IR-induced Foci Research Articles

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

C-Fos-dependent miR-22 targets MDC1 and regulates DNA repair in terminally differentiated cells.

Terminally differentiated cells have a reduced capacity to repair double-stranded breaks (DSB) in DNA, however, the underlying molecular mechanism remains unclear. Here, we show that miR-22 is upregulated during postmitotic differentiation of human breast MCF-7 cells, hematopoietic HL60 and K562 cells. Increased expression of miR-22 in differentiated cells was associated with decreased expression of MDC1, a protein that plays a key role in the response to DSBs. This downregulation of MDC1 was accompanied by reduced DSB repair, impaired recruitment of the protein to the site of DNA damage following IR. Conversely, inhibiting miR-22 enhanced MDC1 protein levels, recovered MDC1 foci, fully rescued DSB repair in terminally differentiated cells. Moreover, MDC1 levels, IR-induced MDC1 foci, and the efficiency of DSB repair were fully rescued by siRNA-mediated knockdown of c-Fos in differentiated cells. These findings indicate that the c-Fos/miR-22/MDC1 axis plays a relevant role in DNA repair in terminally differentiated cells, which may facilitate our understanding of molecular mechanism underlying the downregulating DNA repair in differentiated cells.

Transmission of persistent ionizing radiation-induced foci through cell division in human primary cells

Unrepaired DNA double-strand breaks (DSBs) induced by ionizing radiation are associated with lethal effects and genomic instability. After the initial breaks and chromatin destabilization, a set of post-translational modifications of histones occurs, including phosphorylation of serine 139 of histone H2AX (γH2AX), which leads to the formation of ionizing radiation-induced foci (IRIF). DSB repair results in the disappearance of most IRIF within hours after exposure, although some remain 24h after irradiation. Their relation to unrepaired DSBs is generally accepted but still controversial. This study evaluates the frequency and kinetics of persistent IRIF and analyzes their impact on cell proliferation. We observed persistent IRIF up to 7 days postirradiation, and more than 70% of cells exposed to 5Gy had at least one of these persistent IRIF 24h after exposure. Moreover we demonstrated that persistent IRIF did not block cell proliferation definitively. The frequency of IRIF was lower in daughter cells, due to asymmetric distribution of IRIF between some of them. We report a positive association between the presence of IRIF and the likelihood of DNA missegregation. Hence, the structure formed after the passage of a persistent IRI focus across the S and G2 phases may impede the correct segregation of the affected chromosome's sister chromatids. The ensuing abnormal resolution of anaphase might therefore cause the nature of IRIF in daughter-cell nuclei to differ before and after the first cell division. The resulting atypical chromosomal assembly may be lethal or result in a gene dosage imbalance and possibly enhanced genomic instability, in particular in the daughter cells.

MCL-1 Depletion Impairs DNA Double-Strand Break Repair and Reinitiation of Stalled DNA Replication Forks.

Myeloid cell leukemia 1 (MCL-1) is a prosurvival BCL-2 protein family member highly expressed in hematopoietic stem cells (HSCs) and regulated by growth factor signals that manifest antiapoptotic activity. Here we report that depletion of MCL-1 but not its isoform MCL-1S increases genomic instability and cell sensitivity to ionizing radiation (IR)-induced death. MCL-1 association with genomic DNA increased postirradiation, and the protein colocalized with 53BP1 foci. Postirradiation, MCL-1-depleted cells exhibited decreased γ-H2AX foci, decreased phosphorylation of ATR, and higher levels of residual 53BP1 and RIF1 foci, suggesting that DNA double-strand break (DSB) repair by homologous recombination (HR) was compromised. Consistent with this model, MCL-1-depleted cells had a reduced frequency of IR-induced BRCA1, RPA, and Rad51 focus formation, decreased DNA end resection, and decreased HR repair in the DR-GFP DSB repair model. Similarly, after HU induction of stalled replication forks in MCL-1-depleted cells, there was a decreased ability to subsequently restart DNA synthesis, which is normally dependent upon HR-mediated resolution of collapsed forks. Therefore, the present data support a model whereby MCL-1 depletion increases 53BP1 and RIF1 colocalization at DSBs, which inhibits BRCA1 recruitment, and sensitizes cells to DSBs from IR or stalled replication forks that require HR for repair.

Chromatin organization revealed by nanostructure of irradiation induced \u03b3H2AX, 53BP1 and Rad51 foci

The spatial distribution of DSB repair factors γH2AX, 53BP1 and Rad51 in ionizing radiation induced foci (IRIF) in HeLa cells using super resolution STED nanoscopy after low and high linear energy transfer (LET) irradiation was investigated. 53BP1 and γH2AX form IRIF with same mean size of (540 ± 40) nm after high LET irradiation while the size after low LET irradiation is significantly smaller. The IRIF of both repair factors show nanostructures with partial anti-correlation. These structures are related to domains formed within the chromatin territories marked by γH2AX while 53BP1 is mainly situated in the perichromatin region. The nanostructures have a mean size of (129 ± 6) nm and are found to be irrespective of the applied LET and the labelled damage marker. In contrast, Rad51 shows no nanostructure and a mean size of (143 ± 13) nm independent of LET. Although Rad51 is surrounded by 53BP1 it strongly anti-correlates meaning an exclusion of 53BP1 next to DSB when decision for homologous DSB repair happened.

Validation of JCountPro software for efficient assessment of ionizing radiation-induced foci in human lymphocytes

Purpose: Ionizing radiation-induced foci (IRIF) known also as DNA repair foci represent the most sensitive and specific assay for assessing DNA double-strand break (DSB). IRIF are usually visualized and enumerated with the aid of fluorescence microscopy using antibodies to phosphorylated γH2AX and 53BP1. Although several approaches and software packages were developed for quantification of IRIF, not one of them was commonly accepted and inter-laboratory variability in the outputs was reported. In this study, JCountPro software was validated for IRIF enumeration in two independent laboratories.Materials and methods: Human lymphocytes were γ-irradiated at doses of 0, 2, 5, 10 and 50 cGy. The cells were fixed, permeabilized and IRIF were immunostained using appropriate antibodies. Cell images were acquired with automatic Metafer system. Endogenous and radiation-induced γH2AX and 53BP1 foci were enumerated using JCountPro. This analysis was performed from the same cell galleries by the researchers from two laboratories. Yield of foci was analyzed by either arithmetic mean (AM) value (foci/cell) or principal average (PA) derived from the approximation of foci distribution with Poisson statistics. Statistical analysis was performed using factorial ANOVA.Results: Enumeration of 53BP1, γH2AX and co-localized 53BP1/γH2AX foci by JCountPro was essentially the same between laboratories. IRIF were detected at all doses and linear dose response was obtained in the studied dose range. PA values from Poisson distribution fitted the data better as compared to AM values and were more powerful and sensitive for IRIF analysis than the AM values. All JCountPro data were confirmed by visual focus enumeration.Conclusions: We concluded that the JCountPro software was efficient in objectively enumerating IRIF regardless of an individual researcher’s bias and has a potential for usage in clinics and molecular epidemiology.

Abstract 1644: NF-κB inhibitor DMAPT blocks non-homologous end-joining repair of radiation-induced DSBs in NSCLC

Abstract Background: Despite optimal multimodality therapy, non-small cell lung carcinoma (NSCLC) remains the leading cause of cancer-related death in the United States. A common limitation is our inability to provide sufficient radiotherapy (RT) to eradicate tumor due to risk of toxicities in surrounding tissues. There is thus an unmet need for radiosensitizers that can improve the therapeutic index. Dimethylaminoparthenolide (DMAPT), an orally bioavailable small molecule NF-κB inhibitor, inhibits repair of ionizing radiation (IR)-induced DNA double strand breaks (DSBs) and increases control of subcutaneous mouse NSCLC xenografts. While our prior work focused on inhibition of homologous recombination as a mechanism for radiosensitization, we sought to characterize the effect of DMAPT on a second major DSB repair pathway, non-homologous end-joining (NHEJ). Methods: NHEJ was assessed in NSCLC lines using the pEJ reporter and flow cytometry. The NF-κB super repressor (IκBαS32A,S36A) was used as a control. Immunofluorescence and Western blotting were used to assess NHEJ biomarkers including 53BP1, DNA-PKS2056, Ku70/80 and XRCC4. Cell fractionation was performed to assess Ku chromatin binding. Quantitative RT-PCR was performed to assess gene transcription. Ku complexes were purified to identify binding partners. Results: NSCLC cells treated with IR-sensitizing doses of DMAPT (5-15 μM) or the NF-κB super repressor showed significant decreases in NHEJ. DMAPT increased the persistence of 53BP1 foci, indicating a failure to complete NHEJ. Regardless of exogenous DNA damage, there was reduced Ku70 chromatin binding following DMAPT treatment. DMAPT-treated cells produced fewer distinct IR-induced DNA-PKS2056 foci. Further, there was decreased IR-induced XRCC4 chromatin recruitment, suggesting that repair was impaired prior to ligation. There was no change in Ku70/80 transcription following DMAPT and/or IR. However, Western blotting of purified Ku complexes showed that DMAPT treatment decreased Ku association with RNA binding partners including RPL19. We also observed decreased recruitment of DNA-PKcs to the Ku complex, suggesting decreased Ku affinity for DSBs. Conclusions: These results indicate that DMAPT radiosensitizes NSCLC by perturbing the binding affinity of Ku to RNA, DNA and its complex binding partners, thus blocking NHEJ. Further mechanistic investigation and analysis of NHEJ biomarkers in vivo is needed to identify the precise mechanism. Citation Format: Peter V. Deraska, Colin O’Leary, Jean-Bernard Lazaro, Christopher J. Sweeney, Alan D. D’Andrea, David Kozono. NF-κB inhibitor DMAPT blocks non-homologous end-joining repair of radiation-induced DSBs in NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1644.

Abstract 2756: SMARCAD1 depletion enhances hyperthermia-mediated radiosensitization by decreasing resection and enhancing stalled replication forks

Abstract Hyperthermia is the most effective chemo- and radio-sensitizer in use clinically for containing tumor growth. Hyperthermia is proposed to induce degradation of BRCA2, the protein involved in homologous recombination (HR) repair of DNA double strand breaks and, thus sensitizes cells to poly (ADP-ribose) polymerase (PARP)-1 inhibition. We and other laboratories have reported that hyperthermia inhibits DNA damage repair. Since HR includes single-strand annealing, gene conversion and break-induced replication, it is critical for DNA replication during recovery of stalled replication forks, but it is not yet clear how hyperthermia affects these important steps in HR. Here we report that SMARCAD1 depletion increases hyperthermia induced cell death and chromosome damage in irradiated S-phase cells. Combined SMARCAD1 depletion and hyperthermia treatment decreased repair and recruitment of repairosome proteins to ISce-1 induced DSBs as well as decreased IR-induced repairosome foci formation. This effect was limited to S-phase cells, where homologous recombination is up regulated and the predominant pathway of DSB repair. Hyperthermia decreased the quantity of 5′ single stranded DNA intermediates at endonuclease-generated DNA break sites, an effect that was further decreased by SMARCAD1 depletion. Consistent with these results, simultaneous SMARCAD1 depletion and hyperthermia increased replication fork stalling and affected firing of new replication origins. The results presented here support a mechanism by which SMARCAD1 depletion and hyperthermia mediate radiosensitization through inhibition of the DNA resection step that precedes strand homology search during HR repair of IR-induced DSBs. Citation Format: Sharmistha Chakraborty, Clayton R. Hunt, Shashank Hambarde, Kalpana Mujoo, Nobuo Horikoshi, Raj K. Pandita, Abid Mattoo, Bin Teh, Tej K. Pandita. SMARCAD1 depletion enhances hyperthermia-mediated radiosensitization by decreasing resection and enhancing stalled replication forks. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2756.

Abstract 3719: TAK-243, a small molecule inhibitor of the ubiquitin activating enzyme (UAE), disrupts DNA damage repair and sensitizes tumor cells and xenografts to ionizing radiation

Abstract Radiation therapy, as a primary therapy or as a combination partner, is used in half of all worldwide cancer treatments. Research is ongoing to identify agents which potentiate the effects of ionizing radiation (IR) in tumor cells. Because IR causes DNA double strand breaks (DSBs), inhibition of DNA damage repair mechanisms could enhance the effects of radiation. DNA repair at DSBs is mediated by the non-homologous end-joining (NHEJ) and homologous recombination (HR) pathways, both of which rely on the post-translational modification of proteins by ubiquitin (Ub). A phosphorylation and ubiqutination cascasde at DSBs results in Ub-dependent recruitment of 53BP1 and BRCA1 complexes. We have identified a first in class investigational drug, TAK-243 (MLN7243), which targets the ubiquitin activating enzyme, UAE (UBA1), the enzyme responsible for activating > 99% of all cellular Ub. Previously, TAK-243 was shown to inhibit mono-Ub of PCNA and FANCD2, key proteins within the translesion synthesis (TLS) and Fanconi Anemia (FA) DNA repair pathways, and also to inhibit Ub transfer to UBC13, an E2 ubiquitin-conjugating enzyme utilized in DSB repair. We hypothesized that TAK-243 would prevent repair of DSBs and thereby potentiate IR-induced cell death. Here we show that TAK-243 pre-treatment potentiates the effect of IR on HCT-116 cells in a colony formation assay in vitro. To link this combination benefit to the disruption of DNA damage repair, we demonstrate that TAK-243 pre-treatment blocks the IR-induced recruitment of 53BP1 to sites of DNA damage both in vitro and in vivo. In a patient-derived xenograft (PDX) model of non-small cell lung cancer, formation of IR-induced 53BP1 foci is inhibited when TAK-243 is dosed 1 hour before beam-focused radiation exposure. In contrast, levels of IR-induced pH2Ax are not significantly changed by TAK-243 treatment, suggesting that TAK-243 does not prevent formation or detection of DSBs, but rather acts downstream to prevent DNA damage repair. Additive-to-synergistic effects on tumor growth inhibition were observed in several xenograft models treated with the combination of TAK-243 and beam-focused IR, with persistent tumor regressions noted in some NSCLC and breast cancer models. The results of our experiments provide a mechanistic rationale for combining radiation with TAK-243 in the clinical setting. Currently, TAK-243 is being evaluated in a solid tumor phase I clinical trial evaluating safety, tolerability, pharmacokinetics, pharmacodynamics and anti-tumor activity (NCT02045095). Citation Format: Michael A. Milhollen, Judy Qiuju Shi, Tary Traore, Jessica Huck, Darshan Sappal, Kenichi Iwai, Akihiro Ohashi, Claudia Rabino, Jennifer A. Duffy, Eric Lightcap, Yuko Ishii, Jeffrey Ciavarri, Neil Bence, Allison J. Berger, Marc L. Hyer. TAK-243, a small molecule inhibitor of the ubiquitin activating enzyme (UAE), disrupts DNA damage repair and sensitizes tumor cells and xenografts to ionizing radiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3719.

Tetratricopeptide repeat factor XAB2 mediates the end resection step of homologous recombination.

We examined the influence of the tetratricopeptide repeat factor XAB2 on chromosomal break repair, and found that XAB2 promotes end resection that generates the 3′ ssDNA intermediate for homologous recombination (HR). Namely, XAB2 is important for chromosomal double-strand break (DSB) repair via two pathways of HR that require end resection as an intermediate step, end resection of camptothecin (Cpt)-induced DNA damage, and RAD51 recruitment to ionizing radiation induced foci (IRIF), which requires end resection. Furthermore, XAB2 mediates specific aspects of the DNA damage response associated with end resection proficiency: CtIP hyperphosphorylation induced by Cpt and BRCA1 IRIF. XAB2 also promotes histone acetylation events linked to HR proficiency. From truncation mutation analysis, the capacity for XAB2 to promote HR correlates with its ability to form a complex with ISY1 and PRP19, which show a similar influence as XAB2 on HR. This XAB2 complex localizes to punctate structures consistent with interchromatin granules that show a striking adjacent-localization to the DSB marker γH2AX. In summary, we suggest that the XAB2 complex mediates DNA damage response events important for the end resection step of HR, and speculate that its adjacent-localization relative to DSBs marked by γH2AX is important for this function.

Breaks in the 45S rDNA Lead to Recombination-Mediated Loss of Repeats.

rDNA repeats constitute the most heavily transcribed region in the human genome. Tumors frequently display elevated levels of recombination in rDNA, indicating that the repeats are a liability to the genomic integrity of a cell. However, little is known about how cells deal with DNA double-stranded breaks in rDNA. Using selective endonucleases, we show that human cells are highly sensitive to breaks in 45S but not the 5S rDNA repeats. We find that homologous recombination inhibits repair of breaks in 45S rDNA, and this results in repeat loss. We identify the structural maintenance of chromosomes protein 5 (SMC5) as contributing to recombination-mediated repair of rDNA breaks. Together, our data demonstrate that SMC5-mediated recombination can lead to error-prone repair of 45S rDNA repeats, resulting in their loss and thereby reducing cellular viability.

DNA Damage-Induced Foci of E2 Ubiquitin-Conjugating Enzyme are Detectable upon Co-transfection with an Interacting E3 Ubiquitin Ligase.

DNA damage repair elements accumulate at DNA damage sites to form ionizing radiation-induced foci (IRIF) for damage repair. IRIF, which represent direct evidence of DNA damage response activity, which are conveniently to be observed via immunofluorescence staining. Protein ubiquitination plays an important role in initiating the DNA damage response. Following DNA damage, the substrate binding protein E3 ubiquitin-ligases enzymes are recruited to DNA damage sites, then the E2 ubiquitin-conjugating enzymes are recruited to these sites by the E3 where they catalyze protein ubiquitination. However, IRIF of E2 enzymes are relatively transient and unstable in vivo and difficult to detect. Here, we present a new method for the observation of E2 IRIF. This method is based on the co-transfection of interacting E2 and E3 enzymes into cells and identifies IRIF via immunofluorescence following DNA damage.

Dynamic Behavior of the RNA Polymerase II and the Ubiquitin Proteasome System During the Neuronal DNA Damage Response to Ionizing Radiation.

Neurons are highly vulnerable to genotoxic agents. To restore genome integrity upon DNA lesions, neurons trigger a DNA damage response (DDR) that requires chromatin modifications and transcriptional silencing at DNA damage sites. To study the reorganization of the active RNA polymerase II (Pol II), which transcribes all mRNA-encoding genes, and the participation of the ubiquitin-proteasome system (UPS) in the neuronal DDR, we have used rat sensory ganglion neurons exposed to X-rays (4Gy) ionizing radiation (IR). In control neurons, Pol II appears concentrated in numerous chromatin microfoci identified as transcription factories by the incorporation of 5'-fluorouridine into nascent RNA. Upon IR treatment, numerous IR-induced foci (IRIF), which were immunoreactive for γH2AX and 53BP1, were observed as early as 30min post-IR; their number progressively reduced at 3h, 1day, and 3days post-IR. The formation of IRIF was associated with a decrease in Pol II levels by both immunofluorescence and Western blotting. Treatment with the proteasome inhibitor bortezomib strongly increased Pol II levels in both control and irradiated neurons, suggesting that proteasome plays a proteolytic role by clearing stalled Pol II complexes at DNA damage sites, as a prelude to DNA repair. Neuronal IRIF recruited ubiquitylated proteins, including ubiquitylated histone H2A (Ub-H2A), and the catalytic proteasome 20S. Ub-H2A has been associated with transcriptional silencing at DNA damage sites. On the other hand, the participation of UPS in neuronal DDR may be essential for the ubiquitylation of Pol II and other proteasome substrates of the DNA repair machinery and their subsequent proteasome-mediated degradation.

A genome-wide IR-induced RAD51 foci RNAi screen identifies CDC73 involved in chromatin remodeling for DNA repair.

To identify new regulators of homologous recombination repair, we carried out a genome-wide short-interfering RNA screen combined with ionizing irradiation using RAD51 foci formation as readout. All candidates were confirmed by independent short-interfering RNAs and validated in secondary assays like recombination repair activity and RPA foci formation. Network analysis of the top modifiers identified gene clusters involved in recombination repair as well as components of the ribosome, the proteasome and the spliceosome, which are known to be required for effective DNA repair. We identified and characterized the RNA polymerase II-associated protein CDC73/Parafibromin as a new player in recombination repair and show that it is critical for genomic stability. CDC73 interacts with components of the SCF/Cullin and INO80/NuA4 chromatin-remodeling complexes to promote Histone ubiquitination. Our findings indicate that CDC73 is involved in local chromatin decondensation at sites of DNA damage to promote DNA repair. This function of CDC73 is related to but independent of its role in transcriptional elongation.

Abstract A11: Radio-sensitivities and angiogenic signaling pathway of irradiated endothelial cells isolated from cancer and normal tissue of human breasts in vitro

Abstract Despite the strong possibility that endothelial cells of tumors and normal tissues may be different in various aspects, most of the previous studies on endothelial cells have been conducted using normal endothelial cells. Therefore, we have developed a novel method for harvesting endothelial cells from blood vessels of freshly obtained cancer and adjacent normal tissue of human breast, and further investigated radio-sensitivities and angiogenic signaling pathway in the alternation of angiogenic process observed after the purified cancer-derived endothelial cells (CECs) and normal tissue-derived endothelial cells (NECs) were irradiated in vitro. When human breast tissues including cancer were embedded in Matrigel and cultured in endothelial cell culture medium (ECM) containing growth factors, endothelial cells grew out of the tissues. The endothelial cells were harvested, cultured as monolayer cells in plates coated with gelatin, and exposed to radiation, and then the changes in clonogenic cell survival, tube formation capacity, γH2AX foci, gene expression, and cellular signaling pathways were determined. Both CECs and NECs expressed almost the same levels of surface markers CD31, CD105 and TEM-8 (tumor endothelial marker-8), which are known to be expressed in angiogenic endothelial cells, i.e., mitotically active endothelial cells. CECs were significantly more radiosensitive in treatment of single dose (4 Gy) and hyperfractionation (4 Gy in 2 or 4 equal fractions) of ionizing radiation (IR) than their normal counterparts although hyperfractionated IR reduced clonogenic cell death in both NECs and CECs than single dose of IR. In addition, we found that IR-induced foci formation of γH2AX in NECs increased 1 hr and declined afterwards when the cells were treated with 4 Gy of IR. On the contrary, it was remained up to 4 hr in CECs. In NECs, 4 Gy of IR induced an increase in the capillary-like tube formation, the expression of matrix metalloproteinase-2 (MMP-2), and the activation of ERK pathway. However, in CECs, 4 Gy of IR significantly reduced the capillary-like tube formation, and induced the expression of angiostatin and the activation of both AKT and JNK pathways. Additionally, inhibition of ERK with a pharmacological inhibitor or a small interfering RNA markedly suppressed the capillary-like tube formation and the expression of MMP-2 caused by IR in NECs. In CECs, inhibition of either AKT or JNK with a pharmacological inhibitor or a small interfering RNA clearly attenuated inhibition of the capillary-like tube formation, and the expression of angiostatin caused by IR. Furthermore, hypoxia increased the capillary-like tube formation and radioresistance in both NECs and CECs, but the delineation of the mechanisms remains to be studied. We have developed a simple and efficient new method for isolating endothelial cells from cancer and normal tissue. Moreover, our results collectively demonstrate that there are distinct differences in the radiation responses of NECs and CECs, and might provide important clues for improving the efficacy of radiation therapy. [This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (MRC: NRF-2014R1A5A2009392 and NRF-2013M2A2A7043703] Citation Format: Eun-Taex Oh, Min-Jeong Song, Hyemi Lee, Yun-Jeong Choi, Heon Joo Park. Radio-sensitivities and angiogenic signaling pathway of irradiated endothelial cells isolated from cancer and normal tissue of human breasts in vitro. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A11.

Imaging flow cytometry as a sensitive tool to detect low-dose-induced DNA damage by analyzing 53BP1 and γH2AX foci in human lymphocytes.

Ionizing radiation induced foci (IRIF) are considered the most sensitive indicator for DNA double-strand break (DSB) detection. Monitoring DSB induction by low doses of ionizing radiation is important due to the increasing exposure in the general population. γH2AX and 53BP1 are commonly used molecular markers for in situ IRIF assessment. Imaging flow cytometry (IFC) via ImageStream system provides a new opportunity in this field. We analyzed the formation of 53BP1, γH2AX foci and their co-localization induced by γ-rays (2, 5, 10, 50, 200 cGy) in human lymphocytes using ImageStream and the automated microscopic system Metafer. We observed very similar sensitivity of both systems for the detection of endogenous and low-dose-induced IRIF. Statistically significant induction of γH2AX foci was found at doses of 2 and 10 cGy using ImageStream and Metafer, respectively. Statistically significant induction of 53BP1 foci was evident at doses ≥ 5 cGy when analyzed by IFC. Analysis of the co-localizing foci by ImageStream and Metafer showed statistical significance at doses ≥ 2 cGy, suggesting that foci co-localization is a sensitive parameter for DSB quantification. Assessment of γH2AX, 53BP1 foci and their co-localization by Metafer and ImageStream showed similar linear dose responses in the low-dose range up to 10 cGy, although IFC showed slightly better resolution for IRIF in this dose range. At higher doses, IFC underestimated IRIF numbers. Using the imaging ability of ImageStream, we introduced an optimized assay by gating γH2AX foci positive (with 1 or more γH2AX foci) and negative (cells without foci) cells. This assay resulted in statistically significant IRIF induction at doses ≥ 5cGy and a linear dose response up to 50 cGy. In conclusion, we provide evidence for the use of IFC as an accurate high throughput assay for the prompt detection and enumeration of endogenous and low-dose induced IRIF.

Abstract LB-215: Epigenetic loss-of-function BRCA1 mediates tumor cure by single dose radiotherapy

Abstract The mechanism of tumor cure by ionizing radiation is regarded tumor cell autonomous, effected by misrepair of radiation-induced DNA double strand breaks (DSBs), mainly via the function of error prone non-homologous end joining (NHEJ). Genomic instability yields mitosis-dependent buildup of potentially lethal chromosomal aberrations, with repeated radiation exposures required for tumor ablation. Here we show that at the high dose range (>10Gy), single dose radiotherapy (SDRT) engages an alternative dual target model, inducing in addition to DSBs also an early wave of acid sphingomyelinase (ASMase)-mediated microcirculatory ischemia/reperfusion injury. DSB repair was analyzed in situ by quantitative assessment of the time-dependent buildup and resolution of ionizing radiation-induced foci (IRIF) of specific NHEJ or homology-driven repair (HDR) mediators. Effect of SDRT on the tumor microvasculature was assessed by dynamic contrast-enhanced magnetic resonance imaging. Engagement of microvascular dysfunction in DSB repair was assessed using ASMase-deficient mice, refractory to vascular endothelial injury. Western blot analysis of Small Ubiquitin-like Modifiers (SUMO) in tumor extracts and studies of SUMO conjugating enzymes IRIF in situ were used to evaluate effects of SDRT on SUMOylation. SDRT concomitantly induces DSBs in tumor cells and an early wave (<1 hour) of ASMase-mediated microcirculatory ischemia/reperfusion, synthetically coupling parenchymal tumor cell DNA damage response to re-program tumor lethality. Ischemia/reperfusion induced in reperfused tumor clonogens an oxidative stress, dysfunctioning therein SUMO conjugating enzymes that are critically required for assembly of the inherent DSB repair codex, leading to catastrophic reprograming of DSB repair. While Ku- and 53BP1-mediated NHEJ were not affected, although 53BP1 resolution was delayed, HDR was aborted. We show that SUMO 2/3 dysfunction, specifically induced post reperfusion, impairs recruitment of RAP80, BRCA1, RPA and RAD51 into DSB repair foci. The epigenetic loss-of-function BRCA1/HDR diverted DSB repair to an aberrant lethal NHEJ pathway, yielding massive lethal chromosomal aberrations, reproductive cell death and tumor cure. The dual target microvascular/tumor clonogen model, described here, which functions exclusively at high-dose radiation exposures, constitutes a functional alternative to the classical single target mechanism operating at the low dose range, and provides new targets for modulation of the radiation response, with a potential for yielding new options for tumor ablation in the clinical management of human cancer. Citation Format: Cecile G. CAMPAGNE, Tin H. Thin, John D. Fuller, Ellen Ackerstaff, Jason A. Koutcher, Adriana Haimovitz-Friedman, Simon N. Powell, Richard N. Kolesnick, Zvi Fuks. Epigenetic loss-of-function BRCA1 mediates tumor cure by single dose radiotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-215. doi:10.1158/1538-7445.AM2015-LB-215

Abstract 3860: USP7 deubiquitinase promotes ubiquitin-dependent DNA damage signaling by stabilizing RNF168

Abstract In DNA damage response (DDR), histone ubiquitination by RNF168 is a critical event, which orchestrates the recruitment of downstream DDR factors, e.g. BRCA1 and 53BP1. Here, we report USP7 deubiquitinase regulates the stability of RNF168. We showed that USP7 disruption impairs the monoubiquitination of H2A (uH2A) and ultraviolet radiation (UVR)-induced monoubiquitination of γH2AX, and decreases the levels of pBmi1, Bmi1, RNF168, pChk1 and BRCA1. USP7 silencing by siRNA reduces uH2A and UVR-induced monoubiquititination of γH2AX, as well as the levels of Ring1B, Bmi1, RNF168 and BRCA1, recapitulating the chronic USP7 deficiency. USP7 disruption also compromises the formation of UVR-induced foci (UVRIF) and ionizing radiation-induced foci (IRIF) of uH2A, poly-K63-ubiquititination and BRCA1, but does not affect UVRIF and IRIF of 53BP1. USP7 was shown to physically bind RNF168 in vitro and in vivo. Overexpression of wild-type USP7, but not its catalytic-inactive or interaction-defective mutants, prevents UVR-induced RNF168 degradation. Both USP7 mutants are unable to cleave Ub-conjugates of RNF168 in vivo. Furthermore, ectopic expression of RNF168, or both RNF8 and RNF168 together, rescue the formation of UVRIF and IRIF of poly-K63-ubiquititination and BRCA1. Taken together, these findings reveal an important role of USP7 in regulating ubiquitin-dependent signaling via stabilization of RNF168. Note: This abstract was not presented at the meeting. Citation Format: Qianzheng Zhu, Nidhi Sharma, Jingshan He, Gulzar Wani, Altaf A. Wani. USP7 deubiquitinase promotes ubiquitin-dependent DNA damage signaling by stabilizing RNF168. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3860. doi:10.1158/1538-7445.AM2015-3860

Abstract 3336: NF-κB inhibition by DMAPT radiosensitizes non-small cell lung carcinoma by impairing DNA double strand break repair

Abstract Background: Despite optimal radiation therapy (RT), chemotherapy and/or surgery, non-small cell lung carcinoma (NSCLC) remains the leading cause of cancer-related death in the United States. Previously, we demonstrated that proteasome inhibition blocks the expression of Fanconi anemia/homologous recombination (FA/HR) genes via inhibition of the NF-κB pathway. Dimethylaminoparthenolide (DMAPT) is a water-soluble analog of parthenolide that has been shown to decrease p65/p50 heterodimer DNA binding. We hypothesized that DMAPT may radiosensitize NSCLC cells by impairing NF-κB-mediated expression of DNA double strand break (DSB) repair genes. Materials and Methods: Cytotoxicity and radiosensitivity were assessed using clonogenic assays. IκB kinase (IKK) activity was measured using a substrate phosphorylation assay. HR and canonical non-homologous end joining (cNHEJ) were measured using A549 cells stably expressing reporter constructs that express GFP upon repair of I-SceI-induced DNA DSBs. DNA damage and DSB repair biomarker foci were assessed by immunofluorescence. For in vivo studies, 1×106 A549 cells were injected subcutaneously into the flanks of NCr nude mice. Once tumors reached an average size of 200 mm3, mice were treated concurrently with vehicle or DMAPT (100 mg/kg) ± focal ionizing radiation (4 Gy x 5) using a small animal radiation research platform (SAARP). Tumor growth was followed for three months following treatment. Results: Radiation and NF-κB inhibition with DMAPT showed synergistic cytotoxicity. Treatment of A549 cells with DMAPT resulted in a decrease in constitutive IκBα phosphorylation by IKK. NF-κB inhibition via DMAPT resulted in a 60-70% decrease in HR. In addition, treatment with DMAPT resulted in ≥80% loss of IR-induced BRCA1, FANCD2, and RAD51 foci. NF-κB inhibition with either DMAPT or overexpression of the NF-κB super-repressor IκBα(S32A, S36A) resulted in a 50-70% decrease in cNHEJ. Treatment with DMAPT resulted in the loss of IR-induced phosphoDNA-PKS2056 foci. Further, treatment with DMAPT resulted in the persistence of radiation-induced γH2AX foci over a 24 hour period (100% of DMAPT treated cells showing foci compared to 30% of vehicle treated cells). Compared to either single agent DMAPT or IR alone, combined treatment of A549 xenografts with DMAPT and IR prevented tumor growth over three months (p < 0.0001). Conclusions: DMAPT inhibits NF-κB signaling by inhibiting IKK. This in turn inhibits HR and cNHEJ by preventing the recruitment of DNA repair proteins to sites of IR-induced DNA damage. Both in vitro and in vivo experiments show support for further evaluation of DMAPT as a NSCLC radiosensitizer. Citation Format: Colin O'Leary, Peter Deraska, Christopher Sweeney, Alan D'Andrea, David Kozono. NF-κB inhibition by DMAPT radiosensitizes non-small cell lung carcinoma by impairing DNA double strand break repair. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3336. doi:10.1158/1538-7445.AM2015-3336

Abstract 2852: Torin2 suppresses ionizing radiation induced DNA damage repair

Abstract The phosphatidylinositol 3- kinase like kinase (PIKK) family of signaling proteins play a central role in stress response, DNA damage signaling, and sensing growth factor and nutrient levels to regulate cellular metabolism. In tumor cells, the PIKK signaling is often hyperactivated making it an attractive therapeutic target. In this study, we have focused our efforts on an ATP-competitive mTOR/PIKK inhibitor ‘Torin2′ which has been previously reported to exhibit potent activity against the DNA repair-associated proteins ATM, ATR and DNA-PK in addition to mTOR in biochemical profiling studies. Based on this profiling data, we hypothesized that Torin2 would have synergistic effects with agents or biological processes that cause DNA damage, especially in cases where high levels of replicative stress and non-oncogenic addiction to DNA repair factors are present. In order to understand the practical clinical implications, we evaluated how Torin2 modulates the effects of ionizing radiation (IR) induced DNA damage responses. With a defined set of biochemical and cytogenetic studies along with DNA repair reporter system, we established the role for Torin2 in double strand break repair responses in human tumor cell lines. Torin2 enhances ionizing radiation-induced cell killing in cancer cell lines independent of ATM status, suggesting that Torin2 simultaneously influences multiple PIKK targets. Moreover, Torin2 pre-treatment did not alter the appearance of γH2AX foci post irradiation but significantly delayed the disappearance of IR-induced γH2AX foci indicating a defective DNA repair processes. Pre-treatment of this drug decreased the number of IR-induced G2/S-phase specific chromosomal aberrations, and also reduces the frequency of the foci formation of IR-induced homologous recombination (HR) repair proteins, such as CtIP and Rad51, suggesting that the drug works by blocking HR, a repair process that predominantly works in S/G2 phase of the cell cycle. Consistent with these results, a HR reporter assay showed that Torin2 treatment reduces repair of I-SceI restriction enzyme induced DNA damage. Torin2 also influences DNA replicative stress through inhibition of ATR-Chk1 activation, and contributes to fork stalling during replication. With the above set of studies, we have identified key DNA damage signaling events by which Torin2 affects HR mediated double strand break repair after ionizing radiation treatment. This enhanced radiosensitizing effect of Torin2 merits further pre-clinical evaluation in combination with various radiotherapy modalities. Citation Format: Durga Udayakumar, Raj K. Pandita, Nobuo Horikoshi, Clayton R. Hunt, Qingsong Liu, Kwok-Kin Wong, Nathanael S. Gray, Tej K. Pandita, Kenneth D. Westover. Torin2 suppresses ionizing radiation induced DNA damage repair. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2852. doi:10.1158/1538-7445.AM2015-2852