Requirement of ATM-dependent monoubiquitylation of histone H2B for timely repair of DNA double-strand breaks.

The cellular response to DNA double-strand breaks (DSBs) is mobilized by the protein kinase ATM, which phosphorylates key players in the DNA damage response (DDR) network. A major question is how ATM controls DSB repair. Optimal repair requires chromatin relaxation at damaged sites. Chromatin reorganization is coupled to dynamic alterations in histone posttranslational modifications. Here, we show that in human cells, DSBs induce monoubiquitylation of histone H2B, a modification that is associated in undamaged cells with transcription elongation. We find that this process relies on recruitment to DSB sites and ATM-dependent phosphorylation of the responsible E3 ubiquitin ligase: the RNF20-RNF40 heterodimer. H2B monoubiquitylation is required for timely recruitment of players in the two major DSB repair pathways-nonhomologous end-joining and homologous recombination repair-and optimal repair via both pathways. Our data and previous data suggest a two-stage model for chromatin decondensation that facilitates DSB repair.

Carbon ion radiotherapy of human lung cancer attenuates HIF-1 signaling and acts with considerably enhanced therapeutic efficiency.

Carbon ion irradiation is an emerging therapeutic option for various tumor entities. Radiation resistance of solid tumors toward photon irradiation is caused by attenuation of DNA damage in less oxygenated tumor areas and by increased hypoxia-inducible factor (HIF)-1 signaling. Carbon ion irradiation acts independently of oxygen; however, the role of HIF-1 is unclear. We analyzed the effect of HIF-1 signaling after carbon ions in comparison to photons by using biological equivalent radiation doses in a human non-small-cell cancer model. The studies were performed in cultured A549 and H1299 cell lines and in A549 xenografts. Knockdown of HIF-1α in vivo combined with photon irradiation delayed tumor growth (23 vs. 13 d; P<0.05). Photon irradiation induced HIF-1α and target genes, predominantly in oxygenated cells (1.6-fold; P<0.05), with subsequent enhanced tumor angiogenesis (1.7-fold; P<0.05). These effects were not observed after carbon ion irradiation. Micro-DNA array analysis indicated that photons, but not carbon ions, significantly induced components of the mTOR (mammalian target of rapamycin) pathway (gene set enrichment analysis; P<0.01) as relevant for HIF-1α induction. After carbon ion irradiation in vivo, we observed substantially decreased HIF-1α levels (8.9-fold; P<0.01) and drastically delayed tumor growth (P<0.01), an important finding that indicates a higher relative biological effectiveness (RBE) than anticipated from the cell survival data. Taken together, the evidence showed that carbon ions mediate an improved therapeutic effectiveness without tumor-promoting HIF-1 signaling.

Radiation-induced double-strand breaks require ATM but not Artemis for homologous recombination during S-phase.

Double-strand breaks (DSBs) are repaired by two distinct pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). The endonuclease Artemis and the PIK kinase Ataxia-Telangiectasia Mutated (ATM), mutated in prominent human radiosensitivity syndromes, are essential for repairing a subset of DSBs via NHEJ in G1 and HR in G2. Both proteins have been implicated in DNA end resection, a mandatory step preceding homology search and strand pairing in HR. Here, we show that during S-phase Artemis but not ATM is dispensable for HR of radiation-induced DSBs. In replicating AT cells, numerous Rad51 foci form gradually, indicating a Rad51 recruitment process that is independent of ATM-mediated end resection. Those DSBs decorated with Rad51 persisted through S- and G2-phase indicating incomplete HR resulting in unrepaired DSBs and a pronounced G2 arrest. We demonstrate that in AT cells loading of Rad51 depends on functional ATR/Chk1. The ATR-dependent checkpoint response is most likely activated when the replication fork encounters radiation-induced single-strand breaks leading to generation of long stretches of single-stranded DNA. Together, these results provide new insight into the role of ATM for initiation and completion of HR during S- and G2-phase. The DSB repair defect during S-phase significantly contributes to the radiosensitivity of AT cells.

The alternative end-joining pathway for repair of DNA double-strand breaks requires PARP1 but is not dependent upon microhomologies.

Non-homologous end-joining (NHEJ), the major repair pathway for DNA double-strand breaks (DSB) in mammalian cells, employs a repertoire of core proteins, the recruitment of which to DSB-ends is Ku-dependent. Lack of either of the core components invariably leads to a repair deficiency. There has been evidence that an alternative end-joining operates in the absence of the core components. We used chromosomal reporter substrates to specifically monitor NHEJ of single I-SceI-induced-DSB for detailed comparison of classical and alternative end-joining. We show that rapid repair of both compatible and non-compatible ends require Ku-protein. In the absence of Ku, cells use a slow but efficient repair mode which experiences increasing sequence-loss with time after DSB induction. Chemical inhibition and PARP1-depletion demonstrated that the alternative end-joining in vivo is completely dependent upon functional PARP1. Furthermore, we show that the requirement for PARP1 depends on the absence of Ku but not on DNA-dependent protein kinase (DNA-PKcs). Extensive sequencing of repair junctions revealed that the alternative rejoining does not require long microhomologies. Together, we show that mammalian cells need Ku for rapid and conservative NHEJ. PARP1-dependent alternative route may partially rescue the deficient repair phenotype presumably at the expense of an enhanced mutation rate.

Targeting DNA double-strand break repair: is it the right way for sensitizing cells to 5-fluorouracil?

Inhibition of the repair of 5-fluorouracil (FU)-induced DNA lesions may improve the response of many tumors to this anticancer agent. Despite the identified associations between DNA strand breaks and the lethality of thymidylate synthase inhibitors, the role of DNA double-strand break (DSB) repair pathways in a cellular response to 5-FU treatment has not been studied yet. Isogenic cell lines defective (irs1SF), wild type (AA8), or reconstituted (1SFK8) in the DSB repair protein XRCC3 were used to investigate the effect of defective DSB repair on the overall sensitivity of cells to 5-FU and to see how targeting DSB repair may affect other cellular responses to 5-FU. Treatment with 5-FU resulted in (i) similar induction of DSB in both cell lines as indicated by the formation of gamma-H2AX (a marker for DSB). The repair of these breaks was complete in AA8 but not in irs1SF cells. (ii) Concentration-dependent reduction in the survival of both cell lines. The AA8 cells were six times more sensitive to 5-FU than the irs1SF cells. (iii) An earlier and more prolonged G(1)/S phase arrest in AA8 compared with the irs1SF cells. (iv) Induction of apoptosis as indicated by sub-G(1) cells and caspase-3 activity in AA8 but not in irs1SF cells. XRCC3 complementation of irs1SF cells restored the wild-type phenotype. This result shows that targeting DSB repair is not always associated with increased sensitivity to DNA damaging agents such as 5-FU because it may affect other cellular responses such as cell cycle regulation and induction of apoptosis.

Distinct roles of XRCC4 and Ku80 in non-homologous end-joining of endonuclease- and ionizing radiation-induced DNA double-strand breaks.

Non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSBs) is mediated by two protein complexes comprising Ku80/Ku70/DNA-PKcs/Artemis and XRCC4/LigaseIV/XLF. Loss of Ku or XRCC4/LigaseIV function compromises the rejoining of radiation-induced DSBs and leads to defective V(D)J recombination. In this study, we sought to define how XRCC4 and Ku80 affect NHEJ of site-directed chromosomal DSBs in murine fibroblasts. We employed a recently developed reporter system based on the rejoining of I-SceI endonuclease-induced DSBs. We found that the frequency of NHEJ was reduced by more than 20-fold in XRCC4-/- compared to XRCC4+/+ cells, while a Ku80 knock-out reduced the rejoining efficiency by only 1.4-fold. In contrast, lack of either XRCC4 or Ku80 increased end degradation and shifted repair towards a mode that used longer terminal microhomologies for rejoining. However, both proteins proved to be essential for the repair of radiation-induced DSBs. The remarkably different phenotype of XRCC4- and Ku80-deficient cells with regard to the repair of enzyme-induced DSBs mirrors the embryonic lethality of XRCC4 knock-out mice as opposed to the viability of the Ku80 knock-out. Thus, I-SceI-induced breaks may resemble DSBs arising during normal DNA metabolism and mouse development. The removal of these breaks likely has different genetic requirements than the repair of radiation-induced DSBs.

Hierarchy of nonhomologous end-joining, single-strand annealing and gene conversion at site-directed DNA double-strand breaks.

In mammalian cells, DNA double-strand breaks (DSBs) are repaired by three pathways, nonhomologous end-joining (NHEJ), gene conversion (GC) and single-strand annealing (SSA). These pathways are distinct with regard to repair efficiency and mutagenic potential and must be tightly controlled to preserve viability and genomic stability. Here, we employed chromosomal reporter constructs to characterize the hierarchy of NHEJ, GC and SSA at a single I-SceI-induced DSB in Chinese hamster ovary cells. We discovered that the use of GC and SSA was increased by 6- to 8-fold upon loss of Ku80 function, suggesting that NHEJ is dominant over the other two pathways. However, NHEJ efficiency was not altered if GC was impaired by Rad51 knockdown. Interestingly, when SSA was made available as an alternative mode for DSB repair, loss of Rad51 function led to an increase in SSA activity at the expense of NHEJ, implying that Rad51 may indirectly promote NHEJ by limiting SSA. We conclude that a repair hierarchy exists to limit the access of the most mutagenic mechanism, SSA, to the break site. Furthermore, the cellular choice of repair pathways is reversible and can be influenced at the level of effector proteins such as Ku80 or Rad51.

Establishment of a Transformation Coupled in vitro End Joining Assay to Estimate Radiosensitivity in Tumor Cells.

Here, we present a modified in vitro end-joining (EJ) assay to quantify EJ capacity, accuracy as well as pathway switch to alternative end-joining (Alt-EJ) or single strand annealing (SSA). A novel transformation assay was established to specifically measure circular repair products, which correlate with classical EJ efficiency. The EJ assay was validated using EJ-deficient mammalian cell lines (Ku80, DNA-PKcs, LigIV, or XRCC4 mutants). A pathway switch to Alt-EJ and SSA was seen exclusively in Ku-deficient cells. Circular EJ product formation correlated with cell survival and DSB repair capacity after X-irradiation. Investigation of 14 HNSCC cell lines revealed differences in the total EJ capacity but a broader variation in the amount of circular repair products. Sequencing of repair junctions in HNSCC cells demonstrated a predominance of high-fidelity EJ and an avoidance of both Alt-EJ and SSA. A significant correlation was observed between the amount of circular repair products, repair of IR-induced DSB and radiosensitivity. Collectively, these data indicate that the presented in vitro-EJ-assay can not only estimate the repair capacity of cancer cells to enable the stratification into radiosensitive or radioresistant, but can also identify repair pathway deregulation such as a switch to Alt-EJ or SSA, which enables tumor targeting.

The extreme radiosensitivity of the squamous cell carcinoma SKX is due to a defect in double-strand break repair.

PURPOSE: Squamous cell carcinomas (SCCs) are characterized by moderate radiosensitivity. We have established the human head & neck SCC cell line SKX, which shows an exceptionally high radiosensitivity. It was the aim of this study to understand the underlying mechanisms. MATERIALS & METHODS: Experiments were performed with SKX and FaDu, the latter taken as a control of moderate radiosensitivity. Cell lines were grown as xenografts as well as cell cultures. For xenografts, radiosensitivity was determined via local tumour control assay, and for cell cultures using colony assay. For cell cultures, apoptosis was determined by Annexin V staining and G1-arrest by BrdU labelling. Double-strand breaks (DSBs) were detected by both constant-field gel electrophoresis (CFGE) and gammaH2AX-foci technique; DSB rejoining was also assessed by in vitro rejoining assay; chromosomal damage was determined by G01-assay. RESULTS: Compared to FaDu, SKX cells are extremely radiosensitive as found for both xenografts (TCD(50) for 10 fractions 46.0Gy [95% C.I.: 39; 54 Gy] vs. 18.9 Gy [95% C.I.: 13; 25Gy]) and cell cultures (D(0.01); 7.1 vs. 3.5Gy). Both cell lines showed neither radiation-induced apoptosis nor radiation-induced permanent G1-arrest. For DSBs, there was no difference in the induction but for repair with SKX cells showing a higher level of both, slowly repaired DSBs and residual DSBs. The in vitro DSB repair assay revealed that SKX cells are defective in nonhomologous endjoining (NHEJ), and that more than 40% of DSBs are rejoined by single-strand annealing (SSA). SKX cells also depicted a two-fold higher number of lethal chromosomal aberrations when compared to FaDu cells. CONCLUSIONS: The extreme radiosensitivity of the SCC SKX seen both in vivo and in vitro can be ascribed to a reduced DNA double-strand break repair, resulting from a defect in NHEJ. This defect might be due to preferred usage of other pathways, such as SSA, which prevents efficient endjoining.

"Meeting Report DNA Repair 2006: Ninth Biennial Meeting of the German Society for Research on DNA Repair" [DNA Repair 6 (2007) 140-144].

A comprehensive meeting on current aspects of DNA repair organized by, Jochen Dahm-Daphi, Ekkehard Dikomey, Alexander Bürkle, Marlis Frankenberg-Schwager, Frank Grosse, Andrea Hartwig, George Iliakis, Bernd Kaina, Jürgen Thomale, and Lisa Wiesmüller was held in Hamburg, Germany from 12 to 15 September 2006. This article summarizes information of invited lectures and proferred papers of six plenary sessions.

DNA Repair 2006: ninth biennial meeting of the German Society for Research on DNA repair.

A comprehensive meeting on current aspects of DNA repair organized by, Jochen Dahm-Daphi, Ekkehard Dikomey, Alexander B_rkle, Marlis Frankenberg-Schwager, Frank Grosse, Andrea Hartwig, George Iliakis, Bernd Kaina, J_rgen Thomale, and Lisa Wiesm_ller was held in Hamburg, Germany from September 12 to 15, 2006. This article summarizes information of invited lectures and proferred papers of six plenary sessions.

Radiosensitization of tumour cell lines by the polyphenol Gossypol results from depressed double-strand break repair and not from enhanced apoptosis.

PURPOSE: New drugs are needed to increase the efficiency of radiotherapy in order to improve the therapeutic outcome of tumour patients. In this respect, the polyphenol Gossypol might be of interest, because of its effect on apoptosis and DNA repair, which is either mediated directly or indirectly via the inositol phosphate metabolism. It was investigated, whether these effects result in enhanced radiosensitivity of tumour cells. MATERIAL AND METHODS: Tumour cell lines investigated: A549, FaDu, H1299, MCF7 and Du145. Cell cycle distribution was determined by FACS analysis, apoptosis was measured by DAPI staining and caspase3/7 activity. Double-strand breaks (DSB) were investigated via gammaH2AX-foci and cell survival by colony formation assay. The level of inositol phosphates was determined by HPLC, protein expression by Western blot. RESULTS: In A549 cells, Gossypol at concentrations 1microM strongly affects proliferation with only a modest arrest in the G1-phase, but with no increase in the fraction of apoptotic cells or the number of additional DSB. Additional DSB were only seen in FaDu cells, where Gossypol (2microM) was extremely toxic with a plating efficiency <0.002. When combined with irradiation, incubation with Gossypol (1-2microM) was found to result in an enhanced radiosensitivity with, however, a substantial variation. While there was a strong radiosensitization for FaDu and Du145 cells, there was an intermediate response for A549 cells, but almost no effect for H1299 and MCF7 cells. This sensitization was not caused from an elevated rate of apoptosis, but primarily resulted from reduced DSB repair capacity. The reduction in DSB repair could be ascribed neither to changes in the level of repair proteins relevant for non-homologous end-joining (Ku70, Ku80, DNA-PKcs) nor to changes in the level of higher phosphorylated inositols, whereby the latter were even found to be enhanced by Gossypol. CONCLUSIONS: For some tumour cell lines treatment with low concentrations of Gossypol can be used to inhibit DSB repair capacity and with that to increase the cellular radiosensitivity.

Nonhomologous end-joining of site-specific but not of radiation-induced DNA double-strand breaks is reduced in the presence of wild-type p53.

Nonhomologous end-joining (NHEJ) of DNA double-strand breaks (DSBs) entails two principal mechanisms: modification of DNA ends prior to ligation (error-prone rejoining) or precise ligation without modification if the DNA ends are complementary (error-free repair). Error-prone rejoining is mutagenic, because it can lead to destruction of coding sequence or to chromosomal aberrations, and therefore must be tightly regulated. Previous studies on the role of the p53 tumor suppressor in the regulation of NHEJ have yielded conflicting results, but a rigorous analysis of NHEJ proficiency and fidelity in a purely chromosomal context has not been carried out. To this end, we created novel repair plasmid substrates that integrate into the genome. DSBs generated by the I-SceI endonuclease within these substrates were repaired by either error-prone rejoining or precise ligation. We found that the expression of wild-type p53 inhibited any repair-associated DNA sequence deletion, including a more than 250-fold inhibition of error-prone rejoining events compared to p53-null cells, while any promoting effect of p53 on precise ligation could not be directly evaluated. The role of p53 in NHEJ appeared to involve a direct transactivation-independent mechanism, possibly restricting DNA end-modification by blocking the annealing of single strands along flanking stretches of microhomology. The inhibition of error-prone rejoining by p53 did not apply to the rejoining of DSBs induced by ionizing radiation. In conclusion, our data suggest that p53 restricts the mutagenic effects of NHEJ without compromising repair proficiency or cell survival, thereby maintaining genomic stability.

Short-course radiotherapy is not optimal for spinal cord compression due to myeloma.

PURPOSE: To investigate the suitability of short-course radiotherapy (RT) for spinal cord compression (SCC) in myeloma patients. METHODS AND MATERIALS: Data for 172 myeloma patients irradiated between January 1994 and December 2004 for SCC were retrospectively evaluated. Short-course RT (1 x 8 Gy, 5 x 4 Gy, n = 61) and long-course RT (10 x 3 Gy, 15 x 2.5 Gy, 20 x 2 Gy, n = 111) were compared for functional outcome up to 24 months after RT. In addition, 10 potential prognostic factors were investigated. RESULTS: Improvement of motor function occurred in 90 patients (52%). Forty-seven percent of nonambulatory patients regained the ability to walk. Functional outcome was significantly influenced by the time of developing motor deficits before RT. Improvement of motor function was more frequent after long-course RT than after short-course RT: 59% vs. 39% (p = 0.10) at 1 month, 67% vs. 43% (p = 0.043) at 6 months, 76% vs. 40% (p = 0.003) at 12 months, 78% vs. 43% (p = 0.07) at 18 months, and 83% v 54% (p = 0.33) at 24 months. A subgroup analysis of the long-course RT group demonstrated a similar functional outcome for 10 x 3 Gy when compared with 15 x 2.5 Gy and 20 x 2 Gy. CONCLUSIONS: Long-course RT is preferable for SCC in myeloma patients because it resulted in better functional outcome than short-course RT. Treatment with 10 x 3 Gy can be considered appropriate.

Indicators of late normal tissue response after radiotherapy for head and neck cancer: fibroblasts, lymphocytes, genetics, DNA repair, and chromosome aberrations.

PURPOSE: To investigate the relationship between late tissue response after radiotherapy, cellular sensitivity and DNA repair capacity measured in dermal fibroblasts and chromosomal aberrations measured in lymphocytes. The study was in particular designed to compare cellular parameters of patients with maximum differences in late tissue reactions. MATERIALS AND METHODS: The study was performed with 16 pair-wise matched head and neck cancer patients 2-7 years after curative therapy exhibiting maximum differences (grade 1 vs. grade 3) in late normal tissue reactions. Clinical endpoints were fibrosis, telangiectasia, mucositis and xerostomia using the radiation therapy oncology group score. Patients with grade 3 reactions were tested for mutations in ataxia telangiectasia (AT), Nijmegen Breakage Syndrome (NBS), MRE11, RAD50 and DNA ligase IV genes by means of polymerase chain reaction-single-strand conformation polymorphism and sequencing analysis. Skin fibroblasts obtained from biopsies were used to determine the cellular sensitivity by colony formation and the induction and repair of DNA double-strand breaks (dsb) using constant-field gel electrophoresis. Lymphocytes were taken to measure chromosomal damage either in metaphase using conventional chromosome analysis or in G(0) using premature chromosome condensation (PCC)-technique. RESULTS: Patients with extreme late reactions (grade 3) showed no evidence for an AT, NBS, MRE11 or RAD50 mutation. Studies with fibroblasts revealed that extreme late reactions were associated neither with a pronounced cellular radiosensitivity nor with a difference in dsb repair capacity. In contrast, there was a significant difference in chromosomal damage measured in lymphocytes. After in vitro irradiation with 6Gy, lymphocytes taken from overreacting patients showed on average a significantly higher number of lethal aberrations than lymphocytes isolated from patients with mild reactions (7.2+/-0.8 vs. 5.0+/-0.3). Similar differences were found for PCC fragments. CONCLUSION: This study suggests that lymphocytes are more promising than fibroblasts to predict patient's normal tissue response after radiotherapy.

Inhibition of PARP1-dependent end-joining contributes to Olaparib-mediated radiosensitization in tumor cells.

Poly-ADP-ribose-polymerase inhibitors (PARPi) are considered to be optimal tools for specifically enhancing radiosensitivity. This effect has been shown to be replication-dependent and more profound in HR-deficient tumors. Here, we present a new mode of PARPi-mediated radiosensitization which was observed in four out of six HR-proficient tumor cell lines (responders) investigated, but not in normal cells. This effect is replication-independent, as the radiosensitization remained unaffected following the inhibition of replication using aphidicolin. We showed that responders are radiosensitized by Olaparib because their DSB-repair is switched to PARP1-dependent end-joining (PARP1-EJ), as evident by (i) the significant increase in the number of residual γH2AX foci following irradiation with 3Gy and treatment with Olaparib, (ii) the enhanced enrichment of PARP1 at the chromatin after 3Gy and (iii) the inhibition of end-joining activity measured by a specific reporter substrate upon Olaparib treatment. This is the first study which directly demonstrates the switch to PARP1-EJ in tumor cells and its contribution to the response to Olaparib as a radiosensitizer, findings which could widen the scope of application of PARPi in tumor therapy.

ATM is required for the repair of Topotecan-induced replication-associated double-strand breaks.

PURPOSE: DNA replication is a promising target for anti-cancer therapies. Therefore, the understanding of replication-associated DNA repair mechanisms is of great interest. One key factor of DNA double-strand break (DSB) repair is the PIK kinase Ataxia-Telangiectasia Mutated (ATM) but it is still unclear whether ATM is involved in the repair of replication-associated DSBs. Here, we focused on the involvement of ATM in homology-directed repair (HDR) of indirect DSBs associated with replication. MATERIAL AND METHODS: Experiments were performed using ATM-deficient and -proficient human cells. Replication-associated DSBs were induced with Topotecan (TPT) and compared with γ-irradiation (IR). Cell survival was measured by clonogenic assay. Overall DSB repair and HDR were evaluated by detecting residual γH2AX/53BP1 and Rad51 foci, respectively. Cell cycle distribution was analysed by flow cytometry and protein expression by Western blot. RESULTS: ATM-deficiency leads to enhanced numbers of residual DSBs, resulting in a pronounced S/G2-block and decreased survival upon TPT-treatment. In common with IR, persisting Rad51 foci were detected following TPT-treatment. CONCLUSIONS: These results demonstrate that ATM is essentially required for the completion of HR-mediated repair of TPT-induced DSBs formed indirectly at replication forks.

The absence of Ku but not defects in classical non-homologous end-joining is required to trigger PARP1-dependent end-joining.

Classical-non-homologous end-joining (C-NHEJ) is considered the main pathway for repairing DNA double strand breaks (DSB) in mammalian cells. When C-NHEJ is defective, cells may switch DSB repair to an alternative-end-joining, which depends on PARP1 and is more erroneous. This PARP1-EJ is suggested to be active especially in tumor cells contributing to their genomic instability. Here, we define conditions under which cells would switch the repair to PARP1-EJ. Using the end jining repair substrate pEJ, we revealed that PARP1-EJ is solely used when Ku is deficient but not when either DNA-PKcs or Xrcc4 is lacking. In the latter case, DSB repair, however, could be shuttled to PARP1-EJ after additional Ku80 down-regulation, which partly rescued the DSB repair in these mutants. We demonstrate here that PARP-EJ may work on DSB ends at high fidelity manner, as evident from the unchanged efficiency upon blocking end resection by either roscovitin or mirin. Furthermore, we demonstrate for that PARP-EJ is likewise involved in the repair of multiple DSBs (I-PpoI- and IR-induced). Importantly, we identified a chromatin signature associated with the switch to PARP1-EJ which is characterized by a strong enrichment of both PARP1 and LigIII at damaged chromatin. Together, these data indicate that Ku is the main regulator for the hierarchal organization between C-NHEJ and PARP1-EJ.

Residual γH2AX foci predict local tumour control after radiotherapy.

PURPOSE: Evaluation of micromilieu-dependent quantified γH2AX foci as a potential predictive biomarker in well-oxygenated tumour areas in 9 HNSCC xenograft models in vivo. MATERIALS & METHODS: GammaH2AX foci were quantified in perfused tumour areas 30 min (initial γH2AX foci) and 24 h (residual γH2AX foci) after exposure to a single dose of 4 Gy. The initial and residual normalised γH2AX foci were correlated with TCD50 after single dose irradiation under clamped blood flow (SDclamp) or a fractionated irradiation setting under ambient blood flow (fx). RESULTS: A significant negative correlation between initial and residual normalised γH2AX foci and TCD50 SDclamp and TCD50 fx for 9 HNSCC tumour xenograft models in vivo was found. Residual normalised γH2AX foci showed higher intertumoural variability and their correlation with TCD50 was more robust. CONCLUSIONS: For the first time a significant negative correlation between γH2AX foci and local tumour control after irradiation has been demonstrated. Our results underline the potential of residual γH2AX foci as a predictive biomarker for local tumour control after radiotherapy.

Detection of impaired homologous recombination repair in NSCLC cells and tissues.

INTRODUCTION: Homologous recombination repair (HRR) is a critical pathway for the repair of DNA damage caused by cisplatin or poly-ADP ribose polymerase (PARP) inhibitors. HRR may be impaired by multiple mechanisms in cancer, which complicates assessing the functional HRR status in cells. Here, we monitored the ability of non-small-cell lung cancer (NSCLC) cells to form subnuclear foci of DNA repair proteins as a surrogate of HRR proficiency. METHODS: We assessed clonogenic survival of 16 NSCLC cell lines in response to cisplatin, mitomycin C (MMC), and the PARP inhibitor olaparib. Thirteen tumor explants from patients with NSCLC were subjected to cisplatin ex vivo. Cells were assayed for foci of repair-associated proteins such as BRCA1, FANCD2, RAD51, and γ-H2AX. RESULTS: Four cell lines (25%) showed an impaired RAD51 foci-forming ability in response to cisplatin. Impaired foci formation correlated with cellular sensitivity to cisplatin, MMC and olaparib. Foci responses complemented or superseded genomic information suggesting alterations in the ATM/ATR and FA/BRCA pathways. Because baseline foci in untreated cells did not predict drug sensitivity, we adapted an ex vivo biomarker assay to monitor damage-induced RAD51 foci in NSCLC explants from patients. Ex vivo cisplatin treatment of explants identified two tumors (15%) exhibiting compromised RAD51 foci induction. CONCLUSIONS: A fraction of NSCLC harbors HRR defects that may sensitize the affected tumors to DNA-damaging agents including PARP inhibitors. We propose that foci-based functional biomarker assays represent a powerful tool for prospective determination of treatment sensitivity, but will require ex vivo techniques for induction of DNA damage to unmask the underlying HRR defect.