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1 This article was downloaded by: [Krause, Mechthild] On: 4 November 2009 Access details: Access Details: [subscription number ] Publisher Informa Healthcare Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK International Journal of Radiation Biology Publication details, including instructions for authors and subscription information: Prediction of clonogenic cell survival curves based on the number of residual DNA double strand breaks measured by H2AX staining Apostolos Menegakis ab ; Ala Yaromina ab ; Wolfgang Eicheler ab ; AnnegRet Dörfler ab ; Bettina Beuthien- Baumann bcd ; Howard D. Thames e ; Michael Baumann ab ; Mechthild Krause ab a Department of Radiation Oncology, b OncoRay - Center for Radiation Research in Oncology, c Department of Nuclear Medicine, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Germany d PET Center Dresden-Rossendorf, Research Center Dresden-Rossendorf, Germany e Department of Biostatistics, M.D. Anderson Cancer Centre Houston, Houston, Texas, USA Online Publication Date: 01 November 2009 To cite this Article Menegakis, Apostolos, Yaromina, Ala, Eicheler, Wolfgang, Dörfler, AnnegRet, Beuthien-Baumann, Bettina, Thames, Howard D., Baumann, Michael and Krause, Mechthild(2009)'Prediction of clonogenic cell survival curves based on the number of residual DNA double strand breaks measured by H2AX staining',international Journal of Radiation Biology,85:11, To link to this Article: DOI: / URL: PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: This article may be used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.

2 Int. J. Radiat. Biol., Vol. 85, No. 11, November 2009, pp Prediction of clonogenic cell survival curves based on the number of residual DNA double strand breaks measured by gh2ax staining APOSTOLOS MENEGAKIS 1,2, ALA YAROMINA 1,2, WOLFGANG EICHELER 1,2, ANNEGRET DÖRFLER 1,2, BETTINA BEUTHIEN-BAUMANN 2,3,4, HOWARD D. THAMES 5, MICHAEL BAUMANN 1,2, & MECHTHILD KRAUSE 1,2 Downloaded By: [Krause, Mechthild] At: 09:08 4 November Department of Radiation Oncology, 2 OncoRay Center for Radiation Research in Oncology, 3 Department of Nuclear Medicine, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Germany, 4 PET Center Dresden-Rossendorf, Research Center Dresden-Rossendorf, Germany, and 5 Department of Biostatistics, M.D. Anderson Cancer Centre Houston, Houston, Texas, USA (Received 31 March 2009; Revised 20 July 2009; Accepted 20 July 2009) Abstract Purpose: To assess the potential of using the residual phosphorylation of histone H2AX (gh2ax) after irradiation as a marker of radiosensitivity in vitro. Material and methods: Confluent cell cultures of FaDu and SKX human squamous cell carcinoma lines were irradiated with graded single doses. Twenty-four hours after irradiation cells were seeded for standard colony forming assay (CFA). In parallel, staining for gh2ax was performed to visualise the residual foci. Results: In the CFA, FaDu showed a higher radioresistance than SKX. After analysis of the residual foci data, we constructed predicted survival curves using two different methods. First, the proportion of nuclei with 53 foci was found to correlate closely with the observed surviving fraction (SF) in FaDu, with a slight overestimation of the true SF in SKX. Second, there was a strong linear correlation of the mean number of residual foci and observed 7lnSF. Based on regression analysis, we calculated the SF for both cell lines based on the mean number of residual gh2ax foci. This second approach again led to a good correlation of predicted and observed SF values in FaDu and a (slight) overestimation in SKX. Conclusion: In the two cell lines investigated the mean number of residual foci of gh2ax can be used to predict differences in the radiation dose response relationship in vitro. Keywords: clonogenic cell survival, irradiation, gh2ax, DNA repair, double strand breaks, prediction Introduction Irradiation of eukaryotic cells leads to a variety of deoxyribonucleic acid (DNA) lesions. Among them, double strand breaks (DSB) seem to pose the most serious threat for the genomic integrity. Cells respond to the formation of a DSB with the activation of a sophisticated and well orchestrated mechanism that involves different signalling pathways, aiming to efficiently detect and repair those lesions. A milestone in our knowledge of the different proteins involved in the different steps of DNA damage response has been provided from the observation that many of those proteins, upon specific modifications in their structure, generate distinct nuclear foci easy to detect with immunofluorescence microscopy. One of the earliest events is the phosphorylation of a specific variant of the histone H2A, i.e., H2AX. This histone, which comprises 2 25% of the whole H2A histone in chromatin, becomes rapidly phosphorylated on Serine 139, located 4 amino acids from the carboxy terminus of the protein. The phosphorylation takes place in the vicinity of the DSB generating nuclear foci (usually referred to as gh2ax), which can be detected with phosphor-specific antibodies, thereby visualising the sites of the chromatin where DSBs are present. (Burma et al. 2001, Celeste et al. 2003, Fernandez-Capetillo et al. 2002, Girard et al. 2002, Correspondence: Mechthild Krause, MD, PhD, Department of Radiation Oncology, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, Fetscherstr. 74, Dresden, Germany. Tel: þ Fax: þ mechthild.krause@uniklinikum-dresden.de ISSN print/issn online Ó 2009 Informa UK Ltd. DOI: /

3 Downloaded By: [Krause, Mechthild] At: 09:08 4 November 2009 Prediction of clonogenic cell survival curves 1033 Kang et al. 2005, Pilch et al. 2003). It has been shown that the formation of each focus involves the phosphorylation of numerous H2AX molecules (*2000) and represents one DSB (Sedelnikova et al. 2002, Takahashi & Ohnishi 2005). The foci are formed very rapidly within 3 minutes after irradiation and exhibit plateau levels at minutes after irradiation (Rogakou et al. 1998). It has been proposed that gh2ax phosphorylation is essential for the further recruitment of various repair proteins at the damaged sites of the chromatin (Goldberg et al. 2003, Paull et al. 2000, Rappold et al. 2001, Schultz et al. 2000, Stewart et al. 2003, Ward & Chen 2001), as well as for numerous chromatin remodelling factors aiming to condense the chromatin around the break and (thus) facilitate the processing of the repair by holding the broken ends in close proximity (Downs 2007, Foster & Downs 2005, Morrison & Shen 2005, Stucki & Jackson 2006). Dephosphorylation of the H2AX histone by a phosphatase complex (PP2A) has been reported to facilitate efficient DNA repair and the progression of cells into the cell cycle by removing the checkpoint arrest (Keogh et al. 2006). This offers the opportunity to monitor the induction and the processing of the DNA damage. The use of gh2ax foci as a marker of DSB in many mechanistic studies of DNA repair has provided great insight into the molecular basis of DNA repair. However, despite the overall simplicity and high sensitivity, the specificity of the method is unclear. Although it is generally proven that each DSB generates a gh2ax focus, it is not clear whether each focus corresponds to exactly one DSB (Bonner et al. 2008, Kinner et al. 2008). It has been shown that residual gh2ax foci may persist after the rejoining of the initial DSB (Banath et al. 2004, Kinner et al. 2008, Mirzayans et al. 2006). Furthermore, the presence of a subpopulation of small foci ( microfoci ) (Bonner et al. 2008) appears to be cell cycle dependent and irrelevant for DNA Damage Response (DDR) (McManus & Hendzel 2005). Also, a dependence on the level of condensation of the chromatin has been demonstrated (Goodarzi et al. 2008, Kinner et al. 2008). Persistent phosphorylation of H2AX histone might activate the DDR even without the presence of a DSB (Soutoglou & Misteli 2008). These facts express some of the limitations of the method and should be taken into account when quantification of DSB is performed based only on foci counting. Nevertheless, detection gh2ax foci remains one of the most sensitive methods for monitoring DNA damage, as it can be applied even in cases where only a few DSBs are present in the nucleus, e.g., after low doses of radiation, this technique is highly advantageous compared to the physical techniques of assessing DNA damage. In addition to the detection of the induction and processing of DNA damage, the H2AX assay can be used as an indicator of cell death. This is supported by the observation of a correlation between higher rate of foci loss and a higher clonogenic surviving fraction in ten different cancer cell lines (MacPhail et al. 2003). A linear correlation has been shown between surviving fraction after two Gy (Gray) (SF 2 ) and the slope of the residual DNA damage response measured with comet assay (Wada et al. 2005). In human fibroblast strains with different radiosensitivity (normal, ataxia telangiectasia and Lifraumeni syndrome), a correlation between the residual DSB 24 hours after irradiation measured by neutral comet assay and H2AX fluorescence has been demonstrated (Mirzayans et al. 2006). Residual gh2ax measured semi-quantitatively as flourescence intensity has been shown to correlate with SF 2 values 24 hours after irradiation of different cervical cell lines (Banath et al. 2004), but also with cell survival after etoposide and tirapizamine exposition (Olive et al. 2004). However, there are also negative studies that found no correlation between gh2ax and clonogenic cell survival, showing the limitations of the assay when flourescence intensity (Mahrhofer et al. 2006) or just percentage of positive cells (Yoshikawa et al. 2009) are evaluated in tumour cell lines that usually have higher background levels of gh2ax. Using the maintenance of residual foci as an indicator of radiation-induced cell killing (Klokov et al. 2006) showed that the fraction of cells with background levels of foci 24 hours (53 foci per cell) after irradiation was correlated with the cell survival for two tumour cell lines. The fraction of cells containing 53 residual gh2ax foci per cell measured 24 hours after 1 hour treatment with cisplatin was shown to be predictive of clonogenic cell survival (Olive & Banath 2009). The purpose of the present study was to assess the potential of predicting the surviving fraction of clonogenic cells after irradiation in vitro by evaluation of the residual number of gh2ax foci. For this, two human squamous cell carcinoma (hscc) lines of the head and neck with known and pronounced difference in radiation sensitivity were examined and data obtained from the colony forming assays (CFA) were compared with the predicted survival curves obtained from two different analyses of the gh2ax foci. Materials and methods Cell lines The two cell lines used in the experiments have been previously described (Eicheler et al. 2002). FaDu, an undifferentiated human hypopharyngeal SCC cell line expresses moderate radiation sensitivity.

4 Downloaded By: [Krause, Mechthild] At: 09:08 4 November A. Menegakis et al. The FaDu cell line used in the present experiments is a subline of the original FaDu cell line available in the American Type Culture Collection (ATCC). SKX is a moderately well differentiated SCC from the floor of mouth that is exceptionally radiosensitive. The DNA index is in both cell lines. Cell culture and irradiation The cells were reactivated from a cryo-stock (liquid nitrogen) and maintained as exponential cultures in Dulbecco s Minimum Essential Medium (DMEM) containing 2% Hepes buffer, 1% of non-essential amino acids, 1% sodium pyruvate and 1% antibiotics (Penicillin, Streptomycin) (Biochrom, Berlin, FRG, Germany) and 10% Fetal Calf Serum (FCS) (Sigma Chemical Co., St. Louis, MO, USA). Exponentially growing cells from the third passage after activation were seeded (common seeding number per passage: ) in 25 cm 2 flasks (Nunc, Roskilde, Denmark), and cultivated (4 days for FaDu and 5 days for SKX) until they reached confluence ( cells/ cm 2 ). Cells were irradiated in the flasks with 200 kv x- rays and 20 ma at dose rate * 1 Gy/ min (Isovolt 320/ 13, Fa. Seifert, Ahrensdorf, Germany). Experimental design As the aim of the experiments was the direct correlation of the clonogenic survival with the number of remaining gh2ax foci, cells of the same flask were used for both procedures. Confluent cell cultures were irradiated with doses of 0; 1; 2; 4; 6; and 8 Gy. To allow repair of sublethal damage, irradiated cells were incubated for additional 24 hours (378C and 5% CO 2 ). The timepoint of 24 hours after irradiation has been shown previously to be adequate for evaluation of residual DNA damage in both cell lines (Kasten-Pisula et al. 2009). After preparation of single cell suspensions, cells were split into two parts and the first portion was seeded in petri dishes (5.8 cm diameter; Nunc, Roskilde, Denmark) for standard colony forming assay (CFA). For each dose, three different seeding densities were used in triplicates. The results of three (for FaDu) and four (for SKX) independent experiments are presented. For gh2ax staining, the remaining cells (second portion) from each flask were placed on glass slides by cytospin procedure (75,000 cells per spot; 200 rpm, 2 min) and fixed in neutral buffered 4% formalin for 15 min. Immunohistochemistry The fixed cells were rehydrated in Phosphate Buffer Saline (PBS). After permeabilisation of the cell membranes with Triton X-100 (0.01 % in PBS v/v; three times), bovine serum albumin (BSA; SERVA electrophoresis, Heidelberg, Germany) (1% in PBS, room temperature (RT), 30 min) was used to block unspecific reactions before primary antibody was added for 1 hour (Upstate Millipore; Schwalbach; Germany); clone JBW301; 1:1000 dilution in BSA, RT). As secondary antibody, ALEXA 594 fluorescent probe (red fluorescent) was used (Molecular Probes Invitrogen, Karlsruhe, Germany; 1:400 dilution; RT, 30 min). Nuclei were labelled by 4 0,6- Diamidino-2-phenylindole dihydrochloride (DAPI) (Boehringer Bioproducts Roche, Mannheim, Germany; 25 mg/ml; RT, 10 min) and mounted with fluorescent mounting medium (Dakocytomation, REF S3023, Hamburg, Germany). Evaluation of foci The gh2ax foci were counted manually under 630 magnification (Zeiss Axioplan 2 fluorescence microscope, monochrome digital camera Axiocam MRm Carl Zeiss, Jena, Germany) with red fluorescence signal (filter set: 15, excitation 546 nm, emission: 590 nm). For evaluation, 200 nuclei of each dose group were randomly chosen and the number of foci per nucleus was recorded. The cells were selected according to morphological criteria and only intact nuclei (DAPI, filter set: 2, excitation: 395 nm, emission: 420 nm) were analysed. For establishment of the method, randomly selected nuclei were cochecked by two observers, showing similar results. The results of at least three independent experiments were pooled so that each point represents the mean of at least 600 measurements and their standard deviations. Statistical analysis of data For analysis of the clonogenic cell survival and the gh2ax data, the Prism software (Graphpad Prism, Prism 4 for windows, version 4.03) was used. For correlation of survival fractions and the number of foci, STATA/SE 8.0 software was used (STATA Corporation, College station, TX, USA). Results The surviving fractions (SF) for three and four independent experiments for FaDu and SKX cells are presented in Figure 1. The data were fitted using the linear-quadratic model: lnðsfþ ¼ c ðad þ bd 2 Þ; where c is a constant, a and b are the coefficients of dose (d) and dose squared respectively.

5 Downloaded By: [Krause, Mechthild] At: 09:08 4 November 2009 Figure 1. Clonogenic cell survival curves obtained from standard colony forming assay for FaDu (closed symbols, continuous line) and SKX (open symbols, broken line). Means and standard deviations for three (FaDu) and four (SKX) independent experiments are shown. The exceptional radiosensitivity of SKX required extremely high seeding densities, leading to difficulties in distinguishing colonies with less vs. more than 50 cells and thereby to non-reliable results. Therefore, the 6- and 8-Gy values for SKX are not shown. The equations obtained for the SF of both cell lines were: lnðsf FaDuÞ ¼ 0:0017 0:247d þ 0:0280d 2 ð1þ lnðsf SKXÞ ¼ 0:041 1:131d 0:0388d 2 ð2þ Figure 2 shows a representative example of residual gh2ax staining pattern for controls and irradiated cells with 4 Gy for both cell lines and Figure 3 the relative frequency distributions of the residual number of foci 24 hours after irradiation for the equivalent doses. To evaluate which level of residual foci would best represent cell survival, we correlated the fraction of cells containing residual foci numbers below different cut-off levels with the observed surviving fraction from the CFA. We found that the cut-off level of 53 foci per nucleus best identified a surviving cell. We then evaluated the fraction of the cells with less than 3 residual foci after all radiation doses tested and plotted this proportion against the SF values obtained from the CFA. The regression analysis resulted in a strong linear relationship with no difference between the two cell lines (Figure 4A). As a next step, we constructed cell survival curves for both cell lines based on either the immunofluorescence gh2ax data (predicted SF) or the data obtained from the CFA (observed SF). For FaDu cells, the correlation led to a good prediction of the CFA data by the predicted SF curve (Figure 4B). In the SKX cell line the prediction resulted in a slight overestimation of the true surviving fraction (Figure 4C). As a second approach, we tested whether the mean number of residual foci of gh2ax, i.e., inclusion of all individual data, correlates with the surviving fraction of both cell lines. Given that the radiationinduced cell kill is described by Poisson statistics, the probability of a single cell to survive is determined by the probability that the cell has no residual lethal lesions. This can be described mathematically from the following equations: or SF ¼ probability ð0 lethal lesionsþ ¼ exp ð mean number lethal lesionsþ ln SF ¼ mean number lethal lesions ð3þ Since residual gh2ax foci are considered to reflect unrejoined DSBs, we hypothesised that their number should be related to the mean number of lethal lesions predicted by Poisson statistics (equation 3). Indeed, the fit of both parameters resulted in a significant linear regression for FaDu and SKX (Figure 5A, B). From the slopes of these regressions (mean number of residual foci against 7lnSF), we derived the equations (4, 6), where the mean number of residual foci after various doses could be directly correlated with 7lnSF. Transposing these equations allows estimation of the predicted SF after different doses. From the equations (5, 7) we constructed hypothetical ( predicted ) cell survival curves for both cell lines based only on the mean number of the residual foci after various doses. FaDu: MeanNfoci FaDu ¼ 1:3393 þ 3:111 x ð lnðsf FaDu ÞÞ ð4þ SKX: Prediction of clonogenic cell survival curves 1035 SF FaDu ¼ exp ðð1:3393 MeanNfoci FaDu Þ=3:111 ð5þ MeanNfoci ¼ 0: þ3:651576xð lnðsf SKX ÞÞ ð6þ SF SKX ¼ exp ðð0: MeanNfoci SKX Þ=3:651576Þ ð7þ Figure 5 (C, D) compare the resulting predicted SF values obtained from the above equations

6 1036 A. Menegakis et al. Downloaded By: [Krause, Mechthild] At: 09:08 4 November 2009 Figure 2. Examples of residual immunofluorescence staining (24 h after irradiation) against phosphorylated histone H2AX (gh2ax foci, red) for controls and after irradiation with 4 Gy for FaDu and SKX. The photos are made under 630 magnification which was also the magnification used for evaluation as well. The average number of residual foci in the more radiosensitive cell line (SKX) is higher. Note that in both cell lines two different subpopulations of foci are present. Large and bright residual foci present particularly in irradiated cells (panel C, D) that mirror repair processes and smaller foci equally distributed in both controls and irradiated cells usually referred to as micro-foci (Kinner et al. 2008). Their presence in cycling cells was recorded in the work performed by (McManus & Hendzel 2005) and found to be irrelevant for the processing of DNA repair as they do not colocalise with any of the DNA repair proteins. In both cell lines, a background level of residual (large foci) was observed (panel A, B). This endogeneous expression of gh2ax foci in cancer cell lines appears to be a consequence of chromatid instability rather than connected with DSB processing (Yu et al. 2006). Figure 3. Examples of frequency distribution of residual gh2ax foci 24 h after irradiation for both cell lines. (A, B) FaDu, control and after 4 Gy. (C, D) SKX, control and after 4 Gy.

7 Prediction of clonogenic cell survival curves 1037 Downloaded By: [Krause, Mechthild] At: 09:08 4 November 2009 Figure 4. Prediction of the clonogenic survival curve from the percentage of cells with less than three foci per nucleus 24 hours after irradiation. (A) Correlation between clonogenic cell survival and proportion of cells with less than 3 residual foci. (B, C) Predicted survival curves based on the proportion of cells with less than 3 foci for FaDu (B) and SKX (C). Open symbols show the data obtained for each cell line from the CFA for comparison. Means and standard deviations for three (FaDu) and four (SKX) independent experiments are shown. with the SF values observed in the CFA. The calculation resulted in a good prediction of the different radiosensitivities of both cell lines. In case of FaDu, also the dose-response relationship of the SF values obtained by CFA could accurately be predicted, whereas for SKX the prediction of the true SF values was less accurate. Discussion In the present study, we investigated the relationship between cell survival and unrepaired DSBs assessed by residual gh2ax foci in two different hscc lines with significant difference in radiation sensitivity. It has been shown previously (Kasten-Pisula et al. 2009) that the induction of damage did not differ significantly between both cell lines, whereas the number of foci reached a lower plateau level in FaDu without further decrease 16 hours after irradiation with 4 Gy. The mean number of residual foci after 24 h was constantly significantly higher by a factor of 2 or more in SKX compared to FaDu (Figure 3 and Kasten-Pisula et al. 2009). Since the DNA index of both cell lines was similar, it is justified to directly compare the gh2ax data of both cell lines. Since in this and previous studies (Kasten-Pisula et al. 2009) we observed marked differences in radiation sensitivity when performing CFA (SF 2FaDu ¼ 0.52, SF 2SKX ¼ 0.11) and in the number of unrepaired DSBs between both cell lines, we hypothesised that the differences in radiation dose-response could be predicted by the number of residual foci. To test this hypothesis, we analysed the data using two different approaches. In the first approach, we plotted the frequency distributions of the residual foci number 24 hours after irradiation over all doses. The pattern was similar for both cell lines and for all doses tested (lognormal distribution). Since we hypothesised that there should be a close relation between cell survival and cell death with the number of residual foci per nucleus for each individual cell, we tested different threshold values of residual foci, which, if exceeded, will lead to cell death. The direct comparison of the

8 1038 A. Menegakis et al. Downloaded By: [Krause, Mechthild] At: 09:08 4 November 2009 Figure 5. Calculation of the survival curves based on the mean number of the residual foci. (A, B) Correlation of the mean number of residual foci for both cell lines with the observed 7lnSF (A: FaDu, B: SKX). (C, D) Comparison of the predicted SF obtained from the equations (4, 6) with the observed SF (C: FaDu, D: SKX). Means for three (FaDu) and four (SKX) independent experiments are shown; error bars indicate the standard deviations. frequency distributions with surviving fraction for each dose showed that cell survival for these two cell lines is best represented by the proportion of cells with less than 3 residual foci. Subsequently, we evaluated this proportion over the whole panel of radiation doses examined in the present experiments. Thereby, we were able to construct the survival curves for both cell lines based on the residual gh2ax foci data (Figure 4B, C). Although in SKX the prediction resulted in a slight overestimation of the true surviving fraction (Figure 4C), the model allowed to identify the difference in radiation-dose response between both cell lines Our observations are in line with data published by Klokov et al. (2006), showing also a linear correlation of the fraction of cells with less than three foci with the surviving fraction of two human cervical carcinoma cell lines. Both our data and the results of Klokov et al. (2006) may appear contradictory to the observation that one unrepaired DSB is sufficient to kill a diploid normal tissue cell (Haber 2000, Hoeijmakers 2001, Jackson 2002, Rothkamm et al. 2003). However, it should be pointed out that, as mentioned above, both cell lines in our study are hyperdiploid, as it is the case for many human cancer cell lines. Therefore, one may speculate that one unrepaired DSB is not enough to cause a lethal effect in such cells. This would be in line with findings made by (Kasten-Pisula et al. 2009), where the number of lethal chromosomal aberrations per dose were assessed in these two cell lines and their number was found to be approximately one third of the residual foci number for both cell lines for each dose. This disparity between unrepaired DSB and residual gh2ax foci was also evident in the second analysis, where the data obtained from the survival curves were plotted against the mean number of residual foci. Here, according to the Poisson statistics, the values of the observed 7ln(SF) reflect the expected lethal lesions after specific radiation doses. The latter was shown to be equivalent with

9 Downloaded By: [Krause, Mechthild] At: 09:08 4 November 2009 Prediction of clonogenic cell survival curves 1039 one chromosomal aberration. In (Kasten-Pisula et al. 2009) the authors observed a negative linear correlation between the SF and the number of chromosomal aberrations for both cell lines and a reduction of survival to 37% by one chromosomal aberration, reflecting the lethal lesion expected from the Poisson statistics. These can be translated into unrepaired residual DSB since the latter lead to cell death. If one residual DSB would lead to cell death, a slope of 1 would be expected for this correlation. The fact that a slope of *3 was observed in both cell lines, suggests that *3 residual foci are necessary for cell death. This observation can be interpreted as follows: Given that the real number of lethal lesions is predicted by the Poisson model, it can be speculated that only a small number, i.e., one third, of the observed residual foci represents truly unrepaired DSBs. There is increasing evidence that the disappearance of gh2ax foci is connected to procedures beyond the rejoining of DSB. Thus, there is a significant difference between the half times of DSB disappearance measured with pulsed field gel electrophoresis versus foci dephosphorylation, where the first expressed much faster kinetics (Kinner et al. 2008). This would imply that the term repair of DSB describes something additional to the simple rejoining of DNA-ends. Induction of DSB not only disrupts the structure of chromatin but in addition causes phosphorylation of histone H2AX, thereby generating additional remodelling of the chromatin sites where DSB are introduced. Therefore, one might speculate that the presence of gh2ax foci may not only contribute to the resealing of the gap but additionally to facilitate processes aiming to restore the chromatin integrity to its original status. Thus, residual foci might refer not only to unrejoined DSBs but also to sites where after rejoining of DSB the chromatin processing is still active (Bonner et al. 2008, Kinner et al. 2008). Individualisation of radiation therapy may importantly gain from the development of approaches to predict the cell response to irradiation. The repair capacity and the recovery from DNA DSB after irradiation have a central role in the observed differencies of radiation response of the cells. Although the correlation of residual foci with the SF and the radiation sensitivity might be influenced by different factors for each individual cell line, e.g., different DNA index or repair efficiency, our data suggest that the radiation sensitivity of individual cell lines is closely connected to the number of unrejoined DSB. The present approach of constructing hypothetical cell survival curves based on the evaluation of residual gh2ax foci provides a promising experimental method to predict clonogenic cell survival. The value of this method to compare different radiosensitivities needs to be further tested in panels of different tumour lines in vitro and in vivo. For the use in other cell lines it needs to be considered that a correction might be necessary if DNA indices differ between different cell lines. One step further is the application of the gh2ax assay in vivo with the mid-term aim to predict radiation response. As it is known that additional facors like tumour hypoxia can affect induction and repair of DNA damage in vivo, careful and precise selection of cell subpopulations from different tumour cell compartments is necessary. Also, correction might be needed for the different background levels of gh2ax primarly arising from the differences of cell cycle distributions of different cell populations if digital image analysis is performed (Qvarnstrom et al. 2004). Using appropriate staining procedures, both paraffin-embedded sections as well as cryosections might be used for evaluation (Bonner et al. 2008), however, establishment of the method in paraffin section seems preferable in light of a potential clinical use. Overall, residual gh2ax foci in vitro correlate with clonogenic cell survival. Using this approach in tumour tissue might be a tool to predict radiation response in vivo. This approach is currently being tested in our laboratory. Acknowledgement The authors wish to thank Ms Sigrid Balschukat and Ms Dorothee Pfitzmann for excellent technical assistance. This work was supported in part by the German Federal Ministry of Education and Research (BMBF 03ZIK/OncoRay), the Deutsche Forschungsgemeinschaft (Ba1433) and the 6th framework EU-project BioCare, proposal # This publication does not necessarily reflect the views of the EC. The community is not liable for any use that may be made of the information contained herein. AM holds a scholarship of the State Scholarships Foundation of Greece (IKY). Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References Banath JP, Macphail SH, Olive PL Radiation sensitivity, H2AX phosphorylation, and kinetics of repair of DNA strand breaks in irradiated cervical cancer cell lines. 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