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• W2054104078 abstract "Nitric oxide (NO) is a potent activator of the p53 tumor suppressor protein, thereby inducing cell cycle arrest and apoptosis. However, little is known about the regulation of the two other p53-family members, p63 and p73, by nitrogen oxides. We report here an up-regulation of p73 by NO in p53-null K-562 leukemia cells. Chemical NO prodrugs or macrophage iNOS activity induced an accumulation of the TAp73α isoform in these cells, whereas macrophages from iNOS−/− mice did not. NO also up-regulated TAp73 mRNA expression, suggesting a transcriptional regulation. The checkpoint kinases Chk1 and Chk2 can regulate TAp73 induction after DNA damage. We show that these kinases were rapidly phosphorylated upon NO treatment. Genetic silencing or pharmacological inhibition of Chk1 impaired NO-mediated accumulation of TAp73α. Because NO is known to block DNA synthesis through ribonucleotide reductase inhibition, the up-regulation of TAp73α might be caused by DNA damage induced by an arrest of DNA replication forks. In support of this hypothesis, DNA replication inhibitors such as hydroxyurea and aphidicolin similarly enhanced TAp73α expression and Chk1 phosphorylation. Moreover, inhibition of Chk1 also prevented TAp73α accumulation in response to replication inhibitors. The knockdown of TAp73 with siRNA sensitized K-562 cells to apoptosis induced by a nitrosative (NO) or oxidative (H2O2) injury. Therefore, TAp73α has an unusual cytoprotective role in K-562 cells, contrasting with its pro-apoptotic functions in many other cell models. In conclusion, NO up-regulates several p53 family members displaying pro- and anti-apoptotic effects, suggesting a complex network of interactions and cross-regulations between NO production and p53-related proteins. Nitric oxide (NO) is a potent activator of the p53 tumor suppressor protein, thereby inducing cell cycle arrest and apoptosis. However, little is known about the regulation of the two other p53-family members, p63 and p73, by nitrogen oxides. We report here an up-regulation of p73 by NO in p53-null K-562 leukemia cells. Chemical NO prodrugs or macrophage iNOS activity induced an accumulation of the TAp73α isoform in these cells, whereas macrophages from iNOS−/− mice did not. NO also up-regulated TAp73 mRNA expression, suggesting a transcriptional regulation. The checkpoint kinases Chk1 and Chk2 can regulate TAp73 induction after DNA damage. We show that these kinases were rapidly phosphorylated upon NO treatment. Genetic silencing or pharmacological inhibition of Chk1 impaired NO-mediated accumulation of TAp73α. Because NO is known to block DNA synthesis through ribonucleotide reductase inhibition, the up-regulation of TAp73α might be caused by DNA damage induced by an arrest of DNA replication forks. In support of this hypothesis, DNA replication inhibitors such as hydroxyurea and aphidicolin similarly enhanced TAp73α expression and Chk1 phosphorylation. Moreover, inhibition of Chk1 also prevented TAp73α accumulation in response to replication inhibitors. The knockdown of TAp73 with siRNA sensitized K-562 cells to apoptosis induced by a nitrosative (NO) or oxidative (H2O2) injury. Therefore, TAp73α has an unusual cytoprotective role in K-562 cells, contrasting with its pro-apoptotic functions in many other cell models. In conclusion, NO up-regulates several p53 family members displaying pro- and anti-apoptotic effects, suggesting a complex network of interactions and cross-regulations between NO production and p53-related proteins. NO is a free radical implicated in numerous physiological functions. In cancer biology, both positive and negative actions of NO have been reported. For example, NO was found to promote tumor growth angiogenesis, and metastasis on the one hand and to induce apoptotic cell death or tumor cytostasis on the other hand (1Jenkins D.C. Charles I.G. Thomsen L.L. Moss D.W. Holmes L.S. Baylis S.A. Rhodes P. Westmore K. Emson P.C. Moncada S. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 4392-4396Crossref PubMed Scopus (750) Google Scholar, 2Brüne B. Zhou J. Cardiovasc. Res. 2007; 75: 275-282Crossref PubMed Scopus (108) Google Scholar, 3Thomas D.D. Ridnour L.A. Isenberg J.S. Flores-Santana W. Switzer C.H. Donzelli S. Hussain P. Vecoli C. Paolocci N. Ambs S. Colton C.A. Harris C.C. Roberts D.D. Wink D.A. Free Radic. Biol. Med. 2008; 45: 18-31Crossref PubMed Scopus (709) Google Scholar, 4Roy B. Guittet O. Beuneu C. Lemaire G. Lepoivre M. Free Radic. Biol. Med. 2004; 36: 507-516Crossref PubMed Scopus (26) Google Scholar). These opposite responses are linked to the chemistry of nitric oxide in a cellular context. NO not only reacts directly on target molecules but also exerts its effects through derived species with a higher degree of oxidation and generated from NO interaction with O2 (e.g. N2O3, •NO2) or superoxide anion (ONOO−). Those reactive nitrogen species can act as potent nitrosating, nitrating, or oxidizing agents. Given this complex chemistry, the biological outcome of NO is directed by its concentration, the cellular redox environment, and different target susceptibilities. High fluxes of NO are generated by inducible NO synthase (iNOS), 3The abbreviations used are: iNOSinducible NO synthaseTAtransactivation domainSNAPS-nitrosothiol S-nitroso-N-acetyl-d,l-penicillamineDETA-NO(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]dia-zen-1-ium-1,2-diolateBSOl-buthionine-[S,R]-sulfoximineH2DCFDA2′,7′-dichlorodihydrofluoresceine diacetateJC-15,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodideROSreactive oxygen speciesRnrribonucleotide reductaseΔΨmmitochondrial membrane potential. transcriptionally induced in immune cells such as macrophages by cytokines and bacterial products (e.g. IFN-γ and lipopolysaccharide). Elevated concentrations of NO cause DNA damage, mutation, and apoptotic cell death (5Akaike T. Fujii S. Kato A. Yoshitake J. Miyamoto Y. Sawa T. Okamoto S. Suga M. Asakawa M. Nagai Y. Maeda H. FASEB J. 2000; 14: 1447-1454Crossref PubMed Google Scholar, 6Messmer U.K. Ankarcrona M. Nicotera P. Brüne B. FEBS Lett. 1994; 355: 23-26Crossref PubMed Scopus (388) Google Scholar). The tumor suppressor protein p53 is a key player in the DNA damage response and the onset of apoptosis. NO has been shown to activate p53 at high concentrations, which also promote genotoxic and pro-apoptotic effects (3Thomas D.D. Ridnour L.A. Isenberg J.S. Flores-Santana W. Switzer C.H. Donzelli S. Hussain P. Vecoli C. Paolocci N. Ambs S. Colton C.A. Harris C.C. Roberts D.D. Wink D.A. Free Radic. Biol. Med. 2008; 45: 18-31Crossref PubMed Scopus (709) Google Scholar, 6Messmer U.K. Ankarcrona M. Nicotera P. Brüne B. FEBS Lett. 1994; 355: 23-26Crossref PubMed Scopus (388) Google Scholar, 7Forrester K. Ambs S. Lupold S.E. Kapust R.B. Spillare E.A. Weinberg W.C. Felley-Bosco E. Wang X.W. Geller D.A. Tzeng E. Billiar T.R. Harris C.C. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 2442-2447Crossref PubMed Scopus (395) Google Scholar). Because p53 is a transcription factor regulating the expression of several genes involved in DNA repair, cell cycle arrest, and apoptosis, activation of p53 by NO can be considered as a regulatory mechanism preventing the emergence of NO-induced DNA mutations and, hence, tumorigenesis. Augmentation of mutation rates in p53-deficient cells exposed to high NO doses and repression of iNOS gene transcription by p53 in a negative feedback loop are occurrences that strongly support a major role for p53 in the control of NO-induced genotoxicity (7Forrester K. Ambs S. Lupold S.E. Kapust R.B. Spillare E.A. Weinberg W.C. Felley-Bosco E. Wang X.W. Geller D.A. Tzeng E. Billiar T.R. Harris C.C. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 2442-2447Crossref PubMed Scopus (395) Google Scholar, 8Ambs S. Ogunfusika M.O. Merriam W.G. Bennett W.P. Billiar T.R. Harris C.C. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 8823-8828Crossref PubMed Scopus (141) Google Scholar, 9Li C.Q. Trudel L.J. Wogan G.N. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 10364-10369Crossref PubMed Scopus (72) Google Scholar). inducible NO synthase transactivation domain S-nitrosothiol S-nitroso-N-acetyl-d,l-penicillamine (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]dia-zen-1-ium-1,2-diolate l-buthionine-[S,R]-sulfoximine 2′,7′-dichlorodihydrofluoresceine diacetate 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide reactive oxygen species ribonucleotide reductase mitochondrial membrane potential. Two p53-related genes, p63 and p73, have been identified more than 15 years after the discovery of p53 (10Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1538) Google Scholar, 11Yang A. Kaghad M. Wang Y. Gillett E. Fleming M.D. Dötsch V. Andrews N.C. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1846) Google Scholar). The p53 family members have a similar structural organization comprising an NH2-terminal transactivation domain (TA), a central DNA binding domain, and a COOH-terminal oligomerization domain (12Bénard J. Douc-Rasy S. Ahomadegbe J.C. Hum. Mutat. 2003; 21: 182-191Crossref PubMed Scopus (193) Google Scholar, 13Deyoung M.P. Ellisen L.W. Oncogene. 2007; 26: 5169-5183Crossref PubMed Scopus (216) Google Scholar, 14Vilgelm A. El-Rifai W. Zaika A. Drug Resist. Updat. 2008; 11: 152-163Crossref PubMed Scopus (51) Google Scholar, 15Collavin L. Lunardi A. Del Sal G. Cell Death Differ. 2010; 17: 901-911Crossref PubMed Scopus (178) Google Scholar). In p63 and p73, an additional sterile-α motif and a transcription inhibitory domain may follow the oligomerization domain. Sequence homology among the three members is limited to 25–40% identity between TA and oligomerization domain regions but increases to 60–80% amino acid identity within the DNA binding domain. These structural similarities enable physical and functional interactions between the p53-family proteins. All three p53-family members are expressed as a variety of isoforms, resulting from the usage of two distinct promoters (P1 and P2) and mRNA differential splicing at the 5′- and 3′-ends. There is now no doubt that p73 and p63 are, like p53, involved in tumor suppression (16Tomasini R. Tsuchihara K. Wilhelm M. Fujitani M. Rufini A. Cheung C.C. Khan F. Itie-Youten A. Wakeham A. Tsao M.S. Iovanna J.L. Squire J. Jurisica I. Kaplan D. Melino G. Jurisicova A. Mak T.W. Genes and Dev. 2008; 22: 2677-2691Crossref PubMed Scopus (361) Google Scholar, 17Flores E.R. Sengupta S. Miller J.B. Newman J.J. Bronson R. Crowley D. Yang A. McKeon F. Jacks T. Cancer Cell. 2005; 7: 363-373Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 18Guo X. Keyes W.M. Papazoglu C. Zuber J. Li W. Lowe S.W. Vogel H. Mills A.A. Nat. Cell Biol. 2009; 11: 1451-1457Crossref PubMed Scopus (201) Google Scholar). However, the p73 and p63 homologues have additional important functions. For instance, in mice p73 and p63 are crucial to proper neural and epithelial development, respectively, whereas the embryonic development of p53-null mice is almost normal (19Yang A. Walker N. Bronson R. Kaghad M. Oosterwegel M. Bonnin J. Vagner C. Bonnet H. Dikkes P. Sharpe A. McKeon F. Caput D. Nature. 2000; 404: 99-103Crossref PubMed Scopus (887) Google Scholar, 20Mills A.A. Zheng B. Wang X.J. Vogel H. Roop D.R. Bradley A. Nature. 1999; 398: 708-713Crossref PubMed Scopus (1708) Google Scholar, 21Sah V.P. Attardi L.D. Mulligan G.J. Williams B.O. Bronson R.T. Jacks T. Nat. Genet. 1995; 10: 175-180Crossref PubMed Scopus (497) Google Scholar). The human p73 gene generates two groups of isoforms, one with a complete TA domain comprising nine COOH-terminal splice variants (named TAp73α, β, γ, ..) and the other encompassing at least six other proteins exhibiting a truncated TA domain, produced either from the P1 promoter and NH2-terminal splicing (e.g. ΔEx2p73, ΔEx2/3p73) or from the P2 promoter within intron 3 (ΔNp73). COOH-terminal splice variants such as ΔNp73α and ΔNp73β also exist within the group lacking an entire TA domain. TAp73 isoforms can induce the transcription of an overlapping subset of p53 target genes such as CDKN1A, GADD45, Bax, PUMA, and SFN, but specific targets have been also identified (22Harms K. Nozell S. Chen X. Cell. Mol. Life Sci. 2004; 61: 822-842Crossref PubMed Scopus (256) Google Scholar, 23Fontemaggi G. Kela I. Amariglio N. Rechavi G. Krishnamurthy J. Strano S. Sacchi A. Givol D. Blandino G. J. Biol. Chem. 2002; 277: 43359-43368Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Growth suppression or induction of apoptosis can be accomplished by TAp73 isoforms in cooperation with p53 (24Flores E.R. Tsai K.Y. Crowley D. Sengupta S. Yang A. McKeon F. Jacks T. Nature. 2002; 416: 560-564Crossref PubMed Scopus (725) Google Scholar, 25Gonzalez S. Perez-Perez M.M. Hernando E. Serrano M. Cordon-Cardo C. Cancer Res. 2005; 65: 2186-2192Crossref PubMed Scopus (41) Google Scholar) but also via p53-independent pathways (10Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1538) Google Scholar, 25Gonzalez S. Perez-Perez M.M. Hernando E. Serrano M. Cordon-Cardo C. Cancer Res. 2005; 65: 2186-2192Crossref PubMed Scopus (41) Google Scholar, 26Jost C.A. Marin M.C. Kaelin Jr., W.G. Nature. 1997; 389: 191-194Crossref PubMed Scopus (902) Google Scholar). Several studies have shown that p73 is required for apoptosis induction in response to DNA damage by chemotherapeutic drugs, and these findings can explain the relative effectiveness of anti-cancer agents in p53-defective tumor cells (27Müller M. Schleithoff E.S. Stremmel W. Melino G. Krammer P.H. Schilling T. Drug Resist. Updat. 2006; 9: 288-306Crossref PubMed Scopus (120) Google Scholar). Indeed, mutations in the p73 gene are rare in cancer patients. On the contrary, up-regulation of p73 was frequently observed. In particular, deregulated expression of isoforms containing a truncated TA domain, especially ΔNp73, has been demonstrated in several human cancers (27Müller M. Schleithoff E.S. Stremmel W. Melino G. Krammer P.H. Schilling T. Drug Resist. Updat. 2006; 9: 288-306Crossref PubMed Scopus (120) Google Scholar, 28Domínguez G. García J.M. Peña C. Silva J. García V. Martínez L. Maximiano C. Gómez M.E. Rivera J.A. García-Andrade C. Bonilla F. J. Clin. Oncol. 2006; 24: 805-815Crossref PubMed Scopus (112) Google Scholar). p73 isoforms lacking the TA domain are transactivation-deficient and do not induce growth suppression or cell death. They behave as dominant-negative proteins by competing with p53 and TAp73 variants at specific DNA binding sequences on promoters and by forming heteroduplexes with TAp73 and TAp63 proteins. The antiapoptotic role of ΔNp73 is crucial during mouse neural development, but it can also confer survival advantages and chemotherapeutic drug resistance in tumors by several mechanisms (27Müller M. Schleithoff E.S. Stremmel W. Melino G. Krammer P.H. Schilling T. Drug Resist. Updat. 2006; 9: 288-306Crossref PubMed Scopus (120) Google Scholar, 29Stiewe T. Stanelle J. Theseling C.C. Pollmeier B. Beitzinger M. Pützer B.M. J. Biol. Chem. 2003; 278: 14230-14236Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 30Wilhelm M.T. Rufini A. Wetzel M.K. Tsuchihara K. Inoue S. Tomasini R. Itie-Youten A. Wakeham A. Arsenian-Henriksson M. Melino G. Kaplan D.R. Miller F.D. Mak T.W. Genes Dev. 2010; 24: 549-560Crossref PubMed Scopus (183) Google Scholar). The oncogenic properties of NH2-terminal-truncated p73 isoforms may help to understand the absence of mutations and overexpression of p73 in tumors. Because ΔNp73 expression is transcriptionally activated by p53 and TAp73, it is implicated in a feedback mechanism that attenuates p53 and TAp73 transcriptional functions, including the p53-dependent DNA damage response, as demonstrated in a recent study using ΔNp73−/− mice (30Wilhelm M.T. Rufini A. Wetzel M.K. Tsuchihara K. Inoue S. Tomasini R. Itie-Youten A. Wakeham A. Arsenian-Henriksson M. Melino G. Kaplan D.R. Miller F.D. Mak T.W. Genes Dev. 2010; 24: 549-560Crossref PubMed Scopus (183) Google Scholar). Molecular mechanisms of TAp73 activation by genotoxic agents and γ-irradiation have been investigated and have led to the identification of a variety of transcriptional and post-translational regulatory pathways (Refs. 13Deyoung M.P. Ellisen L.W. Oncogene. 2007; 26: 5169-5183Crossref PubMed Scopus (216) Google Scholar, 14Vilgelm A. El-Rifai W. Zaika A. Drug Resist. Updat. 2008; 11: 152-163Crossref PubMed Scopus (51) Google Scholar and references therein). Among others, phosphorylation of TAp73 by the c-Abl tyrosine kinase, the JNK and p38 MAP kinases (the latter via c-Abl-mediated activation), or the Chk1 checkpoint kinase stabilizes the protein and increases its transcriptional activity, especially toward pro-apoptotic target genes. The ATM serine-protein kinase activated by DNA damage lies upstream in the pathways leading to TAp73 activation by c-Abl and Chk1. TAp73 activity is also regulated by protein degradation. Indeed, the E3 ubiquitin ligases Itch and MDM2 have opposite effects on p73 half-life. Itch-dependent ubiquitination of p73 targets it to proteasomal degradation, whereas physical association of p73 with MDM2 enhances its stability but inhibits its transcriptional activity. Contrasting with p53 regulation, augmentation of p73 mRNA levels is an important aspect of p73 activation in response to DNA damage. The E2F1 transcription factor implicated in cell-cycle regulation binds to E2F sites within the P1 promoter of the p73 gene and activates the transcriptional expression of TAp73α and TAp73β isoforms in response to oncogenic stress. Phosphorylation of E2F1 by the Chk1/Chk2 checkpoint kinases can increase E2F-1 levels and, as a consequence, transcription of TAp73 isoforms (31Urist M. Tanaka T. Poyurovsky M.V. Prives C. Genes Dev. 2004; 18: 3041-3054Crossref PubMed Scopus (195) Google Scholar). Several important players acting in the regulatory network controlling p73 activity have been previously shown to be involved in NO-dependent p53 activation. For instance, ATM/ATR DNA damage sensors, the stress-responsive p38 MAP kinase, and MDM2 have been implicated in p53 regulation by NO (32Wang X. Michael D. de Murcia G. Oren M. J. Biol. Chem. 2002; 277: 15697-15702Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 33Kim S.J. Hwang S.G. Shin D.Y. Kang S.S. Chun J.S. J. Biol. Chem. 2002; 277: 33501-33508Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 34Hofseth L.J. Saito S. Hussain S.P. Espey M.G. Miranda K.M. Araki Y. Jhappan C. Higashimoto Y. He P. Linke S.P. Quezado M.M. Zurer I. Rotter V. Wink D.A. Appella E. Harris C.C. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 143-148Crossref PubMed Scopus (292) Google Scholar). There is, thus, a possibility that those events that activate p53 could also increase p73 transcriptional activity. A number of studies have accumulated that describe the interplay between p53 and nitrogen oxides. However, more than 10 years after the discovery of the other two p53-related proteins and to the best of our knowledge, the potential regulation of p73 or p63 by NO has not been thoroughly investigated. In this report, we started to address this question by examining the effect of genotoxic, high concentrations of NO on TAp73 expression in a p53-deficient human leukemia cell line. Two NO-generating compounds were used. The S-nitrosothiol S-nitroso-N-acetyl-d,l-penicillamine (SNAP) was synthesized according to Field et al. (35Field L. Dilts R.V. Ravichandran R. Lenhert P.G. Carnahan G.E. J. Chem. Soc. Chem. Commun. 1978; 1157: 249-250Crossref Google Scholar). Depending on the reductive power of its environment, SNAP decomposition generates either the •NO radical or a nitrosating species. It is a powerful nitrosating agent via transnitrosation reactions. (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]dia-zen-1-ium-1,2-diolate (DETA-NO) was obtained from Cayman chemicals. DETA-NO decomposition gives rise mostly to the free radical •NO. Hydroxyurea, resveratrol, 4-propoxyphenol, aphidicolin, adriamycin® (doxorubicin), caffeine, UCN-01, 3-methyladenine, l-buthionine-[S,R]-sulfoximine (BSO), propidium iodide, lipopolysaccharide (LPS) from Salmonella enteritidis, and paraquat (methyl viologen) were purchased from Sigma. 2′,7′-Dichlorodihydrofluoresceine diacetate (H2DCFDA), S-ethyl-isothiourea, FITC-annexin V, and gemcitabine were obtained, respectively, from Invitrogen/Molecular Probes, Merck/Calbiochem, BD Pharmingen, and Lilly France. 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was also obtained from Invitrogen/Molecular Probes. Primary antibodies to p73 (IMG-246, Imgenex, and A300-126A, Bethyl Laboratories), Chk1, NQO1, and Crk-L (sc-8408, sc-32793, and sc-319 respectively, Santa Cruz Biotechnology), phospho-Chk1 and phospho-Chk2 (2348 and 2661 respectively, Cell Signaling), β-actin and α-tubulin (A 5316 and T 9026, Sigma) were used for immunoblotting. Appropriate horseradish peroxidase-conjugated anti-mouse (A 8924, Sigma) or anti-rabbit (sc-2030, Santa Cruz Biotechnology) IgG as well as fluorescent Alexa Fluor 680-conjugated anti-mouse (A 21057, Invitrogen/Molecular Probes) or IRDye 800CW-conjugated anti-rabbit (926–32211, LI-COR Biosciences) IgG were used for detection. The human leukemia cell line K-562 was maintained in RPMI 1640 medium supplemented with 5% fetal bovine serum, 2 mm l-glutamine, 25 mm HEPES and antibiotics. Cultures were grown in a 95% air, 5% CO2 atmosphere in a humidified incubator at 37 °C. Cells were plated in 6-well culture plates at 0.75 × 106 cells per well in 5 ml of culture medium and allowed to recover for 24 h. They were then treated from 30 min to 48 h with NO prodrugs or DNA synthesis inhibitors and finally harvested for protein or RNA extraction. In some experiments caffeine, 3-methyladenine, UCN-01, or Chir-124 were added 30 min before other treatments. For GSH depletion, tumor cells were incubated for 18 h with 100 μm BSO, washed, and resuspended in fresh medium, as described previously (36Petit J.F. Nicaise M. Lepoivre M. Guissani A. Lemaire G. Biochem. Pharmacol. 1996; 52: 205-212Crossref PubMed Scopus (42) Google Scholar). Cells were then processed normally for subsequent treatments. Peritoneal macrophages elicited by the intraperitoneal injection of thioglycolate broth were obtained from C57Bl/6 mice and stimulated as previously described (37Guittet O. Tebbi A. Cottet M.H. Vésin F. Lepoivre M. Nitric. Oxide. 2008; 19: 84-94Crossref PubMed Scopus (13) Google Scholar). We also employed macrophages isolated from genetically iNOS-deficient mice (38MacMicking J.D. Nathan C. Hom G. Chartrain N. Fletcher D.S. Trumbauer M. Stevens K. Xie Q.W. Sokol K. Hutchinson N. Cell. 1995; 81: 641-650Abstract Full Text PDF PubMed Scopus (1285) Google Scholar) kindly given by Dr. Charles-Henry Cottart from the Université Paris Descartes, Paris, France). Briefly, macrophages were seeded into 6-well culture plates at a density of 0.3 to 2 × 106 cells/well. Induction of iNOS expression was performed by stimulation for 24 h with mouse IFN-γ and LPS. After elimination of nonadherent peritoneal cells, K-562 cells were added at a concentration of 2 × 106 per well in 5 ml of fresh culture medium containing LPS to maintain iNOS expression. Cocultures were incubated for up to 48 h. Tumor cells were then gently harvested and processed for immunoblotting. Culture supernatant was recovered for nitrite assay with Griess reagent (37Guittet O. Tebbi A. Cottet M.H. Vésin F. Lepoivre M. Nitric. Oxide. 2008; 19: 84-94Crossref PubMed Scopus (13) Google Scholar). Cells were washed twice in ice-cold phosphate-buffered saline (PBS). Crude cell extracts were prepared in a 50 mm Tris.HCl lysis buffer, pH 7.4, supplemented with 150 mm NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mm EDTA, 1 mm DTT, and protease inhibitors including 1 mm Pefabloc (Merck/Calbiochem). The soluble proteins (usually 30 μg) were separated by SDS-PAGE using a 10% polyacrylamide gel and transferred onto nitrocellulose membranes. Blocking of the membrane with 5% skimmed milk, incubation with primary and horseradish peroxidase-conjugated secondary antibodies, washings, and chemiluminescent detection of the antigens were performed as described in Guittet et al. (37Guittet O. Tebbi A. Cottet M.H. Vésin F. Lepoivre M. Nitric. Oxide. 2008; 19: 84-94Crossref PubMed Scopus (13) Google Scholar). To ameliorate the quantification of the immunoblots, we also used the two-color infrared imaging system from LI-COR Biosciences. In these experiments, membranes were blocked with 2.5% skimmed milk or 5% BSA (A3059, Sigma) in PBS containing 0.05% Tween 20 (PBS/Tween). After overnight incubation at 4 °C with primary antibodies and four washings in PBS/Tween, membranes were incubated for 1 h with fluorescent dye-conjugated secondary antibodies. They were then washed four times in PBS/Tween and twice in PBS. The infrared fluorescent signals at 680 and 800 nm were recorded and quantified with an Odyssey scanner (LI-COR Biosciences). Expression of up to three loading controls (Crk-L, β-actin, and α-tubulin) was also analyzed in each experiment. The signal intensity for each band of interest in a treated sample was first normalized relative to the untreated sample. Then, to take into account variations in protein loading and transfer onto the membrane, the mean of all normalized loading control signals was calculated and used to divide the normalized signal intensity for each antigen. Total RNA was prepared using TRIzol (Invitrogen) according to the manufacturer's instructions. A quantity of 2 μg of RNA was used for cDNA synthesis with the High Capacity cDNA Archive kit (Applied Biosystems). Real-time PCR was performed on a Light Cycler 2.0 apparatus (Roche Applied Science) using the LightCycler® FastStart DNA MasterPLUS SYBR Green I kit (Roche Applied Science). Each PCR cycle consisted of 10 s at 95 °C, 10 s at 67 °C, and 15 s at 72 °C. Results were normalized to β-actin RNA levels. The following pairs of oligonucleotides were used: TAp73, 5′-GGCTGCGACGGCTGCAGAGC-3′/5′-GCTCAGCAGATTGAACTGGGCCATG-3′; β-actin, 5′-ACGAGTCCGGCCCCTCCATC-3′/5′-TGGGGGATGCTCGCTCCAAC-3′; ΔNp73, 5′-CAAACGGCCCGCATGTTCCC-3′/5′-TGGTCCATGGTGCTGCTCAGC-3′; and p21CDKN1A, as described by Guittet et al. (37Guittet O. Tebbi A. Cottet M.H. Vésin F. Lepoivre M. Nitric. Oxide. 2008; 19: 84-94Crossref PubMed Scopus (13) Google Scholar). K-562 cells were transiently transfected with siRNA using a Nucleofector kit V and a Nucleofector II device (all from Lonza) set to program T-016 according to the manufacturer's protocol. We used validated siRNA targeted to p53 and TAp73 mRNA (37Guittet O. Tebbi A. Cottet M.H. Vésin F. Lepoivre M. Nitric. Oxide. 2008; 19: 84-94Crossref PubMed Scopus (13) Google Scholar) as well as a siRNA targeted to Chk1 (stealth siRNA HSS101855, Invitrogen), a siRNA against p63, and a scrambled siRNA control (both from Dharmacon). Depletion of TAp73 mRNA and inhibition of protein expression were maximal at 24 h and decreased thereafter. Therefore, transfected cells were used 24 h after nucleofection. FITC-Annexin V (BD Pharmingen) was used to detect apoptosis by flow cytometry. Briefly, 1 × 106 K-562 cells were transfected with siRNA as described above. After 24 h of recovery, half of these cells were treated by 0.5 mm DETA-NO for 24 h. Cells were harvested 48 h post-transfection, washed twice in cold PBS, and resuspended at a concentration of 1 × 106 cells/ml in 100 μl of a binding buffer containing 10 mm HEPES, pH 7.4, 140 mm NaCl, and 2.5 mm CaCl2. Cells were then stained with 5 μl of FITC-annexin V and 50 μg/ml propidium iodide for 15 min at room temperature in the dark. Finally, 400 μl of binding buffer were added, and the cells were subjected to FACS analysis within 1 h. At least 10,000 events were acquired by a FACScan flow cytometer (BD Biosciences). Data were analyzed using the CellQuestTM program. The percentage of apoptotic cells was calculated by scoring for annexin V-positive cells. Variation of mitochondrial membrane potential (ΔΨm) was measured using the JC-1 lipophilic cationic fluorescent dye. K-562 cells were incubated in 0.5 ml of JC-1 solution (10 μg/ml in PBS) and incubated for 20 min at 37 °C in the dark. Cells were analyzed immediately with a FACScan flow cytometer. The green and red fluorescence signals were detected at 530 nm (FL1 channel) and 575 nm (FL2 channel) for JC-1 monomers and aggregates, respectively. We counted 10,000 cells for each sample in acquisition and analyzed them using CellQuestTM software. ΔΨm variations are indicated by fluctuations in the ratio of red to green fluorescence, which is dependent only on the membrane potential. Mitochondrial depolarization is indicated by a decrease in this ratio. The lipophilic fluorogen H2DCFDA is retained in viable cells and converted intracellularly to a fluorescent fluorescein derivative after oxidation by ROS. K-562 cells previously transfected with siRNA and treated for 24 h with 0.1 mm H2O2 or 6 μg/ml paraquat were harvested, washed once with PBS, and resuspended in Hanks' balanced salt solution supplemented with 10 mm HEPES containing 10 μm H2DCFDA. After dye loading for 1 h at room temperature, cells were centrifuged, resuspended in prewarmed complete culture medium, and incubated for 30 min in a humidified incubator at 37 °C to allow oxidation of the dye. The green fluorescence intensity of the cells was analyzed immediately by a FACScan flow cytometer. NO has been reported to activate p53 in various cellular mod" @default.
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• W2054104078 date "2011-03-01" @default.
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• W2054104078 title "TAp73 Induction by Nitric Oxide" @default.
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