scholarly journals Analysis of Residual DSBs in Ataxia-Telangiectasia Lymphoblast Cells Initiating Apoptosis

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Teresa Anglada ◽  
Mariona Terradas ◽  
Laia Hernández ◽  
Anna Genescà ◽  
Marta Martín
Keyword(s):  
Dna Damage ◽  
Ataxia Telangiectasia ◽  
Dna Double Strand Breaks ◽  
Predictive Tool ◽  
Normal Cells ◽  
Strand Breaks ◽  
Radiation Induced ◽  
Late Apoptosis ◽  
Induced Apoptosis ◽  
The Relationship

In order to examine the relationship between accumulation of residual DNA double-strand breaks (DSBs) and cell death, we have used a control and an ATM (Ataxia-Telangiectasia Mutated) defective cell line, as Ataxia-Telangiectasia (AT) cells tend to accumulate residual DSBs at long times after damage infliction. After irradiation, AT cells showed checkpoint impairment and a fraction of cells displayed an abnormal centrosome number and tetraploid DNA content, and this fraction increased along with apoptosis rates. At all times analyzed, AT cells displayed a significantly higher rate of radiation-induced apoptosis than normal cells. Besides apoptosis, 70–85% of the AT viable cells (TUNEL-negative) carried ≥10γH2AX foci/cell, while only 12–27% of normal cells did. The fraction of AT and normal cells undergoing early and late apoptosis were isolated by flow cytometry and residual DSBs were concretely scored in these populations. Half of theγH2AX-positive AT cells undergoing early apoptosis carried ≥10γH2AX foci/cell and this fraction increased to 75% in late apoptosis. The results suggest that retention of DNA damage-inducedγH2AX foci is an indicative of lethal DNA damage, as cells undergoing apoptosis are those accumulating more DSBs. Scoring of residualγH2AX foci might function as a predictive tool to assess radiation-induced apoptosis.

scholarly journals DNA structure-specific priming of ATR activation by DNA-PKcs

2013 ◽  
Vol 202 (3) ◽  
pp. 421-429 ◽  
Author(s):  
Sophie Vidal-Eychenié ◽  
Chantal Décaillet ◽  
Jihane Basbous ◽  
Angelos Constantinou
Keyword(s):  
Dna Damage ◽  
Ataxia Telangiectasia ◽  
Protein A ◽  
Dna Structure ◽  
Specific Response ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Synergistic Activation ◽  
Dependent Protein ◽  
Specific Priming

Three phosphatidylinositol-3-kinase–related protein kinases implement cellular responses to DNA damage. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia-telangiectasia mutated respond primarily to DNA double-strand breaks (DSBs). Ataxia-telangiectasia and RAD3-related (ATR) signals the accumulation of replication protein A (RPA)–covered single-stranded DNA (ssDNA), which is caused by replication obstacles. Stalled replication intermediates can further degenerate and yield replication-associated DSBs. In this paper, we show that the juxtaposition of a double-stranded DNA end and a short ssDNA gap triggered robust activation of endogenous ATR and Chk1 in human cell-free extracts. This DNA damage signal depended on DNA-PKcs and ATR, which congregated onto gapped linear duplex DNA. DNA-PKcs primed ATR/Chk1 activation through DNA structure-specific phosphorylation of RPA32 and TopBP1. The synergistic activation of DNA-PKcs and ATR suggests that the two kinases combine to mount a prompt and specific response to replication-born DSBs.

scholarly journals Quality control in oocytes by p63 is based on a spring-loaded activation mechanism on the molecular and cellular level

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Daniel Coutandin ◽  
Christian Osterburg ◽  
Ratnesh Kumar Srivastav ◽  
Manuela Sumyk ◽  
Sebastian Kehrloesser ◽  
...  
Keyword(s):  
Dna Damage ◽  
Quality Control ◽  
Cellular Level ◽  
Activation Mechanism ◽  
Control Factor ◽  
Dna Double Strand Breaks ◽  
High Concentration ◽  
P53 Family ◽  
Strand Breaks ◽  
Induced Apoptosis

Mammalian oocytes are arrested in the dictyate stage of meiotic prophase I for long periods of time, during which the high concentration of the p53 family member TAp63α sensitizes them to DNA damage-induced apoptosis. TAp63α is kept in an inactive and exclusively dimeric state but undergoes rapid phosphorylation-induced tetramerization and concomitant activation upon detection of DNA damage. Here we show that the TAp63α dimer is a kinetically trapped state. Activation follows a spring-loaded mechanism not requiring further translation of other cellular factors in oocytes and is associated with unfolding of the inhibitory structure that blocks the tetramerization interface. Using a combination of biophysical methods as well as cell and ovary culture experiments we explain how TAp63α is kept inactive in the absence of DNA damage but causes rapid oocyte elimination in response to a few DNA double strand breaks thereby acting as the key quality control factor in maternal reproduction.

scholarly journals The Effect of High-Dose-Rate Pulsed Radiation on the Survival of Clinically Relevant Radioresistant Cells

Life ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1295
Author(s):  
Shingo Terashima ◽  
Hironori Yoshino ◽  
Yoshikazu Kuwahara ◽  
Hiro Sakuraba ◽  
Yoichiro Hosokawa
Keyword(s):  
Cell Survival ◽  
Dose Rate ◽  
High Dose Rate ◽  
High Dose ◽  
Dna Double Strand Breaks ◽  
Single Exposure ◽  
Strand Breaks ◽  
Cell Line Cell ◽  
Radiation Induced ◽  
Induced Apoptosis

We demonstrated that low dose pulsed radiation (0.25 Gy) at a high-dose-rate, even for very short intervals (10 s), decreases cell survival to a greater extent than single exposure to a similar total dose and dose rate. The objective of this study was to clarify whether high-dose-rate pulsed radiation is effective against SAS-R, a clinically relevant radioresistant cell line. Cell survival following high-dose-rate pulsed radiation was evaluated via a colony assay. Flow cytometry was utilized to evaluate γH2AX, a molecular marker of DNA double-strand breaks and delayed reactive oxygen species (ROS) associated with radiation-induced apoptosis. Increased cytotoxicity was observed in SAS-R and parent SAS cells in response to high dose rate pulsed radiation compared to single dose, as determined by colony assays. Residual γH2AX in both cells subjected to high-dose-rate pulsed radiation showed a tendency to increase, with a significant increase observed in SAS cells at 72 h. In addition, high-dose-rate pulsed radiation increased delayed ROS more than the single exposure did. These results indicate that high-dose-rate pulsed radiation was associated with residual γH2AX and delayed ROS, and high-dose-rate pulsed radiation may be used as an effective radiotherapy procedure against radioresistant cells.

scholarly journals H2A.X Phosphorylation in Oxidative Stress and Risk Assessment in Plasma Medicine

2021 ◽  
Vol 2021 ◽  
pp. 1-18
Author(s):  
Clarissa S. Schütz ◽  
Matthias B. Stope ◽  
Sander Bekeschus
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Dna Double Strand Breaks ◽  
Plasma Medicine ◽  
Mutagenic Potential ◽  
Strand Breaks ◽  
Radiation Induced ◽  
And Function ◽  
Physical Plasma ◽  
Suitable Marker

At serine139-phosphorylated gamma histone H2A.X (γH2A.X) has been established over the decades as sensitive evidence of radiation-induced DNA damage, especially DNA double-strand breaks (DSBs) in radiation biology. Therefore, γH2A.X has been considered a suitable marker for biomedical applications and a general indicator of direct DNA damage with other therapeutic agents, such as cold physical plasma. Medical plasma technology generates a partially ionized gas releasing a plethora of reactive oxygen and nitrogen species (ROS) simultaneously that have been used for therapeutic purposes such as wound healing and cancer treatment. The quantification of γH2A.X as a surrogate parameter of direct DNA damage has often been used to assess genotoxicity in plasma-treated cells, whereas no sustainable mutagenic potential of the medical plasma treatment could be identified despite H2A.X phosphorylation. However, phosphorylated H2A.X occurs during apoptosis, which is associated with exposure to cold plasma and ROS. This review summarizes the current understanding of γH2A.X induction and function in oxidative stress in general and plasma medicine in particular. Due to the progress towards understanding the mechanisms of H2A.X phosphorylation in the absence of DSB and ROS, observations of γH2A.X in medical fields should be carefully interpreted.

scholarly journals Increased activity of poly(ADP-ribose)-polymerase in PML-depleted cells - novel perspectives for cancer therapy

2013 ◽  
Vol 94 (1) ◽  
pp. 75-79
Author(s):  
S V Boichuk ◽  
B R Ramazanov ◽  
I G Mustafin ◽  
Gjoerup O
Keyword(s):  
Dna Damage ◽  
Ionizing Radiation ◽  
Genotoxic Stress ◽  
Dna Double Strand Breaks ◽  
Compensatory Response ◽  
Strand Breaks ◽  
Fetal Bovine ◽  
Parp Activity ◽  
The Relationship ◽  
Increased Activity

Aim. To investigate the relationship between PML expression and poly(ADP-ribose)-polymerase (PARP) activity in physiological conditions and at genotoxic stress induced by chemotherapy and ionizing radiation. Methods. The study was conducted on BJ fibroblasts cultured in DMEM/199 medium supplemented with fetal bovine serum, L-glutamine and antibiotics. PML down-regulation was achieved by short interfering ribonucleic acid transfection. To induce deoxyribonucleic acid (DNA) damage in BJ fibroblasts, doxorubicin and hydroxyurea or ionizing radiation were used. PARP activity, formation of DNA double-strand breaks and DNA damage response were examined by Western blotting and immunofluorescence microscopy. Results. PML knockdown was accomplished with an increased PARP activity, confirmed by an increased expression of poly-ADP-ribose (PAR) polymers. At PML knockdown ant DNA damage caused by chemotherapy and ionizing radiation, there is a significant increase in PAR polymers expression as well as increase in the number of cells containing PAR foci. Conclusion. Increased activity of poly(ADP-ribose)-polymerase at PML knockdown and DNA damaging conditions indicates the compensatory response due to insufficiency of the homologous recombination mechanisms. The phenomenon found widens the spectrum of malignancies that might be potentially sensitive to the therapy with poly(ADP-ribose)-polymerase inhibitors.

scholarly journals Fusion Tyrosine Kinases Induce Drug Resistance by Stimulation of Homology-Dependent Recombination Repair, Prolongation of G2/M Phase, and Protection from Apoptosis

2002 ◽  
Vol 22 (12) ◽  
pp. 4189-4201 ◽  
Author(s):  
Artur Slupianek ◽  
Grazyna Hoser ◽  
Ireneusz Majsterek ◽  
Agnieszka Bronisz ◽  
Maciej Malecki ◽  
...  
Keyword(s):  
Dna Damage ◽  
Drug Resistance ◽  
Tyrosine Kinases ◽  
Transformed Cells ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Recombination Repair ◽  
M Phase ◽  
Induced Apoptosis ◽  
In Cells

ABSTRACT Fusion tyrosine kinases (FTKs) such as BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGFβR, TEL/TRKC(L), and NPM/ALK arise from reciprocal chromosomal translocations and cause acute and chronic leukemias and non-Hodgkin's lymphoma. FTK-transformed cells displayed drug resistance against the cytostatic drugs cisplatin and mitomycin C. These cells were not protected from drug-mediated DNA damage, implicating activation of the mechanisms preventing DNA damage-induced apoptosis. Various FTKs, except TEL/TRKC(L), can activate STAT5, which may be required to induce drug resistance. We show that STAT5 is essential for FTK-dependent upregulation of RAD51, which plays a central role in homology-dependent recombinational repair (HRR) of DNA double-strand breaks (DSBs). Elevated levels of Rad51 contributed to the induction of drug resistance and facilitation of the HRR in FTK-transformed cells. In addition, expression of antiapoptotic protein Bcl-xL was enhanced in cells transformed by the FTKs able to activate STAT5. Moreover, cells transformed by all examined FTKs displayed G2/M delay upon drug treatment. Individually, elevated levels of Rad51, Bcl-xL, or G2/M delay were responsible for induction of a modest drug resistance. Interestingly, combination of these three factors in nontransformed cells induced drug resistance of a magnitude similar to that observed in cells expressing FTKs activating STAT5. Thus, we postulate that RAD51-dependent facilitation of DSB repair, antiapoptotic activity of Bcl-xL, and delay in progression through the G2/M phase work in concert to induce drug resistance in FTK-positive leukemias and lymphomas.

scholarly journals The BRCA2-Interacting Protein BCCIP Functions in RAD51 and BRCA2 Focus Formation and Homologous Recombinational Repair

2005 ◽  
Vol 25 (5) ◽  
pp. 1949-1957 ◽  
Author(s):  
Huimei Lu ◽  
Xu Guo ◽  
Xiangbing Meng ◽  
Jingmei Liu ◽  
Chris Allen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Genome Stability ◽  
Recombinational Repair ◽  
Dna Double Strand Breaks ◽  
Focus Formation ◽  
Interacting Protein ◽  
Strand Breaks ◽  
Homologous Recombinational Repair ◽  
Radiation Induced ◽  
Nuclear Foci

ABSTRACT Homologous recombinational repair (HRR) of DNA damage is critical for maintaining genome stability and tumor suppression. RAD51 and BRCA2 colocalization in nuclear foci is a hallmark of HRR. BRCA2 has important roles in RAD51 focus formation and HRR of DNA double-strand breaks (DSBs). We previously reported that BCCIPα interacts with BRCA2. We show that a second isoform, BCCIPβ, also interacts with BRCA2 and that this interaction occurs in a region shared by BCCIPα and BCCIPβ. We further show that chromatin-bound BRCA2 colocalizes with BCCIP nuclear foci and that most radiation-induced RAD51 foci colocalize with BCCIP. Reducing BCCIPα by 90% or BCCIPβ by 50% by RNA interference markedly reduces RAD51 and BRCA2 foci and reduces HRR of DSBs by 20- to 100-fold. Similarly, reducing BRCA2 by 50% reduces RAD51 and BCCIP foci. These data indicate that BCCIP is critical for BRCA2- and RAD51-dependent responses to DNA damage and HRR.

Radiation induced apoptosis in ataxia telangiectasia homozygote, heterozygote and normal cells

2001 ◽  
Vol 476 (1-2) ◽  
pp. 13-20 ◽  
Author(s):  
D.Gwyn Bebb ◽  
Pamela J Warrington ◽  
Gary de Jong ◽  
Zhe Yu ◽  
Joyce A Moffat ◽  
...  
Keyword(s):  
Ataxia Telangiectasia ◽  
Normal Cells ◽  
Radiation Induced ◽  
Induced Apoptosis

scholarly journals 53BP1 deficiency combined with telomere dysfunction activates ATR-dependent DNA damage response

2012 ◽  
Vol 197 (2) ◽  
pp. 283-300 ◽  
Author(s):  
Paula Martínez ◽  
Juana M. Flores ◽  
Maria A. Blasco
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Ataxia Telangiectasia ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Damage Response

TRF1 protects mammalian telomeres from fusion and fragility. Depletion of TRF1 leads to telomere fusions as well as accumulation of γ-H2AX foci and activation of both the ataxia telangiectasia mutated (ATM)– and the ataxia telangiectasia and Rad3 related (ATR)–mediated deoxyribonucleic acid (DNA) damage response (DDR) pathways. 53BP1, which is also present at dysfunctional telomeres, is a target of ATM that accumulates at DNA double-strand breaks and favors nonhomologous end-joining (NHEJ) repair over ATM-dependent resection and homology-directed repair (homologous recombination [HR]). To address the role of 53BP1 at dysfunctional telomeres, we generated mice lacking TRF1 and 53BP1. 53BP1 deficiency significantly rescued telomere fusions in mouse embryonic fibroblasts (MEFs) lacking TRF1, but they showed evidence of a switch from the NHEJ- to HR-mediated repair of uncapped telomeres. Concomitantly, double-mutant MEFs showed evidence of hyperactivation of the ATR-dependent DDR. In intact mice, combined 53BP1/TRF1 deficiency in stratified epithelia resulted in earlier onset of DNA damage and increased CHK1 phosphorylation during embryonic development, leading to aggravation of skin phenotypes.

Sign in / Sign up

Export Citation Format

Share Document