Inhibition of p53 Function Prevents Renin-Angiotensin System Activation and Stretch-Mediated Myocyte Apoptosis
2000; Elsevier BV; Volume: 157; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)64598-1
ISSN1525-2191
AutoresAnnarosa Leri, Fabio Fiordaliso, Manabu Setoguchi, Federica Limana, Nanette H. Bishopric, Jan Kajstura, Keith A. Webster, Piero Anversa,
Tópico(s)RNA modifications and cancer
ResumoTo determine whether stretch-induced activation of p53 is necessary for the up-regulation of the local renin-angiotensin system and angiotensin II (Ang II)-induced apoptosis, ventricular myocytes were infected with an adenoviral vector carrying mutated p53, Adp53m, before 12 hours of stretch. Noninfected myocytes and myocytes infected with AdLacZ served as controls. Stretching of Adp53m-infected myocytes prevented stimulation of p53 function that conditioned the expression of p53-dependent genes; quantity of angiotensinogen (Aogen), AT1, and Bax decreased, whereas Bcl-2 increased. Ang II generation was not enhanced by stretch. Conversely, stretch produced opposite changes in noninfected and AdLacZ-infected myocytes: Aogen increased twofold, AT1 increased 2.1-fold, Bax increased 2.5-fold, and Ang II increased 2.4-fold. These responses were coupled with 4.5-fold up-regulation of wild-type p53. Stretch elicited comparable adaptations in p53-independent genes, in the presence or absence of mutated p53; renin increased threefold, angiotensin-converting enzyme increased ninefold, and AT2 increased 1.7-fold. Infection with Adp53m inhibited myocyte apoptosis after stretch. Conversely, stretch increased apoptosis by 6.2-fold in myocytes with elevated endogenous wild-type p53. Thus, a competitor of p53 function interfered with both stretch-induced Ang II formation and apoptosis, indicating that p53 is a major modulator of myocyte renin-angiotensin system and cell survival after mechanical deformation. To determine whether stretch-induced activation of p53 is necessary for the up-regulation of the local renin-angiotensin system and angiotensin II (Ang II)-induced apoptosis, ventricular myocytes were infected with an adenoviral vector carrying mutated p53, Adp53m, before 12 hours of stretch. Noninfected myocytes and myocytes infected with AdLacZ served as controls. Stretching of Adp53m-infected myocytes prevented stimulation of p53 function that conditioned the expression of p53-dependent genes; quantity of angiotensinogen (Aogen), AT1, and Bax decreased, whereas Bcl-2 increased. Ang II generation was not enhanced by stretch. Conversely, stretch produced opposite changes in noninfected and AdLacZ-infected myocytes: Aogen increased twofold, AT1 increased 2.1-fold, Bax increased 2.5-fold, and Ang II increased 2.4-fold. These responses were coupled with 4.5-fold up-regulation of wild-type p53. Stretch elicited comparable adaptations in p53-independent genes, in the presence or absence of mutated p53; renin increased threefold, angiotensin-converting enzyme increased ninefold, and AT2 increased 1.7-fold. Infection with Adp53m inhibited myocyte apoptosis after stretch. Conversely, stretch increased apoptosis by 6.2-fold in myocytes with elevated endogenous wild-type p53. Thus, a competitor of p53 function interfered with both stretch-induced Ang II formation and apoptosis, indicating that p53 is a major modulator of myocyte renin-angiotensin system and cell survival after mechanical deformation. The tumor suppressor gene, p53, has been implicated in the modulation of cardiac myocyte apoptosis in vitro1Long X Boluyt MO Hipolito ML Lundberg MS Zheng JS O'Neill L Cirielli C Lakatta EG Crow MT p53 and the hypoxia-induced apoptosis of cultured neonatal rat cardiac myocytes.J Clin Invest. 1997; 99: 2635-2643Crossref PubMed Scopus (275) Google Scholar, 2Leri A Claudio PP Li Q Wang X Reiss K Wang S Malhotra A Kajstura J Anversa P Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.J Clin Invest. 1998; 101: 1326-1342Crossref PubMed Google Scholar, 3Long X Crow MT Sollott SJ O'Neill L Menees DS de Lourdes Hipolito M Boluyt MO Asai T Lakatta EG Enhanced expression of p53 and apoptosis induced by blockade of the vacuolar proton ATPase in cardiomyocytes.J Clin Invest. 1998; 101: 1453-1461Crossref PubMed Scopus (61) Google Scholar and in vivo.4Leri A Li Y Malhotra A Li Q Stiegler P Claudio PP Giordano A Kajstura J Hintze TH Anversa P Pacing-induced heart failure in dogs enhances the expression of p53 and p53-dependent genes in ventricular myocytes.Circulation. 1998; 97: 194-203Crossref PubMed Scopus (106) Google Scholar However, not all results are in agreement; forced re-entry of myocytes into the cell cycle by infection of cells with E2F-1 leads to the stimulation of the endogenous cell death pathway, independently from p53.5Agah R Kirshenbaum LA Abdellatif M Truong LD Chakraborty S Michael LH Schneider MD Adenoviral delivery of E2F-1 directs cell cycle reentry and p53-independent apoptosis in postmitotic adult myocardium in vivo.J Clin Invest. 1997; 100: 2722-2728Crossref PubMed Scopus (186) Google Scholar Conversely, hypoxia1Long X Boluyt MO Hipolito ML Lundberg MS Zheng JS O'Neill L Cirielli C Lakatta EG Crow MT p53 and the hypoxia-induced apoptosis of cultured neonatal rat cardiac myocytes.J Clin Invest. 1997; 99: 2635-2643Crossref PubMed Scopus (275) Google Scholar or disruption of the vacuolar proton-ATPase system3Long X Crow MT Sollott SJ O'Neill L Menees DS de Lourdes Hipolito M Boluyt MO Asai T Lakatta EG Enhanced expression of p53 and apoptosis induced by blockade of the vacuolar proton ATPase in cardiomyocytes.J Clin Invest. 1998; 101: 1453-1461Crossref PubMed Scopus (61) Google Scholar, 6Karwatowska-Prokopczuk E Nordberg JA Li HL Engler RL Gottlieb RA Effect of vacuolar proton ATPase on pHi, Ca2+, and apoptosis in neonatal cardiomyocytes during metabolic inhibition/recovery.Circ Res. 1998; 82: 1139-1144Crossref PubMed Scopus (76) Google Scholar initiates myocyte apoptosis in which p53 seems to play a significant role.3Long X Crow MT Sollott SJ O'Neill L Menees DS de Lourdes Hipolito M Boluyt MO Asai T Lakatta EG Enhanced expression of p53 and apoptosis induced by blockade of the vacuolar proton ATPase in cardiomyocytes.J Clin Invest. 1998; 101: 1453-1461Crossref PubMed Scopus (61) Google Scholar p53 is a transcriptional activator of the proapoptotic gene product Bax7Miyashita T Reed J Tumor suppressor p53 is a direct transcriptional activator of the human bax gene.Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (305) Google Scholar and a p53-negative response element has been identified in the promoter region of the antiapoptotic gene product Bcl-2.8Miyashita T Harigai M Hanada M Reed JC Identification of a p53-dependent negative response element in the bcl-2 gene.Cancer Res. 1994; 54: 3131-3135PubMed Google Scholar However, in nonmyocytes, increases in p53 quantity and activity and decreases in Bcl-2:Bax protein ratios enhance the susceptibility of cells to apoptotic death signals. In adult ventricular myocytes, p53 has been reported to up-regulate the cellular renin-angiotensin system (RAS), resulting in the synthesis of the octapeptide angiotensin II (Ang II) in vitro2Leri A Claudio PP Li Q Wang X Reiss K Wang S Malhotra A Kajstura J Anversa P Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.J Clin Invest. 1998; 101: 1326-1342Crossref PubMed Google Scholar, 9Leri A Liu Y Claudio PP Kajstura J Wang X Wang S Kang P Malhotra A Anversa P Insulin-like growth factor-1 induces Mdm2 and down-regulates p53, attenuating the myocyte renin-angiotensin system and stretch-mediated apoptosis.Am J Pathol. 1999; 154: 567-580Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar and in vivo.10Leri A Liu Y Wang X Kajstura J Malhotra A Meggs LG Anversa P Overexpression of insulin-like growth factor-1 attenuates the myocyte renin-angiotensin system in transgenic mice.Circ Res. 1999; 84: 752-762Crossref PubMed Scopus (90) Google Scholar Ang II, in turn, activates p53, creating a positive feed-back loop in which hormone production and p53 function are intimately related in myocytes.2Leri A Claudio PP Li Q Wang X Reiss K Wang S Malhotra A Kajstura J Anversa P Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.J Clin Invest. 1998; 101: 1326-1342Crossref PubMed Google Scholar, 9Leri A Liu Y Claudio PP Kajstura J Wang X Wang S Kang P Malhotra A Anversa P Insulin-like growth factor-1 induces Mdm2 and down-regulates p53, attenuating the myocyte renin-angiotensin system and stretch-mediated apoptosis.Am J Pathol. 1999; 154: 567-580Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 10Leri A Liu Y Wang X Kajstura J Malhotra A Meggs LG Anversa P Overexpression of insulin-like growth factor-1 attenuates the myocyte renin-angiotensin system in transgenic mice.Circ Res. 1999; 84: 752-762Crossref PubMed Scopus (90) Google Scholar Indirect evidence favoring this possibility, has been obtained by applying physical forces to myocytes in vitro,2Leri A Claudio PP Li Q Wang X Reiss K Wang S Malhotra A Kajstura J Anversa P Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.J Clin Invest. 1998; 101: 1326-1342Crossref PubMed Google Scholar or by adding IGF-1 to this preparation.9Leri A Liu Y Claudio PP Kajstura J Wang X Wang S Kang P Malhotra A Anversa P Insulin-like growth factor-1 induces Mdm2 and down-regulates p53, attenuating the myocyte renin-angiotensin system and stretch-mediated apoptosis.Am J Pathol. 1999; 154: 567-580Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar In the first case, stretching was associated with release of Ang II, up-regulation of p53 and p53-inducible genes, enhanced cellular RAS, and myocyte apoptosis. In the second case, IGF-1 inhibited p53 via the formation of Mdm2-p53 protein complexes, decreasing Ang II synthesis and cell death. A more causal relationship between the local RAS and p53 was obtained by infecting myocytes with an adenovirus carrying wild-type p53; p53 activated the cellular RAS, decreased the Bcl-2:Bax ratio and produced extensive apoptosis.11Pierzchalski P Reiss K Cheng W Cirielli C Kajstura J Nitahara JA Rizk M Capogrossi MC Anversa P p53 induces myocyte apoptosis via the activation of the renin-angiotensin system.Exp Cell Res. 1997; 234: 57-65Crossref PubMed Scopus (125) Google Scholar Despite these findings, the postulated effects of sarcomere stretching on p53, p53-dependent genes, myocyte RAS, and apoptosis2Leri A Claudio PP Li Q Wang X Reiss K Wang S Malhotra A Kajstura J Anversa P Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.J Clin Invest. 1998; 101: 1326-1342Crossref PubMed Google Scholar, 9Leri A Liu Y Claudio PP Kajstura J Wang X Wang S Kang P Malhotra A Anversa P Insulin-like growth factor-1 induces Mdm2 and down-regulates p53, attenuating the myocyte renin-angiotensin system and stretch-mediated apoptosis.Am J Pathol. 1999; 154: 567-580Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar remain to be unequivocally clarified. This is a relevant issue because acute changes in diastolic loading and sarcomere elongation occur immediately after myocardial infarction, resulting in wall thinning and chamber dilation.12Pfeffer MA Braunwald E Ventricular remodeling after myocardial infarction.Circulation. 1990; 81: 1161-1172Crossref PubMed Scopus (2611) Google Scholar Myocyte apoptosis is a critical event in acute ventricular remodeling,13Cheng W Li B Kajstura J Li P Wolin MS Sonnenblick EH Hintze TH Olivetti G Anversa P Stretch induced programmed myocyte cell death.J Clin Invest. 1995; 96: 2247-2259Crossref PubMed Scopus (591) Google Scholar, 14Cheng W Kajstura J Nitahara JA Li B Reiss K Liu Y Clark WA Krajewski S Reed JC Olivetti G Anversa P Programmed myocyte cell death affects the viable myocardium after infarction in rats.Exp Cell Res. 1996; 226: 316-327Crossref PubMed Scopus (321) Google Scholar and the decrease in Bcl-2 and increase in Bax in the spared myocytes of the postinfarcted heart suggest involvement of p53 in this setting.13Cheng W Li B Kajstura J Li P Wolin MS Sonnenblick EH Hintze TH Olivetti G Anversa P Stretch induced programmed myocyte cell death.J Clin Invest. 1995; 96: 2247-2259Crossref PubMed Scopus (591) Google Scholar Moreover, increases in end-diastolic pressure and sarcomere stretching characterize ventricular dysfunction and failure of different origin.12Pfeffer MA Braunwald E Ventricular remodeling after myocardial infarction.Circulation. 1990; 81: 1161-1172Crossref PubMed Scopus (2611) Google Scholar To obtain direct, functional evidence for the role of p53 in stretch-mediated Ang II generation and apoptotic signaling, adult ventricular myocytes were infected with a recombinant adenovirus expressing a dominant-negative form of p53 before stretching. Results indicate that induction of p53 by mechanical deformation of myocytes for a period of 12 hours is causally related to both phenomena. Infection with dominant-negative p53 was induced before mechanical deformation to avoid activation of endogenous wild-type p53 with stretch in the absence of its mutated inhibitory form. Myocytes were isolated from 3-month-old Sprague-Dawley rats.2Leri A Claudio PP Li Q Wang X Reiss K Wang S Malhotra A Kajstura J Anversa P Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.J Clin Invest. 1998; 101: 1326-1342Crossref PubMed Google Scholar, 9Leri A Liu Y Claudio PP Kajstura J Wang X Wang S Kang P Malhotra A Anversa P Insulin-like growth factor-1 induces Mdm2 and down-regulates p53, attenuating the myocyte renin-angiotensin system and stretch-mediated apoptosis.Am J Pathol. 1999; 154: 567-580Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 11Pierzchalski P Reiss K Cheng W Cirielli C Kajstura J Nitahara JA Rizk M Capogrossi MC Anversa P p53 induces myocyte apoptosis via the activation of the renin-angiotensin system.Exp Cell Res. 1997; 234: 57-65Crossref PubMed Scopus (125) Google Scholar, 14Cheng W Kajstura J Nitahara JA Li B Reiss K Liu Y Clark WA Krajewski S Reed JC Olivetti G Anversa P Programmed myocyte cell death affects the viable myocardium after infarction in rats.Exp Cell Res. 1996; 226: 316-327Crossref PubMed Scopus (321) Google Scholar From each left ventricle, 6.0 × 106 myocytes were obtained. Cells were plated at a density of 2 × 104 per cm2 in a device with a silicon substrate coated with 0.5 μg/cm2 laminin.2Leri A Claudio PP Li Q Wang X Reiss K Wang S Malhotra A Kajstura J Anversa P Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.J Clin Invest. 1998; 101: 1326-1342Crossref PubMed Google Scholar, 9Leri A Liu Y Claudio PP Kajstura J Wang X Wang S Kang P Malhotra A Anversa P Insulin-like growth factor-1 induces Mdm2 and down-regulates p53, attenuating the myocyte renin-angiotensin system and stretch-mediated apoptosis.Am J Pathol. 1999; 154: 567-580Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar The entire study included 44 animals and a total of ∼300 stretch devices. Cells were infected with a replication-deficient adenovirus, containing human mutated p53, Adp53m, under the control of the human cytomegalovirus immediate early gene promoter.15Bacchetti S Graham FL Inhibition of cell proliferation by an adenovirus vector expressing the human wild type p53 protein.Int J Oncol. 1993; 3: 781-788PubMed Google Scholar, 16Webster KA Discher DJ Kaiser S Hernandez O Sato B Bishopric NH Hypoxia-activated apoptosis of cardiac myocytes requires reoxygenation or a pH shift and is independent of p53.J Clin Invest. 1999; 104: 239-252Crossref PubMed Google Scholar p53mutations consisted of substitution of cysteine in position 135 with serine, and proline in position 72 with arginine.15Bacchetti S Graham FL Inhibition of cell proliferation by an adenovirus vector expressing the human wild type p53 protein.Int J Oncol. 1993; 3: 781-788PubMed Google Scholar A second vector, expressing β-galactosidase, AdLacZ, was used as control. Myocytes were infected 2 hours after plating and maintained in serum-free medium (SFM) for 12 hours; infection was terminated by removing the medium and adding fresh SFM. This phase lasted 36 hours. Myocytes were infected for 48 hours with 100, 300, 500, and 1,000 pfu/cell of AdLacZ; infection efficiency was 60 ± 9% (n = 3), 87 ± 5% (n = 5), 88 ± 3% (n = 5), and 91 ± 3% (n = 5), respectively. In all studies, infection with 300 pfu/cell was used. After 48 hours of infection, a 20% strain was applied to a stretch device for 12 hours.2Leri A Claudio PP Li Q Wang X Reiss K Wang S Malhotra A Kajstura J Anversa P Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.J Clin Invest. 1998; 101: 1326-1342Crossref PubMed Google Scholar, 9Leri A Liu Y Claudio PP Kajstura J Wang X Wang S Kang P Malhotra A Anversa P Insulin-like growth factor-1 induces Mdm2 and down-regulates p53, attenuating the myocyte renin-angiotensin system and stretch-mediated apoptosis.Am J Pathol. 1999; 154: 567-580Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar In each preparation, sarcomere length was measured in 150 myocytes before and after stretch. To assess whether stretch injured cells, ethidium monoazide bromide (EMB; Molecular Probes, Eugene, OR), 5 μg/ml, was used. EMB binds to nucleic acids only in cells with membrane breakage.17Riedy MC Muirhead KA Jensen CP Stewart CC Use of a photolabeling technique to identify nonviable cells in fixed homologous or heterologous cell populations.Cytometry. 1991; 12: 133-139Crossref PubMed Scopus (115) Google Scholar After incubation with EMB and exposure to light, cells were fixed in 1% paraformaldehyde. EMB-labeled cells were measured by confocal microscopy. Two synthetic oligonucleotides, 5′-AGCCTCTGTACAGAGTAGCC-TGGGAATAGATCCATCTTC-3′ and 5′-GAAGATGGATCTATTCCCAGGCTACTCTGTAC-AGAGGCT-3′, corresponding to two half-sites of the p53 motif in the rat angiotensinogen (Aogen) promoter were used. Two synthetic oligonucleotides, 5′-GCTGAGCTTGGATCTGGAAGGCGACACTGGG-3′ and 5′-CCCAGTGTCGCCTTCCAGATCCAAGCTCAGC-3′, corresponding to two half-sites of the p53 motif in the rat AT1 promoter were used. Two synthetic oligonucleotides, 5′-AGCTTGCTCACAAGTTAGAGACAAGCCTGGGCGTGGCTATATTGA-3′ and 5′-AGCTTCAATATAGCCCACGCCCAGGCTTGTCTCTAACTTGTGAGCA-3′, corresponding to two half-sites of the p53 motif in the human bax promoter were used. Two additional sites of the p53 motif are located in the 3′-region of the perfect p53 element at 0 and 6 bp where they partially overlap.7Miyashita T Reed J Tumor suppressor p53 is a direct transcriptional activator of the human bax gene.Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (305) Google Scholar Nuclear extracts were obtained by incubating myocytes in hypotonic buffer and in high-salt buffer2Leri A Claudio PP Li Q Wang X Reiss K Wang S Malhotra A Kajstura J Anversa P Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.J Clin Invest. 1998; 101: 1326-1342Crossref PubMed Google Scholar, 4Leri A Li Y Malhotra A Li Q Stiegler P Claudio PP Giordano A Kajstura J Hintze TH Anversa P Pacing-induced heart failure in dogs enhances the expression of p53 and p53-dependent genes in ventricular myocytes.Circulation. 1998; 97: 194-203Crossref PubMed Scopus (106) Google Scholar, 9Leri A Liu Y Claudio PP Kajstura J Wang X Wang S Kang P Malhotra A Anversa P Insulin-like growth factor-1 induces Mdm2 and down-regulates p53, attenuating the myocyte renin-angiotensin system and stretch-mediated apoptosis.Am J Pathol. 1999; 154: 567-580Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 10Leri A Liu Y Wang X Kajstura J Malhotra A Meggs LG Anversa P Overexpression of insulin-like growth factor-1 attenuates the myocyte renin-angiotensin system in transgenic mice.Circ Res. 1999; 84: 752-762Crossref PubMed Scopus (90) Google Scholar; 30 μg of proteins were diluted in binding buffer with 1 to 2 μl of labeled probe. Nuclear extracts were exposed to p53 antibodies consisting of 0.5 μg pAb 240 and DO-1 (Santa Cruz, Santa Cruz, CA), or 0.5 μg pAb 246 (Ab-4, Calbiochem, San Diego, CA). Controls included unlabeled Aogen, AT1, and bax probes as competitors and unlabeled mutated Aogen (5′-AGCCTCTATAAAGAGTAGCCTGGGAATAGATCCATCTTC-3′), AT1 (5′-GCTGAGATTAGATCTGGAAGGCGACACTGGG3′), and bax (5′-AAGTTAGAGATAATGCTGGGCGAG-3′) as noncompetitors. Myocytes were lysed in the presence of protease inhibitors. Samples were incubated on ice, centrifuged, and 50 μg proteins were separated by 8 to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose and exposed to mouse monoclonal anti-human p53 (pAb 240, Santa Cruz), rabbit polyclonal anti-human Bax (P19, Santa Cruz), rabbit polyclonal anti-human Bcl-2 (ΔC21, Santa Cruz), mouse anti-rat Aogen (Swant, Bellinzona, Switzerland), mouse anti-rat renin (Swant), mouse anti-rat ACE (gift from Dr. David E. Dostal), rabbit polyclonal anti-human AT1 (306, Santa Cruz), and goat polyclonal anti-human AT2 (C-18, Santa Cruz) antibodies. Bound antibodies were identified by horseradish peroxidase-conjugated anti-mouse, anti-rabbit, anti-goat IgG, or anti-mouse IgM and recognized by a peroxidase chemiluminescent detection reagent.2Leri A Claudio PP Li Q Wang X Reiss K Wang S Malhotra A Kajstura J Anversa P Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.J Clin Invest. 1998; 101: 1326-1342Crossref PubMed Google Scholar, 4Leri A Li Y Malhotra A Li Q Stiegler P Claudio PP Giordano A Kajstura J Hintze TH Anversa P Pacing-induced heart failure in dogs enhances the expression of p53 and p53-dependent genes in ventricular myocytes.Circulation. 1998; 97: 194-203Crossref PubMed Scopus (106) Google Scholar, 9Leri A Liu Y Claudio PP Kajstura J Wang X Wang S Kang P Malhotra A Anversa P Insulin-like growth factor-1 induces Mdm2 and down-regulates p53, attenuating the myocyte renin-angiotensin system and stretch-mediated apoptosis.Am J Pathol. 1999; 154: 567-580Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 10Leri A Liu Y Wang X Kajstura J Malhotra A Meggs LG Anversa P Overexpression of insulin-like growth factor-1 attenuates the myocyte renin-angiotensin system in transgenic mice.Circ Res. 1999; 84: 752-762Crossref PubMed Scopus (90) Google Scholar Two hundred μg of proteins were incubated with 3 μg of rabbit polyclonal anti-human Bax antibody (P19, Santa Cruz) and 250 μl of HNTG buffer (20 mmol/L Hepes, pH 7.5, 150 mmol/L NaCl, 0.1% Triton X-100, 10% glycerol), containing protease inhibitors at 4°C overnight. Fifty μl of protein A-agarose was added. Proteins were separated by 12% polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes and exposed to rabbit polyclonal anti-human Bcl-2 (ΔC21, Santa Cruz) at a concentration of 1 μg/ml Tris-buffered saline/Tween 20 (TBST). An identical procedure was followed, with the exception that rabbit polyclonal anti-human Bcl-2 antibody (ΔC21, Santa Cruz) was used to immunoprecipitate myocyte lysates9Leri A Liu Y Claudio PP Kajstura J Wang X Wang S Kang P Malhotra A Anversa P Insulin-like growth factor-1 induces Mdm2 and down-regulates p53, attenuating the myocyte renin-angiotensin system and stretch-mediated apoptosis.Am J Pathol. 1999; 154: 567-580Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 10Leri A Liu Y Wang X Kajstura J Malhotra A Meggs LG Anversa P Overexpression of insulin-like growth factor-1 attenuates the myocyte renin-angiotensin system in transgenic mice.Circ Res. 1999; 84: 752-762Crossref PubMed Scopus (90) Google Scholar and that membranes were exposed to rabbit polyclonal anti-human Bcl-2 and rabbit polyclonal anti-human Bax. Cells were fixed in methanol and acetone (2:1) at −20°C and were incubated with two p53 antibodies: DO-1 (Santa Cruz) which recognizes human p53 and pAb 246 (Ab-4; Calbiochem, San Diego, CA) which binds to rodent wild-type p53. The fraction of p53-positive myocyte nuclei was evaluated by confocal microscopy;4Leri A Li Y Malhotra A Li Q Stiegler P Claudio PP Giordano A Kajstura J Hintze TH Anversa P Pacing-induced heart failure in dogs enhances the expression of p53 and p53-dependent genes in ventricular myocytes.Circulation. 1998; 97: 194-203Crossref PubMed Scopus (106) Google Scholar 500 cells were examined in each case. Cultures were incubated with 50 μl of solution, containing 5 U of TdT, 2.5 mmol/L CoCl2, and 0.5 nmol/L biotin-16-dUTP. Myocytes were stained with 5 μg/ml of fluorescein isothiocyanate-extravidin in 4× standard saline citrate buffer. Cells were treated with α-sarcomeric actin antibody and with rhodamine-labeled anti-mouse IgG. Nuclei were labeled by propidium iodide (PI).2Leri A Claudio PP Li Q Wang X Reiss K Wang S Malhotra A Kajstura J Anversa P Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.J Clin Invest. 1998; 101: 1326-1342Crossref PubMed Google Scholar, 4Leri A Li Y Malhotra A Li Q Stiegler P Claudio PP Giordano A Kajstura J Hintze TH Anversa P Pacing-induced heart failure in dogs enhances the expression of p53 and p53-dependent genes in ventricular myocytes.Circulation. 1998; 97: 194-203Crossref PubMed Scopus (106) Google Scholar, 9Leri A Liu Y Claudio PP Kajstura J Wang X Wang S Kang P Malhotra A Anversa P Insulin-like growth factor-1 induces Mdm2 and down-regulates p53, attenuating the myocyte renin-angiotensin system and stretch-mediated apoptosis.Am J Pathol. 1999; 154: 567-580Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 18Li B Setoguchi M Wang X Andreoli AM Leri A Malhotra A Kajstura J Anversa P Insulin-like growth factor-1 attenuates the detrimental impact of nonocclusive coronary artery constriction on the heart.Circ Res. 1999; 84: 1007-1019Crossref PubMed Scopus (126) Google Scholar Myocytes were stained with 50 mmol/L Tris/HCl, pH 7.8, 10 mmol/L MgCl2, 10 mmol/L dithiothreitol, 1 mmol/L ATP, 15% polyethylene glycol 8,000, 1 U/μl T4 ligase, and 35 ng/μl hairpin probe with single-base 3′ overhang.19Didenko VV Tunstead JR Hornsby PJ Biotin-labeled hairpin oligonucleotides: probes to detect double-strand breaks in DNA in apoptotic cells.Am J Pathol. 1998; 152: 897-902PubMed Google Scholar Cells were exposed to fluorescein isothiocyanate-extravidin in bicarbonate buffer and stained for confocal microscopy.2Leri A Claudio PP Li Q Wang X Reiss K Wang S Malhotra A Kajstura J Anversa P Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.J Clin Invest. 1998; 101: 1326-1342Crossref PubMed Google Scholar, 18Li B Setoguchi M Wang X Andreoli AM Leri A Malhotra A Kajstura J Anversa P Insulin-like growth factor-1 attenuates the detrimental impact of nonocclusive coronary artery constriction on the heart.Circ Res. 1999; 84: 1007-1019Crossref PubMed Scopus (126) Google Scholar Ang II in conditioned medium was measured by enzyme-linked immunosorbent assay (Peninsula, Belmont, CA). Conditioned medium was treated with 10% trifluoroacetic acid and centrifuged. Supernatant was dried, residue dissolved in 0.1% trifluoroacetic acid, and purified in a C-18 Sep-Pak column. Ang II fraction was eluted with 30% acetonitrile in 5 ml of 0.1% trifluoroacetic acid, dried, and dissolved in 0.25 ml of TBST. Samples, 50 μl, were analyzed in a microtiter plate using Ang II antibody (1:32,000) and a tracer, biotinylated Ang II. Color reaction was developed with tetramethyl-benzidine. Absorbance was recorded at 450 nm and concentration calculated from standard curves.9Leri A Liu Y Claudio PP Kajstura J Wang X Wang S Kang P Malhotra A Anversa P Insulin-like growth factor-1 induces Mdm2 and down-regulates p53, attenuating the myocyte renin-angiotensin system and stretch-mediated apoptosis.Am J Pathol. 1999; 154: 567-580Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 10Leri A Liu Y Wang X Kajstura J Malhotra A Meggs LG Anversa P Overexpression of insulin-like growth factor-1 attenuates the myocyte renin-angiotensin system in transgenic mice.Circ Res. 1999; 84: 752-762Crossref PubMed Scopus (90) Google Scholar Results are presented as mean ± SD. Autoradiograms were assessed by an image analyzer. Significance between two measurements, P < 0.05, was determined by Student's t-test. Significance among several preparations was established by the Bonferroni method.20Wallenstein S Zucker CL Fleiss JL Some statistical methods useful in circulation research.Circ Res. 1980; 47: 1-9Crossref PubMed Scopus (3662) Google Scholar pAb 240 p53 antibody recognized both wild-type and mutated p53; small amounts of p53 were detectable in control AdLacZ-infected myocytes. In Adp53m-infected cells, p53 increased from 24 to 48 hours when 100 and 300 pfu/cell were added. In comparison with AdLacZ-infected cells for 48 hours
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