Use of the γ-H2AX Assay to Investigate DNA Repair Dynamics Following Multiple Radiation Exposures

Radiation therapy is one of the most common and effective strategies used to treat cancer. The irradiation is usually performed with a fractionated scheme, where the dose required to kill tumour cells is given in several sessions, spaced by specific time intervals, to allow healthy tissue recovery. In this work, we examined the DNA repair dynamics of cells exposed to radiation delivered in fractions, by assessing the response of histone-2AX (H2AX) phosphorylation (γ-H2AX), a marker of DNA double strand breaks. γ-H2AX foci induction and disappearance were monitored following split dose irradiation experiments in which time interval between exposure and dose were varied. Experimental data have been coupled to an analytical theoretical model, in order to quantify key parameters involved in the foci induction process. Induction of γ-H2AX foci was found to be affected by the initial radiation exposure with a smaller number of foci induced by subsequent exposures. This was compared to chromatin relaxation and cell survival. The time needed for full recovery of γ-H2AX foci induction was quantified (12 hours) and the 1:1 relationship between radiation induced DNA double strand breaks and foci numbers was critically assessed in the multiple irradiation scenarios.     

Ciprofloxacin as a potential radio-sensitizer to tumor cells and a radio-protectant for normal cells: differential effects on γ-H2AX formation,p53 phosphorylation,Bcl-2 production,and cell death

Ionizing radiation increases cell mortality in a dose-dependent manner. Increases in DNA double strand breaks, γ-H2AX, p53 phophorylation, and protein levels of p53 and Bax also occur. We investigated the ability of ciprofloxacin (CIP), a widely prescribed antibiotic, to inhibit DNA damage induced by ionizing radiation. Human tumor TK6, NH32 (p53 ^?/? of TK6) cells, and human normal peripheral blood mononuclear cells (PBMCs) were exposed to 2–8 Gy ⁶⁰Co-γ-photon radiation. γ-H2AX (an indicator of DNA strand breaks), phosphorylated p53 (responsible for cell-cycle arrest), Bcl-2 (an apoptotic protein, and cell death were measured. Ionizing irradiation increased γ-H2AX amounts in TK6 cells (p53^+/+) within 1 h in a radiation dose-dependent manner. CIP pretreatment and posttreatment effectively inhibited the increase in γ-H2AX. CIP pretreatment reduced Bcl-2 production but promoted p53 phosphorylation, caspase-3 activation and cell death. In NH32 cells, CIP failed to significantly inhibit the radiation-induced γ-H2AX increase, suggesting that CIP inhibition involves in p53-dependent mechanisms. In normal healthy human PBMCs, CIP failed to block the radiation-induced γ-H2AX increase but effectively increased Bcl-2 production, but blocked the phospho-p53 increase and subsequent cell death. CIP increased Gadd45α, and enhanced p21 protein 24 h postirradiation. Results suggest that CIP exerts its effect in TK6 cells by promoting p53 phosphorylation and inhibiting Bcl-2 production and in PBMCs by inhibiting p53 phosphorylation and increasing Bcl-2 production. Our data are the first to support the view that CIP may be effective to protect normal tissue cells from radiation injury, while enhancing cancer cell death in radiation therapy.     

Resveratrol Induced Premature Senescence Is Associated with DNA Damage Mediated SIRT1 and SIRT2 Down-Regulation

The natural polyphenolic compound resveratrol (3,4,5-trihydroxy-trans-stilbene) has broad spectrum health beneficial activities including antioxidant, anti-inflammatory, anti-aging, anti-cancer, cardioprotective, and neuroprotective effects. Remarkably, resveratrol also induces apoptosis and cellular senescence in primary and cancer cells. Resveratrol’s anti-aging effects both in vitro and in vivo attributed to activation of a (NAD)-dependent histone deacetylase family member sirtuin-1 (SIRT1) protein. In mammals seven members (SIRT1-7) of sirtuin family have been identified. Among those, SIRT1 is the most extensively studied with perceptive effects on mammalian physiology and suppression of the diseases of aging. Yet no data has specified the role of sirtuins, under conditions where resveratrol treatment induces senescence. Current study was undertaken to investigate the effects of resveratrol in human primary dermal fibroblasts (BJ) and to clarify the role of sirtuin family members in particular SIRT1 and SIRT2 that are known to be involved in cellular stress responses and cell cycle, respectively. Here, we show that resveratrol decreases proliferation of BJ cells in a time and dose dependent manner. In addition the increase in senescence associated β-galactosidase (SA-β-gal) activity and methylated H3K9-me indicate the induction of premature senescence. A significant increase in phosphorylation of γ-H2AX, a surrogate of DNA double strand breaks, as well as in levels of p53, p21^CIP1 and p16^INK4A is also detected. Interestingly, at concentrations where resveratrol induced premature senescence we show a significant decrease in SIRT1 and SIRT2 levels by Western Blot and quantitative RT-PCR analysis. Conversely inhibition of SIRT1 and SIRT2 via siRNA or sirtinol treatment also induced senescence in BJ fibroblasts associated with increased SA-β-gal activity, γ-H2AX phosphorylation and p53, p21^CIP1 and p16^INK4A levels. Interestingly DNA damaging agent doxorubicin also induced senescence in BJ fibroblasts associated with decreased SIRT1/2 levels. In conclusion our data reveal that resveratrol induced premature senescence is associated with SIRT1 and SIRT2 down regulation in human dermal fibroblasts. Here we suggest that the concomitant decline in SIRT1/2 expression in response to resveratrol treatment may be a cause for induction of senescence, which is most likely mediated by a regulatory mechanism activated by DNA damage response.     

Analysis of Lymphocytic DNA Damage in Early Multiple Sclerosis by Automated Gamma-H2AX and 53BP1 Foci Detection: A Case Control Study

Background

In response to DNA double-strand breaks, the histone protein H2AX becomes phosphorylated at its C-terminal serine 139 residue, referred to as γ-H2AX. Formation of γ-H2AX foci is associated with recruitment of p53-binding protein 1 (53BP1), a regulator of the cellular response to DNA double-strand breaks. γ-H2AX expression in peripheral blood mononuclear cells (PBMCs) was recently proposed as a diagnostic and disease activity marker for multiple sclerosis (MS).

Objective

To evaluate the significance of γ-H2AX and 53BP1 foci in PBMCs as diagnostic and disease activity markers in patients with clinically isolated syndrome (CIS) and early relapsing-remitting MS (RRMS) using automated γ-H2AX and 53BP1 foci detection.

Methods

Immunocytochemistry was performed on freshly isolated PBMCs of patients with CIS/early RRMS (n = 25) and healthy controls (n = 27) with γ-H2AX and 53BP1 specific antibodies. Nuclear γ-H2AX and 53BP1 foci were determined using a fully automated reading system, assessing the numbers of γ-H2AX and 53BP1 foci per total number of cells and the percentage of cells with foci. Patients underwent contrast enhanced 3 Tesla magnetic resonance imaging (MRI) and clinical examination including expanded disability status scale (EDSS) score. γ-H2AX and 53BP1 were also compared in previously frozen PBMCs of each 10 CIS/early RRMS patients with and without contrast enhancing lesions (CEL) and 10 healthy controls.

Results

The median (range) number of γ-H2AX (0.04 [0–0.5]) and 53BP1 (0.005 [0–0.2]) foci per cell in freshly isolated PBMCs across all study participants was low and similar to previously reported values of healthy individuals. For both, γ-H2AX and 53BP1, the cellular focus number as well as the percentage of positive cells did not differ between patients with CIS/RRMS and healthy controls. γ-H2AX and 53BP1 levels neither correlated with number nor volume of T2-weighted lesions on MRI, nor with the EDSS. Although γ-H2AX, but not 53BP1, levels were higher in previously frozen PBMCs of patients with than without CEL, γ-H2AX values of both groups overlapped and γ-H2AX did not correlate with the number or volume of CEL.

Conclusion

γ-H2AX and 53BP1 foci do not seem to be promising diagnostic or disease activity biomarkers in patients with early MS. Lymphocytic DNA double-strand breaks are unlikely to play a major role in the pathophysiology of MS.     

γ-H2AX as a Marker for Dose Deposition in the Brain of Wistar Rats after Synchrotron Microbeam Radiation

Objective

Synchrotron radiation has shown high therapeutic potential in small animal models of malignant brain tumours. However, more studies are needed to understand the radiobiological effects caused by the delivery of high doses of spatially fractionated x-rays in tissue. The purpose of this study was to explore the use of the γ-H2AX antibody as a marker for dose deposition in the brain of rats after synchrotron microbeam radiation therapy (MRT).

Methods

Normal and tumour-bearing Wistar rats were exposed to 35, 70 or 350 Gy of MRT to their right cerebral hemisphere. The brains were extracted either at 4 or 8 hours after irradiation and immediately placed in formalin. Sections of paraffin-embedded tissue were incubated with anti γ-H2AX primary antibody.

Results

While the presence of the C6 glioma does not seem to modulate the formation of γ-H2AX in normal tissue, the irradiation dose and the recovery versus time are the most important factors affecting the development of γ-H2AX foci. Our results also suggest that doses of 350 Gy can trigger the release of bystander signals that significantly amplify the DNA damage caused by radiation and that the γ-H2AX biomarker does not only represent DNA damage produced by radiation, but also damage caused by bystander effects.

Conclusion

In conclusion, we suggest that the γ-H2AX foci should be used as biomarker for targeted and non-targeted DNA damage after synchrotron radiation rather than a tool to measure the actual physical doses.     

Middle Infrared Radiation Induces G2/M Cell Cycle Arrest in A549 Lung Cancer Cells

There were studies investigating the effects of broadband infrared radiation (IR) on cancer cell, while the influences of middle-infrared radiation (MIR) are still unknown. In this study, a MIR emitter with emission wavelength band in the 3–5 µm region was developed to irradiate A549 lung adenocarcinoma cells. It was found that MIR exposure inhibited cell proliferation and induced morphological changes by altering the cellular distribution of cytoskeletal components. Using quantitative PCR, we found that MIR promoted the expression levels of ATM (ataxia telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3-related and Rad3-related), TP53 (tumor protein p53), p21 (CDKN1A, cyclin-dependent kinase inhibitor 1A) and GADD45 (growth arrest and DNA-damage inducible), but decreased the expression levels of cyclin B coding genes, CCNB1 and CCNB2, as well as CDK1 (Cyclin-dependent kinase 1). The reduction of protein expression levels of CDC25C, cyclin B1 and the phosphorylation of CDK1 at Thr-161 altogether suggest G₂/M arrest occurred in A549 cells by MIR. DNA repair foci formation of DNA double-strand breaks (DSB) marker γ-H2AX and sensor 53BP1 was induced by MIR treatment, it implies the MIR induced G₂/M cell cycle arrest resulted from DSB. This study illustrates a potential role for the use of MIR in lung cancer therapy by initiating DSB and blocking cell cycle progression.     

TPX2 Impacts Acetylation of Histone H4 at Lysine 16: Implications for DNA Damage Response

During interphase, the spindle assembly factor TPX2 is compartmentalized in the nucleus where its roles remain largely uncharacterized. Recently, we found that TPX2 regulates the levels of serine 139-phosphoryated H2AX (γ-H2AX) at chromosomal breaks induced by ionizing radiation. Here, we report that TPX2 readily associates with the chromatin in the absence of ionizing radiation. Overexpression of TPX2 alters the DAPI staining pattern of interphase cells and depletion of TPX2 constitutively decreases the levels of histone H4 acetylated at lysine16 (H4K16ac) during G1-phase. Upon ionizing irradiation, this constitutive TPX2 depletion-dependent decrease in H4K16ac levels correlates with increased levels of γ-H2AX. The inversely correlated levels of H4K16ac and γ-H2AX can also be modified by altering the levels of SIRT1, herein identified as a novel protein complex partner of TPX2. Furthermore, we find that TPX2 depletion also interferes with formation of 53BP1 ionizing radiation-induced foci, known to depend on γ-H2AX and the acetylation status of H4K16. In brief, our study is the first indication of a constitutive control of TPX2 on H4K16ac levels, with potential implications for DNA damage response.     

Ribonucleotide reductase inhibition enhances chemoradiosensitivity of human cervical cancers

For repair of damaged DNA, cells increase de novo synthesis of deoxyribonucleotide triphosphates through the rate-limiting, p53-regulated ribonucleotide reductase (RNR) enzyme. In this study we investigated whether pharmacological inhibition of RNR by 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, NSC #663249) enhanced chemoradiation sensitivity through a mechanism involving sustained DNA damage. RNR inactivation by 3-AP and resulting chemoradiosensitization were evaluated in human cervical (CaSki, C33-a) cancer cells through study of DNA damage (γ-H2AX signal) by flow cytometry, RNR subunit p53R2 and p21 protein steady-state levels by Western blot analysis and laser scanning imaging cytometry, and cell survival by colony formation assays. 3-AP treatment led to sustained radiation- and cisplatin-induced DNA damage (i.e. increased γ-H2AX signal) in both cell lines through a mechanism of inhibited RNR activity. Radiation, cisplatin and 3-AP exposure resulted in significantly elevated numbers and persistence of γ-H2AX foci that were associated with reduced clonogenic survival. DNA damage was associated with a rise in p53R2 but not p21 protein levels 6 h after treatment with radiation and/or cisplatin plus 3-AP. We conclude that blockage of RNR activity by 3-AP impairs DNA damage responses that rely on deoxyribonucleotide production and thereby may substantially increase chemoradiosensitivity of human cervical cancers.     

Calibration of the γ-H2AX DNA Double Strand Break Focus Assay for Internal Radiation Exposure of Blood Lymphocytes

DNA double strand break (DSB) formation induced by ionizing radiation exposure is indicated by the DSB biomarkers γ-H2AX and 53BP1. Knowledge about DSB foci formation in-vitro after internal irradiation of whole blood samples with radionuclides in solution will help us to gain detailed insights about dose-response relationships in patients after molecular radiotherapy (MRT). Therefore, we studied the induction of radiation-induced co-localizing γ-H2AX and 53BP1 foci as surrogate markers for DSBs in-vitro, and correlated the obtained foci per cell values with the in-vitro absorbed doses to the blood for the two most frequently used radionuclides in MRT (I-131 and Lu-177). This approach led to an in-vitro calibration curve. Overall, 55 blood samples of three healthy volunteers were analyzed. For each experiment several vials containing a mixture of whole blood and radioactive solutions with different concentrations of isotonic NaCl-diluted radionuclides with known activities were prepared. Leukocytes were recovered by density centrifugation after incubation and constant blending for 1 h at 37°C. After ethanol fixation they were subjected to two-color immunofluorescence staining and the average frequencies of the co-localizing γ-H2AX and 53BP1 foci/nucleus were determined using a fluorescence microscope equipped with a red/green double band pass filter. The exact activity was determined in parallel in each blood sample by calibrated germanium detector measurements. The absorbed dose rates to the blood per nuclear disintegrations occurring in 1 ml of blood were calculated for both isotopes by a Monte Carlo simulation. The measured blood doses in our samples ranged from 6 to 95 mGy. A linear relationship was found between the number of DSB-marking foci/nucleus and the absorbed dose to the blood for both radionuclides studied. There were only minor nuclide-specific intra- and inter-subject deviations.     

High Throughput Measurement of γH2AX DSB Repair Kinetics in a Healthy Human Population

The Columbia University RABiT (Rapid Automated Biodosimetry Tool) quantifies DNA damage using fingerstick volumes of blood. One RABiT protocol quantifies the total γ-H2AX fluorescence per nucleus, a measure of DNA double strand breaks (DSB) by an immunofluorescent assay at a single time point. Using the recently extended RABiT system, that assays the γ-H2AX repair kinetics at multiple time points, the present small scale study followed its kinetics post irradiation at 0.5 h, 2 h, 4 h, 7 h and 24 h in lymphocytes from 94 healthy adults. The lymphocytes were irradiated ex vivo with 4 Gy γ rays using an external Cs-137 source. The effect of age, gender, race, ethnicity, alcohol use on the endogenous and post irradiation total γ-H2AX protein yields at various time points were statistically analyzed. The endogenous γ-H2AX levels were influenced by age, race and alcohol use within Hispanics. In response to radiation, induction of γ-H2AX yields at 0.5 h and peak formation at 2 h were independent of age, gender, ethnicity except for race and alcohol use that delayed the peak to 4 h time point. Despite the shift in the peak observed, the γ-H2AX yields reached close to baseline at 24 h for all groups. Age and race affected the rate of progression of the DSB repair soon after the yields reached maximum. Finally we show a positive correlation between endogenous γ-H2AX levels with radiation induced γ-H2AX yields (RIY) (r=0.257, P=0.02) and a negative correlation with residuals (r=-0.521, P=<0.0001). A positive correlation was also observed between RIY and DNA repair rate (r=0.634, P<0.0001). Our findings suggest age, race, ethnicity and alcohol use influence DSB γ-H2AX repair kinetics as measured by RABiT immunofluorescent assay.     

Arsenite induces premature senescence via p53/p21 pathway as a result of DNA damage in human malignant glioblastoma cells

In this study, we investigate whether arsenite-induced DNA damage leads to p53-dependent premature senescence using human glioblastoma cells with p53-wild type (U87MG-neo) and p53 deficient (U87MG-E6). A dose dependent relationship between arsenite and reduced cell growth is demonstrated, as well as induced γH2AX foci formation in both U87MG-neo and U87MG-E6 cells at low concentrations of arsenite. Senescence was induced by arsenite with senescence-associated β-galactosidase staining. Dimethyl- and trimethyl-lysine 9 of histone H3 (H3DMK9 and H3TMK9) foci formation was accompanied by p21 accumulation only in U87MG-neo but not in U87MG-E6 cells. This suggests that arsenite induces premature senescence as a result of DNA damage with heterochromatin forming through a p53/p21 dependent pathway. p21 and p53 siRNA consistently decreased H3TMK9 foci formation in U87M G-neo but not in U87MG-E6 cells after arsenite treatment. Taken together, arsenite reduces cell growth independently of p53 and induces premature senescence via p53/p21-dependent pathway following DNA damage. [BMB Reports 2014; 47(10): 575-580]     

γ-H2AX Kinetic Profile in Mouse Lymphocytes Exposed to the Internal Emitters Cesium-137 and Strontium-90

In the event of a dirty bomb scenario or an industrial nuclear accident, a significant dose of volatile radionuclides such as ¹³⁷Cs and ⁹⁰Sr may be dispersed into the atmosphere as a component of fallout and inhaled or ingested by hundreds and thousands of people. To study the effects of prolonged exposure to ingested radionuclides, we have performed long-term (30 day) internal-emitter mouse irradiations using soluble-injected ¹³⁷CsCl and ⁹⁰SrCl₂ radioisotopes. The effect of ionizing radiation on the induction and repair of DNA double strand breaks (DSBs) in peripheral mouse lymphocytes in vivo was determined using the γ-H2AX biodosimetry marker. Using a serial sacrifice experimental design, whole-body radiation absorbed doses for ¹³⁷Cs (0 to 10 Gy) and ⁹⁰Sr (0 to 49 Gy) were delivered over 30 days following exposure to each radionuclide. The committed absorbed doses of the two internal emitters as a function of time post exposure were calculated based on their retention parameters and their derived dose coefficients for each specific sacrifice time. In order to measure the kinetic profile for γ-H2AX, peripheral blood samples were drawn at 5 specific timed dose points over the 30-day study period and the total γ-H2AX nuclear fluorescence per lymphocyte was determined using image analysis software. A key finding was that a significant γ-H2AX signal was observed in vivo several weeks after a single radionuclide exposure. A mechanistically-motivated model was used to analyze the temporal kinetics of γ-H2AX fluorescence. Exposure to either radionuclide showed two peaks of γ-H2AX: one within the first week, which may represent the death of mature, differentiated lymphocytes, and the second at approximately three weeks, which may represent the production of new lymphocytes from damaged progenitor cells. The complexity of the observed responses to internal irradiation is likely caused by the interplay between continual production and repair of DNA damage, cell cycle effects and apoptosis.     

Decay of γ-H2AX foci correlates with potentially lethal damage repair and P53 status in human colorectal carcinoma cells

The influence of p53 status on potentially lethal damage repair (PLDR) and DNA double-strand break (DSB) repair was studied in two isogenic human colorectal carcinoma cell lines: RKO (p53 wild-type) and RC10.1 (p53 null). They were treated with different doses of ionizing radiation, and survival and the induction of DNA-DSB were studied. PLDR was determined by using clonogenic assays and then comparing the survival of cells plated immediately with the survival of cells plated 24 h after irradiation. Doses varied from 0 to 8 Gy. Survival curves were analyzed using the linear-quadratic formula: S(D)/S(0) = exp-(αD+βD²). The γ-H2AX foci assay was used to study DNA DSB kinetics. Cells were irradiated with single doses of 0, 0.5, 1 and 2 Gy. Foci levels were studied in non-irradiated control cells and 30 min and 24 h after irradiation. Irradiation was performed with gamma rays from a ¹³⁷Cs source, with a dose rate of 0.5 Gy/min. The RKO cells show higher survival rates after delayed plating than after immediate plating, while no such difference was found for the RC10.1 cells. Functional p53 seems to be a relevant characteristic regarding PLDR for cell survival. Decay of γ-H2AX foci after exposure to ionizing radiation is associated with DSB repair. More residual foci are observed in RC10.1 than in RKO, indicating that decay of γ-H2AX foci correlates with p53 functionality and PLDR in RKO cells.     

The HINT1 tumor suppressor regulates both gamma-H2AX and ATM in response to DNA damage

Hint1 is a haploinsufficient tumor suppressor gene and the underlying molecular mechanisms for its tumor suppressor function are unknown. In this study we demonstrate that HINT1 participates in ionizing radiation (IR)–induced DNA damage responses. In response to IR, HINT1 is recruited to IR-induced foci (IRIF) and associates with γ-H2AX and ATM. HINT1 deficiency does not affect the formation of γ-H2AX foci; however, it impairs the removal of γ-H2AX foci after DNA damage and this is associated with impaired acetylation of γ-H2AX. HINT1 deficiency also impairs acetylation of ATM and activation of ATM and its downstream effectors, and retards DNA repair, in response to IR. HINT1-deficient cells exhibit resistance to IR-induced apoptosis and several types of chromosomal abnormalities. Our findings suggest that the tumor suppressor function of HINT1 is caused by, at least in part, its normal role in enhancing cellular responses to DNA damage by regulating the functions of both γ-H2AX and ATM.     

Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates γ-Histone 2AX (γ-H2AX) Levels upon Ionizing Radiation

The microtubule-associated protein targeting protein for Xenopus kinesin-like protein 2 (TPX2) plays a key role in spindle assembly and is required for mitosis in human cells. In interphase, TPX2 is actively imported into the nucleus to prevent its premature activity in microtubule organization. To date, no function has been assigned to nuclear TPX2. We now report that TPX2 plays a role in the cellular response to DNA double strand breaks induced by ionizing radiation. Loss of TPX2 leads to inordinately strong and transient accumulation of ionizing radiation-dependent Ser-139-phosphorylated Histone 2AX (γ-H2AX) at G₀ and G₁ phases of the cell cycle. This is accompanied by the formation of increased numbers of high intensity γ-H2AX ionizing radiation-induced foci. Conversely, cells overexpressing TPX2 have reduced levels of γ-H2AX after ionizing radiation. Consistent with a role for TPX2 in the DNA damage response, we found that the protein accumulates at DNA double strand breaks and associates with the mediator of DNA damage checkpoint 1 (MDC1) and the ataxia telangiectasia mutated (ATM) kinase, both key regulators of γ-H2AX amplification. Pharmacologic inhibition or depletion of ATM or MDC1, but not of DNA-dependent protein kinase (DNA-PK), antagonizes the γ-H2AX phenotype caused by TPX2 depletion. Importantly, the regulation of γ-H2AX signals by TPX2 is not associated with apoptosis or the mitotic functions of TPX2. In sum, our study identifies a novel and the first nuclear function for TPX2 in the cellular responses to DNA damage.     

The induction of thioredoxin-1 by epinephrine withdraws stress via interaction with β-arrestin-1

Stress regulates a panel of important physiological functions and disease states. Epinephrine is produced under stresses threaten to homeostasis. Thioredoxin-1(Trx-1) is a redox regulating protein which is induced to resist stresses and related with various diseases. Thus, it is important to examine whether Trx-1 is induced by epinephrine and to understand the underlying molecular mechanisms that Trx-1 modulates epinephrine stress. Here, we show that the expression of Trx-1 was induced by epinephrine via β-adrenergic receptor/Cyclic AMP/protein kinase A (PKA) signaling pathway in PC12 cells. The down-regulation of Trx-1 by siRNA aggravated accumulation of γ-H2AX and further decreased expression of p53 by epinephrine. Accordingly, Trx-1 overexpression alleviated accumulation of γ-H2AX and restored the expressions of p53 and C/EBP homologous protein (CHOP) in the cortex, hippocampus and thymus of mice. Moreover, Trx-1 overexpression reduced the malondialdehyde concentration by epinephrine. We further explored the mechanism on p53 and γ-H2AX regulated by Trx-1. We found that overexpression of Trx-1 suppressed β-arrestin-1 expression through interaction with β-arrestin-1. Consequently, the downregulation of β-arrestin-1 suppressed the cell viability and the expressions of γ-H2AX and cyclin D1, and increased p53 expression. Taken together, our data suggest that Trx-1/β-arrestin-1 interaction may represent a novel endogenous mechanism on protecting against stress.     

The Role of Hypoxia Inducible Factor-1 Alpha in Bypassing Oncogene-Induced Senescence

Oncogene induced senescence (OIS) is a sustained anti-proliferative response acutely induced in primary cells via activation of mitogenic oncogenes such as Ras/BRAF. This mechanism acts as an initial barrier preventing normal cells transformation into malignant cell. Besides oncogenic activation and DNA damage response (DDR), senescence is modulated by a plethora of other factors, and one of the most important one is oxygen tension of the tissue. The aim of this study was to determine the impact of hypoxia on Ras^V12-induced senescence in human diploid fibroblasts (HDFs). We showed here that hypoxia prevents execution of oncogene induced senescence (OIS), through a strong down-regulation of senescence hallmarks, such as SA- β-galactosidase, H3K9me3, HP1γ, p53, p21^CIP1 and p16^INK4a in association with induction of hypoxia inducible factor-1α (HIF-1α). In addition, hypoxia also decreased marks of H-Ras^V12-induced DDR in both cell lines through down-regulation of ATM/ATR, Chk1 and Chk2 phosphorylation as well as decreased γ-H2AX positivity. Utilizing shRNA system targeting HIF-1α we show that HIF-1α is directly involved in down regulation of p53 and its target p21^CIP1 but not p16^INK4a. In line with this finding we found that knock down of HIF-1α leads to a strong induction of apoptotic response, but not restoration of senescence in Ras expressing HDFs in hypoxia. This indicates that HIF-1α is an important player in early steps of tumorigenesis, leading to suppression of senescence through its negative regulation of p53 and p21^CIP1. In our work we describe a mechanism through which hypoxia and specifically HIF-1α preclude cells from maintaining senescence-driven anti proliferative response. These findings indicate the possible mechanism through which hypoxic environment helps premalignant cells to evade impingement of cellular failsafe pathways.     

Human papilloma virus type16 E6 deregulates CHK1 and sensitizes human fibroblasts to environmental carcinogens independently of its effect on p53

After treatment with ultraviolet radiation (UV), human fibroblasts that express the HPV type 16 E6 oncoprotein display defects in repair of cyclobutane pyrimidine dimers, hypersensitivity to inactivation of clonogenic survival and an inability to sustain DNA replication. To determine whether these effects are specific to depletion of p53 or inactivation of its function , fibroblast lines were constructed with ectopic expression of a dominant-negative p53 allele (p53-H179Q) to inactivate function or a short-hairpin RNA (p53-RNAi) to deplete expression of p53. Only the expression of HPV16E6 sensitized fibroblasts to UV or the chemical carcinogen, benzo[a]pyrene diolepoxide I (BPDE). Carcinogen-treated cells expressing p53-H179Q or p53-RNAi were resistant to inactivation of colony formation and did not suffer replication arrest. CHK1 is a key checkpoint kinase in the response to carcinogen-induced DNA damage. Control and p53-RNAi-expressing fibroblasts displayed phosphorylation of Ser345 on CHK1 45-120 min after carcinogen treatment with a return to near baseline phosphorylation by 6 h after treatment. HPV16E6-expressing fibroblasts displayed enhanced and sustained phosphorylation of CHK1. This was associated with enhanced phosphorylation of Thr68 on CHK2 and Ser139 on H2AX, both markers of severe replication stress and DNA double strand breaks. Incubation with the phosphatase inhibitor okadaic acid produced more phosphorylation of CHK1 in UV-treated HPV16E6-expressing cells than in p53-H179Q-expressing cells suggesting that HPV16E6 may interfere with the recovery of coupled DNA replication at replication forks that are stalled at [6-4]pyrimidine-pyrimidone photoproducts and BPDE-DNA adducts. The results indicate that HPV16E6 targets a protein or proteins other than p53 to deregulate the activity of CHK1 in carcinogen-damaged cells.     

HDAC6 inhibitor ACY1215 inhibits the activation of NLRP3 inflammasome in acute liver failure by regulating the ATM/F-actin signalling pathway

Acute liver failure (ALF) is a rare and critical medical condition. This study was designed to investigate the protective effects and underlying mechanism of ACY1215 in ALF mice. Our findings suggested that ACY1215 treatment ameliorates the pathological hepatic damage of ALF and decreases the serum levels of ALT and AST. Furthermore, ACY1215 pretreatment increased the level of ATM, γ-H2AX, Chk2, p53, p21, F-actin and vinculin in ALF. Moreover, ACY1215 inhibited the level of NLRP3, ASC, caspase-1, IL-1β and IL-18 in ALF. The ATM inhibitor KU55933 could decrease the level of ATM, γ-H2AX, Chk2, p53, p21, F-actin and vinculin in ALF with ACY1215 pretreatment. The F-actin inhibitor cytochalasin B decreased the level of F-actin and vinculin in ALF with ACY1215 pretreatment. However, cytochalasin B had no effect on protein levels of ATM, Chk2, p53 and p21 in ALF with ACY1215 pretreatment. Cytochalasin B could dramatically increase the level of NLRP3, ASC, caspase-1, IL-1β and IL-18 in ALF with ACY1215 pretreatment. These results indicated that ACY1215 exhibited hepatoprotective properties, which was associated with the inhibition of NLRP3 inflammasome, and this effect of ACY1215 was connected with upregulation of the ATM/F-actin mediated signalling pathways.     

Death receptor-induced activation of the Chk2- and histone H2AX-associated DNA damage response pathways

TRAIL is an endogenous death receptor ligand also used therapeutically because of its selective proapoptotic activity in cancer cells. In the present study, we examined chromatin alterations induced by TRAIL and show that TRAIL induces a rapid activation of DNA damage response (DDR) pathways with histone H2AX, Chk2, ATM, and DNA-PK phosphorylations. Within 1 h of TRAIL exposure, immunofluorescence confocal microscopy revealed γ-H2AX peripheral nuclear staining (γ-H2AX ring) colocalizing with phosphorylated/activated Chk2, ATM, and DNA-PK inside heterochromatin regions. The marginal distribution of DDR proteins in early apoptotic cells is remarkably different from the focal staining seen after DNA damage. TRAIL-induced DDR was suppressed upon caspase inhibition or Bax inactivation, demonstrating that the DDR activated by TRAIL is downstream from the mitochondrial death pathway. H2AX phosphorylation was dependent on DNA-PK, while Chk2 phosphorylation was dependent on both ATM and DNA-PK. Downregulation of Chk2 decreased TRAIL-induced cell detachment; delayed the activation of caspases 2, 3, 8, and 9; and reduced TRAIL-induced cell killing. Together, our findings suggest that nuclear activation of Chk2 by TRAIL acts as a positive feedback loop involving the mitochondrion-dependent activation of caspases, independently of p53.