Double-strand DNA Damage Research Articles

The effect of gamma radiation on the Common carp (Cyprinus carpio): In vivo genotoxicity assessment with the micronucleus and comet assays

Radioactive wastes may be leached into freshwater, either accidentally or in industrial effluents. We have studied gamma radiation-induced DNA damage in the freshwater fish Cyprinus carpio. Fish were irradiated with 2–10Gy gamma radiation and genotoxic effects in blood cells were studied with the micronucleus (MN) and comet assays. Micronuclei and a dose-dependent increase in comet-tail DNA were seen in dose- and time-dependent studies. The highest % tail DNA was observed at 24h, declining until 72h, which may indicate the repair of radiation-induced DNA single-strand breaks after gamma radiation. However, double-stranded DNA damage may not have been repaired, as indicated by increased micronuclei at later periods. A positive correlation was observed between the comet and micronucleus assay results. This study confirms the mutagenic/genotoxic potential of gamma radiation in the Common carp, as well as the possible combined use of the micronucleus and comet assays for in vivo laboratory studies with fresh-water fish for screening the genotoxic potential of radioactive pollution.

Functions of the MRE11 complex in the development and maintenance of oocytes.

The MRE11 complex (MRE11, RAD50, and NBS1) is a central component of the DNA damage response, governing both double-strand break repair and DNA damage response signaling. To determine the functions of the MRE11 complex in the development and maintenance of oocytes, we analyzed ovarian phenotypes of mice harboring the hypomorphic Mre11ATLD1 allele. Mre11ATLD1/ATLD1 females exhibited premature oocyte elimination attributable to defects in homologous chromosome pairing and double-strand break repair during meiotic prophase. Other aspects of meiotic progression, including attachment of telomeres to the nuclear envelope and recruitment of RAD21L, a component of the meiotic cohesin complex to the synaptonemal complex, were normal. Unlike Dmc1−/− and Trp13Gt/Gt mice which exhibit comparable defects in double-strand break repair and oocyte depletion by 5 days post-partum, we found that oocyte attrition occurred by 12 weeks in Mre11ATLD1/ATLD1. Disruption of the oocyte checkpoint pathway governed by Chk2 gene further enhanced the survival of Mre11ATLD1/ATLD1 follicles. Together our data suggest that the MRE11 complex influences the elimination of oocytes with unrepaired meiotic double-strand breaks post-natally, in addition to its previously described role in double-strand break repair and homologous synapsis during female meiosis.Electronic supplementary materialThe online version of this article (doi:10.1007/s00412-015-0535-8) contains supplementary material, which is available to authorized users.

Mouse Tafazzin Is Required for Male Germ Cell Meiosis and Spermatogenesis.

Barth syndrome is an X-linked mitochondrial disease, symptoms of which include neutropenia and cardiac myopathy. These symptoms are the most significant clinical consequences of a disease, which is increasingly recognised to have a variable presentation. Mutation in the Taz gene in Xq28 is thought to be responsible for the condition, by altering mitochondrial lipid content and mitochondrial function. Male chimeras carrying a targeted mutation of Taz on their X-chromosome were infertile. Testes from the Taz knockout chimeras were smaller than their control counterparts and this was associated with a disruption of the progression of spermatocytes through meiosis to spermiogenesis. Taz knockout ES cells also showed a defect when differentiated to germ cells in vitro. Mutant spermatocytes failed to progress past the pachytene stage of meiosis and had higher levels of DNA double strand damage and increased levels of endogenous retrotransposon activity. Altogether these data revealed a novel role for Taz in helping to maintain genome integrity in meiosis and facilitating germ cell differentiation. We have unravelled a novel function for the Taz protein, which should contribute to an understanding of how a disruption of the Taz gene results in the complex symptoms underlying Barth Syndrome.

Atrazine Triggers DNA Damage Response and Induces DNA Double-Strand Breaks in MCF-10A Cells.

Atrazine, a pre-emergent herbicide in the chloro-s-triazine family, has been widely used in crop lands and often detected in agriculture watersheds, which is considered as a potential threat to human health. Although atrazine and its metabolites showed an elevated incidence of mammary tumors in female Sprague–Dawley (SD) rats, no molecular evidence was found relevant to its carcinogenesis in humans. This study aims to determine whether atrazine could induce the expression of DNA damage response-related proteins in normal human breast epithelial cells (MCF-10A) and to examine the cytotoxicity of atrazine at a molecular level. Our results indicate that a short-term exposure of MCF-10A to an environmentally-detectable concentration of atrazine (0.1 µg/mL) significantly increased the expression of tumor necrosis factor receptor-1 (TNFR1) and phosphorylated Rad17 in the cells. Atrazine treatment increased H2AX phosphorylation (γH2AX) and the formation of γH2AX foci in the nuclei of MCF-10A cells. Atrazine also sequentially elevated DNA damage checkpoint proteins of ATM- and RAD3-related (ATR), ATRIP and phospho-Chk1, suggesting that atrazine could induce DNA double-strand breaks and trigger the DNA damage response ATR-Chk1 pathway in MCF-10A cells. Further investigations are needed to determine whether atrazine-triggered DNA double-strand breaks and DNA damage response ATR-Chk1 pathway occur in vivo.

Prevalence of PALB2 mutations in the Creighton University breast cancer family registry.

e12519 Background: PALB2 (i.e., the partner and localizer of BRCA2) plays an important role in double-strand DNA damage repair through interaction with BRCA1, BRCA2, and RAD51. Xia et al. (Nat Gene...

Augmentation of response to chemotherapy by microRNA-506 through regulation of RAD51 in serous ovarian cancers.

Chemoresistance is a major challenge in cancer treatment. miR-506 is a potent inhibitor of the epithelial-to-mesenchymal transition (EMT), which is also associated with chemoresistance. We characterized the role of miR-506 in chemotherapy response in high-grade serous ovarian cancers. We used Kaplan-Meier and log-rank methods to analyze the relationship between miR-506 and progression-free and overall survival in The Cancer Genome Atlas (TCGA) (n = 468) and Bagnoli (n = 130) datasets, in vitro experiments to study whether miR-506 is associated with homologous recombination, and response to chemotherapy agents. We used an orthotopic ovarian cancer mouse model (n = 10 per group) to test the effect of miR-506 on cisplatin and PARP inhibitor sensitivity. All statistical tests were two-sided. MiR-506 was associated with better response to therapy and longer progression-free and overall survival in two independent epithelial ovarian cancer patient cohorts (PFS: high vs low miR-506 expression; Bagnoli: hazard ratio [HR] = 3.06, 95% confidence interval [CI] = 1.90 to 4.70, P < .0001; TCGA: HR = 1.49, 95% CI = 1.00 to 2.25, P = 0.04). MiR-506 sensitized cells to DNA damage through directly targeting the double-strand DNA damage repair gene RAD51. Systemic delivery of miR-506 in 8-12 week old female athymic nude mice statistically significantly augmented the cisplatin and olaparib response (mean tumor weight ± SD, control miRNA plus cisplatin vs miR-506 plus cisplatin: 0.36±0.05g vs 0.07±0.02g, P < .001; control miRNA plus olaparib vs miR-506 plus olaparib: 0.32±0.13g vs 0.05±0.02g, P = .045, respectively), thus recapitulating the clinical observation. MiR-506 is a robust clinical marker for chemotherapy response and survival in serous ovarian cancers and has important therapeutic value in sensitizing cancer cells to chemotherapy.

Tu1227 Effect of Oral Administration of Methylene Blue MMx® Tablets on Double-Stranded DNA Damage Assessed by γH2AX Analysis of Colon Biopsy Samples - A Single Center, Open Label, Safety, Phase II Study

Tu1227 Effect of Oral Administration of Methylene Blue MMx® Tablets on Double-Stranded DNA Damage Assessed by γH2AX Analysis of Colon Biopsy Samples - A Single Center, Open Label, Safety, Phase II Study

The nuclear oncogene SET controls DNA repair by KAP1 and HP1 retention to chromatin.

Cells experience damage from exogenous and endogenous sources that endanger genome stability. Several cellular pathways have evolved to detect DNA damage and mediate its repair. Although many proteins have been implicated in these processes, only recent studies have revealed how they operate in the context of high-ordered chromatin structure. Here, we identify the nuclear oncogene SET (I2PP2A) as a modulator of DNA damage response (DDR) and repair in chromatin surrounding double-strand breaks (DSBs). We demonstrate that depletion of SET increases DDR and survival in the presence of radiomimetic drugs, while overexpression of SET impairs DDR and homologous recombination (HR)-mediated DNA repair. SET interacts with the Kruppel-associated box (KRAB)-associated co-repressor KAP1, and its overexpression results inthe sustained retention of KAP1 and Heterochromatin protein 1 (HP1) on chromatin. Our results are consistent with a model in which SET-mediated chromatin compaction triggers an inhibition of DNA end resection and HR.

Radiation-induced formation of purine lesions in single and double stranded DNA: revised quantification.

The formation of oxidative lesions arising from double stranded DNA damage is of major significance to chemical biology from the perspective of application to human health. The quantification of purine lesions arising from γ-radiation-induced hydroxyl radicals (HO•) has been the subject of numerous studies, with discrepancies on the measured 5′,8-cyclo-2′-deoxyadenosine (cdA) and 5′,8-cyclo-2′-deoxyguanosine (cdG) lesions reported by different groups. Here we applied an ameliorative protocol for the analysis of DNA damage with quantitative determination of these lesions via isotope dilution liquid chromatography coupled with tandem mass spectrometry. Tandem-type purine lesions were quantified along with 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) and 7,8-dihydro-8-oxo-2′-deoxyadenosine (8-oxo-dA) in single and double stranded DNA, generated during DNA exposure to diffusible HO• radicals in the absence or presence of physiological levels of oxygen. The cdA and cdG lesions in absence of oxygen were found ~2 times higher in single than double stranded DNA, with 5′R being ~6.5 and ~1.5 times more predominant than 5′S in cdG and cdA, respectively. Interestingly, in the presence of 5% molecular oxygen the R/S ratios are retained with substantially decreased yields for cdA and cdG, whereas 8-oxo-dA and 8-oxo-dG remain nearly constant. The overall lesion formation follows the order: 8-oxo-dG >> 8-oxo-dA > 5′R-cdG > 5′R-cdA > 5′S-cdA > 5′S-cdG. By this method, there was a conclusive evaluation of radiation-induced DNA purine lesions.

Molecular determinants of polyubiquitin recognition by continuous ubiquitin-binding domains of Rad18.

Rad18 is a key factor in double-strand break DNA damage response (DDR) pathways via its association with K63-linked polyubiquitylated chromatin proteins through its bipartite ubiquitin-binding domains UBZ and LRM with extra residues between them. Rad18 binds K63-linked polyubiquitin chains as well as K48-linked ones and monoubiquitin. However, the detailed molecular basis of polyubiquitin recognition by UBZ and LRM remains unclear. Here, we examined the interaction of Rad18(201-240), including UBZ and LRM, with linear polyubiquitin chains that are structurally similar to the K63-linked ones. Rad18(201-240) binds linear polyubiquitin chains (Ub2-Ub4) with affinity similar to that of a K63-linked one for diubiquitin. Ab initio modeling suggests that LRM and the extra residues at the C-terminus of UBZ (residues 227-237) likely form a continuous helix, termed the "extended LR motif" (ELRM). We obtained a molecular envelope for Rad18 UBZ-ELRM:linear Ub2 by small-angle X-ray scattering and derived a structural model for the complex. The Rad18:linear Ub2 model indicates that ELRM enhances the binding of Rad18 with linear polyubiquitin by contacting the proximal ubiquitin moiety. Consistent with the structural analysis, mutational studies showed that residues in ELRM affect binding with linear Ub2, not monoubiquitin. In cell data support the idea that ELRM is crucial in the localization of Rad18 to DNA damage sites. Specifically, E227 seems to be the most critical in polyubiquitin binding and localization to nuclear foci. Finally, we reveal that the ubiquitin-binding domains of Rad18 bind linear Ub2 more tightly than those of RAP80, providing a quantitative basis for blockage of RAP80 at DSB sites. Taken together, our data demonstrate that Rad18(201-240) forms continuous ubiquitin-binding domains, comprising UBZ and ELRM, and provides a structural framework for polyubiquitin recognition by Rad18 in the DDR pathway at a molecular level.

DNA damage and ER stress contribute to oblongifolin C-induced cell killing in Bax/Bak-deficient cells

A key clinical problem in oncology is the treatment of apoptosis-resistant tumors. Tumor cells deficient in both of the proapoptotic proteins Bax and Bak are protected against most chemotherapeutic drug-induced apoptosis. We report here that a natural compound, oblongifolin C (OC), effectively eliminates Bax/Bak-deficient murine embryonic fibroblasts and colon carcinoma HCT116 cells. OC not only triggers DNA double-strand breaks and DNA damage response, but also inhibits repair of DNA damage. In addition, OC induces ER stress through upregulation of the transcription factor CHOP and activation of JNK kinases. Upon treatment with OC, cells undergo Bax/Bak-independent, caspase-mediated apoptosis. Taken together, our data establish a rationale for the broad use of OC to treat apoptosis deficient tumors.

Inhibition of the BER Factor APE1 Disrupts Repair of Double-Strand DNA Damage in Cells Treated with Low Dose-Rate, but Not High Dose-Rate XRadiation

Introduction: Radiotherapy is utilised in the treatment of many cancers, but its efficacy is limited by normal tissue toxicity and new radiotherapy techniques are thus urgently sought. The AP endonuclease APE1 is involved in repair of single strand DNA damage through the break excision repair (BER) pathway and altered levels of APE1 have been found in some cancers. In this study, we investigated the effects of APE1 inhibition, using the APE1-specific inhibitor CRT0044876 (CRT), in tumour cells following exposure to either high dose-rate (HDR) or low dose-rate (LDR) X-irradiation. Materials and Methods: Treatment efficacy was assessed by clonogenic assay followed by isobologram analysis to assess potential synergy. Cell cycle distribution was assessed by propidium iodide staining followed by flow cytometry. Induction of DNA damage and repair was assessed by single cell gel electrophoresis and by H2A.X phosphorylation. Results: In isobologram analysis of clonogenic assays, combinations of CRT and both HDR and LDR X-irradiation resulted in supra-additive levels of cytotoxicity. Cell cycle analysis showed that, while CRT had no effect on cell cycle distribution, HDR or LDR X-irradiation, and CRT-HDR or CRT-LDR combination treatment induced significant G2/M arrest. However, CRT-HDR combinations induced significantly less G2/M accumulation than HDR alone. Analysis of DNA damage indicated that treatment with HDR or LDR X-irradiation and CRT-HDR and CRT-LDR combinations induced significant double-strand DNA damage. Cells treated with CRT-HDR exhibited a significant reduction in γH2A.X foci 24 h after treatment compared to 1 h, suggesting induction of DNA repair mechanisms. However, in cells treated with CRT-LDR, there was no significant difference between H2A.X phosphorylation at 24 h compared to 1 h, suggesting disruption of dsDNA repair pathways. Conclusions: Pharmacological inhibition of APE1 enhances the cytotoxicity of high dose-rate and low dose-rate X-irradiation by different mechanisms.

Abnormal Expression of DNA Double-Strand Breaks Related Genes, ATM and GammaH2AX, in Thyroid Carcinoma.

ATM and γH2AX play a vital role in the detection of DNA double-strand breaks (DSB) and DNA damage response (DDR). This study aims to investigate ATM and γH2AX expression in thyroid cancer and discuss possible relationship between thyroid function tests and DNA damage. The expression of ATM and γH2AX was detected by immunohistochemistry in 30 cases of benign nodular goiter, 110 cases of well differentiated thyroid cancer, 22 cases of poorly differentiated thyroid cancer, and 21 cases of anaplastic thyroid cancer. Clinicopathological features, including differentiation stages, distant metastasis, lymph node metastasis, T classification, TNM stage, and tests of thyroid functions (TPOAb, Tg Ab, T3, FT3, T4, FT4, TSH, and Tg), were reviewed and their associations with γH2AX and ATM were analyzed. γH2AX and ATM expressed higher in thyroid cancer tissues than in benign nodular goiter and normal adjacent tissues. γH2AX was correlated with ATM in thyroid cancer. Both γH2AX and ATM expression were associated with FT3. γH2AX was also associated with T classification, TNM stage, FT4, TSH, and differentiation status. Therefore both of ATM and γH2AX seem to correlate with thyroid hormones and γH2AX plays a role in the differentiation status of thyroid cancer.

Human PIF1 helicase supports DNA replication and cell growth under oncogenic-stress.

Unwinding duplex DNA is a critical processing step during replication, repair and transcription. Pif1 are highly conserved non-processive 5'->3' DNA helicases with well-established roles in maintenance of yeast genome stability. However, the function of the sole member of Pif1 family in humans remains unclear. Human PIF1 is essential for tumour cell viability, particularly during replication stress, but is dispensable in non-cancerous cells and Pif1 deficient mice. Here we report that suppression of PIF1 function slows replication fork rates and increases arrested forks during normal cycling conditions. Importantly, PIF1-dependent replication impediments impair S-phase progression and reduce proliferation rates of RAS oncogene-transformed fibroblasts, where replication fork slowing is exacerbated, but not parental, non-cancerous cells. Disrupted fork movement upon PIF1-depletion does not enhance double-stranded break formation or DNA damage responses but affects resumption of DNA synthesis after prolonged replication inhibitor exposure, accompanied by diminished new origin firing and mainly S-phase entry. Taken together, we characterised a functional role for human PIF1 in DNA replication that becomes important for cell growth under oncogenic stress. Given that oncogenes induce high levels of replication stress during the early stages of tumorigenesis, this function of PIF1 could become critical during cancer development.

Partner and Localizer of BRCA-2 (PALB-2) Mutation Analysis Is Rapidly Being Adopted into Clinical Practice.

To the Editor, Breast-Cancer Risk in Families with Mutations in PALB2: A.C. Antoniou, S. Casadei, T. Heikkinen, et al. N Engl J Med. 2014;371:497-506. In a study published in N Engl J Med. August 2014, researchers from 14 centers in eight countries investigated information from 362 family members from 154 families who had an abnormal partner and localizer of BRCA2 (PALB2) gene, but did not have an abnormal BRCA1 or BRCA2 gene. Their aim was to demonstrate the importance of loss-of-function mutations in PALB2 as a cause of hereditary breast cancer. The study included 311 women with an abnormal PALB2 gene; 229 of these women had been diagnosed with breast cancer and 51 men with an abnormal PALB2 gene; seven of whom had been diagnosed with breast cancer. The researchers compared the breast cancer risk of the study population to the general population. They found out that overall, women with an abnormal PALB2 gene had a risk of breast cancer that was 9.47 times higher than average. Women with an abnormal PALB2 gene had a 14% risk of developing breast cancer by age 50 and a 35% risk of developing breast cancer by age 70. The average lifetime risk of breast cancer is about 12%. This increase in risk linked to an abnormal PALB2 gene was dependent on the women’s age and family history. They have demonstrated that the relative risk of breast cancer among PALB2 mutation carriers increased significantly as birth cohort became more recent (P<0.001). In women with an abnormal PALB2 gene, breast cancer risk was eight to nine times higher than average in women aged 20 to 39, six to eight times higher than average in women aged 40 to 60, and five times higher than average in women older than 60 years of age. By the age of 70, women with an abnormal PALB2 gene with no family history of breast cancer had a 33% risk of developing breast cancer whereas women with two or more first-degree relatives with breast cancer at 50 years of age had a 58% risk of developing the disease. The authors concluded that based on their estimates, breast cancer risk for a PALB2 mutation carrier, even in the absence of a family history of breast cancer, would be classified as high according to various guidelines. The researchers also reported that this level of risk may justify adding PALB2 to genetic testing for BRCA1 and BRCA2. A Genetic counselor’s perspective Partner and localizer of BRCA2 (PALB) is a gene whose protein facilitates the function of BRCA2 and interacts with BRCA1 to aid in repairing double strand and interstrand cross-link DNA damage. Biallelic PALB2 mutations cause Fanconi Anemia complement group N (FANCN) and monoallellic mutations in the PALB2 gene are associated with increased breast and pancreas cancer risks (1). PALB2 mutations may account for as much as 3% of familial breast cancer risk (2). A recent study of 362 members from PALB2 mutation positive families found that the risk for breast cancer in female mutation carriers ranged between 33% to 58% by age 70, with the higher risks relating to those women with a family history of early onset (<50) breast cancer in 2 first degree family members (3). The study also identified an 8 fold increase in male breast cancer risk and a 2 fold increase in ovarian cancer risk, both of which need to be confirmed in additional studies due a lack of statistical significance. These data suggest that female PALB2 mutation carriers may have breast cancer risks that are similar to those conferred by mutations in the BRCA2 gene. These findings suggest that women who carry PALB2 gene mutations may warrant similar breast cancer screening and prevention strategies as those offered to BRCA2 mutation carriers. PALB2 mutation analysis is available and is rapidly being adopted into clinical practice. As the data continues to accumulate it is likely that PALB2 mutation analysis will become a standard part of genetic testing in the realm of hereditary breast cancer predisposition. Whether testing will be done in addition to BRCA1/2 or as part of a larger breast cancer gene panel remains to be determined. In addition, more data will provide evidence to support the development of specific management guidelines to aid in the care of these at risk patients.

Abstract 437: Novel function of the BAP1 nuclear deubiquitinase in the non-homologous end joining (NHEJ) pathway of double strand DNA repair

Abstract Background- The deubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with increased risk of malignant pleural mesothelioma (MPM) and uveal melanoma and in subsets of sporadic cases of these cancers, as well as in clear cell renal carcinoma and intrahepatic cholangiocarcinoma. Methods and Results- We found that BAP1 knockdown is associated with increased DNA damage and that this effect of BAP1 knockdown is further enhanced by both X-irradiation and concomitant DNA-PKcs inhibitor II treatment in normal human mesenchymal stem cells and the MPM cell line H-Meso. A BAP1 mutant deficient in deubiquitinase activity is also associated with increased DNA damage in the H-Meso cells. Interactional total peptide (ITOP) analysis based on proteins identified by Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS/MS) of BAP1 protein complexes in H-Meso MPM cells and 293T cells identified known binding partners [HCF1, ASXL2, H2A] as well as possible novel interaction partners, notably PRKDC which encodes the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) and functions with the Ku70/Ku80 heterodimer in the non-homologous end joining (NHEJ) pathway of DNA repair. Double stranded DNA damage resulted in prominent nuclear expression of BAP1 protein in H-Meso cells and phosphorylation of BAP1 at Serine 395. Overexpression of dominant negative BAP1 C91W mutant in MPM cells caused instability of DNA-PKcs. Although strong polyubiquitination of DNA-PKcs was observed upon DNA damage in MPM cells expressing BAP1 C91W, this polyubiquitination was not observed in the presence of wild type BAP1. A plasmid-based NHEJ assay also confirmed a significant effect of BAP1 on NHEJ activity. Conclusion- Functional complexes between BAP1 and the NHEJ family components point to a novel role in DNA repair and may shed light on BAP1's role as an important tumor suppressor Citation Format: Tatsuo Ito, Shigehisa Kitano, Hediye Erdjument-Bromage, Marc Ladanyi. Novel function of the BAP1 nuclear deubiquitinase in the non-homologous end joining (NHEJ) pathway of double strand DNA repair. [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 437. doi:10.1158/1538-7445.AM2014-437

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

Rho GTPase independent regulation of ATM activation and cell survival by the RhoGEF Net1A

ATM activation following DNA damage is a critical event which is required for efficient DNA repair and cell survival, yet signaling mechanisms controlling its activation are incompletely understood. The RhoGEF Net1 has previously been reported to control Rho GTPase activation and downstream cell survival outcomes following double strand DNA damage. However the role of Net1 isoforms in controlling ATM-dependent cell signaling has not been assessed. In the present work we show that expression of the Net1A isoform is specifically required for efficient activation of ATM but not the related kinase DNA-PK after ionizing radiation. Surprisingly Net1A overexpression also potently suppresses ATM activation and phosphorylation of its substrate H2AX. This effect does not require catalytic activity towards RhoA or RhoB, and neither Rho GTPase affects ATM activation, on its own. Consistent with a role in controlling ATM activation, Net1A knockdown also impairs DNA repair and cell survival. Taken together these data indicate that Net1A plays a plays a previously unrecognized, Rho GTPase-independent role in controlling ATM activity and downstream signaling after DNA damage to impact cell survival.

Evaluation of in vitro cytotoxicity and genotoxicity of copper–zinc alloy nanoparticles in human lung epithelial cells

In the present study, in vitro cytotoxic and genotoxic effect of copper–zinc alloy nanoparticles (Cu–Zn ANPs) on human lung epithelial cells (BEAS-2B) were investigated. XTT test and clonogenic assay were used to determine cytotoxic effects. Cell death mode and intracellular reactive oxygen species formations were analyzed using M30, M65 and ROS Elisa assays. Genotoxic effects were evaluated using micronucleus, comet and γ-H2AX foci assays. Cu–Zn ANPs were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential measurements. Characterization of Cu–Zn ANPs showed an average size of 200nm and zeta potential of −22mV. TEM analyses further revealed the intracellular localization of Cu–Zn ANPs in cytoplasm within 24h. Analysis of micronucleus, comet and γ-H2AX foci counts showed that exposure to Cu–Zn ANPs significantly induced chromosomal damage as well as single and double stranded DNA damage in BEAS-2B cells. Our results further indicated that exposure to Cu–Zn ANPs significantly induced intracellular ROS formation. Evaluation of M30:M65 ratios suggested that cell death was predominantly due to necrosis.

Profiling dose-dependent activation of p53-mediated signaling pathways by chemicals with distinct mechanisms of DNA damage.

As part of a larger effort to provide proof-of-concept in vitro-only risk assessments, we have developed a suite of high-throughput assays for key readouts in the p53 DNA damage response toxicity pathway: double-strand break DNA damage (p-H2AX), permanent chromosomal damage (micronuclei), p53 activation, p53 transcriptional activity, and cell fate (cell cycle arrest, apoptosis, micronuclei). Dose-response studies were performed with these protein and cell fate assays, together with whole genome transcriptomics, for three prototype chemicals: etoposide, quercetin, and methyl methanesulfonate. Data were collected in a human cell line expressing wild-type p53 (HT1080) and results were confirmed in a second p53 competent cell line (HCT 116). At chemical concentrations causing similar increases in p53 protein expression, p53-mediated protein expression and cellular processes showed substantial chemical-specific differences. These chemical-specific differences in the p53 transcriptional response appear to be determined by augmentation of the p53 response by co-regulators. More importantly, dose-response data for each of the chemicals indicate that the p53 transcriptional response does not prevent micronuclei induction at low concentrations. In fact, the no observed effect levels and benchmark doses for micronuclei induction were less than or equal to those for p53-mediated gene transcription regardless of the test chemical, indicating that p53's post-translational responses may be more important than transcriptional activation in the response to low dose DNA damage. This effort demonstrates the process of defining key assays required for a pathway-based, in vitro-only risk assessment, using the p53-mediated DNA damage response pathway as a prototype.