H2AX Phosphorylation In Response Research Articles

Silencing of RUNX2 enhances gemcitabine sensitivity of p53-deficient human pancreatic cancer AsPC-1 cells through the stimulation of TAp63-mediated cell death.

It has been well-known that human pancreatic cancer represents the fourth and fifth leading causes of cancer-related deaths in the United States and Japan, respectively.1, 2 Notably, pancreatic cancer is characterized by high metastatic potential, resistance to chemotherapy and thus its prognosis is extremely poor with 5-year survival <5%. At diagnosis, more than 80% cases are already advanced and non-resectable.3 Therefore, chemotherapy and/or radiotherapy is the only option. Despite improvements in the treatments, the survival rate has not been significantly ameliorated over the last few decades. For chemotherapy, a deoxycytidine analog termed gemcitabine (GEM) is the first line of standard treatment given to most of the patients bearing advanced pancreatic cancer.4 Unfortunately, GEM treatment provides limited clinical benefits, especially in advanced and metastatic disease.5 Hence, the extensive efforts to clarify the precise molecular mechanisms behind GEM-resistant phenotype of malignant pancreatic cancer and also to develop the promising strategies to enhance the efficacy of GEM should be required.

A kinome-targeted RNAi-based screen links FGF signaling to H2AX phosphorylation in response to radiation.

A general radioprotective effect by fibroblast growth factor (FGF) has been extensively described since the early 1990s; however, the molecular mechanisms involved remain largely unknown. Radiation-induced DNA double-strand breaks (DSBs) lead to a complex set of responses in eukaryotic cells. One of the earliest consequences is phosphorylation of histone H2AX to form nuclear foci of the phosphorylated form of H2AX (γH2AX) in the chromatin adjacent to sites of DSBs and to initiate the recruitment of DNA-repair molecules. Upon a DSB event, a rapid signaling network is activated to coordinate DNA repair with the induction of cell-cycle checkpoints. To date, three kinases (ATM, ATR, and DNA-PK) have been shown to phosphorylate histone H2AX in response to irradiation. Here, we report a kinome-targeted small interfering RNA (siRNA) screen to characterize human kinases involved in H2AX phosphorylation. By analyzing γH2AX foci at a single-nucleus level, we identified 46 kinases involved either directly or indirectly in H2AX phosphorylation in response to irradiation in human keratinocytes. Furthermore, we demonstrate that in response to irradiation, the FGFR4 signaling cascade promotes JNK1 activation and direct H2AX phosphorylation leading, in turn, to more efficient DNA repair. This can explain, at least partially, the radioprotective effect of FGF.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-015-1901-7) contains supplementary material, which is available to authorized users.

Stella preserves maternal chromosome integrity by inhibiting 5hmC-induced γH2AX accumulation.

In the mouse zygote, Stella/PGC7 protects 5-methylcytosine (5mC) of the maternal genome from Tet3-mediated oxidation to 5-hydroxymethylcytosine (5hmC). Although ablation of Stella causes early embryonic lethality, the underlying molecular mechanisms remain unknown. In this study, we report impaired DNA replication and abnormal chromosome segregation (ACS) of maternal chromosomes in Stella-null embryos. In addition, phosphorylation of H2AX (γH2AX), which has been reported to inhibit DNA replication, accumulates in the maternal chromatin of Stella-null zygotes in a Tet3-dependent manner. Cell culture assays verified that ectopic appearance of 5hmC induces abnormal accumulation of γH2AX and subsequent growth retardation. Thus, Stella protects maternal chromosomes from aberrant epigenetic modifications to ensure early embryogenesis.

RM-04 * RETINOBLASTOMA BINDING PROTEIN 4 (RBBP4) MODULATES TEMOZOLOMIDE RESPONSE THROUGH REGULATION OF MGMT EXPRESSION IN GLIOBLASTOMA

Through shRNA library screen we identified RBBP4 as a modulator of TMZ response in glioblastoma (GBM). Consequently, we investigated the mechanisms whereby RBBP4 modulates TMZ response using shRNA to silence this gene in MGMT-expressing T98G and U138 GBM cells. The cytotoxicity was evaluated using fluorescence-based CYQUANT proliferation assay. A total of 4 shRNA constructs significantly suppressed RBBP4 in both T98G and U138. Cells expressing non-specific targeting shRNA (NT-shRNA) were used as control. RBBP4 knockdown significantly sensitized TMZ both in T98G and U138 cells; the relative fluorescence for the TMZ-treated (100 µM) control T98NT-shRNA cells was 1.17 ± 0.15, whereas for T98RBBP4-shRNA clones were 0.54 ± 0.02, 0.29 ± 0.03, 0.36 ± 0.05, and 0.34 ± 0.03, respectively (p < 0.001). Similar sensitization was observed in U138 cells; relative fluorescence for the TMZ-treated (300 µM) control U138NT-shRNA cells was 0.70 ± 0.05 and for U138RBBP4-shRNA clones were 0.42 ± 0.06, 0.27 ± 0.01, 0.28 ± 0.02, and 0.30 ± 0.01, respectively (p < 0.001). Interestingly, knockdown of RBBP4 in T98G was accompanied with a synthetic lethality to PARP inhibition and increased response to TMZ-induced DNA damage, as evidenced by increased phosphorylation of KAP1, CHK1 and CHK2. Moreover, phosphorylation of H2AX in response to TMZ treatment was significantly higher in T98RBBP4-shRNA clones. Consistent with deficient homologous recombination (HR), T98RBBP4-shRNA clones significantly expressed less RAD51 compared with the control T98NT-shRNA cells. Even more interesting, RBBP4 knockdown silenced MGMT expression in both T98G and U138, which was accompanied by decreased recruitment of acetylated H3K9 coupled with increased recruitment of tri-methylated H3K9. Moreover, RBBP4 knockdown was coupled with loss of p300 recruitment to bind MGMT promoter region. Collectively, these findings suggest that RBBP4 modulates TMZ response in GBM cells through epigenetic regulation of MGMT, and perhaps through RAD51-mediated HR repair of DNA damage in selected GBM cells.

Abstract 2423: Development of a validated high throughput ELISA assay for gamma H2AX as a pharmacodynamic marker

Abstract Histone H2AX is a 14 kDa ubiquitous member of the H2A histone family that becomes rapidly phosphorylated at Serine 139 by ATM and ATR kinases to yield a form known as γ-H2AX in response to double-strand DNA damage and apoptosis. γ-H2AX is an ideal Pharmacodynamic (PD) surrogate marker to measure molecular responses to a large number of drugs; however, methods such as western blots and immunohistochemistry are widely used but are difficult to validate to regulatory standards, and not suitable for high throughput screening applications. To address this need, we have developed a novel high throughput ELISA assay to measure γ-H2AX levels in cellular extracts and phosphorylation of H2AX in response to therapeutic intervention. This assay documents differences of γ-H2AX levels in PBMC, cultured cells, tissue biopsies, and will be useful in future clinical trials providing one of many needed tools to enable hypothesis-driven preclinical drug design strategies.p Background Histone H2AX is a 14 kDa ubiquitous member of the H2A histone family that contains an evolutionarily conserved SQ motif at the C-terminus in eukaryotes. Serine 139 within this motif becomes rapidly phosphorylated by ATM and ATR kinases to yield a form known as γ-H2AX in response to double-strand DNA damage and apoptosis (1). During the past year investigators have confirmed the value of γ-H2AX as an important Pharmacodynamic (PD) marker (2) and genotoxicity endpoint (3). There are over 21 anticancer drugs that are known to result in γ-H2AX formation. As a result, γ-H2AX is an ideal Pharmacodynamic PD surrogate marker to measure molecular responses to a large number of drugs (4,5,6). While many of these drugs have already garnered regulatory approval, and are currently being used to manage various types of cancers, they are the subject of ongoing clinical studies to evaluate their efficacy when used alone or in combination with molecularly targeted drugs. While methods such as western blots and immunohistochemistry are widely used but are difficult to validate to regulatory standards, the ELISA method is the most quantifiable and easiest to validate, and not suitable for high throughput screening applications. To address this need Trevigen's quantitative pharmacodynamic HT γ-H2AX ELISA assay measures γ-H2AX levels in cellular extracts and phosphorylation of H2AX in response to therapeutic intervention. This assay documents differences of γ-H2AX levels in PBMC, cultured cells, tissue biopsies, and will be useful in future clinical trials providing one of many needed tools to enable hypothesis-driven preclinical drug design strategies. Citation Format: Jay George, Hai Xu. Development of a validated high throughput ELISA assay for gamma H2AX as a pharmacodynamic marker. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2423. doi:10.1158/1538-7445.AM2014-2423

Distinct roles for DNA-PK, ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress

DNA damage encountered by DNA replication forks poses risks of genome destabilization, a precursor to carcinogenesis. Damage checkpoint systems cause cell cycle arrest, promote repair and induce programed cell death when damage is severe. Checkpoints are critical parts of the DNA damage response network that act to suppress cancer. DNA damage and perturbation of replication machinery causes replication stress, characterized by accumulation of single-stranded DNA bound by replication protein A (RPA), which triggers activation of ataxia telangiectasia and Rad3 related (ATR) and phosphorylation of the RPA32, subunit of RPA, leading to Chk1 activation and arrest. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [a kinase related to ataxia telangiectasia mutated (ATM) and ATR] has well characterized roles in DNA double-strand break repair, but poorly understood roles in replication stress-induced RPA phosphorylation. We show that DNA-PKcs mutant cells fail to arrest replication following stress, and mutations in RPA32 phosphorylation sites targeted by DNA-PKcs increase the proportion of cells in mitosis, impair ATR signaling to Chk1 and confer a G2/M arrest defect. Inhibition of ATR and DNA-PK (but not ATM), mimic the defects observed in cells expressing mutant RPA32. Cells expressing mutant RPA32 or DNA-PKcs show sustained H2AX phosphorylation in response to replication stress that persists in cells entering mitosis, indicating inappropriate mitotic entry with unrepaired damage.

Lymphocyte subsets and their H2AX phosphorylation in response to in vivo irradiation in rats

Purpose: The objective of the study was to investigate differences in the radiosensitivity of rat peripheral blood lymphocyte subsets identified by expression of surface clusters of differentiation markers (CD3, CD4, CD8, CD45RA, CD161) after whole-body in vivo gamma-ray irradiation and to assess their individual histone H2AX phosphorylation as an early cell response to irradiation.Materials and methods: The relative representations of CD45RA B-lymphocytes, CD161 natural killer cells (NK cells), CD3CD4 T-lymphocyte subset and CD3CD8 T-lymphocyte subset in the rat peripheral blood were studied 24–72 hours after irradiation in a dose range of 0–5 Gy. Their intracellular H2AX phosphorylation (γ-H2AX) after 4 Gy and 9 Gy whole-body in vivo irradiation was assessed by multicolour flow cytometry.Results: We determined the linear dose response of radioresistant CD161 NK cells (24 h), both radiosensitive T-lymphocyte subsets (24 h) and CD45RA B-lymphocytes (72 h) after in vivo irradiation. CD45RA B-lymphocytes showed the highest radiosensitivity and we observed pronounced H2AX phosphorylation which remained expressed in these cells for over 4 h after irradiation.Conclusion: The combination of the surface immunophenotyping together with intracellular detection of γ-H2AX offers the possibility to assess the absorbed dose of ionizing irradiation with high sensitivity post irradiation and could be successfully applied to biodosimetry.

Abstract 4345: Inadequate repair of ionizing radiation damage: A consequence of the invasive tumor phenotype

Abstract A major characteristic of the transition of prostatic intraepithelial neoplasia (PIN) to invasive prostate cancer is the progressive loss of laminin 322 in the basal lamina and loss of A6B4 integrin, a laminin 322 adhesion receptor in the basal cells. Previous work from our group reported that the loss of interaction with laminin 322 decreased the ability of normal prostate cells to respond to DNA damage. The defect was manifested as an alteration of an ionizing radiation (IR) induced G2 progression block. In this study, the goal was to determine if an invasive phenotype of human tumor cells, expressing A6B1 integrin, a laminin 511 adhesion receptor, influenced a DNA damage response (DDR) to IR. DU145 tumor cells were grown on laminin 511 and DDR was determined under conditions of stimulated invasion via HGF. The DDR endpoint chosen was the production, resolution and persistence of nuclear H2AX phosphorylation foci as an indicator of damage, repair and inadequate repair, respectively. Similar to a recent report, we observed a fast and transient phosphorylation of H2AX in response to IR within the basal cells of the normal human prostate epithelium that was both dose and time dependent. Utilizing tissue culture conditions, we determined that under optimal growth conditions on laminin 511 there was a dose and time response of H2AX phosphorylation in response to IR treatment. Under conditions of HGF stimulation, the persistence of H2AX foci 24 hours after IR was two to four fold higher as compared to unstimulated cells. The amplified persistence of H2AX foci was observed at 1, 2, 4, 6 and 8 Gy at both 24 and 36 hours after IR treatment. Current studies are determining if the inadequate repair response to IR under conditions of stimulated invasion results in an increased tumor cell killing or an increased genomic instability of surviving tumor cells. (Supported in part by NIH Cancer Center Core Grant CA 23074 and DOD grant W81XWH-10-1-0496) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4345. doi:1538-7445.AM2012-4345

20-O-IngenolEZ, a catalytic topoisomerase II inhibitor, specifically inhibits cell proliferation and induces double-strand DNA breaks in BLM-/- cells

Bis-dioxopiperazines have been termed catalytic inhibitors to distinguish them from topoisomerase poisons that induce DNA double-strand breaks (DSBs). However, it has been reported that ICRF-193 acts as a poison in cells containing mutated genes related to checkpoint mechanisms. We also showed previously that 20-O-ingenolEZ acts as a catalytic inhibitor to inhibit the ATPase activity of topoisomerase IIα, inducing the G2 arrest of mouse mammary tumor (MMT) cells. In this study, we observed the effects of 20-O-ingenolEZ on cells containing a mutation in the RecQ helicase gene. 20-O-IngenolEZ completely inhibited the proliferation of BLM-/-cells in BLM-/- and WRN-/-DT40 cells and wild-type DT40 cells. This inhibition induced the phosphorylation of H2AX in response to agents that introduce topoisomerase II-mediated DSBs. Following DNA damage, the induction of apoptosis in the BLM-/-cells by 20-O-ingenolEZ showed the characteristics of a topoisomerase II poison.

H2AX phosphorylation in response to DNA double-strand break formation during bystander signalling: effect of microRNA knockdown

Upon DNA double-strand break (DSB) formation, hundreds of H2AX molecules in the chromatin flanking the break site are phosphorylated on serine residue 139, termed gamma-H2AX, so that virtually every DSB site in a nucleus can be visualised within 10 min of its formation using an antibody to gamma-H2AX. One application of this sensitive assay is to examine the induction of DNA double-strand damage in subtle non-targeted cellular effects such as the bystander effect. Here whether microRNA (miRNA) serve as a primary signalling mechanism for bystander effect propagation by comparing matched human colon carcinoma cell lines with wild-type or depleted levels of mature miRNAs was investigated. No major differences were found in the levels of induced gamma-H2AX foci in the tested cell lines, indicating that though miRNAs play a role in bystander effect manifestation, they appear not to be the primary bystander signalling molecules in the formation of bystander effect-induced DSBs.

Rosiglitazone enhances the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells

Combined-modality treatment has improved the outcome in cases of various solid tumors, and radiosensitizers are used to enhance the radiotherapeutic efficiency. Rosiglitazone, a synthetic ligand of peroxisome proliferator-activated receptors γ used in the treatment of type-2 diabetes, has been shown to reduce tumor growth and metastasis in human cancer cells, and may have the potential to be used as a radiosensitizer in radiotherapy for human colorectal cancer cells. In this study, rosiglitazone treatment significantly reduced the cell viability of p53-wild type HCT116 cells but not p53-mutant HT-29 cells. Interestingly, rosiglitazone pretreatment enhanced radiosensitivity in p53-mutant HT-29 cells but not HCT116 cells, and prolonged radiation-induced G 2/M arrest and enhanced radiation-induced cell growth inhibition in HT-29 cells. Pretreatment with rosiglitazone also suppressed radiation-induced H2AX phosphorylation in response to DNA damage and AKT activation for cell survival; on the contrary, rosiglitazone pretreatment enhanced radiation-induced caspase-8, -9, and -3 activation and PARP cleavage in HT-29 cells. In addition, pretreatment with a pan-caspase inhibitor, zVAD-fmk, attenuated the levels of caspase-3 activation and PARP cleavage in radiation-exposed cancer cells in combination with rosiglitazone pretreatment. Our results provide proof for the first time that rosiglitazone suppresses radiation-induced survival signals and DNA damage response, and enhances the radiation-induced apoptosis signaling cascade. These findings can assist in the development of rosiglitazone as a novel radiosensitizer.

DNA-PKcs plays a dominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression

BackgroundWhen DNA double-strand breaks (DSB) are induced by ionizing radiation (IR) in cells, histone H2AX is quickly phosphorylated into γ-H2AX (p-S139) around the DSB site. The necessity of DNA-PKcs in regulating the phosphorylation of H2AX in response to DNA damage and cell cycle progression was investigated.ResultsThe level of γH2AX in HeLa cells increased rapidly with a peak level at 0.25 - 1.0 h after 4 Gy γ irradiation. SiRNA-mediated depression of DNA-PKcs resulted in a strikingly decreased level of γH2AX. An increased γH2AX was also induced in the ATM deficient cell line AT5BIVA at 0.5 - 1.0 h after 4 Gy γ rays, and this IR-increased γH2AX in ATM deficient cells was dramatically abolished by the PIKK inhibitor wortmannin and the DNA-PKcs specific inhibitor NU7026. A high level of constitutive expression of γH2AX was observed in another ATM deficient cell line ATS4. The alteration of γH2AX level associated with cell cycle progression was also observed. HeLa cells with siRNA-depressed DNA-PKcs (HeLa-H1) or normal level DNA-PKcs (HeLa-NC) were synchronized at the G1 phase with the thymidine double-blocking method. At ~5 h after the synchronized cells were released from the G1 block, the S phase cells were dominant (80%) for both HeLa-H1 and HeLa-NC cells. At 8 - 9 h after the synchronized cells released from the G1 block, the proportion of G2/M population reached 56 - 60% for HeLa-NC cells, which was higher than that for HeLa H1 cells (33 - 40%). Consistently, the proportion of S phase for HeLa-NC cells decreased to ~15%; while a higher level (26 - 33%) was still maintained for the DNA-PKcs depleted HeLa-H1 cells during this period. In HeLa-NC cells, the γH2AX level increased gradually as the cells were released from the G1 block and entered the G2/M phase. However, this γH2AX alteration associated with cell cycle progressing was remarkably suppressed in the DNA-PKcs depleted HeLa-H1 cells, while wortmannin and NU7026 could also suppress this cell cycle related phosphorylation of H2AX. Furthermore, inhibition of GSK3β activity with LiCl or specific siRNA could up-regulate the γH2AX level and prolong the time of increased γH2AX to 10 h or more after 4 Gy. GSK3β is a negative regulation target of DNA-PKcs/Akt signaling via phosphorylation on Ser9, which leads to its inactivation. Depression of DNA-PKcs in HeLa cells leads to a decreased phosphorylation of Akt on Ser473 and its target GSK3β on Ser9, which, in other words, results in an increased activation of GSK3β. In addition, inhibition of PDK (another up-stream regulator of Akt/GSK3β) by siRNA can also decrease the induction of γH2AX in response to both DNA damage and cell cycle progression.ConclusionDNA-PKcs plays a dominant role in regulating the phosphorylation of H2AX in response to both DNA damage and cell cycle progression. It can directly phosphorylate H2AX independent of ATM and indirectly modulate the phosphorylation level of γH2AX via the Akt/GSK3 β signal pathway.

Abstract A161: Acquired resistance to combination treatment with TMZ and ABT-888 is mediated by both BER and HR DNA repair pathways

Abstract Many established cancer therapies involve DNA-damaging chemotherapy or radiotherapy. Gain of DNA repair capacity of the tumor represents a common mechanism employed by cancer cells to survive DNA-damaging therapy. Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that is activated by DNA-damage and plays a critical role in base excision repair (BER). Inhibition of PARP represents an attractive approach for the treatment of cancer. Previously, we have described the discovery and characterization of a potent PARP inhibitor, ABT-888. ABT-888 potentiates the activity of DNA-damaging agents such as temozolomide (TMZ) in a variety of preclinical models. Previously, we demonstrated that ABT-888 potentiated the cytotoxic effect of TMZ by converting TMZ-induced single strand DNA breaks (SSB) to double strand breaks (DSB). We report here the generation of HCT116 cells resistant to treatment with TMZ and ABT-888 (HCT116R cells). HCT116R cells exhibit decreased H2AX phosphorylation in response to the treatment with TMZ and ABT-888 relative to parental HCT116 cells. Microarray and western blot studies indicate that HCT116R cells have decreased PARP-1 and elevated Rad51 expression levels. HCT116R cells are dependent on Rad51 for proliferation and survival, as demonstrated by inhibition of proliferation and induction of apoptosis upon treatment with Rad51 siRNA. In addition, HCT116R cells are more resistant to radiation than the parental HCT116 cells. Our study suggests that cancer cells up-regulate the homologous recombination DNA repair pathway to compensate for the loss of BER, which may account for the observed resistance to the treatment with TMZ and ABT-888. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A161.

Acquired Resistance to Combination Treatment with Temozolomide and ABT-888 Is Mediated by Both Base Excision Repair and Homologous Recombination DNA Repair Pathways

Many established cancer therapies involve DNA-damaging chemotherapy or radiotherapy. Gain of DNA repair capacity of the tumor represents a common mechanism used by cancer cells to survive DNA-damaging therapy. Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that is activated by DNA damage and plays a critical role in base excision repair. Inhibition of PARP represents an attractive approach for the treatment of cancer. Previously, we have described the discovery and characterization of a potent PARP inhibitor, ABT-888. ABT-888 potentiates the activity of DNA-damaging agents such as temozolomide (TMZ) in a variety of preclinical models. We report here the generation of HCT116 cells resistant to treatment with TMZ and ABT-888 (HCT116R cells). HCT116R cells exhibit decreased H2AX phosphorylation in response to treatment with TMZ and ABT-888 relative to parental HCT116 cells. Microarray and Western blot studies indicate that HCT116R cells have decreased PARP-1 and elevated Rad51 expression levels. HCT116R cells are dependent on Rad51 for proliferation and survival, as shown by inhibition of proliferation and induction of apoptosis upon treatment with Rad51 small interfering RNA. In addition, HCT116R cells are more resistant to radiation than the parental HCT116 cells. Our study suggests that cancer cells upregulate the homologous recombination DNA repair pathway to compensate for the loss of base excision repair, which may account for the observed resistance to treatment with TMZ and ABT-888.

Structural basis for recognition of H3K4 methylation status by the DNA methyltransferase 3A ATRX–DNMT3–DNMT3L domain

DNMT3 proteins are de novo DNA methyltransferases that are responsible for the establishment of DNA methylation patterns in mammalian genomes. Here, we have determined the crystal structures of the ATRX-DNMT3-DNMT3L (ADD) domain of DNMT3A in an unliganded form and in a complex with the amino-terminal tail of histone H3. Combined with the results of biochemical analysis, the complex structure indicates that DNMT3A recognizes the unmethylated state of lysine 4 in histone H3. This finding indicates that the recruitment of DNMT3A onto chromatin, and thereby de novo DNA methylation, is mediated by recognition of the histone modification state by its ADD domain. Furthermore, our biochemical and nuclear magnetic resonance data show mutually exclusive binding of the ADD domain of DNMT3A and the chromodomain of heterochromatin protein 1alpha to the H3 tail. These results indicate that de novo DNA methylation by DNMT3A requires the alteration of chromatin structure.

Protective role of Purα to cisplatin

BACKGROUND: The nucleic acid-binding protein Purα is involved at stalled DNA replication forks, in double-strand break (DSB) DNA repair and the cellular response to DNA replication stress. Purα also regulates homologous recombination-directed DNA repair (HRR). METHODS: We investigated the effects of the DNA damage-inducing cancer chemotherapeutic agent cisplatin on mouse embryo fibroblasts (MEFs) from PURA-/- knockout mice that lack Purα. RESULTS: Cells lacking Purα showed enhanced sensitivity to cisplatin as evaluated by assays for cell viability and cell clonogenicity. This was seen both in Purα-negative MEFs and in human glioblastoma cells treated with siRNA directed against Purα. MEFs lacking Purα also showed enhanced H2AX phosphorylation in response to cisplatin. Repair of a reporter plasmid that had been treated with cisplatin was decreased in a reactivation assay using Purα-negative MEFs and the capacity of nuclear extracts from Purα-negative MEFs to perform non-homologous end-joining in vitro was also impaired. CONCLUSIONS: Purα has a role in the cellular response to cisplatin-induced DNA damage and may provide new therapeutic modalities for cisplatin-resistant tumors.

Histone H2AX phosphorylation in response to changes in chromatin structure induced by altered osmolarity

DNA strand breaks trigger marked phosphorylation of histone H2AX (i.e. gamma-H2AX). While DNA double-strand breaks (DSBs) provide a strong stimulus for this event, the accompanying structural alterations in chromatin may represent the actual signal that elicits gamma-H2AX. Our data show that changes in chromatin structure are sufficient to elicit extensive gamma-H2AX formation in the relative absence of DNA strand breaks. Cells subjected to hypotonic (0.05 M) treatment exhibit gamma-H2AX levels that are equivalent to those found after the induction of 80-200 DNA DSBs (i.e. 2-5 Gy). Despite this significant increase in phosphorylation, cell survival remains relatively unaffected (<10% cytotoxicity), and there is no significant increase in apoptosis. Nuclear staining profiles indicate that gamma-H2AX-positive cells induced under altered tonicity exhibit variable levels of staining, ranging from uniform pan staining to discrete punctate foci more characteristic of DNA strand breakage. The capability to induce significant gamma-H2AX formation under altered tonicity in the relative absence of DNA strand breaks suggests that this histone modification evolved in response to changes in chromatin structure.

Autophosphorylation of H2AX in a cell-specific frozen dependent way

Effective cryopreservation is a highly desired outcome in many laboratories including clinical and research centers. Cryopreservation-induced cellular damage through necrosis and apoptosis has been well characterized. However, our knowledge of the mechanism of induction of these cell death modalities is limited. H2AX histone phosphorylation to γ-H2AX is a DNA damage response and is an excellent indicator of DNA double stranded break formation. In this study we examined and detected significant phosphorylation of H2AX in response to cryopreservation of HELF and B16 cell lines. The data provide strong evidence of intrinsic alterations in DNA repair pathway members for which the impact is yet to be fully understood. While further investigation will be necessary to characterize this response, our findings show a clear linkage between freezing resulting in phosphorylation and activation of the key DNA repair enzyme H2AX.

Differential roles of ATR and ATM in p53, Chk1, and histone H2AX phosphorylation in response to hyperoxia: ATR-dependent ATM activation

Elevated level of oxygen (hyperoxia) is widely used in critical care units and in respiratory insufficiencies. In addition, hyperoxia has been implicated in many diseases such as bronchopulmonary dysplasia or acute respiratory distress syndrome. Although hyperoxia is known to cause DNA base modifications and strand breaks, the DNA damage response has not been adequately investigated. We have investigated the effect of hyperoxia on DNA damage signaling and show that hyperoxia is a unique stress that activates the ataxia telangiectasia mutant (ATM)- and Rad3-related protein kinase (ATR)-dependent p53 phosphorylations (Ser6, -15, -37, and -392), phosphorylation of histone H2AX (Ser139), and phosphorylation of checkpoint kinase 1 (Chk1). In addition, we show that phosphorylation of p53 (Ser6) and histone H2AX (Ser139) depend on both ATM and ATR. We demonstrate that ATR activation precedes ATM activation in hyperoxia. Finally, we show that ATR is required for ATM activation in hyperoxia. Taken together, we report that ATR is the major DNA damage signal transducer in hyperoxia that activates ATM.

H2AX phosphorylation marks gemcitabine-induced stalled replication forks and their collapse upon S-phase checkpoint abrogation

Gemcitabine is a nucleoside analogue that is incorporated into replicating DNA, resulting in partial chain termination and stalling of replication forks. The histone variant H2AX is phosphorylated on Ser(139) (gamma-H2AX) and forms nuclear foci at sites of DNA damage. Here, we characterize the concentration- and time-dependent phosphorylation of H2AX in response to gemcitabine-induced stalled replication forks. The number of gamma-H2AX foci increased with time up to 2 to 6 h after exposure to gemcitabine, whereas longer exposures did not cause greater phosphorylation or increase cell death. The percentage of gamma-H2AX-positive cells increased with concentrations of gemcitabine up to 0.1 micromol/L, and gamma-H2AX was most evident in the S-phase fraction. Phosphorylation of ataxia-telangiectasia mutated (ATM) on Ser(1981) was also associated with S-phase cells and colocalized in the nucleus with phosphorylated H2AX foci after gemcitabine exposure. Chemical inhibition of ATM, ATM- and Rad3-related, and DNA-dependent protein kinase blocked H2AX phosphorylation. H2AX and ATM phosphorylation were associated with inhibition of DNA synthesis, S-phase accumulation, and activation of the S-phase checkpoint pathway (Chk1/Cdc25A/cyclin-dependent kinase 2). Exposure of previously gemcitabine-treated cultures to the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01) caused a 10-fold increase in H2AX phosphorylation, which was displayed as an even pan-nuclear staining. This increased phosphorylation was not due to apoptosis-induced DNA fragmentation and was associated with the S-phase fraction and decreased reproductive viability. Thus, H2AX becomes phosphorylated and forms nuclear foci in response to gemcitabine-induced stalled replication forks, and this is greatly increased upon checkpoint abrogation.