H2AX In Response Research Articles

Establishment of Cellular Quiescence Together with H2AX Downregulation and Genome Stability Maintenance

H2AX is required for genome stability. In response to DNA double-strand breaks (DSBs), H2AX is rapidly phosphorylated to form γH2AX foci, which mediate DNA repair and checkpoint signaling. This process is regulated by modifications and molecular interactions of H2AX. In addition, the rapid stabilization of H2AX in response to DSBs facilitates γH2AX foci formation. Although H2AX is markedly downregulated in many cellular states, γH2AX foci can still efficiently form upon DSB generation. Here, we review the regulation of H2AX in response to DSBs.

ASCIZ/ATMIN is dispensable for ATM signaling in response to replication stress

The ATM kinase plays critical roles in the response to DNA double-strand breaks, and can also be activated by prolonged DNA replication blocks. It has recently been proposed that replication stress-dependent ATM activation is mediated by ASCIZ (also known as ATMIN, ZNF822), an essential developmental transcription factor. In contrast, we show here that ATM activation, and phosphorylation of its substrates KAP1, p53 and H2AX in response to the replication blocking agent aphidicolin was unaffected in both immortalized and primary ASCIZ/ATMIN-deficient murine embryonic fibroblasts compared to control cells. Similar results were also obtained in human ASCIZ/ATMIN-deleted lymphoma cells. The results demonstrate that ASCIZ/ATMIN is dispensable for ATM activation, and contradict the previously reported dependence of ATM on ASCIZ/ATMIN.

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.

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

USP3 counteracts RNF168 via deubiquitinating H2A and γH2AX at lysine 13 and 15

Histone ubiquitination plays a vital role in DNA damage response (DDR), which is important for maintaining genomic integrity in eukaryotic cells. In DDR, ubiquitination of histone H2A and γH2AX by the concerted action of ubiquitin (Ub) ligases, RNF168 and RNF8, generates a cascade of ubiquitination signaling. However, little is known about deubiquitinating enzymes (DUBs) that may catalyze the removal of Ub from these histones. This study demonstrated that USP3, an apparent DUB for mono-ubiquitinated H2A, is indeed the enzyme for deubiquitinating Ub conjugates of γH2AX and H2A from lysine sites, where the ubiquitination is initiated by RNF168. Here, we showed that ectopic expression of USP3 led to the deubiquitination of both H2A and γH2AX in response to UV-induced DNA damage. Moreover, ectopic USP3 expression abrogated FK2 antibody-reactive Ub-conjugate foci, which co-localize with damage-induced γH2AX foci. In addition, USP3 overexpression impaired the accumulation of downstream repair factors BRCA1 and 53BP1 at the damage sites in response to both UV and γ-irradiation. We further identified that the USP3 removes Ub at lysine 13 and 15 of H2A and γH2AX, as well as lysine 118 and 119 of H2AX in response to DNA damage. Taken together, the results suggested that USP3 is a negative regulator of ubiquitination signaling, counteracting RNF168- and RNF8-mediated ubiquitination.

An autonomous chromatin/DNA-PK mechanism for localized DNA damage signaling in mammalian cells

Rapid phosphorylation of histone variant H2AX proximal to DNA breaks is an initiating event and a hallmark of eukaryotic DNA damage responses. Three mammalian kinases are known to phosphorylate H2AX in response to DNA damage. However, the mechanism(s) for damage-localized phosphorylation remains incompletely understood. The DNA-dependent protein kinase (DNA-PK) is the most abundant H2AX-modifying kinases and uniquely activated by binding DNA termini. Here, we have developed a novel approach to examine enzyme activity and substrate properties by executing biochemical assays on intact cellular structures. We apply this approach to examine the mechanisms of localized protein modification in chromatin within fixed cells. DNA-PK retains substrate specificity and independently generates break-localized γH2AX foci in chromatin. In situ DNA-PK activity recapitulates localization and intensity of in vivo H2AX phosphorylation and requires no active cellular processes. Nuclease treatments or addition of exogenous DNA resulted in genome-wide H2AX phosphorylation, showing that DNA termini dictated the locality of H2AX phosphorylation in situ. DNA-PK also reconstituted focal phosphorylation of structural maintenance of chromatin protein 1, but not activating transcription factor 2. Allosteric regulation of DNA-PK by DNA termini protruding from chromatin constitutes an autonomous mechanism for break-localized protein phosphorylation that generates sub-nuclear foci. We discuss generalized implications of this mechanism in localizing mammalian DNA damage responses.

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

Abstract B78: A novel EWS/Fli1 target gene, Eya3, is upregulated in Ewing's sarcoma to mediate chemoresistance.

Abstract Ewing's sarcoma is an aggressive pediatric cancer of the bone and soft tissue, in which patients' whose tumors have a poor histological response to initial chemotherapy have a poor overall prognosis. Importantly, childhood cancer survivors are faced with long-term chemotherapy-associated toxicities many years after they have been cured of their primary disease. Therefore, targeting molecules involved in resistance to chemotherapy should benefit patients with resistant disease and allow lower doses of toxic chemotherapies to be used in treatment regimens, thus improving outcomes for children suffering from Ewing's sarcoma on multiple fronts. The vast majority of Ewing's sarcomas harbor a characteristic translocation, t(11;22), encoding the EWS/Fli1 fusion protein. We have identified the DNA-repair protein and transcriptional cofactor, Eya3, as a downstream target of EWS/Fli1. Eya3 is highly expressed in Ewing's sarcoma tumor samples and cell lines compared with mesenchymal stem cells, the presumed cell of origin of Ewing's sarcoma. We demonstrate that EWS/Fli1 mediates upregulation of Eya3 via repression of miR-145 and miR-708, microRNAs that target the Eya3 3'UTR. Furthermore, replacement of miR-145 and miR-708 in a Ewing's sarcoma cell line decreases Eya3 levels. It was recently shown that Eya3 promotes DNA repair by dephosphorylating a critical tyrosine residue on H2AX in response to DNA damage, resulting in the recruitment of DNA repair factors to the sites of DNA damage. Thus we tested whether knockdown of Eya3 in Ewing's sarcoma cells would decrease cell survival and sensitize cells to DNA-damaging chemotherapeutics used in the treatment of Ewing's sarcoma: etoposide and doxorubicin. Indeed, Eya3 knockdown led to increased cell death and sensitization to chemotherapy, and, as expected, Eya3 knockdown cells contained more DNA damage following treatment with etoposide than their control counterparts. These data strongly suggest that Eya3 mediates chemoresistance in Ewing's sarcoma through its ability to enhance DNA repair. Because Eya3 mediates DNA repair and survival via its intrinsic phosphatase activity, and because the phosphatase activity of Eya is also known to play a role in metastasis of other cancers, we have begun to develop inhibitors targeting the Eya phosphatase activity and to demonstrate their efficacy both in vitro and in cell culture. In closing, Eya phosphatase inhibitors or synthetic mimics of the microRNAs targeting Eya3 have the potential to improve outcomes for Ewing's sarcoma patients by improving therapeutic efficacy and decreasing associated toxicities of conventional chemotherapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B78.

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.

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.

Sirt1 physically interacts with Tip60 and negatively regulates Tip60-mediated acetylation of H2AX

Sirt1 appear to be NAD(+)-dependent deacetylase that deacetylates histones and several non-histone proteins. In this study, we identified Sirt1 as a physical interaction partner of Tip60, which is a mammalian MYST-type histone acetyl-transferase that specifically acetylates histones H2A and H4. Although Tip60 also acetylates DNA damage-specific histone H2A variant H2AX in response to DNA damage, which is a process required for appropriate DNA damage response, overexpression of Sirt1 represses Tip60-mediated acetylation of H2AX. Furthermore, Sirt1 depletion by RNAi causes excessive acetylation of H2AX, and enhances accumulation of γ-ray irradiation-induced MDC1, BRCA1, and Rad51 foci in nuclei. These findings suggest that Sirt1 functions as negative regulator of Tip60-mediated acetylation of H2AX. Moreover, Sirt1 deacetylates an acetylated Tip60 in response to DNA damage and stimulates proteasome-dependent Tip60 degradation in vivo, suggesting that Sirt1 negatively regulates the protein level of Tip60 in vivo. Sirt1 may thus repress excessive activation of the DNA damage response and Rad51-homologous recombination repair by suppressing the function of Tip60.

Calmodulin Mediates DNA Repair Pathways Involving H2AX in Response to Low-Dose Radiation Exposure of RAW 264.7 Macrophages

Understanding the molecular mechanisms that modulate macrophage radioresistance is necessary for the development of effective radiation therapies, as tumor-associated macrophages promote both angiogenesis and matrix remodeling that, in turn, enhance tumor metastasis. In this respect, we have identified a dose-dependent increase in the abundance (i.e., expression level) of the calcium regulatory protein calmodulin (CaM) in RAW 264.7 macrophages upon irradiation. At low doses of irradiation there are minimal changes in the abundance of other cellular proteins detected using mass spectrometry, indicating that increases in CaM levels are part of a specific radiation-dependent cellular response. CaM overexpression results in increased macrophage survival following radiation exposure, acting to diminish the sensitivity to low-dose radiation exposures. Following macrophage irradiation, increases in CaM abundance also result in an increase in the number of phosphorylated histone H2AX foci, associated with DNA repair, with no change in the extent of double-stranded DNA damage. In comparison, when nuclear factor kappaB (NFkappaB)-dependent pathways are inhibited, through the expression of a dominant-negative IkappaB construct, there is no significant increase in phosphorylated histone H2AX foci upon irradiation. These results indicate that the molecular basis for the up-regulation of histone H2AX-mediated DNA repair pathways is not the result of nonspecific NFkappaB-dependent pathways or a specific threshold of DNA damage. Rather, increases in CaM abundance act to minimize the low-dose hypersensitivity to radiation by enhancing macrophage radioresistance through processes that include the up-regulation of DNA repair pathways involving histone H2AX phosphorylation.

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.

ATM activation accompanies histone H2AX phosphorylation in A549 cells upon exposure to tobacco smoke.

BackgroundIn response to DNA damage or structural alterations of chromatin, histone H2AX may be phosphorylated on Ser139 by phosphoinositide 3-kinase related protein kinases (PIKKs) such as ataxia telangiectasia mutated (ATM), ATM-and Rad-3 related (ATR) kinase, or by DNA dependent protein kinase (DNA-PKcs). When DNA damage primarily involves formation of DNA double-strand breaks (DSBs), H2AX is preferentially phosphorylated by ATM rather than by the other PIKKs. We have recently reported that brief exposure of human pulmonary adenocarcinoma A549 cells or normal human bronchial epithelial cells (NHBE) to cigarette smoke (CS) induced phosphorylation of H2AX.ResultsWe report here that H2AX phosphorylation in A549 cells induced by CS was accompanied by activation of ATM, as revealed by ATM phosphorylation on Ser1981 (ATM-S1981P) detected immunocytochemically and by Western blotting. No cell cycle-phase specific differences in kinetics of ATM activation and H2AX phosphorylation were observed. When cells were exposed to CS from cigarettes with different tobacco and filter combinations, the expression levels of ATM-S1981P correlated well with the increase in expression of phosphorylated H2AX (γH2AX) (R = 0.89). In addition, we note that while CS-induced γH2AX expression was localized within discrete foci, the activated ATM was distributed throughout the nucleoplasm.ConclusionThese data implicate ATM as the PIKK that phosphorylates H2AX in response to DNA damage caused by CS. Based on current understanding of ATM activation, expression and localization, these data would suggest that, in addition to inducing potentially carcinogenic DSB lesions, CS may also trigger other types of DNA lesions and cause chromatin alterations. As checkpoint kinase (Chk) 1, Chk2 and the p53 tumor suppressor gene are known to be phosphorylated by ATM, the present data indicate that exposure to CS may lead to their phosphorylation, with the downstream consequences related to the halt in cell cycle progression and increased propensity to undergo apoptosis. Defining the nature and temporal sequence of molecular events that are disrupted by CS through activation and eventual dysregulation of normal defense mechanisms such as ATM and its downstream effectors may allow a more precise understanding of how CS promotes cancer development.

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.

A role for WRN in telomere-based DNA damage responses.

Telomeres cap the ends of eukaryotic chromosomes and prevent them from being recognized as DNA breaks. We have shown that certain DNA damage responses induced during senescence and, at times of telomere uncapping, also can be induced by treatment of cells with small DNA oligonucleotides homologous to the telomere 3' single-strand overhang (T-oligos), implicating this overhang in generation of these telomere-based damage responses. Here, we show that T-oligo-treated fibroblasts contain gammaH2AX foci and that these foci colocalize with telomeres. T-oligos with nuclease-resistant 3' ends are inactive, suggesting that a nuclease initiates T-oligo responses. We therefore examined WRN, a 3'-->5' exonuclease and helicase mutated in Werner syndrome, a disorder characterized by aberrant telomere maintenance, premature aging, chromosomal rearrangements, and predisposition to malignancy. Normal fibroblasts and U20S osteosarcoma cells rendered deficient in WRN showed reduced phosphorylation of p53 and histone H2AX in response to T-oligo treatment. Together, these data demonstrate a role for WRN in processing of telomeric DNA and subsequent activation of DNA damage responses. The T-oligo model helps define the role of WRN in telomere maintenance and initiation of DNA damage responses after telomere disruption.

H2AX Is a Target of the JNK Signaling Pathway that Is Required For Apoptotic DNA Fragmentation

The mechanism of apoptotic signaling by JNK is incompletely understood. In the July 7 issue of Molecular Cell, Lu et al. (2006) report that JNK phosphorylation of H2AX at a noncanonical site is required for caspase-induced DNA fragmentation.

Expression of disease-causing lamin A mutants impairs the formation of DNA repair foci

A-type lamins are components of the nuclear lamina. Mutations in the gene encoding lamin A are associated with a range of highly degenerative diseases termed laminopathies. To evaluate sensitivity to DNA damage, GFP-tagged lamin A cDNAs with disease-causing mutations were expressed in HeLa cells. The inner nuclear membrane protein emerin was mislocalised upon expression of the muscular dystrophy mutants G232E, Q294P or R386K, which aberrantly assembled into nuclear aggregates, or upon expression of mutants causing progeria syndromes in vivo (lamin A del50, R471C, R527C and L530P). The ability of cells expressing these mutants to form DNA repair foci comprising phosphorylated H2AX in response to mild doses of cisplatin or UV irradiation was markedly diminished, unlike the nearly normal response of cells expressing wild-type GFP-lamin A or disease-causing H222P and R482L mutants. Interestingly, mutants that impaired the formation of DNA repair foci mislocalised ATR (for ;ataxia telangiectasia-mutated and Rad3-related') kinase, which is a key sensor in the response to DNA damage. Our results suggest that a subset of lamin A mutants might hinder the response of components of the DNA repair machinery to DNA damage by altering interactions with chromatin.