Changes in phosphorylation of histone H2A.X and p53 in response of peripheral blood lymphocytes to gamma irradiation

• Autorzy: Vilasova Z. , Rezacova M. , Vavrova J. , Tichy A. , Vokurkova D. , Zoelzer F. , Rehakova Z. , Osterreicher J. , Lukasova E.
• Języki publikacji: EN

Abstrakty

EN

The main aim of this study was to compare the reaction of quiescent and proliferating, i.e. phytohemagglutinin (PHA)-stimulated, human peripheral blood mononuclear cells (PBMCs) to γ-radiation, and analyse changes of proteins related to repair of DNA damage and apoptosis, such as γH2A.X, p53, p53 phosphorylation at serines-15 and -392, and p21 and their dose dependence. Freshly isolated PBMCs in peripheral blood are predominantly quiescent, in G0phase, and with very low amounts of proteins p53 and p21. Using confocal microscopy we detected dose dependent (0.5–5 Gy) induction of foci containing γH2A.X (1 h after γ-ray exposure), which are formed around radiation-induced double strand breaks of DNA. Apoptosis was detected from 24 h after irradiation by the dose of 4 Gy onwards by Annexin V binding and lamin B cleavage. Seventy two hours after irradiation 70% of CD3+lymphocytes were A+. Neither increase in p53 nor its phosphorylation on serine-392 after irradiation was detected in these cells. However, massive increase in p21 (cyclin-dependent kinase inhibitor 1A) was detected after irradiation, which can be responsible for late occurrence of apoptosis in these quiescent cells. PHA-stimulation itself (72 h) caused an increase in early apoptosis (A+PI–) in comparison to non-stimulated PBMCs (38% A+resp. 13.4%). After PHA-stimulation also the amount of γH2A.X, p53, and p21 increased, but no phosphorylation of p53 on serine-392 or -15 was detected. Reaction to γ-radiation was different in PHA-stimulated lymphocytes: the p53 pathway was activated and p53 was phosphorylated on serines-15 and -392 4 h after irradiation by the dose of 4 Gy. Phosphorylation of p53 at serine-15 increased in a dose-dependent manner in the studied dose range 0.2–7.5 Gy. Also the amount of p21 increased after irradiation. Seventy two hours after irradiation of PHA-stimulated CD3+T lymphocytes by the dose of 4 Gy 65% of cells were A+.

Słowa kluczowe

PL

lymphocyte; phosphorylation; histone; phytohemagglutinin; apoptosis; DNA damage; gamma-irradiation; peripheral blood lymphocyte

• Wydawca: -
• Czasopismo: Acta Biochimica Polonica
• Rocznik: 2008
• Tom: 55
• Numer: 2
• Opis fizyczny: p.381-390,fig.,ref.

Twórcy

autor

Vilasova Z.

• University of Defence, Faculty of Medicine in Hradec Kralove, Simkova 870, 500 01 Hradec Kralove, Czech Republic

autor

Rezacova M.

autor

Vavrova J.

autor

Tichy A.

autor

Vokurkova D.

autor

Zoelzer F.

autor

Rehakova Z.

autor

Osterreicher J.

autor

Lukasova E.

Bibliografia

• Bakkenist CJ, Kastan MB (2003) DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421:499-506.
• Banath JP, MacPhail SH, Olive PL (2004) Radiation sensitivity, H2A.X phosphorylation, and kinetics of repair of DNA strand breaks in irradiated cervical cancer cell lineas. Cancer Res 64:7144-7149.
• Bekker-Jensen S, Lukas C, Kitagawa R, Melander F, Kastan MB, Bartek J, Lukas J (2006) Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks. J Cell Biol 173: 195-206.
• Cornelissen M, Philippe J, De Sitter S, De Ridder L (2002) Annexin V expression in apoptotic peripheral blood lymphocytes. An electron microscopic evaluation. Apoptosis 7:41-47.
• Fernandez-Capetillo O, Chen HT, Celeste A, Ward I, Romanienko PJ, Morales JC, Naka K, Xia Z, Camerini-Otero RD, Motoyama N, Carpenter PB, Bonner WM, Chen J, Nussenzweig A (2002) DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1. Nat Cell Biol 4:993-997.
• Fukao T, Kaneko H, Birrell G, Gatei M, Tashita H, Yoshida T, Cross S, Kedar P, Watters D, Khana KK, Misko I, Kondo N, Lavin MF (1999) ATM is upregulated during the mitogenic response in peripheral blood mononuclear cells. Blood 94:1998-2006.
• Gartel AL, Tyner AL (1999) Transcriptional regulation of the p21(WAF1/CIP1)gene. Exp Cell Res 246: 280-289.
• Gorospe M, Cirielli C, Wang X, Seth P, Capogrossi M, Holbrook N (1997) p21 (WAF1/CIP1) protects against p53-mediated apoptosis of human melanoma cells. Oncogene 14: 929-935.
• Hamasaki K, Imai K, Nakachi K, Takahashi N, Kodama Y, Kusunoki Y( 2007) Short-term culture and gammaH2AX flow cytometry determine differences in individual radiosensitivity in human peripheral T lymphocytes. Environ Mol Mutagen 48:38-47.
• Hertveld TK, Philippe J, Thierens H, Cornelissen M, Vral A, De Ridder L (1997) Flow cytometry as a quantitative and sensitive methods to evaluate low dose radiation induced apoptosis in vitro in human peripheral blood lymphocytes. Int J Radiat Biol 71:429-433.
• Jones GG, Reaper PM, Pettitt AR, Sherrington PD (2004) The ATR-p53 pathway is suppressed in noncycling normal and malignant lymphocytes. Oncogene 23:1911-1921.
• Ju R, Muller MT (2003) Histone deacetylase inhibitors activate p21(WAF1) expression via ATM Cancer Res 63:2891-2897.
• Kang J, Ferguson D, Song H, Bassing C, Eckersdorff M, Alt FW, Xu Y (2005) Functional interaction of H2AX, NBS1, and p53 in ATM-dependent DNA damage responses and tumor suppression. Mol Cell Biol 25:661-670.
• Kapoor M, Lozano G (1998) Functional activation of p53 viaphosphorylation following DNA damage by UV but not γ-radiation. Proc Natl Acad Sci USA 95:2834-2837.
• Lakin ND, Jackson SP (1999) Regulation of p53 in response to DNA damage. Oncogene 18:7644-7655.
• Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. J Am Chem Soc 56:658-666.
• Mukherjee B, Kessinger C , Kobayashi J, Chen BP, Chen DJ, Chatterjee A, Burma S (2006) DNA-PK phosphorylates histone H2A.X during apoptotic DNA fragmentation in mammalian cells. DNA Repair (Amst) 5:575-590.
• Ocker M, Schneider-Stock R (2007) Histone deacetylase inhibitors: signalling towards p21cip1/waf1. Int J Biochem Cell Biol 39:1367-1374.
• Pettitt AR, Sherrington PD, Stewart G, Cawley JC, Malcolm A, Taylor R, Stankovic T (2001) p53 dysfunction in B-cell chronic lymphocytic leukemia: inactivation of ATM as an alternative to TP53 mutation. Blood 98:814-822.
• Philippe J, Louagie H, Thierens H, Vral A, Cornelissen M, De Ridder L (1997) Quantification of apoptosis in lymphocyte subsets and effect of apoptosis on apparent expression of membrane antigens. Cytometry 29:242-249.
• Porcedda P, Turinetto V, Lantelme E, Fontanella E, Chrzanowska K, Ragona R, De Marchi M, Delia D, Giachino C (2006) Impaired elimination of DNA double-strand break-containing lymphocytes in ataxia telangiectasia and Nijmegen breakage syndrome. DNA Repair 5:904-913.
• Rezacova M, Tichy A, Vavrova J, Vokurkova D, Lukaova E (2008) Is defect in phosphorylation of Nbs1 responsible for high radiosensitivity of T-lymphocyte leukemia cells MOLT-4? Leuk Res 32:1259-1267.
• Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM (1998) DNA double-stranded breaks induce histone H2AX phosphorylation on serine-139. J Biol Chem 273:5858-5868.
• Schultz LB, Chehab NH, Malikzay A, Halazonetis TD (2000) p53 binding protein (53BP1) is an early participant in cellular response to DNA double-strand breaks. J Cell Biol 151:1381-1390.
• Shieh SY, Ikeda M, Taya Y, Prives C (1997) DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell 91:325-334.
• Siliciano JD, Canman CE, Taya Y, Sakaguchi K, Appella E, Kastan MB (1997) DNA damage induces phosphorylation of the amino terminus of p53. Genes Dev 11: 3471-3481.
• Stiff T, Driscoll MO, Rief N, Iwabuchi K, Lobrich M, Jeggo PA (2004) ATM and DNA-PK function redundantly to phosphorylate H2A.X after exposure to ionizing radiation. Cancer Res 64:2390-2396.
• Szkanderova S, Vavrova J, Rezacova M, Vokurkova D, Pavlova S, Smardova J, Stulik J (2003) Gamma irradiation results in phosphorylation of p53 at serine-392 in human T-lymphocyte leukaemia cell line MOLT-4. Folia Biol (Praha) 49:191-196.
• Szkanderova S, Vavrova J, Hernychova L, Neubauerova V, Lenco J, Stulik J (2005) Proteome alterations in gamma-irradiated human T-lymphocyte leukemia cells. Radiat Res 163:307-315.
• Tichy A, Zaskodova D, Rezacova M, Vavrova J, Vokurkova D, Pejchal J, Vilasova Z, Cerman J, Osterreicher J (2007) Gamma-radiation-induced ATM-dependent signaling in human T-lymphocyte leukemic cells, MOLT-4. Acta Biochim Polon 54:281-287.
• Vavrova J, Marekova M, Vokurkova D, Psutka J (2004) Cell cycle alteration and response to low-dose-rate gamma radiation in leukemic cell lines. Physiol Res 53:335-342.
• Voelkerding KV, Steffen DW, Zaidi SHE, Malter JS (1995) Post-transcriptional regulation of the p53 tumor suppressor gene during growth-induction of human peripheral blood mononuclear cells. Oncogene 10:515-521.
• Vokurkova D, Sinkora J, Vavrova J, Rezacova M, Knizek J, Osterreicher J (2006) CD8+natural killer cells have a potential of a sensitive and reliable biodosimetric marker in vitro. Physiol Res 55:689-698.
• Weinberg RA (2007) The biology of cancer. Garland Science, Taylor and Francis Group, LLC. New York, 795 p.
• Zhao Y, Lu S, Wu L, Chai G, Wang H, Chen Y, Sun J, Yu Y, Zhou W, Zheng Q, Wu M, Otterson GA, Zhu WG (2006) Acetylation of p53 at lysine 373/382 by the histone deacetylase inhibitor depsipeptide induces expression of p21Waf1/Cip1. Mol Cell Biol 26:2782-2790.