scholarly journals Persistent Amplification of DNA Damage Signal Involved in Replicative Senescence of Normal Human Diploid Fibroblasts

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Masatoshi Suzuki ◽  
Keiji Suzuki ◽  
Seiji Kodama ◽  
Shunichi Yamashita ◽  
Masami Watanabe
Keyword(s):  
Dna Damage ◽  
Replicative Senescence ◽  
Kinase Inhibitor ◽  
Fish Analysis ◽  
Hypoxic Cell ◽  
Dna Double Strand Breaks ◽  
Histone H2ax ◽  
Diploid Fibroblasts ◽  
Human Diploid Fibroblasts ◽  
Normal Human

Foci of phosphorylated histone H2AX and ATM are the surrogate markers of DNA double strand breaks. We previously reported that the residual foci increased their size after irradiation, which amplifies DNA damage signals. Here, we addressed whether amplification of DNA damage signal is involved in replicative senescence of normal human diploid fibroblasts. Large phosphorylated H2AX foci (>1.5 μm diameter) were specifically detected in presenescent cells. The frequency of cells with large foci was well correlated with that of cells positive for senescence-associatedβ-galactosidase staining. Hypoxic cell culture condition extended replicative life span of normal human fibroblast, and we found that the formation of large foci delayed in those cells. Our immuno-FISH analysis revealed that large foci partially localized at telomeres in senescent cells. Importantly, large foci of phosphorylated H2AX were always colocalized with phosphorylated ATM foci. Furthermore, Ser15-phosphorylated p53 showed colocalization with the large foci. Since the treatment of senescent cells with phosphoinositide 3-kinase inhibitor, wortmannin, suppressed p53 phosphorylation, it is suggested that amplification of DNA damage signaling sustains persistent activation of ATM-p53 pathway, which is essential for replicative senescence.

scholarly journals DNA double-strand break repair: a theoretical framework and its application

2016 ◽  
Vol 13 (114) ◽  
pp. 20150679 ◽  
Author(s):  
Philip J. Murray ◽  
Bart Cornelissen ◽  
Katherine A. Vallis ◽  
S. Jon Chapman
Keyword(s):  
Dna Damage ◽  
Auger Electron ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Histone H2ax ◽  
Dna Double Strand Break ◽  
A Cell

DNA double-strand breaks (DSBs) are formed as a result of genotoxic insults, such as exogenous ionizing radiation, and are among the most serious types of DNA damage. One of the earliest molecular responses following DSB formation is the phosphorylation of the histone H2AX, giving rise to γ H2AX. Many copies of γ H2AX are generated at DSBs and can be detected in vitro as foci using well-established immuno-histochemical methods. It has previously been shown that anti- γ H2AX antibodies, modified by the addition of the cell-penetrating peptide TAT and a fluorescent or radionuclide label, can be used to visualize and quantify DSBs in vivo . Moreover, when labelled with a high amount of the short-range, Auger electron-emitting radioisotope, 111 In, the amount of DNA damage within a cell can be increased, leading to cell death. In this report, we develop a mathematical model that describes how molecular processes at individual sites of DNA damage give rise to quantifiable foci. Equations that describe stochastic mean behaviours at individual DSB sites are derived and parametrized using population-scale, time-series measurements from two different cancer cell lines. The model is used to examine two case studies in which the introduction of an antibody (anti- γ H2AX-TAT) that targets a key component in the DSB repair pathway influences system behaviour. We investigate: (i) how the interaction between anti- γ H2AX-TAT and γ H2AX effects the kinetics of H2AX phosphorylation and DSB repair and (ii) model behaviour when the anti- γ H2AX antibody is labelled with Auger electron-emitting 111 In and can thus instigate additional DNA damage. This work supports the conclusion that DSB kinetics are largely unaffected by the introduction of the anti- γ H2AX antibody, a result that has been validated experimentally, and hence the hypothesis that the use of anti- γ H2AX antibody to quantify DSBs does not violate the image tracer principle. Moreover, it provides a novel model of DNA damage accumulation in the presence of Auger electron-emitting 111 In that is supported qualitatively by the available experimental data.

scholarly journals MDC1 PST-repeat region promotes histone H2AX-independent chromatin association and DNA damage tolerance

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Israel Salguero ◽  
Rimma Belotserkovskaya ◽  
Julia Coates ◽  
Matylda Sczaniecka-Clift ◽  
Mukerrem Demir ◽  
...  
Keyword(s):  
Dna Damage ◽  
Double Strand Breaks ◽  
Repeat Region ◽  
Independent Function ◽  
Dna Double Strand Breaks ◽  
Dna Damage Tolerance ◽  
Strand Breaks ◽  
Histone H2ax ◽  
Independent Association ◽  
Dna Damage Signalling

AbstractHistone H2AX and MDC1 are key DNA repair and DNA-damage signalling proteins. When DNA double-strand breaks (DSBs) occur, H2AX is phosphorylated and then recruits MDC1, which in turn serves as a docking platform to promote the localization of other factors, including 53BP1, to DSB sites. Here, by using CRISPR-Cas9 engineered human cell lines, we identify a hitherto unknown, H2AX-independent, function of MDC1 mediated by its PST-repeat region. We show that the PST-repeat region directly interacts with chromatin via the nucleosome acidic patch and mediates DNA damage-independent association of MDC1 with chromatin. We find that this region is largely functionally dispensable when the canonical γH2AX-MDC1 pathway is operative but becomes critical for 53BP1 recruitment to DNA-damage sites and cell survival following DSB induction when H2AX is not available. Consequently, our results suggest a role for MDC1 in activating the DDR in areas of the genome lacking or depleted of H2AX.

scholarly journals Repair of radiation-induced DNA damage in nondividing populations of human diploid fibroblasts

1980 ◽  
Vol 30 (3) ◽  
pp. 399-413 ◽  
Author(s):  
G.J. Kantor ◽  
R.S. Petty ◽  
C. Warner ◽  
D.J. Phillips ◽  
D.R. Hull
Keyword(s):  
Dna Damage ◽  
Diploid Fibroblasts ◽  
Human Diploid Fibroblasts ◽  
Radiation Induced

scholarly journals Reversible cellular senescence: implications for immortalization of normal human diploid fibroblasts.

1989 ◽  
Vol 9 (7) ◽  
pp. 3088-3092 ◽  
Author(s):  
W E Wright ◽  
O M Pereira-Smith ◽  
J W Shay
Keyword(s):  
Cellular Senescence ◽  
Rare Event ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Diploid Fibroblasts ◽  
Human Diploid Fibroblasts ◽  
Normal Human ◽  
Immortal Cells ◽  
Cellular Dna

IMR-90 normal human diploid fibroblasts, transfected with a steroid inducible mouse mammary tumor virus-driven simian virus 40 T antigen, were carried through crisis to yield an immortal cell line. Growth was dependent on the presence of the inducer (dexamethasone) during both the extended precrisis life span of the cells and after immortalization. After dexamethasone removal, immortal cells divided once or twice and then accumulated in G1. These results are best explained by a two-stage model for cellular senescence. Mortality stage 1 (M1) causes a loss of mitogen responsiveness and arrest near the G1/S interface and can be bypassed or overcome by the cellular DNA synthesis-stimulating activity of T antigen. Mortality stage 2 (M2) is an independent mechanism that is responsible for the failure of cell division during crisis. The inactivation of M2 is a rare event, probably of mutational origin in human cells, independent of or only indirectly related to the expression of T antigen. Under this hypothesis, T-antigen-immortalized cells contain an active but bypassed M1 mechanism and an inactivated M2 mechanism. These cells are dependent on the continued expression of T antigen for the maintenance of immortality for the same reason that precrisis cells are dependent on T antigen for growth: both contain an active M1 mechanism.

scholarly journals Differentiation-Induced Radioresistance in Muscle Cells

2004 ◽  
Vol 24 (14) ◽  
pp. 6350-6361 ◽  
Author(s):  
Lucia Latella ◽  
Jiri Lukas ◽  
Cristiano Simone ◽  
Pier Lorenzo Puri ◽  
Jiri Bartek
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Double Strand Breaks ◽  
Proliferating Cells ◽  
Histone H2ax ◽  
Selective Blockade ◽  
Differentiated Cells ◽  
Dna Damage Signaling ◽  
Ataxia Teleangiectasia ◽  
Associated Proteins

ABSTRACT DNA damage induces cell cycle arrest and DNA repair or apoptosis in proliferating cells. Terminally differentiated cells are permanently withdrawn from the cell cycle and partly resistant to apoptosis. To investigate the effects of genotoxic agents in postmitotic cells, we compared DNA damage-activated responses in mouse and human proliferating myoblasts and their differentiated counterparts, the myotubes. DNA double-strand breaks caused by ionizing radiation (IR) induced rapid activating autophosphorylation of ataxia-teleangiectasia-mutated (ATM), phosphorylation of histone H2AX, recruitment of repair-associated proteins MRE11 and Nbs1, and activation of Chk2 in both myoblasts and myotubes. However, IR-activated, ATM-mediated phosphorylation of p53 at serine 15 (human) or 18 (mouse) [Ser15(h)/18(m)], and apoptosis occurred in myoblasts but was impaired in myotubes. This phosphorylation could be enforced in myotubes by the anthracycline derivative doxorubicin, leading to selective activation of proapoptotic genes. Unexpectedly, the abundance of autophosphorylated ATM was indistinguishable after exposure of myotubes to IR (10 Gy) or doxorubicin (1 μM/24 h) despite efficient phosphorylation of p53 Ser15(h)/18(m), and apoptosis occurred only in response to doxorubicin. These results suggest that radioresistance in myotubes might reflect a differentiation-associated, pathway-selective blockade of DNA damage signaling downstream of ATM. This mechanism appears to preserve IR-induced activation of the ATM-H2AX-MRE11/Rad50/Nbs1 lesion processing and repair pathway yet restrain ATM-p53-mediated apoptosis, thereby contributing to life-long maintenance of differentiated muscle tissues.

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