Mechanism of cell killing after ionizing radiation by a dominant negative DNA polymerase beta.

Several types of DNA lesion are induced after ionizing irradiation (IR) of which double strand breaks (DSBs) are expected to be the most lethal, although single strand breaks (SSBs) and DNA base damages are quantitatively in the majority. Proteins of the base excision repair (BER) pathway repair these numerous lesions. DNA polymerase beta has been identified as a crucial enzyme in BER and SSB repair (SSBR). We showed previously that inhibition of BER/SSBR by expressing a dominant negative DNA polymerase beta (polbetaDN) resulted in radiosensitization. We hypothesized increased kill to result from DSBs arising from unrepaired SSBs and BER intermediates. We find here higher numbers of IR-induced chromosome aberrations in polbetaDN expressing cells, confirming increased DSB formation. These aberrations did not result from changes in DSB induction or repair of the majority of lesions. SSB conversion to DSBs has been shown to occur during replication. We observed an increased induction of chromatid aberrations in polbetaDN expressing cells after IR, suggesting such a replication-dependence of secondary DSB formation. We also observed a pronounced increase of chromosomal deletions, the most likely cause of the increased kill. After H(2)O(2) treatment, polbetaDN expression only resulted in increased chromatid (not chromosome) aberrations. Together with the lack of sensitization to H(2)O(2), these data further suggest that the additional secondarily induced lethal DSBs resulted from repair attempts at complex clustered damage sites, unique to IR. Surprisingly, the polbetaDN induced increase in residual gammaH2AX foci number was unexpectedly low compared with the radiosensitization or induction of aberrations. Our data thus demonstrate the formation of secondary DSBs that are reflected by increased kill but not by residual gammaH2AX foci, indicating an escape from gammaH2AX-mediated DSB repair. In addition, we show that in the polbetaDN expressing cells secondary DSBs arise in a radiation-specific and partly replication-dependent manner.

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.

In normal human fibroblasts variation in DSB repair capacity cannot be ascribed to radiation-induced changes in the localisation, expression or activity of major NHEJ proteins.

BACKGROUND AND PURPOSE: The aim of the present study was to test whether for normal human fibroblasts the variation in double-strand break (DSB) repair capacity results from radiation-induced differences in localisation, expression or activity of major non-homologous end-joining (NHEJ) proteins. MATERIALS AND METHODS: Experiments were performed with 11 normal human fibroblast strains AF01-11. NHEJ proteins were determined by Western blot and DNA-PK activity by pulldown-assay. RESULTS: The four NHEJ proteins tested (Ku70, Ku80, XRCC4 and DNA-PKcs) were found to be localised almost exclusively in the nucleus with no detectable amount in the cytoplasm. This distribution was not altered upon irradiation. In non-irradiated cells the level of these proteins varied with a CV ranging between 16% and 20%, but there was no correlation with the respective cellular DSB repair capacity. Irradiation (3.5 and 15 Gy) did not alter the expression of these proteins and there was also no change in the DNA-PK activity. These results indicate that the variation in DSB repair capacity determined for these fibroblasts can be ascribed to differences neither in the localisation or expression of Ku70, Ku80 and XRCC4 nor in the activity of the DNA-PK complex induced upon irradiation. CONCLUSIONS: For normal human fibroblasts, the level or activity of NHEJ proteins measured prior to or after irradiation cannot be used to predict the DSB repair capacity or cellular radiosensitivity.

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.

EGFR-targeted anti-cancer drugs in radiotherapy: preclinical evaluation of mechanisms.

Preclinical and clinical results indicate that the EGFR can mediate radioresistance in different solid human tumours. Combination of radiotherapy and EGFR inhibitors can improve local tumour control compared to irradiation alone and has been introduced into clinical radiotherapy practice. So far several mechanisms have been identified in preclinical studies to contribute to improved local tumour control after radiation combined with EGFR inhibitors. These include direct kill of cancer stem cells by EGFR inhibitors, cellular radiosensitization through modified signal transduction, inhibition of repair of DNA damage, reduced repopulation and improved reoxygenation during fractionated radiotherapy. Effects and mechanisms may differ for different classes of EGFR inhibitors, for different tumours and for normal tissues. The mechanisms underlying this heterogeneity are currently poorly understood, and predictive assays are not available yet. Importantly, mechanisms and predictors for the combined effects of radiation with EGFR inhibitors appear to be considerably different to those for application of EGFR inhibitors alone or in combination with chemotherapy. Therefore to further evaluate the efficacy and mechanisms of EGFR-inhibition in combined treatments, radiotherapy-specific preclinical research strategies, which include in vivo experiments using local tumour control as an endpoint, as well as animal studies on normal tissue toxicity are needed.

Blockage of epidermal growth factor receptor-phosphatidylinositol 3-kinase-AKT signaling increases radiosensitivity of K-RAS mutated human tumor cells in vitro by affecting DNA repair.

PURPOSE: It is known that blockage of epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K) activity enhances radiation sensitivity of human tumor cells presenting a K-RAS mutation. In the present study, we investigated whether impaired repair of DNA double-strand breaks (DSB) is responsible for the radiosensitizing effect of EGFR and PI3K inhibition in K-RAS mutated (K-RAS(mt)) cells. EXPERIMENTAL DESIGN: The effect of the EGFR tyrosine kinase inhibitor BIBX1382BS (BIBX) on cellular radiosensitivity was determined in K-RAS(mt) (A549) and K-RAS(wt) (FaDu) cell lines by clonogenic survival assay. Radiation-induced phosphorylation of H2AX (Ser139), ATM (Ser1981), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs; Thr2609) was analyzed by immunoblotting. Twenty-four hours after irradiation, residual DSBs were quantified by identification of gammaH2AX foci and frequency of micronuclei. RESULTS: BIBX reduced clonogenic survival of K-RAS(mt)-A549 cells, but not of K-RAS(wt)-FaDu cells, after single-dose irradiation. Analysis of the radiation-induced H2AX phosphorylation revealed that BIBX, as well as the PI3K inhibitor LY294002, leads to a marked reduction of P-H2AX in K-RAS(mt)-A549 and MDA-MB-231 cells, but not in K-RAS(wt)-FaDu and HH4ded cells. Likewise, radiation-induced autophosphorylation of DNA-PKcs at Thr2609 was only blocked in A549 cells by these two inhibitors and AKT1 small interfering RNA transfection. However, neither in K-RAS(mt) nor in K-RAS(wt) cells the inhibitors did affect radiation-induced ATM phosphorylation. As a consequence of inhibitor treatment, a significant enhancement of both residual DSBs and frequency of micronuclei was apparent only in A549 but not in FaDu cells following radiation. CONCLUSION: Targeting of the EGFR-dependent PI3K-AKT pathway in K-RAS-mutated A549 cells significantly affects postradiation survival by affecting the activation of DNA-PKcs, resulting in a decreased DSB repair capacity.

Radiosensitization of HNSCC cells by EGFR inhibition depends on the induction of cell cycle arrests.

The increase in cellular radiosensitivity by EGF receptor (EGFR) inhibition has been shown to be attributable to the induction of a G1-arrest in p53-proficient cells. Because EGFR targeting in combination with radiotherapy is used to treat head and neck squamous cell carcinomas (HNSCC) which are predominantly p53 mutated, we tested the effects of EGFR targeting on cellular radiosensitivity, proliferation, apoptosis, DNA repair and cell cycle control using a large panel of HNSCC cell lines. In these experiments EGFR targeting inhibited signal transduction, blocked proliferation and induced radiosensitization but only in some cell lines and only under normal (pre-plating) conditions. This sensitization was not associated with impaired DNA repair (53BP1 foci) or induction of apoptosis. However, it was associated with the induction of a lasting G2-arrest. Both, the radiosensitization and the G2-arrest were abrogated if the cells were re-stimulated (delayed plating) with actually no radiosensitization being detectable in any of the 14 tested cell lines. Therefore we conclude that EGFR targeting can induce a reversible G2 arrest in p53 deficient HNSCC cells, which does not consequently result in a robust cellular radiosensitization. Together with recent animal and clinical studies our data indicate that EGFR inhibition is no effective strategy to increase the radiosensitivity of HNSCC cells.

Radiosensitization of NSCLC cells by EGFR inhibition is the result of an enhanced p53-dependent G1 arrest.

PURPOSE: How EGF receptor (EGFR) inhibition induces cellular radiosensitization and with that increase in tumor control is still a matter of discussion. Since EGFR predominantly regulates cell cycle and proliferation, we studied whether a G1-arrest caused by EGFR inhibition may contribute to these effects. MATERIALS AND METHODS: We analyzed human non-small cell lung cancer (NSCLC) cell lines either wild type (wt) or mutated in p53 (A549, H460, vs. H1299, H3122) and HCT116 cells (p21 wt and negative). EGFR was inhibited by BIBX1382BS, erlotinib or cetuximab; p21 was knocked down by siRNA. Functional endpoints analyzed were cell signaling, proliferation, G1-arrest, cell survival as well as tumor control using an A549 tumor model. RESULTS: When combined with IR, EGFR inhibition enhances the radiation-induced permanent G1 arrest, though solely in cells with intact p53/p21 signaling. This increase in G1-arrest was always associated with enhanced cellular radiosensitivity. Strikingly, this effect was abrogated when cells were re-stimulated, suggesting the initiation of dormancy. In line with this, only a small non-significant increase in tumor control was observed for A549 tumors treated with fractionated RT and EGFR inhibition. CONCLUSION: For NSCLC cells increase in radiosensitivity by EGFR inhibition results from enhanced G1-arrest. However, this effect does not lead to improved tumor control because cells can be released from this arrest by re-stimulation.

Molecular mechanisms of individual radiosensitivity studied in normal diploid human fibroblasts.

The molecular mechanisms of individual radiosensitivity were studied in normal diploid human fibroblasts. For fibroblasts irradiated with X-rays in G1-phase the individual radiosensitivity was shown to be correlated with the extent of double-strand break (dsb) repair. The number of residual dsbs (including both non- and mis-rejoined dsbs) varied between 2 and 5% of the initial number induced and was low for resistant and high for sensitive strains. In the G1-phase dsbs are considered to be mostly repaired via the non-homologous end-joining pathway (NHEJ). However, so far none of the parameters tested for this pathway was found to be correlated with the number of residual dsbs. The parameters tested were mRNA expression, protein level and localisation and activity of the DNA-PK, which is the central complex of NHEJ. The dsb-repair capacity is also not regulated by the differentiation status, which varies substantially among fibroblast strains, whereas there is some indication that dsb repair might depend on the chromatin structure, with more efficient repair in cells with condensed DNA. Residual dsbs are converted into lethal chromosome aberrations finally leading to the loss of clonogenic activity, when cells pass through mitosis. Beside this so-called mitotic death, X-irradiated human fibroblasts are also inactivated via the TP53-dependent permanent G1-arrest, while apoptosis appears to be not important. On average, mitotic death and G1-arrest are equally effective, but there is a broad variation from one strain to the other, with a negative correlation between these two pathways. Fibroblast strains exhibiting only a moderate G1-arrest showed a high number of lethal aberrations and vice versa. This result points to a common regulator of both G1-arrest and dsb repair, which is presently under investigation.

Inactivation of HNSCC cells by 90Y-labeled cetuximab strictly depends on the number of induced DNA double-strand breaks.

UNLABELLED: Radioimmunotherapy is considered to have great potential for efficient and highly specific treatment of tumors. The aim of this study was to determine the efficacy of radioimmunotherapy when using (90)Y-labeled cetuximab and to determine to what degree induction and repair of DNA double-strand breaks (DSBs) are decisive for this approach. METHODS: This study was performed with 9 cell lines of squamous cell carcinoma of the head and neck (HNSCC) differing strongly in epidermal growth factor receptor (EGFR) expression. The radionuclide (90)Y was coupled by the chelator trans-cyclohexyl-diethylene-triamine-pentaacetic acid (CHX-A″-DTPA)/linker construct to the EGFR-directed antibody cetuximab to yield (90)Y-Y-CHX-A″-DTPA-cetuximab with a specific activity of approximately 1.2 GBq/mg. EGFR expression was determined by immunofluorescence and Western blotting, cetuximab binding by fluorescence-activated cell sorter analysis, the number of DSBs by immunofluorescence staining γH2AX/53BP1-positive repair foci, and cell survival by colony formation. RESULTS: For the 9 HNSCC cell lines, cetuximab binding correlated with the amount of EGFR present in the cell membrane (r(2) = 0.967, P < 0.001). When cells were exposed to (90)Y-Y-CHX-A″-DTPA-cetuximab, the number of induced DSBs increased linearly with time (r(2) = 0.968, P = 0.016). This number was found to correlate with the amount of membranous EGFR (r(2) = 0.877, P = 0.006). Most DSBs were repaired during incubation at 37°C, but the small number of remaining DSBs still correlated with the amount of membranous EGFR (24 h: r(2) = 0.977, P < 0.001; 48 h: r(2) = 0.947, P < 0.001). Exposure to (90)Y-Y-CHX-A″-DTPA-cetuximab also resulted in efficient cell killing, whereby the extent of cell killing correlated strongly with the respective number of remaining DSBs (r(2) = 0.989, P < 0.001) and with the amount of membranous EGFR (r(2) = 0.967, P < 0.001). No cell killing was observed for UTSCC15 cells with low EGFR expression, in contrast to the strong reduction of 86% measured for UTSCC14 cells showing a strong overexpression of EGFR. CONCLUSION: (90)Y-Y-CHX-A″-DTPA-cetuximab affected cell survival through the induction of DSBs. This treatment was especially efficient for HNSCC cells strongly overexpressing EGFR, whereas no effect was seen for cells with low levels of EGFR expression. Therefore, EGFR-directed radioimmunotherapy using (90)Y-Y-CHX-A″-DTPA-cetuximab appears to be a powerful tool that can be used to inactivate tumors with strong EGFR overexpression, which are often characterized by a pronounced radioresistance.

Aberrant overexpression of miR-421 downregulates ATM and leads to a pronounced DSB repair defect and clinical hypersensitivity in SKX squamous cell carcinoma.

BACKGROUND: Cellular and clinical sensitivity to ionizing radiation (IR) is determined by DNA double-strand breaks (DSB) repair. Here, we investigate the molecular mechanism underlying the extreme response of a head and neck tumor case (SKX) to standard radiotherapy. METHODS: Immunofluorescence (IF) was used for the assessment of DSB repair, Western blot and real-time PCR for protein and mRNA expression, respectively. RESULTS: SKX cells exhibited a pronounced radiosensitivity associated with numerous residual γ-H2AX foci after IR. This was not associated with lacking canonical repair proteins. SKX cells did not express any ATM protein. Accordingly, immunoblotting revealed no ATM kinase activity toward substrates such as p-SMC1, p-CHK2 and p-KAP1. Sequencing of all 66 exons of ATM showed no mutation. ATM mRNA level was moderately reduced, which could be reverted by 5'-Aza-C treatment but without restoring protein levels. Importantly, we demonstrated a post-transcriptional regulation in SKX cells via 6-fold enhanced levels of miR-421, which targets the 3'-UTR of ATM mRNA. Transfection of SKX cells with either anti-miR-421 inhibitor or a microRNA-insensitive ATM vector recovered ATM expression and abrogated the hyper-radiosensitivity. CONCLUSION: This is the first report describing microRNA-mediated down-regulation of ATM leading to clinically manifest tumor radiosensitivity.

Fully automated interpretation of ionizing radiation-induced γH2AX foci by the novel pattern recognition system AKLIDES®.

PURPOSE: Assessment of phosphorylated histone H2AX (γH2AX) foci as a measure for double-strand breaks (DSB) is a common technique. Since visual interpretation is time-consuming and influenced by subjective factors, we adapted the pattern recognition algorithms of autoantibodies to automated reading of γH2AX foci. MATERIALS AND METHODS: DSB formation was assessed by detection of γH2AX foci after exposition of thyreocyte rat cell line to (188)Re. We used pattern recognition algorithms of the automated fluorescence interpretation system AKLIDES(®) for evaluation of γH2AX foci. Manual investigation was performed by three laboratories involving five observers. The results were compared by determining correlation and inter-laboratory variability. RESULTS: The study confirmed the adaptation of automated interpretation system AKLIDES® to automated assessment of γH2AX foci in irradiated cells. Both manual and automated quantification resulted in increasing focus numbers depending on dose. Comparison of automated reading with visual assessment for five manual observers resulted in a determination coefficient of R(2) = 0.889. The inter-laboratory variability for five manual investigators of three laboratories was 38.4 %. CONCLUSION: The interpretation system AKLIDES(®) demonstrated a high correlation with visually observed results. High inter-laboratory variability found for manual investigations revealed the usefulness for a standardized technique for evaluation of γH2AX foci.

In tumor cells regulation of DNA double strand break repair through EGF receptor involves both NHEJ and HR and is independent of p53 and K-Ras status.

PURPOSE: The purpose of this study was to examine whether the epidermal growth factor receptor (EGFR) may be used as a general target to modulate DNA double strand break (DSB) repair in tumor cells. MATERIAL AND METHODS: Experiments were performed with human tumor cell lines A549, H1299 and HeLa and primate cell line CV1. EGF, ARG and TGFα were used for EGFR activation, cetuximab or erlotinib for inhibition. Overall DSB repair was assessed by γH2AX/53BP1 co-immunostaining and non-homologous end-joining (NHEJ) and homologous recombination (HR) by using NHEJ and HR reporter cells; cell cycle distribution was determined by flow cytometry and protein expression by Western blot. RESULTS: EGFR activation was found to stimulate overall DSB repair as well as NHEJ regardless of the ligand used. This stimulation was abolished when EGFR signaling was blocked. This regulation was found for all cell lines tested, irrespective of their p53 or K-Ras status. Stimulation and inhibition of EGFR were also found to affect HR. CONCLUSIONS: Regulation of DSB repair by EGFR involves both the NHEJ and HR pathway, and appears to occur in most tumor cell lines regardless of p53 and K-Ras mutation status.

Cellular and tumor radiosensitivity is correlated to epidermal growth factor receptor protein expression level in tumors without EGFR amplification.

PURPOSE: There is conflicting evidence for whether the expression of epidermal growth factor receptor in human tumors can be used as a marker of radioresponse. Therefore, this association was studied in a systematic manner using squamous cell carcinoma (SCC) cell lines grown as cell cultures and xenografts. METHODS AND MATERIALS: The study was performed with 24 tumor cell lines of different tumor types, including 10 SCC lines, which were also investigated as xenografts on nude mice. Egfr gene dose and the length of CA-repeats in intron 1 were determined by polymerase chain reaction, protein expression in vitro by Western blot and in vivo by enzyme-linked immunosorbent assay, and radiosensitivity in vitro by colony formation. Data were correlated with previously published tumor control dose 50% data after fractionated irradiation of xenografts of the 10 SCC. RESULTS: EGFR protein expression varies considerably, with most tumor cell lines showing moderate and only few showing pronounced upregulation. EGFR upregulation could only be attributed to massive gene amplification in the latter. In the case of little or no amplification, in vitro EGFR expression correlated with both cellular and tumor radioresponse. In vivo EGFR expression did not show this correlation. CONCLUSIONS: Local tumor control after the fractionated irradiation of tumors with little or no gene amplification seems to be dependent on in vitro EGFR via its effect on cellular radiosensitivity.

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.