Radiotherapy and Oncology xxx (2013) xxx–xxx

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Radiot herapy and Oncology

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Original article

HNSCC cell lines positive for HPV and p16 possess higher cellular radiosensitivity due to an impaired DSB repair capacity

Thorsten Rieckmann a,⇑, Silke Tribius b, Tobias J. Grob c, Felix Meyer a, Chia-Jung Busch d,Cordula Petersen b, Ekkehard Dikomey a, Malte Kriegs a

a Laboratory of Radiobiology & Experimental Radiooncology; b Department of Radiotherapy and Radiooncology; c Department of Pathology; and d Department of Otolaryngology and Head and Neck Surgery, University Medical Center Hamburg Eppendorf, Hamburg, Germany

a r t i c l e i n f o

Article history:Received 11 September 2012 Received in revised form 12 March 2013 Accepted 13 March 2013 Available online xxxx

Keywords:HNSCCHPVp16INK4a

Radiosensitivity

0167-8140/$ - see front matter � 2013 Elsevier Irelanhttp://dx.doi.org/10.1016/j.radonc.2013.03.013

⇑ Corresponding author. Address: Laboratory of RRadiooncology, Department of Radiotherapy and RadioCenter Hamburg-Eppendorf, Martinistr. 52, D-20246

E-mail address: t.rieckmann@uke.uni-hamburg.de

Please cite this article in press as: Rieckmann TDSB repa ir capacity. Rad iother Oncol (2013), ht

a b s t r a c t

Background and purpose: When treated by radiotherapy, patien ts with squamous cell carcinomas of the head and neck (HNSCC) positive for HPV and p16 INK4a possess a clearly favorable prognosis as compared to those with HPV-negative HNSCC. The aim of this work was to study whether the better outcomes might be caused by an enhanced cellular radiosensitivity.Materials and methods: The radiation response of five HPV/p16 INK4a-positive and five HPV-negative cell lines was characterized with regard to cellular radio sensitivity by colony formation assay. Furthermore G1- and G2-arrest, apoptosis and residual DNA double-strand breaks (DSB) were analyzed by the colce- mid-based G1-efflux assay, propidium iodide staining, the detection of PARP cleavage, the fluorescence- based detection of caspase activity and the immunofluorescence staining of cH2AX and 53BP1 foci.Results: On average, the cellular radiosensitivity of the HNSCC cell lines positive for HPV and p16 INK4a washigher as comp ared to the sensitivity of a panel of five HPV-negative HNSCC cell lines (SF3 = 0.2827 vs.0.4455). The higher sensitivity does not result from increased apoptosis or the execution of a permanent G1-arrest, but is rather associated with both, elevated leve ls of residual DSBs and extensive G2-arrest.Conclusions: Increased cellular radiosensitivity due to compromised DNA repair capacity is likely to con- tribute to the improved outcome of patien ts with HPV/p16 INK4a-positive tumors when treated by radiotherapy.

� 2013 Elsevier Ireland Ltd. All rights reserved. Radiothe rapy and Oncology xxx (2013) xxx–xxx

Classical risk factors for squamous cell carcinomas of the head and neck (HNSCC) are the extensive consumptio n of tobacco and alcohol. In recent years, it has become apparent that a distinct HNSCC entity exists which is depende nt on infection with high-risk types of human papillomavir us (HPV), especially HPV type 16 (HPV-16) [1]. HPV-dependent HNSCC are mostly localized in the oropharynx and account for 20–60% of cases of oropharyng eal car- cinomas. While the incidence of tobacco/alcoho l-induced HNSCC is currently stabile or even declining, cases of HPV-positive tumors are on the rise [2].

Oncogenic transformation by HPV is primarily caused by the viral oncoproteins E6 and E7. Besides a number of other functions, E6 de- grades p53 and E7 inactivates members of the retinobla stoma (Rb)protein family (RB, p107, p130), thereby liberating transcriptio nfactor E2F from Rb-mediated inhibition [3]. This results in the deregulation of S-phase entry and induces the expression of the tumor suppressor p16 INK4a (in the following p16). In contrast,the disruption of p16 expression in HPV-neg ative HNSCC is most of-

d Ltd. All rights reserved.

adiobiology & Experimental oncology, University Medical

Hamburg, Germany.(T. Rieckmann).

et al. HNSCC cell line s pos itive ftp://dx.doi.org/1 0.1016/ j.radon

ten an early step during carcinogene sis. Therefore, the analysis of p16 expression by immunoh istochemistry is a widely used surro- gate marker to identify HPV-pos itive HNSCC.

Recently it was demonstrated that the presence of HPV and the expression of p16 confer excellent prognosis for HNSCC patients when treated with regimens containing radiotherapy , while the prognosis of patients with HPV-negativ e tumors remains poor [4–8]. The cellular and molecular mechanism s underlying the favorable outcome of HPV/p16 -positive patients have yet to be unraveled . Among the known parameters, cellular radiosensitivity is one of the main factors determining tumor radiosensitivity [9].So far, reports on the cellular radiosensitivi ty of HPV-positive HNSCC cell lines are sparse with only two such strains (UM-SCC-47 and UPCI-SCC-90) having been tested for cellular radiosen- sitivity in colony formation assay. The results obtained conflictwith enhanced sensitivity observed in two reports [10,11] butreduced sensitivity in another [12]. The fact that to our knowledge only eight HPV-positive HNSCC cell lines (UT-SCC-45, UD-SCC-2,UPCI-SCC- 90, UPCI-SCC-152, UPCI-SCC- 154, UM-SCC-47, UM- SCC-104 and 93-VU-1 47T) are currently available, represents one of the main obstacles hampering research on this distinct tumor entity.

o r HPV and p16 possess higher cellula r radi osensitivity due to an imp aired c.20 13.03.013

2 HPV-positive HNSCC and radiosensitivity

Here we have compared the cellular radiosens itivities of fiveHPV/p16-pos itive HNSCC cell lines (UT-SCC-45, UD-SCC-2, UPCI- SCC-154, UM-SCC-47 and 93-VU-147T) with those of a panel of fiveverified HPV-negative HNSCC cell lines (HSC4, Cal33, UT-SCC-5,FaDu and SAT). There was a clear difference in average cellular radiosensitivi ty, with HPV-positive strains being more radiosensi- tive (SF3 = 0.2827 vs. 0.4455). This increase in radiosensitivity does not result from enhanced apoptosis or permanent G1-arrest. The HPV/p16-pos itive strains, however, did show an extensive G2- arrest as well as a high number of residual DNA double-strand breaks (DSB). These data strongly suggest that compromised DSB repair capacity leading to enhanced cellular radiosensitivi ty is akey component contributing to the improved outcome of patients with HPV/p16-posit ive HNSCC when treated by radiotherapy .

Materials and methods

Cells and cell culture

All cell lines were grown in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (Biochrome AG) and 2 mM glutamin e(Gibco) at 37 �C, 10% CO 2 and 100% humidification. HPV-positive cell lines: UT-SCC-45 (UT-45) were a kind gift from Prof. R. Gren- man, Turku, Finland; UD-SCC-2 (UD-2) were a kind gift from Prof.T. Hoffman n, Essen, Germany; UM-SCC-47 (UM-47) were a kind gift from Prof. T. Carey, Michigan, USA; 93-VU-147T (93-VU) were a kind gift from Prof. J. de Winter, Amsterdam , Netherlands and UPCI-SCC-154 (UPCI-154) were obtained from the German Collec- tion of Microorgani sms and Cell Lines (DSMZ), originally deposited by S. Gollin, Pittsburgh, USA. All HPV negative cell lines utilized were described previously [13].

HPV-status

Whole DNA was extracted using the Tissue XS Kit (MachereyNagel). All strains were tested for the presence of the L1 oncogene by genomic PCR utilizing the MY09/11 primer set. Resulting PCR products were sequenced to determine the respective HPV-type.In HPV-negativ e strains the absence of the viral oncogenes E6 and E7 of the most abundan t high-risk type HPV-16 was additionally verified by genomic PCR. In HPV-positive strains, active transcrip- tion of E6 and E7 was analyzed by reverse transcriptio n PCR. In brief, whole RNA was extracted using the RNeasy Kit (Qiagen)followed by digestion with DNAseI (Qiagen). Reverse transcriptio nwas performed utilizing the ‘‘High Capacity cDNA Reverse Transcriptio n Kit’’ (Applied Biosystems). PCR was performed employing the primers (MWG Eurofins Operon) listed in Supplement ary Table 1.

X-irradiation

Cells were irradiate d at room temperat ure with 200 kV X-rays (Gulmay RS225, Gulmay Medical Ltd.; 200 kV, 15 mA, 0.8 mmBe + 0.5 mm Cu filtering; dose rate of 1.2 Gy/min).

Colony formation assay

Cell cultures were X-irradiated with the doses indicated. Cell survival was determined by colony formation after delayed plating.Briefly, subconfluent cultures were irradiated and incubated for 24 h, followed by trypsinization and seeding into T25 cell culture flasks (Sarstedt) for colony formation. Incubation varied between two to four weeks depending on the respective cell line. The num- ber of colonies containing more than 50 cells was assessed .

In the case of UM-SCC-47, all samples were seeded with 5000 feeder cells per flask (AT1BR fibroblasts, 30 Gy) to support plating efficiency.

Please cite this ar ticle in press as: Riec kmann T et al. HNSCC cell lines pos itive fDSB repa ir capacity. Radio ther Oncol (2013), http://dx.doi.org/1 0.1016/j.rad on

G1-arrest

Cells were seeded in 6-well dishes and treated the next day with colcemid (0.2 lg/ml, Merck) 30 min prior to X-irradiation with 0 or 6 Gy. Cells were harvested at the indicated time points,fixed with 70% ethanol, briefly washed with 0.2% Triton X-100 in PBS and subsequent ly incubated with 100 ng/ml RNAseA and 10 lg/ml propidium iodide plus 0.2% Triton X-100 in PBS for 30 min at room temperature in the dark. Flow cytometric analysis was performed on a FACS Canto with FACS Diva Software (BectonDickinso n). The portion of cells in the respective cell cycle phases was calculated using ModFit LTTM software (Verity Software House, Inc.).

G2-arrest

Cells were seeded in 6-well dishes and irradiated the next day with various doses of X-irradiation. After 24 h the cells were fixed,stained and analyzed as described for G1-arrest.

Caspase activity

Detection of caspase activity was performed utilizing the FAM- FLICA™ Poly Caspases Assay Kit (Immunochemistry Technolo gies)accordin g to the manufac turer’s instructions. Flow cytometric analysis was performed on a FACS Canto with FACS Diva Software (Becton Dickinso n).

Western blotting

Proteins from whole cell extracts were detected by Western blot according to standard protocols. Signals were detected via ECL™ system (Amersham) and exposure to X-ray films. Antibodies utilized are listed in Supplement ary Table 2.

Immunofluorescence

Cells grown on glass cover slips were fixed with 4% formalde- hyde in PBS for 10 min and permeab ilized with 0.2% Triton X-100 in PBS before blocking for 30 min with 3% BSA plus 0.2% Triton X-100 in PBS. The cells were subsequently incubate d for 1 h at RT with the primary antibody in blocking solution, washed four times with 0.1% Tween20 in PBS before incubation with the sec- ondary antibody and were then washed again four times before mounting with Vectashield mounting medium (Vector Laborato -ries). Cells were inspected using a Zeiss AxioObserv er.Z1 fluores-cence microscope with ApoTome . Antibodies utilized are listed in Supplement ary Table 2.

Data evaluation

Data analysis and statistical evaluation were performed using GraphPad Prism (GraphPad Software). Experiments were per- formed at least three times and values presente d are mean ± SD unless noted otherwis e.

Results

Characteri zation of HPV-positive cell lines

The HPV-status of all cell lines utilized in this study was as- sessed by genomic PCR using the MY09/11 primer set. Sequencing of the respective PCR products revealed that the strains 93-VU- 147T, UD-SCC-2, UM-SCC- 47 and UPCI-SCC- 154 contain sequence sof the most abundant high-risk type HPV-16, while UT-SCC-45 cells contain sequence s of a different high-risk type, HPV-33. In HPV-neg ative strains we additional ly validated the absence of

or HPV and p16 possess higher cellula r radi osensitivity due to an imp aired c.2013.03 .013

T. Rieckmann et al. / Radiotherapy and Oncology xxx (2013) xxx–xxx 3

the main viral oncogenes E6 and E7 of HPV-16 (not shown),whereas in HPV-pos itive strains we confirmed active transcriptio nof E6 and E7 (Fig. 1A). Expression of the HPV-related surrogate marker p16 was analyzed by immunofluorescence and Western blot (Fig. 1B and Supplementary Fig. S1 ). All HPV-positive cell lines proved to be HPV/p16-doub le-positive HNSCC tumor cells, while all HPV-negativ e strains lacked the expression of p16. Therefore,with regard to HPV and p16 status, all cell lines utilized match the characterist ics of typical HPV-positive and -negative head and neck tumors.

HPV/p16-pos itive cell lines possess enhanced radiosensitivi ty

Fig. 2 shows the cellular radiosensitivi ty of all five HPV/p16- positive cell lines tested. Great variation in radiosensitivity was observed, with the surviving fraction at 6 Gy (SF6) ranging be- tween 0.0081 and 0.0831 (Fig. 2, left). While high variation in radiosensitivi ty was also observed for a panel of five HPV-negativ eHNSCC cell lines (Fig. 2, right) there was a clear difference in aver- age radiosensitivity with HPV-negative strains being more radiore- sistant (SF3 = 0.283 vs. 0.446, p = 0.028 and SF6 = 0.042 vs. 0.147,p = 0.048; Mann–Whitney one-taile d t-test). There was, however,some overlap between the two panels, with the most sensitive HPV/p16-ne gative cell line (FaDu) lying in the middle of the range of the HPV-positive.

Fig. 1. Characterization of HPV-positive cell lines. (A) RT-PCR utilizing primers targeting sequences of the oncogenes E6 and E7 from HPV-16 (left) or HPV-33 (right), respectively. UT-SCC-5 was used as an HPV-negative control. Primers targeting GAPDH were used as a quality control. (B) Immunofluorescence analysis utilizing an anti-human p16 antibody.

Fig. 2. Cellular radiosensitivity of HPV/p16-positive and HPV-negative HNSCC cell lines. Clonogenic survival of HPV-positive (left) and HPV-negative (right) cell lines after X-irradiation.

Please cite this article in press as: Rieckmann T et al. HNSCC cell line s pos itive fDSB repa ir capacity. Rad iother Oncol (2013), http://dx.doi.org/1 0.1016/ j.radon

No induction of apoptosis or G1-arrest in HPV/p16- positive cells

In contrast to the vast majority of HPV-negativ e HNSCC, most of the tumors positive for HPV and p16 harbor wtp53 [14,15] and the same had already been shown for three of the HPV/p16 -positive cell lines utilized [16–18]. We now additionally sequenced the whole open reading frame of p53 in UT-SCC-45 and 93-VU-147T utilizing reverse transcriptio n PCR. Taken together, UT-SCC-45,UD-SCC-2, UM-SCC-47 and UPCI-SCC- 154 harbor wtp53; only 93- VU-147T harbors both wt. and mutant p53 (L257R). While the viral oncoprotein E6 mediates p53 degradation, the stabilization of p53 by IR may counteract this function and hence provide a possibility for the induction of apoptosis or cell cycle arrest. Therefore, apop- tosis was analyzed by measuring PARP cleavage as well as caspase activity. However, the apoptosis-speci fic 89 kd PARP fragment was not detected in any of the HPV-positive or -negative cell lines,neither after short (6 h, not shown) nor prolonged incubation (24 h) following irradiation with 10 Gy (Supplement ary Fig. S2A ).In accordance with these data, we also observed no more than 5% of cells displaying increased caspase activity when irradiate dwith 10 Gy (Supplementary Fig. S2B ).

Radiation- induced G1-arrest was analyzed using the colcemid- based G1-efflux assay (Supplementary Fig. S3A and B). No induc- tion of a permanent or transient G1-arrest was found in any of the HNSCC strains when analyzing the efflux from G1 after short (6 h, not shown) or prolonged incubation (24 h, Supplement ary Fig. 3C ) contrasting the situation in some other tumor cells harbor- ing wtp53 (Kriegs et al., unpublished data). These data demon- strate that neither apoptosis nor permane nt cell cycle arrest in G1 can account for the enhanced cellular radiosens itivity of the HPV/p16 -positive HNSCC cell lines.

HPV/p16- positive cell lines show increased G2-arrest and elevated numbers of residual DSBs

We further analyzed the occurrence of a radiation-indu ced G2- arrest 24 h after X-irradiation. While we could hardly detect any arrest in most of the HPV-negativ e cell lines at 24 h after doses of up to 6 Gy, we observed substantial amounts of cells arrested in G2 in four out of five HPV/p16-posit ive cell lines (Fig. 3). For the latter, the extent of cells in G2 matches the observed order of sensitivit y, with the more sensitive cell lines UPCI-SCC-154, UM- SCC-47 and UD-SCC-2 also demonst rating the most pronounced ar- rest readily visible even after low doses.

We next tested whether the sustained G2-arrest might result from a persistent G2/M-ch eckpoint activation caused by unrepaired DSBs. To this end, cells were irradiated with 2 Gy and incubated at 37 �C for 24 h before the detection of residual DSBs via cH2AX/53BP1-posit ive repair foci. When analyzed quantitatively (Fig. 4Aand Supplementary Fig. S4 ), the numbers of foci in the HPV-positive cells match the degree of G2-arrest with the strongest increase in both, foci and arrest in UPCI-SCC-154, UM-SCC-47 and UD-SCC-2,a more moderate arrest and lower number of foci in 93-VU-147T and again a less pronounced arrest and lower number of foci in UT-SCC-4 5 cells. The numbers of foci observed in HPV-pos itive cells negatively correlate with survival as determined in colony forma- tion assay indicative of a dominan t role for DSB repair capacity for cell survival upon X-irradiation in these cells (Fig. 4B). For the HPV-neg ative panel no such correlation was found. Due to the smaller differences in residual foci a considerably higher number of cell lines is necessary to properly test a possible association in this group. This issue needs to be readdres sed in further studies.

Discussion

The aim of this project was to determine the cellular radiosen- sitivity of HPV/p16 -positive HNSCC cell lines and to unravel the

o r HPV and p16 possess higher cellula r radi osensitivity due to an imp aired c.20 13.03.013

Fig. 3. Radiation induced G2-arrest in HPV/p16-positive and -negative HNSCC cell lines. Cells were X-irradiated with the indicated doses and fixed after 24 h. Cell cycle analysis was performed by flow cytometry after propidium iodide staining.

Fig. 4. DSB repair capacity in HPV/p16-positive and -negative HNSCC cell lines after X-irradiation. Cells were X-irradiated with 2 Gy and fixed after 24 h, followed by immunofluorescence staining with anti-human cH2AX and anti-human 53BP1 antibodies (see examples). Nuclei were counterstained with DAPI. (A) Quantification of residual DSBs. After correcting the numbers of residual DSBs for the respective DNA content of each cell line the number of foci measured for non-irradiated cells was subtracted. Quantification was performed for at least 150 nuclei per sample in three separate experiments by three individual researchers with very similar results. One representative experiment is shown. Data presented are mean values + SEM. (B) Association between residual DSBs and cellular radiosensitivity for HPV-positive HNSCC cell lines. Cellular radiosensitivity as measured at 6 Gy, SF6, is plotted against the number of residual DSBs at 2 Gy; data were taken from Fig. 2 and Fig. 4A. Data were fitted by linear regression analysis.

4 HPV-positive HNSCC and radiosensitivity

underlying mechanis ms. We found that, on average, the radiosen- sitivity of the HPV/p16-posit ive cells is higher as compared to HPV- negative strains (Fig. 2B). However, both panels are characteri zed by considerable variation in radiosens itivity resulting in overlap- ping ranges of survival. This overlap may in part explain the contradictor y results regarding the cellular radiosensitivity of HPV-positive HNSCC cells lines obtained so far [10–12].

The observation that the higher radiosensitivity results neither from apoptosis nor from cell cycle arrest in G1, despite the fact that four out of five strains exclusively harbor wtp53 suggests the effi-cient suppressi on of the cellular p53 response by E6. This makes any contribution of wtp53 to the observed cellular radiosensitivity highly unlikely, especiall y since we could recently show that p53 is also unlikely to exert a transactivati on independen t effect on the repair of direct DSBs by homologous recombinati on [19]. We found, however, that for the five HPV/p16-posit ive cell lines tested,the order of radiosensitivi ty is reflected in the extent of G2-arrest and the number of residual DSBs after irradiation. Interestingly,in the case of UD-SCC-2 cells, the extensive G2-arrest and large number of residual DSBs are only partially reflected by the degree

Please cite this ar ticle in press as: Riec kmann T et al. HNSCC cell lines pos itive fDSB repa ir capacity. Radio ther Oncol (2013), http://dx.doi.org/1 0.1016/j.rad on

of radiosensitivi ty (Supplementary Fig. S5 ). This indicates that, at least in this cell line, the sustained G2-arrest exerts a protective function by providing the cells with additional repair time before the onset of mitosis. Overall, our data strongly suggest that an impaired DSB repair capacity is responsib le for the elevated radio- sensitivit y of HPV/p16 -positive cells.

The project described here was initiated because a number of recent studies provided compelli ng evidence that patients with HPV-pos itive HNSCC show drastically increased rates of survival when compare d to those with HPV-negative [4–8]. HPV-positive HNSCC often present with large cystic metastatic adenopathy of the neck, resulting in locally advanced stage III/IV disease. The cur- rent standard of care consists of multimod al therapy which nearly always includes radiotherapy , combined with surgery, chemother- apy or immunotherap y. Our data suggest that the observed effec- tiveness of such treatment may result from an enhanced cellular radiosens itivity. However , we cannot rule out the possibility that other factors such as the lack of hypoxic radioresistance or ther- apy-relat ed anti-HPV immune responses add to this effect in vivo [12,20].

or HPV and p16 possess higher cellula r radi osensitivity due to an imp aired c.2013.03 .013

T. Rieckmann et al. / Radiotherapy and Oncology xxx (2013) xxx–xxx 5

Incidences of HPV-pos itive HNSCC are rapidly rising and these patients are generally younger than those suffering from classical tobacco-induce d HNSCC [2]. These findings together with the im- proved outcome s of such patients motivate the desire for less toxic regimens with fewer severe side effects [21,22]. However , our data also demonst rate that HPV/p16-posit ive HNSCC cell lines are quite heterogeneous and not all of them demonstrat e exceptional ly high radiosensitivi ty. Therefore, caution should be urged when considering the deintensification of therapy via dose reduction,for example. Additional stratification within the subentity of HPV/p16-pos itive HNSCC may be necessar y to more precisely predict individual tumor sensitivit y. Ang et al. [4] reported that abetter classification is achieved when combining the HPV/p16 sta- tus with smoking habits and lymph node status. HPV-pos itive tu- mors in patients with a history of heavy smoking may arise upon HPV-infection in pre-neoplastic epithelia already harboring a num- ber of oncogeni c alterations such as p53 mutations or an increase in EGFR expression. We speculate that such mutations acquired prior to HPV-infection may confer some of the characterist ics of classical, HPV-negative HNSCC, thus resulting in less pronounced cellular radiosensitivity and poor outcome. In this way, although our data do not support a direct functional role for p53 in the cellular radiosens itivity of HPV-pos itive HNSCC, p53 status could still serve as a stratification marker indicating that HPV infection took place when a pre-neoplas tic state was already present.Notably, from the four HPV-16-positive cell lines only 93- VU-147T carries a mutation in p53 and only this strain lacks the high level of residual DSBs after irradiation (Fig. 5B). Unfortunate ly,due to the small number of cases described, data confirming the poor outcome of patients with HPV+/mutp53 HNSCC remain preliminary [6,23]. Other markers which are known to characteri ze HPV-negativ e tumors such as the lack of p16 or the overexpression of EGFR may also prove useful in this context [8,24,25].

In summary , the data presented here indicate that on average,HPV/p16-pos itive HNSCC cell lines are characterized by a higher cellular radiosens itivity than HPV-negative strains, a fact which we attribute to diminished DSB repair capacity. Future studies should concentrate on a more detailed characterizati on of this DSB repair defect in order to identify predictive markers for the better classification of HPV/p16-posit ive tumors and to establish novel targeting approaches that make use of the inherent repair defect rendering such tumors even more radiosens itive to allow deintensification of therapy without compromising outcome .

Conflict of interest statement

The authors declare no conflicts of interest.

Acknowled gments

The authors greatly acknowled ge the technical assistance of K.Hoffer, F. Gatzemeier, T. Fritzen and B. Riepen. We further thank L. Farnebo for information on the mutational status of p53 in UT- SCC-45. This project was in part supported by Deutsche Fors- chungsgem einschaft (DFG PAK 190, Di 457/8-1; E.D., M.K.).

Appendix A. Supplemen tary data

Supplement ary data associated with this article can be found, in the online version, at http://dx.doi .org/10.1016/j.radonc.2013.03.013.

Please cite this article in press as: Rieckmann T et al. HNSCC cell line s pos itive fDSB repa ir capacity. Rad iother Oncol (2013), http://dx.doi.org/1 0.1016/ j.radon

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o r HPV and p16 possess higher cellula r radi osensitivity due to an imp aired c.20 13.03.013