Jochen Dahm-Daphi

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.

Correlation between cellular radiosensitivity and non-repaired double-strand breaks studied in nine mammalian cell lines.

PURPOSE To test the relationship between cell killing and non-repaired DNA strand breaks both in repair proficient and deficient cell lines. MATERIALS AND METHODS Five of the cell lines used are repair competent (CHO, CHO K1, rat rhabdomyosarcoma R1H, mouse balb and normal human fibroblasts), while four display a reduced repair capacity (scid, xrs1, xrs5, AT). Cell survival was determined by colony formation assay. The total number of strand breaks was measured by the alkaline unwinding technique and the numbers of double-strand breaks by constant-field gel electrophoresis. RESULTS The nine cell lines showed a broad spectrum in radiosensitivity with SF2 values ranging from 0.018 to 0.58. The cell lines did not vary in the number of induced strand breaks, neither for all strand breaks nor for double-strand breaks alone. In contrast, there was a large variation in the number of non-repaired strand breaks measured 24 h after irradiation. Comparison of cell killing with the number of non-repaired breaks measured after a dose of 90 Gy showed no correlation for single-strand breaks (r2=0.29) but a fairly good correlation for double-strand breaks (r2=0.87). This correlation was found to hold both for repair proficient and deficient cell lines. CONCLUSIONS The results obtained strongly suggest that the number of non-repaired double-strand breaks measured 24h after irradiation can be used as an indicator of cellular radiosensitivity.

Repair of radiation damage to DNA

DNA double-strand breaks constitute the most dangerous type of DNA damage induced by ionising radiation (IR). Accordingly, the resistance of cells to IR is modulated by three intimately related cellular processes: DNA repair, recombination, and replication. Significant discoveries in this field of research have been made over the last few years. A picture seems to be emerging in which perturbations of recombination in cancer cells are a more widespread cause of genomic instability than previously appreciated. Conversely, such cells may also be more sensitive to certain chemotherapeutic drugs and to IR. Thus, the alterations in recombination that promote carcinogenesis by causing genomic instability may also be the weakness of the tumours that arise in this setting, a concept which could hold great promise for the advancement of cancer treatment in the not too distant future.

The epidermal growth factor receptor modulates DNA double-strand break repair by regulating non-homologous end-joining.

In mammalian cells repair of radiation-induced DNA damage appears to be also controlled by the epidermal growth factor receptor (EGFR) with a special impact on DNA double-strand break (DSB) repair. Aim of this study was to demonstrate this interaction between EGFR signalling and DNA DSB repair and to identify the underlying downstream pathways. We especially wanted to know in how far non-homologous end-joining (NHEJ) as the most important DSB repair pathway is involved in this interaction. Overall DSB repair was determined by counting gammaH2AX foci remaining 24 after irradiation, while NHEJ activity was monitored by using a specially designed repair construct stably integrated into the genome. The overall DSB repair capacity was clearly enhanced when EGFR was activated by its natural ligand EGF and, vice versa, was reduced when EGFR was blocked either by the specific antibody Cetuximab or the tyrosine kinase inhibitor erlotinib, whereby reduction was clearly stronger for erlotinib. There was also a difference in the pathways affected. While erlotinib lead to a block of both, MAPK as well as AKT signalling, Cetuximab only affected MAPK. As demonstrated by specific inhibitors (PD98059, AKTIII) EGFR interacts with DSB repair mostly via MAPK pathway. Also for NHEJ activity, there was a substantial increase, when EGFR was activated by EGF as determined for two different reporter cell lines (A549.EJ and H1299.EJ) and, vice versa, a reduction was seen when EGFR signalling was blocked by Cetuximab or erlotinib. There was, however, no difference for the two inhibitors used. This regulation of NHEJ by EGFR was only blocked when ERK was affected by siRNA but not when AKT was knocked down. These data indicate that EGFR modulates DSB repair by regulating NHEJ via MAPK signalling.

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 patients normal tissue response after radiotherapy.

Comparison of biological effects of DNA damage induced by ionizing radiation and hydrogen peroxide in CHO cells

Purpose: Free OH radicals are considered to be the common mediator of DNA damage after ionizing radiation and oxidative stress. In particular, double-strand breaks (dsb) have a major impact on cell killing after irradiation, while the mechanism of cell killing is less clear for oxidative injury. The latter not only affects DNA, but also equally other cell compartments, such as membranes and mitochondria, which may trigger cell death. This study intended to clarify the relationship between DNA damage induction, repair and cell inactivation for hydrogen peroxide and ionizing radiation. Materials and methods: Chinese hamster ovary (CHO) cells were treated with H 2 O 2 in serum-free medium in combination with/without X-irradiation. DNA damage was measured using the alkaline unwinding method or neutral constant-field gel electrophoresis. Cell survival was recorded using the colony-formation assay. Results: Hydrogen peroxide induced a large number of single-strand breaks (ssb>36000/cell) without impairing cell survival. This number reached a maximum (36Gy-equiv. at 3x10 -4 mol/dm3) without further increase after higher concentrations. Repair kinetics of ssb were similar to those after irradiation. Dsb were found only after very high concentrations of H 2 O 2 (>3x10 -2 mol/dm 3) , which is different from irradiation which generated ssb and dsb in the same dose range. A linear-quadratic increase of dsb was found with increasing concentrations of H 2 O 2 suggesting a single or a pairwise action of OH radicals to form a dsb. After either irradiation or peroxide treatment cell killing was observed only after doses which also allowed dsb detection. The number of dsb calculated per lethal event was in the same range but slightly higher after irradiation (1.7-fold) than after H 2 O 2 treatment. Conclusions: Cell killing after irradiation or hydrogen peroxide appears to be due to dsb, whereas cells withstand large numbers of single-strand lesions and other types of non-DNA damage occurring at lower concentrations of hydrogen peroxide. The number of ssb saturates at intermediate concentrations of H 2 O 2 suggesting that a limited amount of chromatin-bound metal ions is available for OH radical generation.PURPOSE Free OH radicals are considered to be the common mediator of DNA damage after ionizing radiation and oxidative stress. In particular, double-strand breaks (dsb) have a major impact on cell killing after irradiation, while the mechanism of cell killing is less clear for oxidative injury. The latter not only affects DNA, but also equally other cell compartments, such as membranes and mitochondria, which may trigger cell death. This study intended to clarify the relationship between DNA damage induction, repair and cell inactivation for hydrogen peroxide and ionizing radiation. MATERIALS AND METHODS Chinese hamster ovary (CHO) cells were treated with H2O2 in serum-free medium in combination with/ without X-irradiation. DNA damage was measured using the alkaline unwinding method or neutral constant-field gel electrophoresis. Cell survival was recorded using the colony-formation assay. RESULTS Hydrogen peroxide induced a large number of single-strand breaks (ssb>36000/cell) without impairing cell survival. This number reached a maximum (36 Gy-equiv. at 3 x 10(-4) mol/dm3) without further increase after higher concentrations. Repair kinetics of ssb were similar to those after irradiation. Dsb were found only after very high concentrations of H2O2 (>3 x 10(-2) mol/dm3), which is different from irradiation which generated ssb and dsb in the same dose range. A linear-quadratic increase of dsb was found with increasing concentrations of H2O2 suggesting a single or a pairwise action of OH radicals to form a dsb. After either irradiation or peroxide treatment cell killing was observed only after doses which also allowed dsb detection. The number of dsb calculated per lethal event was in the same range but slightly higher after irradiation (1.7-fold) than after H2O2 treatment. CONCLUSIONS Cell killing after irradiation or hydrogen peroxide appears to be due to dsb, whereas cells withstand large numbers of single-strand lesions and other types of non-DNA damage occurring at lower concentrations of hydrogen peroxide. The number of ssb saturates at intermediate concentrations of H2O2 suggesting that a limited amount of chromatin-bound metal ions is available for OH radical generation.

EGF Receptor Inhibition Radiosensitizes NSCLC Cells by Inducing Senescence in Cells Sustaining DNA Double-Strand Breaks

The mechanisms by which inhibition of the epidermal growth factor receptor (EGFR) sensitizes non-small cell lung cancer (NSCLC) cells to ionizing radiation remain poorly understood. We set out to characterize the radiosensitizing effects of the tyrosine kinase inhibitor erlotinib and the monoclonal antibody cetuximab in NSCLC cells that contain wild-type p53. Unexpectedly, EGFR inhibition led to pronounced cellular senescence but not apoptosis of irradiated cells, both in vitro and in vivo. Senescence was completely dependent on wild-type p53 and associated with a reduction in cell number as well as impaired clonogenic radiation survival. Study of ten additional NSCLC cell lines revealed that senescence is a prominent mechanism of radiosensitization in 45% of cell lines and occurs not only in cells with wild-type p53 but also in cells with mutant p53, where it is associated with an induction of p16. Interestingly, senescence and radiosensitization were linked to an increase in residual radiation-induced DNA double-strand breaks irrespective of p53/p16 status. This effect of EGFR inhibition was at least partially mediated by disruption of the MEK-ERK pathway. Thus, our data indicate a common mechanism of radiosensitization by erlotinib or cetuximab across diverse genetic backgrounds. Our findings also suggest that assays that are able to capture the initial proliferative delay that is associated with senescence should be useful for screening large cell line panels to identify genomic biomarkers of EGFR inhibitor-mediated radiosensitization.

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.

Comparison of Short-Course versus Long-Course Whole-Brain Radiotherapy in the Treatment of Brain Metastases

Background and Purpose:Whole-brain radiotherapy (WBRT) is the most common treatment for brain metastases. Most of these patients have a poor survival prognosis. Therefore, a short radiation program is preferred, if it provides a similar outcome as longer programs. This study compares 20 Gy in five fractions (treatment time: 1 week) to longer programs, with higher doses including 30 Gy in ten fractions (2 weeks) and 40 Gy in 20 fractions (4 weeks).Patients and Methods:Data regarding 1,085 patients treated with WBRT for brain metastases were retrospectively analyzed. 387 patients received 20 Gy in five fractions, and 698 patients received higher doses (30 Gy in ten fractions, n = 527, or 40 Gy in 20 fractions, n = 171). In addition, eight potential prognostic factors were investigated including age, sex, Karnofsky Performance Score (KPS), tumor type, interval from tumor diagnosis to WBRT, number of brain metastases, extracranial metastases, and recursive partitioning analysis (RPA) class. Subgroup analyses were performed for each RPA class individually.Results:The WBRT schedule had no significant impact on survival (p = 0.415). On multivariate analysis, improved survival was significantly associated with age ≤ 60 years (risk ratio [RR]: 1.28; p < 0.001), KPS ≥ 70 (RR: 1.73; p = 0.002), lack of extracranial metastases (RR: 1.27; p = 0.007), interval from tumor diagnosis to WBRT > 8 months (RR: 1.19; p = 0.011), and lower RPA class (RR: 1.56; p < 0.001). The subgroup analyses for each RPA class did not reveal a significant association between WBRT schedule and survival.Conclusion:Short-course WBRT with 20 Gy in five fractions is preferable for most patients, because it is associated with similar survival as longer programs and is less time-consuming.Hintergrund und Ziel:Die Ganzhirnbestrahlung ist die häufigste Therapieform bei der Behandlung von Hirnmetastasen. Die meisten dieser Patienten haben eine schlechte Überlebensprognose. Deshalb wäre eine Kurzzeittherapie zu bevorzugen, vorausgesetzt, sie ist ähnlich effektiv wie Langzeitschemata. Diese Studie vergleicht 20 Gy in fünf Fraktionen (Behandlungszeit: 1 Woche) mit Langzeitschemata wie 30 Gy in zehn Fraktionen (2 Wochen) und 40 Gy in 20 Fraktionen (4 Wochen).Patienten und Methodik:Daten von 1 085 Patienten mit Hirnmetastasen, die eine Ganzhirnbestrahlung erhielten, wurden retrospektiv analysiert (Tabelle 1). 387 Patienten erhielten 20 Gy in fünf Fraktionen, 698 Patienten höhere Gesamtdosen (30 Gy in zehn Fraktionen, n = 527, oder 40 Gy in 20 Fraktionen, n = 171). Acht weitere potentielle Prognosefaktoren wurden untersucht: Alter, Geschlecht, Karnofsky-Performance-Score (KPS), Art des Primärtumors, Intervall von der Erstdiagnose des Tumors bis zur Ganzhirnbestrahlung, Anzahl der Hirnmetastasen, Vorliegen extrakranieller Metastasen und die „recursive partitioning analysis“-(RPA-)Klasse. Für jede der drei RPA-Klassen wurden Subgruppenanalysen durchgeführt.Ergebnisse:Das Strahlentherapieregime hatte keinen signifikanten Einfluss auf das Überleben (p = 0,415; Tabelle 2, Abbildung 1). In der multivariaten Analyse (Tabelle 3) war ein besseres Überleben signifikant mit einem Alter ≤ 60 Jahre (Risiko-Ratio [RR]: 1,28; p < 0,001), einem KPS ≥ 70 (RR: 1,73; p = 0,002), Nichtvorliegen extrakranieller Metastasen (RR: 1,27; p = 0,007), einem Intervall von der Erstdiagnose des Tumors bis zur Ganzhirnbestrahlung > 8 Monate (RR: 1,19; p = 0,011) und einer niedrigeren RPA-Klasse (RR: 1,56; p < 0,001) assoziiert. Die Subgruppenanalysen der einzelnen RPA-Klassen (Tabelle 4) zeigten ebenfalls keinen signifikanten Einfluss des Strahlentherapieregimes auf das Überleben.Schlussfolgerung:Die Kurzzeit-Ganzhirnbestrahlung mit 20 Gy in fünf Fraktionen ist bei den meisten Patienten zu bevorzugen, da sie mit ähnlichen Überlebensraten einhergeht wie die Langzeit-Ganzhirnbestrahlung, aber weniger zeitintensiv ist.

Radiosensitivity of human tumour cells is correlated with the induction but not with the repair of DNA double-strand breaks

Nine human tumour cell lines (four mammary, one bladder, two prostate, one cervical, and one squamous cell carcinoma) were studied as to whether cellular radiosensitivity is related to the number of initial or residual double-strand breaks (dsb). Cellular sensitivity was measured by colony assay and dsb by means of constant- and graded-field gel electrophoresis (CFGE and GFGE, respectively). The nine tumour cell lines showed a broad variation in cellular sensitivity (SF2 0.17–0.63). The number of initial dsb as measured by GFGE ranged between 14 and 27 dsb/Gy/diploid DNA content. In contrast, normal fibroblasts raised from skin biopsies of seven individuals showed only a marginal variation with 18–20 dsb/Gy/diploid DNA content. For eight of the nine tumour cell lines, there was a significant correlation between the number of initial dsb and the cellular radiosensitivity. The tumour cells showed a broad variation in the amount of dsb measured 24 h after irradiation by CFGE, which, however, was not correlated with the cellular sensitivity. This residual damage was found to be influenced not only by the actual number of residual dsb, but also by apoptosis and cell cycle progression which had impact on CFGE measurements. Some cell line strains were able to proliferate even after exposure to 150 Gy while others were found to degrade their DNA. Our results suggest that for tumour cells, in contrast to normal cells, the variation in sensitivity is mainly determined by differences in the initial number of dsb induced.