagy is a conserved mechanism throughout evolution. Our present findings demonstrate that CDK-stimulated expression of p53 promoted bile acid toxicity by causing p53 to translocate from the cytoplasm to the nucleus and to increase the expression of BAX, PUMA, NOXA, and CD95. Overexpression of p21 or p27Kip-1 enhanced bile acid-induced autophagy signaling in an acidic sphingomyelinase- and CD95-dependent fashion, which was a protective event, compared with bile acid-induced acidic sphingomyelinase- and CD95-dependent apoptosis. Collectively, these findings argue that CDK inhibitors can promote a protective autophagy response in response to toxic bile acid treatment. Materials—All bile acids were obtained from Sigma. Phospho-/total-ERK1/2 were purchased from Cell Signaling Technologies (Worcester, MA). Jo2 hamster anti-mouse CD95 IgG was from Pharmingen. All the other secondary antibodies (anti-rabbit, anti-mouse, and anti-goat horseradish peroxidase) and rhodamine-conjugated goat anti-Armenian hamster IgGs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). 3-Methyladenine was supplied by Calbiochem as powder, dissolved in sterile PBS, and stored frozen under light-protected conditions at –80 °C. Enhanced chemiluminescence (ECL) kits were purchased from Amersham Biosciences and PerkinElmer Life Sciences. Trypsin-EDTA, Williams Medium E, and penicillin/streptomycin were purchased from Invitrogen. Other reagents were as described previously (9Qiao L. McKinstry R. Gupta S. Gilfor D. Windle J.J. Hylemon P.B. Grant S. Fisher P.B. Dent P. Hepatology. 2002; 36: 39-48Crossref PubMed Scopus (44) Google Scholar, 10Qiao L. Yacoub A. Studer E. Gupta S. Pei X.Y. Grant S. Hylemon P.B. Dent P. Hepatology. 2002; 35: 779-789Crossref PubMed Scopus (130) Google Scholar, 11Qiao L. Studer E. Leach K. McKinstry R. Gupta S. Decker R. Kukreja R. Valerie K. Nagarkatti P. El Deiry W. Molkentin J. Schmidt-Ullrich R. Fisher P.B. Grant S. Hylemon P.B. Dent P. Mol. Biol. Cell. 2001; 12: 2629-2645Crossref PubMed Scopus (203) Google Scholar, 23Yacoub A. Park M.A. Gupta P. Rahmani M. Zhang G. Hamed H. Hanna D. Sarkar D. Lebedeva I.V. Emdad L. Sauane M. Vozhilla N. Spiegel S. Koumenis C. Graf M. Curiel D.T. Grant S. Fisher P.B. Dent P. Mol. Cancer Ther. 2008; 7: 297-313Crossref PubMed Scopus (69) Google Scholar, 24Park M. Yacoub A. Rahmani M. Zhang G. Hart L. Hagan M. Calderwood S. Sherman M. Koumenis C. Spiegel S. Chen C.S. Graf M. Curiel D. Fisher P. Grant S. Dent P. Mol. Pharmacol. 2008; 73: 1168-1184Crossref PubMed Scopus (61) Google Scholar, 25Zhang G. Park M.A. Mitchell C. Hamed H. Rahmani M. Martin A.P. Curiel D.T. Yacoub A. Graf M. Lee R. Roberts J.D. Fisher P.B. Grant S. Dent P. Clin. Cancer Res. 2008; (in press)Google Scholar, 26Park M.A. Zhang Z. Mitchell C. Hamed H. Rahmani M. Martin A.P. Yacoub A. Koumenis C. Spiegel S. Norris J. Hylemon P.B. Graf M. Fisher P.B. Grant S. Dent P. Cancer Biol. Ther. 2008; (in press)Google Scholar). Primary Culture of Rodent Hepatocytes—Hepatocytes were isolated from adult male Sprague-Dawley rats and wild type or CD95–/– or ASMase–/– or p21–/– or p53–/– C57/BL6 mice by the two-step collagenase perfusion technique. The freshly isolated hepatocytes were plated on rat tail collagen (Vitrogen)-coated plate at a density of 2 × 105 cells/well of a 12-well plate, and cultured in Williams E medium supplemented with 0.1 nm dexamethasone, 1 nm thyroxine, and 100 μg/ml of penicillin/streptomycin, at 37 °C in a humidified atmosphere containing 5% CO2. The initial medium change was performed 3 h after cell seeding to minimize the contamination of dead or mechanically damaged cells. In some studies, where noted, hepatocytes were cultured in the presence of 50 nm insulin, 0.1 nm dexamethasone, 1 nm thyroxine, and 100 μg/ml of penicillin/streptomycin. Recombinant Adenoviral Vectors; Generation and Infection in Vitro—Two adenoviral technologies were used. Replication-defective adenovirus was conjugated to poly-l-lysine and a cDNA plasmid construct, as indicated in each legend and as described previously (9Qiao L. McKinstry R. Gupta S. Gilfor D. Windle J.J. Hylemon P.B. Grant S. Fisher P.B. Dent P. Hepatology. 2002; 36: 39-48Crossref PubMed Scopus (44) Google Scholar, 10Qiao L. Yacoub A. Studer E. Gupta S. Pei X.Y. Grant S. Hylemon P.B. Dent P. Hepatology. 2002; 35: 779-789Crossref PubMed Scopus (130) Google Scholar, 11Qiao L. Studer E. Leach K. McKinstry R. Gupta S. Decker R. Kukreja R. Valerie K. Nagarkatti P. El Deiry W. Molkentin J. Schmidt-Ullrich R. Fisher P.B. Grant S. Hylemon P.B. Dent P. Mol. Biol. Cell. 2001; 12: 2629-2645Crossref PubMed Scopus (203) Google Scholar). Next we used recombinant adenoviruses. Hepatocytes were transfected/infected with these adenoviruses at an approximate multiplicity of infection of 250 and 30, respectively. Cells were further incubated for 24 h to ensure adequate expression of transduced gene products prior to bile acid/drug treatments. SDS-PAGE and Western Blot Analysis—At various time points after indicated treatment, hepatocytes were lysed in whole-cell lysis buffer (0.5 m Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 0.02% bromphenol blue), and the samples were boiled for 30 min. The boiled samples were loaded onto 14% SDS-PAGE, and electrophoresis was run overnight. Proteins were electrophoretically transferred onto 0.22-μm nitrocellulose and immunoblotted with various primary antibodies against different proteins. All immunoblots were visualized by ECL. For presentation, immunoblots were digitally scanned at 600 dpi using Adobe PhotoShop CS2; the color was removed, and figures were generated in Microsoft PowerPoint. LC3-GFP Visualization—Cells were plated as described above and transfected 24 h after plating. For mouse embryonic fibroblasts (2–5 μg) or other hepatocytes (0.5 μg), plasmids expressing a specific mRNA (or siRNA) or appropriate vector control plasmid DNA were diluted in 50 μl of serum-free and antibiotic-free medium (1 portion for each sample). Concurrently, 2 μl of Lipofectamine 2000 (Invitrogen) was diluted into 50 μl of serum-free and antibiotic-free medium (1 portion for each sample). Diluted DNA was added to the diluted Lipofectamine 2000 for each sample and incubated at room temperature for 30 min. This mixture was added to each well/dish of cells containing 200 μl of serum-free and antibiotic-free medium for a total volume of 300 μl, and the cells were incubated for 4 h at 37 °C. An equal volume of 2× medium was then added to each well. Cells were incubated for 48 h and then treated with DCA ± PD184352. For analysis of cells transfected with GFP-LC3 constructs, the GFP-LC3-positive vacuolated cells were examined under the ×40 objective of a Zeiss Axiovert fluorescent microscope. In control studies, expression of β-galactosidase using Ad.β-gal did not induce GFP-LC3 vacuoles in hepatocytes in the presence or absence of DCA ± PD184352. 3G. Zhang, M. A. Park, and P. Dent, unpublished observations. Forty LC3-GFP-positive cells were analyzed per condition. Each vacuole was counted, and the average number of vacuoles per cell for each, including cells that did not exhibit vacuolization, was calculated. Immunofluorescence—Hepatocytes were cultured at 10–20% confluence on collagen-coated glass coverslips in 4-well dishes for 24 h. After treatment, the cells were washed with PBS and fixed in cold methanol:acetone (1:1) for 10 min at –20 °C. After washing three times in PBS for 5 min each, the cells were blocked in 0.5% bovine serum albumin in PBS for 30 min at room temperature and incubated with the primary antibody, Jo2, diluted in PBS, 0.5% bovine serum albumin (1:500) overnight at 4 °C. For measurements of total cellular CD95, hepatocytes were fixed in 3.7% formaldehyde in PBS for 10 min at 4 °C and permeabilized with 0.1% Triton X-100 in PBS for 3 min at 4 °C prior to incubation with the primary antibody. Cells were washed again with PBS and then incubated with rhodamine-conjugated goat anti-Armenian hamster IgG at 1:100 dilution for 60 min at room temperature. After washing with PBS, the cells were stained with 5 μm 4′, 6′-diamidino-2-phenylindole dihydrochloride in PBS for 5 min. The cells were washed again with PBS, followed by distilled water, and mounted in Crystal-Mount. Fluorescence staining was viewed with a Zeiss Axiovert fluorescent microscope. Fluorescence was quantified via Image-Pro® Plus analysis software and expressed as integrated optical density. Measurement of Endogenous Ceramide—Lipids were extracted, and mass amounts of ceramide in cellular extracts were measured by the diacylglycerol kinase enzymatic method. Briefly, hepatocytes were washed with PBS and scraped into 1 ml of cold methanol containing 2.5 μl of concentrated HCl. Lipids were extracted by adding 2 ml of chloroform, 1 m NaCl (1:1, v/v), and phases were separated. An aliquot (100 μl) of the chloroform phase was dried under nitrogen gas. Bovine brain type III ceramide was used as a standard. The enzymatic reaction was started by the addition of 100 μl of 50 mm imidazole, 1 mm diethylenetriaminepentaacetic acid, 12.5 mm MgCl2, 50 mm NaCl, 1 mm EGTA, 10 mm dithiothreitol, 1 mm ATP, 1.5% N-octyl-β-d-glucopyranoside, 1 mm cardiolipin, diacylglycerol kinase (0.01 unit), and [γ-32P]ATP (1 μCi). After incubation at 37 °C for 35 min with 15 min of sonication at room temperature in-between, lipids were extracted by the addition of 500 μl of chloroform:methanol:HCl (100:100:1) and 100 μl of 1 m NaCl. Labeled ceramide 1-phosphate and phosphatidic acid (50-μl organic layer) were resolved by TLC with chloroform:acetone: methanol:acetic acid:water (10:4:3:2:1). Spots corresponding to ceramide were quantified with a Bio-Rad PhosphorImager. Detection of Cell Death by Giemsa and/or Hoescht 33342 Assay—Cells were harvested by trypsinization with trypsin/EDTA for ∼10 min at 37 °C. As some apoptotic cells detached from the culture substratum into the medium, these cells were also collected by centrifugation of the medium at 1,500 rpm for 5 min. The pooled cell pellets were resuspended and spun onto glass slides (cytospin). Cells were stained using a commercial Giemsa kit (DiffQuick) and visualized under light microscopy (×40 magnification), scoring the number of cells exhibiting the “classic” morphological features of apoptosis and necrosis. For each condition, 10 randomly selected fields per slide were evaluated, encompassing at least 1500 cells. In parallel for confirmation, cells fixed to slides were stained with Hoechst 33342 dye followed by PBS washing to remove excess dye, air-dried, and mounted in Fluoro-Guard Antifade (Bio-Rad). Nuclear morphology was evaluated. Apoptotic cells were identified as those whose nuclei exhibited brightly staining condensed chromatin or nuclear fragmentation. Data Analysis—Comparison of the effects of various treatments was performed using one-way analysis of variance and a two-tailed t test. Differences with a p value of < 0.05 were considered statistically significant. Experiments shown are the means of multiple points (±S.E.). Bile Acid Toxicity Is CD95-dependent and Is Enhanced by Overexpression of p21 or p27—Initial studies characterized the regulation of rodent hepatocyte survival in vitro after exposure to the cell-permeable bile acid DCA and a MEK1/2 inhibitor. Treatment of mouse hepatocytes with DCA and a MEK1/2 inhibitor enhanced cell killing within 6 h that was blocked by expression of dominant negative FADD or dominant negative caspase 8, overexpression of the caspase 8 inhibitor c-FLIP-s, or was blocked in CD95–/– hepatocytes (Fig. 1A). Hence, the primary death signal generated by DCA + MEK1/2 inhibitor treatment emanated from CD95 (11Qiao L. Studer E. Leach K. McKinstry R. Gupta S. Decker R. Kukreja R. Valerie K. Nagarkatti P. El Deiry W. Molkentin J. Schmidt-Ullrich R. Fisher P.B. Grant S. Hylemon P.B. Dent P. Mol. Biol. Cell. 2001; 12: 2629-2645Crossref PubMed Scopus (203) Google Scholar). Loss of basal p21 expression in p21–/– hepatocytes lowered the toxicity of DCA ± MEK1/2 inhibitor, and overexpression of p21 in hepatocytes enhanced DCA ± MEK1/2 inhibitor lethality (Fig. 1B). We next determined whether expression of a member of the same CDK inhibitor family as p21 would also enhance bile acid lethality, p27Kip-1 (p27). Overexpression of p27 also promoted DCA lethality and DCA + MEK1/2 inhibitor lethality in primary rat hepatocytes (Fig. 1, C and D). The Promotion of Bile Acid Toxicity by CDK Inhibitors Is p53-dependent and Correlates with Reduced p53 Ubiquitination, with Increased p53 Nuclear Localization, and Increased CD95, BAX, PUMA, and NOXA Levels—The tumor suppressor p53 is widely regarded as a key transcriptional regulator of p21 expression. In p53–/– hepatocytes, loss of p53 expression reduced DCA lethality, whereas overexpression of p21 did not promote DCA ± MEK1/2 inhibitor killing in p53–/– cells (Fig. 2A). Overexpression of p21 in rat hepatocytes enhanced the expression of p53 (Fig. 2B). In cells overexpressing p21, the majority of p53 was located in the nuclear fraction, whereas in vector control infected cells, p53 was mostly cytosolic. Treatment of hepatocytes with DCA + MEK1/2 inhibitor promoted modest nuclear translocation of p53, regardless of p21 overexpression. Overexpression of p21 increased the protein levels of several established/bona fide p53 target genes that have been implicated by many investigators in enhanced cell death responses, including the BH3 domain proteins BAX, PUMA, and NOXA (Table 1). In contrast to prior studies from this laboratory, which did not observe any significant changes in CD95 expression during these treatments, more definitive analyses using different antibody reagents that generated immunoblots with less nonspecific background bands demonstrated that overexpression of p21 increased CD95 protein levels (Table 1) (9Qiao L. McKinstry R. Gupta S. Gilfor D. Windle J.J. Hylemon P.B. Grant S. Fisher P.B. Dent P. Hepatology. 2002; 36: 39-48Crossref PubMed Scopus (44) Google Scholar).TABLE 1CDK inhibitors enhance the expression of multiple BH3 domain proteinsInfection/treatmentCMV/VEHCMV/PD + DCAp21/VEHp21/PD + DCAp27/VEHp27/PD + DCABAX1.001.05 ± 0.031.55 ± 0.06ap < 0.05 expression value greater than corresponding value in vector control (CMV) infected cells.3.01 ± 0.42bp < 0.05 expression value is greater than corresponding value in parallel untreated cells.1.40 ± 0.17ap < 0.05 expression value greater than corresponding value in vector control (CMV) infected cells.2.88 ± 0.16bp < 0.05 expression value is greater than corresponding value in parallel untreated cells.NOXA1.001.52 ± 0.13bp < 0.05 expression value is greater than corresponding value in parallel untreated cells.1.67 ± 0.05ap < 0.05 expression value greater than corresponding value in vector control (CMV) infected cells.2.59 ± 0.08bp < 0.05 expression value is greater than corresponding value in parallel untreated cells.1.91 ± 0.24ap < 0.05 expression value greater than corresponding value in vector control (CMV) infected cells.3.11 ± 0.40bp < 0.05 expression value is greater than corresponding value in parallel untreated cells.CD951.001.51 ± 0.33bp < 0.05 expression value is greater than corresponding value in parallel untreated cells.1.80 ± 0.20ap < 0.05 expression value greater than corresponding value in vector control (CMV) infected cells.3.03 ± 0.36bp < 0.05 expression value is greater than corresponding value in parallel untreated cells.3.09 ± 0.09ap < 0.05 expression value greater than corresponding value in vector control (CMV) infected cells.4.23 ± 0.27bp < 0.05 expression value is greater than corresponding value in parallel untreated cells.p531.001.08 ± 0.172.09 ± 0.26ap < 0.05 expression value greater than corresponding value in vector control (CMV) infected cells.5.27 ± 1.01bp < 0.05 expression value is greater than corresponding value in parallel untreated cells.3.91 ± 0.69ap < 0.05 expression value greater than corresponding value in vector control (CMV) infected cells.5.42 ± 1.22bp < 0.05 expression value is greater than corresponding value in parallel untreated cells.PUMA1.007.42 ± 0.07bp < 0.05 expression value is greater than corresponding value in parallel untreated cells.6.24 ± 0.54ap < 0.05 expression value greater than corresponding value in vector control (CMV) infected cells.12.97 ± 0.14bp < 0.05 expression value is greater than corresponding value in parallel untreated cells.7.87 ± 0.07ap < 0.05 expression value greater than corresponding value in vector control (CMV) infected cells.10.91 ± 0.59bp < 0.05 expression value is greater than corresponding value in parallel untreated cells.a p < 0.05 expression value greater than corresponding value in vector control (CMV) infected cells.b p < 0.05 expression value is greater than corresponding value in parallel untreated cells. Open table in a new tab In prior studies overexpression of p21 in p21–/– hepatocytes decreased the expression of MDM2 and increased the expression of p53 (Fig. 2C, upper panel) (9Qiao L. McKinstry R. Gupta S. Gilfor D. Windle J.J. Hylemon P.B. Grant S. Fisher P.B. Dent P. Hepatology. 2002; 36: 39-48Crossref PubMed Scopus (44) Google Scholar). We have now noted that overexpression of p21 correlated with decreased ubiquitination of p53 in immunoprecipitates of p53 (Fig. 2C, lower panel). These findings suggest that p21 interferes with the ability of MDM2 to ubiquitinate p53. As p21 overexpression increased the levels of the pro-apoptotic proteins BAX, PUMA, and NOXA, we determined whether the enhanced expression of these proteins played any role in the elevated levels of the cell killing we were observing. Knockdown of BAX, NOXA, or PUMA expression significantly reduced p21-stimulated DCA + MEK1/2 inhibitor toxicity (Fig. 2D). Knockdown of NOXA expression reduced DCA + MEK1/2 inhibitor lethalit" @default.
• W2078841413 created "2016-06-24" @default.
• W2078841413 creator A5007992168 @default.
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• W2078841413 title "Multiple Cyclin Kinase Inhibitors Promote Bile Acid-induced Apoptosis and Autophagy in Primary Hepatocytes via p53-CD95-dependent Signaling" @default.
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