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The radioprotector Bowman–Birk proteinase inhibitor stimulates DNA repair via epidermal growth factor receptor phosphorylation and nuclear transport
Radiotherapy and Oncology 86 (2008) 375–382 www.thegreenjournal.com
DNA repair
The radioprotector Bowman–Birk proteinase inhibitor stimulates DNA repair via epidermal growth factor receptor phosphorylation and nuclear transport Klaus Dittmanna,*, Claus Mayera, Rainer Kehlbachb, H. Peter Rodemanna a
¨bingen, Tu ¨bingen, Germany, bDepartment of Radiology, Tu ¨bingen, Germany Department of Radiation Oncology, University of Tu
Abstract Background and purpose: The purpose of the study was to elucidate the underlying molecular mechanism of the radioprotector, Bowman–Birk proteinase inhibitor (BBI), and its interaction with EGFR nuclear transport. Materials and methods: Molecular effects of BBI at the level of EGFR responses were investigated in vitro with wt. TP53 bronchial carcinoma cell line A549 and the transformed fibroblast cell line HH4dd characterized by a mt. TP53. EGFR and associated protein expression were quantified by Western blotting and confocal microscopy in the cytoplasmic and nuclear cell fraction. Residual DNA double strand breaks were quantified by means of a cH2AX focus assay. Results: Both irradiation and BBI-treatment stimulated EGFR internalization into the cytoplasm. This process involved src kinase activation, EGFR phosphorylation at Y845, and caveolin 1 phosphorylation at Y14. EGFR internalization correlated with nuclear EGFR transport and was associated with phosphorylation of EGFR at T654. Nuclear EGFR was linked with DNA-PK complex formation and activation. Furthermore, nuclear EGFR was found in complex with TP53, phosphorylated at S15, and with MDC1, following irradiation and BBI treatment. It is noteworthy that MDC1 was strongly decreased in the nuclear EGFR complex in cells with mt. TP53 and failed to be increased by either BBI treatment or irradiation. Interestingly, in cells with mt. TP53 the BBI mediated stimulation of double strand break repair was hampered significantly. Conclusion: These data indicate that BBI stimulates complex formation between EGFR, TP53 and MDC1 protein in wt. TP53 cells only. Since MDC1 is essential for recruitment of DNA repair foci, this observation may explain how BBI selectively stimulated repair of DNA double strand breaks in wt. TP53 cells. c 2008 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 86 (2008) 375–382.
Keywords: BBI; EGFR; TP53; DNA repair; MDC1
Several studies have shown that BBI is able to prevent the development of malignancies in different animal tumor model systems [1,2]. To perform clinical trials, a crude extract from soybeans was developed (BBI Concentrate, BBIC) [3], and in 1992 approved by the FDA for ‘‘Investigational New Drug’’ status. BBIC was used in a phase II study in patients with oral leukoplakia [4]. It was well tolerated by the patients, and no clinical or laboratory evidence of toxicity or drug allergy was observed other than infrequent reporting of nausea, diarrhea, or epigastric discomfort, which did not seem related to drug ingestion [5]. The molecular mode of BBI is not resolved so far. Our in vitro studies on radiation-induced fibroblast differentiation, a cellular process leading to radiation-induced fibrosis [6–8], suggested that pre-treatment of fibroblasts with BBI prevented the radiation-induced terminal differentiation and thus increased clonogenic survival up to 30% [9]. Subsequent studies showed that the BBI increased clonogenic
activity after irradiation only in cells representing functional TP53, but not in cells lacking functional TP53 [10,11]. This characterized BBI as a selective radioprotector for normal cells, but not for tumor cells presenting mt. TP53, which is commonly present in many malignancies [12]. Moreover, by means of a mouse fibrosis model system (leg contracture assay), we were able to prove in vivo that BBI reduced radiation-induced fibrosis by about 50% [13]. In the same study we showed that BBI exerted no radioprotective effect upon tumors [13]. Thus, BBI selectively protects normal tissue and may therefore improve the therapeutic index of radiotherapy. Studies to elucidate the molecular mechanism of BBI revealed that BBI-mediated radioprotection is linked to DNA repair processes by means of TP53 in two ways. First, BBI induces stabilization of the TP53 protein and this process is associated with the expression of DNA repair-relevant genes
0167-8140/$ - see front matter c 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.radonc.2008.01.007
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[14]. Second, BBI stimulates activity of DNA-PK kinase [15], a key player in non-homologous end joining (NHEJ) DNA repair. The BBI treatment significantly reduced formation of radiation-induced dicentric chromosomes [15], implying that BBI improved the fidelity of the NHEJ process. BBI, a soybean-derived polypeptide, was shown to act anticarcinogenicly in a variety of in vitro- and in vivo-carcinogenesis assay systems [16]. The anticarcinogenic activity of BBI is localized in the chymotrypsin inhibitory region of the protein molecule [17]. In addition, we also localized the amino acids responsible for the radioprotective effect within this region [18]. We identified the phosphorylated tyrosine residue within this region as the essential amino acid and could show that the application of phosphotyrosin (P-Tyr) alone is sufficient to generate the radioprotective effect [19]. Ongoing investigations to elucidate the molecular mechanism showed that P-Tyr induced transport of the cytoplasmic EGFR into the cell nucleus [20]. Nuclear EGFR is linked with regulation of DNA-dependent protein kinase (DNA-PK) and thereby stimulates DNA repair after irradiation [21]. Previous data [22] demonstrated that the radioprotective effect of BBI is also mediated via EGFR. Data presented herein show that the molecular mechanism of BBI includes EGFR internalization and activation of DNA-PK. On the basis of these in vitro and in vivo experiments we tried to shed light onto the molecular mechanism of BBI, with particular emphasis on its effect upon DNA repair processes. We performed these experiments with A549 cells, which are radioprotected by BBI due to expression of wt. TP53, and compared the results obtained with data for HH4dd cells, which are characterized by expression of mt.TP53 and are not radioprotected by BBI.
Materials and methods
Western blot analysis and immune-precipitation Cell cultures were irradiated as described above; cells were lysed and proteins were resolved by SDS–PAGE. Western blotting was performed according to standard procedures. The primary antibodies were diluted as follows: anti-EGFR (BD Transduction Laboratories, clone 13) 1:1000; anti EGFR pT654 (nanotools, clone 12A3) 1:1000; anti-src (Santa Cruz, clone H-12) 1:1000; anti-src Y416 (cell signaling, polyclonal), 1:1000: anti-caveolin 1 (BD Transduction Laboratories, clone 2297) 1:1000; anti-caveolin pY14 (BD Transduction Laboratories, clone 56) 1:1000; antiDNA-PK (PharMingen, clone 4F10C5) 1:500: anti-DNA-PK phospho-Thr No 2609 (Rockland) 1:1000; anti-lamin B1 (Biozol, clone ZL-5) 1:1000. Quantification of binding was achieved by incubation with a secondary peroxidase-conjugated antibody with the ECL system (Amersham). EGFR was immune-precipitated from cytoplasmic and nuclear protein fractions obtained from 20 · 106 cells with antiEGFR antibody clone 13 (BD Transduction Laboratories).
Subcellular fractionation Cytoplasmic and nuclear extracts were prepared according to the instructions of the NE-PER nuclear and cytoplasmic extraction kit (Pierce, Rockford, IL, USA).
Quantification of cH2AX-foci formation Cells were cultivated on CultureSlides (Becton–Dickinson), incubated with BBI (10 lM) for 16 h, irradiated and fixed with 70% ice-cold ethanol after 24 h. For immune-fluorescence analysis cells were incubated with cH2AX antibody (Upstate, clone JBW301) (1:500) for 2 h at room temperature. Positive foci were visualized by incubation with a 1:500 dilution of Alexa488-labelled goat anti-mouse serum (Molecular Probes) for 30 min. Coverslips were mounted in Vectashield/DAPI (Vector Laboratories). For each data point, 300–500 nuclei were evaluated.
Cell culture and irradiation The human bronchial carcinoma cell line, designated A549 (wt. TP53) (ATCC), and transformed fibroblast cell line, HH4dd (mt. TP53) [10], were used. Trypsinized cells were seeded for the colony formation assay at a density of 250 per 9.6 cm2 well and 24 h after plating cells were treated with BBI (10 lM). Subsequently, cells were irradiated with 225-kV photons (Gulmay RS 225) with a dose rate of 1 Gy/min at 37 C. BBI was purchased from Sigma and used at a concentration of 10 lM for 16 h before irradiation.
Confocal microscopy A549 cells were cultivated on CultureSlides (Becton– Dickinson, Franklin Lakes, NJ, USA), irradiated with 4 Gy, and after 20 min fixed with periodate-lysine-paraformaldehyde (PLP). For immunofluorescence analysis, cells were incubated with Anti-EGFR (BD Bioscience, clone 13) (1:20) for 1 h at 4 C. Bound antibodies were visualized by incubation with a 1:500 dilution of a Cy3-donkey anti–mouse serum (Dianova, Hamburg, Germany) for 1 h. DNA was stained with YO-PRO (Molecular Probes, Leiden, The Netherlands). The nuclei were analyzed with a confocal laser scanning microscope (Leica TCS SP, Leica Microsystems, Bensheim, Germany).
Results BBI treatment induced nuclear EGFR accumulation To elucidate the subcellular localization of EGFR after BBI- and radiation-treatment, we performed confocal microscopy. We failed to detect EGFR (red) within the nuclear region (DNA = green) of non-irradiated bronchial carcinoma line A549 (Fig. 1). However, there was a strong signal for EGFR in the peri-nuclear cell compartment in these cells. Twenty min after irradiation with 4 Gy, a clear accumulation of EGFR protein within the nuclear region and a co-localization with DNA was observed (merged colour yellow) (Fig. 1). A 16 h pretreatment with BBI resulted in a nuclear EGFR accumulation even in non-irradiated cells. Subsequent irradiation stimulated nuclear EGFR accumulation further.
BBI treatment activated src kinase and triggered EGFR phosphorylation at residue Y845 To elucidate the molecular mechanism of BBI on nuclear EGFR accumulation, we incubated confluent A549 and
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Fig. 1. Nuclear EGFR transport induced by BBI treatment and irradiation. Confluent A549 cells were either treated with BBI for 16 h and/or irradiated with 4 Gy. Twenty minutes after irradiation, cells were fixed and immune-stained with anti-EGFR antibody. Binding was visualized by a secondary antibody labelled with Cy3 (red). DNA was stained with YO-PRO (green). Analysis of nuclei was performed by confocal microscopy; fluorescence signals of Cy3 and YO-PRO were superimposed (yellow).
HH4dd cells with BBI and immune-precipitated EGFR from a fraction of cytoplasmic proteins for various treatment times (Fig. 2). In A549 cells, BBI treatment resulted in an increase of EGFR protein over the time with a maximum after 6 h. This increase was associated with phosphorylation of EGFR at tyrosine residue 845 (Fig. 2A). Since it was reported, that phosphorylation of EGFR at Y845 results from src kinase activity [23] and is essential for EGFR transactivation, we screened for activated src kinase bound to EGFR. Indeed, we observed, that BBI-induced EGFR phosphorylation at Y845 correlated to complex formation with activated src, characterized by Y416 phosphorylation (Fig. 2A) during the first 6 hours of BBI treatment. However, longer treatment times resulted in a loss of this correlation and EGFR phosphorylation at Y845 decreased, whereas activated src increased within the complex with EGFR. To understand the functional importance of this complex formation, we screened for binding of caveolin 1, which is reported to be involved in EGFR internalization following transactivation [24]. As shown in Fig. 2A, caveolin 1 is part of this BBI-induced protein complex. Moreover, bound caveolin 1 is phosphorylated at tyrosine residue 14, which represents the activated status of caveolin 1. This phosphorylation is also performed by src kinase [24]. For transformed HH4dd cells, comparable reaction patterns were observed (Fig. 2B), but it is noteworthy, that in these cells the src response upon BBI treatment is much more pronounced. This effect is consequently associated with both increased phosphorylation of EGFR at Y845 and increased phosphorylation of caveolin 1 at Y14.
Fig. 2. BBI treatment stimulated src kinase and triggers EGFR phosphorylation at residue Y845. Confluent A549 (A) and HH4dd cells (B) were treated with 10 lM BBI for 16 h. At the time points given, cells were lysed and cytoplasmic proteins were isolated. The EGFR was immune-precipitated from the fraction of cytoplasmic proteins. Proteins were separated by SDS–PAGE and proteins were transferred to nitrocellulose membranes by blotting. The expression of EGFR, EGFR phosphorylated at Y845, src, src phosphorylated at Y416, caveolin 1 and caveolin 1 phosphorylated at Y14 were detected. The experiment was performed three times; representative results are shown.
BBI amplified radiation-induced effects on EGFR Irradiation with 4 Gy resulted in an increase in EGFR protein within the cytoplasmic protein fraction of A549 cells (Fig. 3A). This process correlated with EGFR phosphorylation at Y845, binding of activated src and binding of activated caveolin. As shown above (Fig. 2) BBI treatment alone for 16 h increased the amount of EGFR in the absence of irradiation. In addition, BBI treatment per se increased EGFR phosphorylation at Y845, binding of activated src and binding of activated caveolin. Combined treatment with BBI and irradiation further increased the EGFR amount in the fraction of cytoplasmic proteins and this process correlated with EGFR phosphorylation at Y845, binding of activated src and binding of activated caveolin. For HH4dd cells, combined treatment of BBI and irradiation resulted in the same reaction patterns (Fig. 3B); however, as already described in Fig. 2, the response upon radiation alone and
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Fig. 3. BBI amplified radiation-induced molecular effects upon EGFR. Confluent A549 (A) and HH4dd cells (B) were incubated with BBI (10 lM) for various times and irradiated with a dose of 4 Gy. Cytoplasmic proteins were isolated from 20 · 106 cells, EGFR immune-precipitation was performed and proteins were separated by SDS–PAGE. After transfer to nitrocellulose, amounts of EGFR, EGFR phosphorylated at T654, src, src phosphorylated at Y416, caveolin 1 and caveolin 1 phosphorylated at Y14 were detected with the help of specific antibodies. Experiments were performed three times; representative results are shown.
also for combined treatment with BBI was much more pronounced.
BBI- and radiation-treatment resulted in EGFR accumulation within the cell nucleus As reported earlier [21], the EGFR protein was accumulated within the cell nucleus following irradiation. Fig. 4A shows a clear increase of nuclear EGFR 10–20 min after irradiation. As reported recently [20] and also shown herein, this nuclear accumulation was associated with phosphorylation of EGFR at T654 [20]. Nuclear EGFR seems to be involved in regulation of DNA-PK kinase activity [21,25–27]. In agreement with that nuclear EGFR accumulation correlated with the appearance of activated DNA-PK phosphorylated at residue T2609 [28]. As already observed for cytoplasmic proteins (Fig. 3), BBI treatment also increased nuclear EGFR protein (Fig. 4A). This process correlated with increased phosphorylation of EGFR at T654 and phosphorylation of DNA-PK at T2609. In combination with irradiation these accumulation and phosphorylation processes were even further increased. Comparable effects of BBI treatment were observed with HH4dd cells (Fig. 4B).
Fig. 4. BBI mediated modulation of radiation-induced nuclear EGFR accumulation. A549 cells (A) and HH4dd cells (B) were incubated with BBI (10 lM) for 16 h and subsequently irradiated with 4 Gy. At the time points given after irradiation, nuclear proteins were isolated, separated by SDS–PAGE and blotted. With help of specific antibodies the amounts of EGFR, EGFR phosphorylated at T654, DNA-PK and DNA-PK phosphorylated at T2609 were detected. Lamin B1 protein was detected as a loading control. Experiments were performed three times; representative results are shown.
BBI treatment stimulated DNA-double strand break repair in A549 cells, but failed in HH4dd cells Radiation induced a dose dependent increase in residual DNA-double strand breaks in A549 and HH4dd cells (Fig. 5). BBI pre-treatment, however, reduced this residual damage only in A549 cells, but not in HH4dd cells. These data fit the observation, published earlier [10], that BBI acts as a radioprotector for TP53 wt. A549 cells but not for HH4dd cells with mt. TP53.
BBI increased radiation induced complex formation between EGFR, TP53 and MDC1 To elucidate a possible link between EGFR and TP53, which may explain the different biological effects of BBI upon A549 and HH4dd cells reported earlier [10], we performed an EGFR immune-precipitation and tested for binding of TP53 to EGFR. As presented in Fig. 6A, TP53 is bound to nuclear EGFR upon incubation with BBI (Fig. 6). This complex formation increased in A549 with prolonged BBI incubation time. Complex formation could be observed for TP53 protein as well as for activated TP53 phosphorylated at residue Ser15. For HH4dd cells, a complex formation between EGFR and TP53 was also observed. However, the mutated TP53 of HH4dd cells [10] and also its Ser15 phosphorylated
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Fig. 5. BBI decreases radiation induced residual DNA damage. Confluent A549 cells (A) and HH4dd cells (B) were treated with BBI (10 lM) for 16 h. Subsequently cells were irradiated with 2, 4 and 6 Gy and after 24 h cells were fixed. Residual damage was visualized by incubation with cH2AX antibody. Each bar represents means ± SE of residual repair foci positive for cH2AX per cell. For each data point 300–500 nuclei were evaluated. Asterisks indicate significant differences (Student’s t-test *p < 0.05).
form were bound in excess to EGFR and BBI treatment failed to increase complex formation. Most interestingly, BBI increased complex formation between EGFR and TP53 in A549 cells was associated with increased binding of MDC1 protein, whereas in HH4dd cells MDC1 binding was weak and not increased by BBI (Fig. 6A). In combination with irradiation, BBI pre-treatment strengthened the radiation induced complex formation between EGFR, TP53 and MDC1 in A549 cells characterized by a wt. TP53 (Fig. 6B). In contrast, HH4dd cells characterized by a mt. TP53, failed to respond upon BBI or irradiation treatment with a formation of the EGFR/MDC1 complex (Fig. 6C).
Discussion Several recent reports describe EGFR accumulation within the cell nucleus [29–31]. These reports link an increased nuclear EGFR with increased treatment resistance and poor prognosis for patients [32–34]. One explanation could be that nuclear accumulation of EGFR is part of the cellular stress response and ensures increased cell survival. This idea is strengthened by the observation that nuclear EGFR
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Fig. 6. BBI and irradiation modulate complex formation of nuclear EGFR, TP53 and MDC1 Confluent A549- and HH4dd cells were treated with 10 lM BBI up to 16 h (A). In different experiments, A549 cells (B) and HH4dd cells (C) were pretreated with BBI for 16 h and subsequently cells were irradiated with 4 Gy. At the time points given, cells were lysed and nuclear proteins were isolated. The EGFR was immune-precipitated from the fraction of nuclear proteins. Proteins were separated by SDS–PAGE and proteins were transferred to nitrocellulose membranes by blotting. The expressions of EGFR, TP53, TP53 phosphorylated at S15 and MDC1 were visualized by means of specific antibodies. Experiments were performed three times; representative results are shown.
accumulation can be stimulated by cellular treatment with stressors, e.g., radiation, heat, radicals [21]. In addition, it could be shown that nuclear EGFR was linked with activation of DNA-PK, which plays a crucial role during DNA repair processes [28]. Nuclear accumulation of EGFR could be correlated to the pool of cytoplasmic and membrane-bound EGFR, suggesting that both compartments can serve as a protein reservoir for nuclear transport [35]. Membranebound EGFR is activated upon treatment with stress factors and is then sorted into caveolae and internalized [24]. Within caveolae, EGFR is stable and accumulates in a peri-nuclear localization [24]. Earlier observations have shown that BBI interacts with EGFR [36] and that BBI is able to induce
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expression of repair relevant genes [14]. In addition, BBI treatment prevented formation of dicentric chromosomes by activation of DNA-PK [15]. All together these data argue for a link between BBI, EGFR, and DNA repair. Herein we demonstrate that BBI treatment induced EGFR nuclear accumulation, which could also be observed following radiation exposure [37]. Thus, as already suggested earlier [10], BBI-triggered cell signalling runs parallel to radiation-induced activation of DNA repair and increased cellular survival [21]. Indeed, as a consequence of BBI treatment alone, we observed internalization of EGFR via caveolae to a similar extent as observed after irradiation. EGFR internalization seems to be src-kinase dependent as shown by us and others [24] and is associated with phosphorylation of caveolin 1 at Y14. Src activity is also thought to be responsible for EGFR phosphorylation at residue Y845 [38], however this phosphorylation seems not to be essential for EGFR internalization following stress exposure [24]. It is suggested that EGFR phosphorylation at Y845 is involved in regulation of tyrosine kinase activity of EGFR [39]. Both irradiation and BBI treatment induced EGFR internalization. However, BBI treatment did not induce DNA-damage or reduced clonogenic survival [10,15], which suggests a regulation of EGFR internalization independent from DNA damage. BBI-induced EGFR internalization was linked to nuclear EGFR accumulation, which could be followed by increased protein amount and phosphorylation at T654 [20]. As reported earlier [21], nuclear EGFR accumulation was associated with increased phosphorylation of DNA-PK at residue T2609, which correlated with enzyme activation. Activation of DNA-PK is essential for the endonuclease Artemis to ensure DNA end-joining [28] during repair of double strand breaks. These data are in agreement with previous reports [15] that BBI stimulated DNA-PK, reduced the amount of radiation-induced dicentric chromosomes and increased post-irradiation clonogenic survival [9]. Dicentric chromosomes are derived from errors during non-homologous end-joining and are linked with increased cell death [40]. Data presented earlier show that the radioprotective effect of BBI depends on the presence of a wt. TP53 [10]. In the present study we show that BBI induced molecular changes linked to EGFR signalling were independent of the TP53 status. One explanation for this observation would be that BBI-triggered EGFR signalling acts upstream of TP53. In agreement with this idea, we observed that nuclear EGFR is in complex with TP53 after BBI treatment independent from TP53 cellular status. Since TP53 can play an essential role during DNA repair [41,42], we assume that TP53 links BBI mediated effects on EGFR with DNA repair processes. In agreement with that idea, data presented in Fig. 6 provide the first mechanistic evidence for BBI-mediated TP53-dependent radioprotection. In cells with wt. TP53, we observed that BBI-induced nuclear EGFR accumulation was linked with stimulated complex formation of EGFR, TP53, and the protein MDC1. MDC1 was reported to translocate to sites of DNA lesions, where it collaborates with other proteins and with phosphorylated histone H2AX to mediate the accumulation of checkpoint and repair factors into nuclear foci [43]. Thus MDC1 can act as a positive regulator of NHEJ-repair [44] and is regarded as a tumor suppressor [45]. Generally, TP53 and MDC1 can be involved
in both non-homologous end-joining [46] and homologous recombination [47]; however, the use of confluent cells – which repair double strand breaks by NHEJ predominantly – argue for an involvement of both factors in NHEJ under the experimental conditions used. Nevertheless, we cannot exclude an effect upon homologous recombination also. Interestingly, both irradiation and BBI modulated nuclear complex formation between EGFR and MDC1 in cells with wt. TP53. Nevertheless, despite comparable protein expression of MDC1 in HH4dd cells and A549 cells (data not shown), the complex formation between EGFR and MDC1 failed in HH4dd cells with both treatments and may explain the lack of enhancement of double strand break repair by BBI following irradiation. This observation may imply that the presence of a wt. TP53 is mandatory for binding of MDC1 to the EGFR/TP53 complex and double strand break repair. The TP53 mutation in HH4dd cells is located within exon 5, which codes for the core domain of the molecule and is involved in sequence-specific DNA-binding [10]. Indeed this mutation is linked with altered DNA-binding and with failure to induce TP53 dependent gene transcription by ionizing radiation [10]. How this mutation is involved in the failure of EGFR/MDC1 complex formation will be the subject of future investigations. In summary, treatment of cells with BBI resulted in an increased internalization of EGFR into the cytoplasm as well as stimulated nuclear EGFR transport. Increased nuclear EGFR is associated with activation of DNA-PK and binding of TP53 and MDC1. In cells with mt. TP53, MDC1 binding is impaired, which may explain the dependence of BBI mediated radioprotection on the presence of functional TP53.
Acknowledgements We thank Nadine Hoffmann for excellent technical assistance. This work was supported by grants from Deutsche Forschungsgemeinschaft (DI 402/9-1) and Deutsche Krebshilfe (No. 106401). * Corresponding author. Klaus Dittmann, Division of Radiobiology and Environmental Research, Department of Radiation Oncology University of Tu ¨ntgenweg 11, 72076 Tu ¨bingen, Ro ¨bingen, Germany. E-mail address: [email protected] Received 31 October 2007; received in revised form 28 December 2007; accepted 3 January 2008; Available online 30 January 2008
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[43] Stucki M, Jackson SP. MDC1/NFBD1: a key regulator of the DNA damage response in higher eukaryotes. DNA Repair (Amst) 2004;3:953–7. [44] Dimitrova N, de Lange T. MDC1 accelerates nonhomologous endjoining of dysfunctional telomeres. Genes Dev 2006;20:3238–43. [45] Bartkova J, Horej Sbreve IZ, Sehested M, et al. DNA damage response mediators MDC1 and 53BP1: constitutive activation
and aberrant loss in breast and lung cancer, but not in testicular germ cell tumours. Oncogene 2007. [46] Akyuz N, Boehden GS, Susse S, et al. DNA substrate dependence of p53-mediated regulation of double-strand break repair. Mol Cell Biol 2002;22:6306–17. [47] Gatz SA, Wiesmuller L. p53 in recombination and repair. Cell Death Differ 2006;13:1003–16.
DNA repair
The radioprotector Bowman–Birk proteinase inhibitor stimulates DNA repair via epidermal growth factor receptor phosphorylation and nuclear transport Klaus Dittmanna,*, Claus Mayera, Rainer Kehlbachb, H. Peter Rodemanna a
¨bingen, Tu ¨bingen, Germany, bDepartment of Radiology, Tu ¨bingen, Germany Department of Radiation Oncology, University of Tu
Abstract Background and purpose: The purpose of the study was to elucidate the underlying molecular mechanism of the radioprotector, Bowman–Birk proteinase inhibitor (BBI), and its interaction with EGFR nuclear transport. Materials and methods: Molecular effects of BBI at the level of EGFR responses were investigated in vitro with wt. TP53 bronchial carcinoma cell line A549 and the transformed fibroblast cell line HH4dd characterized by a mt. TP53. EGFR and associated protein expression were quantified by Western blotting and confocal microscopy in the cytoplasmic and nuclear cell fraction. Residual DNA double strand breaks were quantified by means of a cH2AX focus assay. Results: Both irradiation and BBI-treatment stimulated EGFR internalization into the cytoplasm. This process involved src kinase activation, EGFR phosphorylation at Y845, and caveolin 1 phosphorylation at Y14. EGFR internalization correlated with nuclear EGFR transport and was associated with phosphorylation of EGFR at T654. Nuclear EGFR was linked with DNA-PK complex formation and activation. Furthermore, nuclear EGFR was found in complex with TP53, phosphorylated at S15, and with MDC1, following irradiation and BBI treatment. It is noteworthy that MDC1 was strongly decreased in the nuclear EGFR complex in cells with mt. TP53 and failed to be increased by either BBI treatment or irradiation. Interestingly, in cells with mt. TP53 the BBI mediated stimulation of double strand break repair was hampered significantly. Conclusion: These data indicate that BBI stimulates complex formation between EGFR, TP53 and MDC1 protein in wt. TP53 cells only. Since MDC1 is essential for recruitment of DNA repair foci, this observation may explain how BBI selectively stimulated repair of DNA double strand breaks in wt. TP53 cells. c 2008 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 86 (2008) 375–382.
Keywords: BBI; EGFR; TP53; DNA repair; MDC1
Several studies have shown that BBI is able to prevent the development of malignancies in different animal tumor model systems [1,2]. To perform clinical trials, a crude extract from soybeans was developed (BBI Concentrate, BBIC) [3], and in 1992 approved by the FDA for ‘‘Investigational New Drug’’ status. BBIC was used in a phase II study in patients with oral leukoplakia [4]. It was well tolerated by the patients, and no clinical or laboratory evidence of toxicity or drug allergy was observed other than infrequent reporting of nausea, diarrhea, or epigastric discomfort, which did not seem related to drug ingestion [5]. The molecular mode of BBI is not resolved so far. Our in vitro studies on radiation-induced fibroblast differentiation, a cellular process leading to radiation-induced fibrosis [6–8], suggested that pre-treatment of fibroblasts with BBI prevented the radiation-induced terminal differentiation and thus increased clonogenic survival up to 30% [9]. Subsequent studies showed that the BBI increased clonogenic
activity after irradiation only in cells representing functional TP53, but not in cells lacking functional TP53 [10,11]. This characterized BBI as a selective radioprotector for normal cells, but not for tumor cells presenting mt. TP53, which is commonly present in many malignancies [12]. Moreover, by means of a mouse fibrosis model system (leg contracture assay), we were able to prove in vivo that BBI reduced radiation-induced fibrosis by about 50% [13]. In the same study we showed that BBI exerted no radioprotective effect upon tumors [13]. Thus, BBI selectively protects normal tissue and may therefore improve the therapeutic index of radiotherapy. Studies to elucidate the molecular mechanism of BBI revealed that BBI-mediated radioprotection is linked to DNA repair processes by means of TP53 in two ways. First, BBI induces stabilization of the TP53 protein and this process is associated with the expression of DNA repair-relevant genes
0167-8140/$ - see front matter c 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.radonc.2008.01.007
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[14]. Second, BBI stimulates activity of DNA-PK kinase [15], a key player in non-homologous end joining (NHEJ) DNA repair. The BBI treatment significantly reduced formation of radiation-induced dicentric chromosomes [15], implying that BBI improved the fidelity of the NHEJ process. BBI, a soybean-derived polypeptide, was shown to act anticarcinogenicly in a variety of in vitro- and in vivo-carcinogenesis assay systems [16]. The anticarcinogenic activity of BBI is localized in the chymotrypsin inhibitory region of the protein molecule [17]. In addition, we also localized the amino acids responsible for the radioprotective effect within this region [18]. We identified the phosphorylated tyrosine residue within this region as the essential amino acid and could show that the application of phosphotyrosin (P-Tyr) alone is sufficient to generate the radioprotective effect [19]. Ongoing investigations to elucidate the molecular mechanism showed that P-Tyr induced transport of the cytoplasmic EGFR into the cell nucleus [20]. Nuclear EGFR is linked with regulation of DNA-dependent protein kinase (DNA-PK) and thereby stimulates DNA repair after irradiation [21]. Previous data [22] demonstrated that the radioprotective effect of BBI is also mediated via EGFR. Data presented herein show that the molecular mechanism of BBI includes EGFR internalization and activation of DNA-PK. On the basis of these in vitro and in vivo experiments we tried to shed light onto the molecular mechanism of BBI, with particular emphasis on its effect upon DNA repair processes. We performed these experiments with A549 cells, which are radioprotected by BBI due to expression of wt. TP53, and compared the results obtained with data for HH4dd cells, which are characterized by expression of mt.TP53 and are not radioprotected by BBI.
Materials and methods
Western blot analysis and immune-precipitation Cell cultures were irradiated as described above; cells were lysed and proteins were resolved by SDS–PAGE. Western blotting was performed according to standard procedures. The primary antibodies were diluted as follows: anti-EGFR (BD Transduction Laboratories, clone 13) 1:1000; anti EGFR pT654 (nanotools, clone 12A3) 1:1000; anti-src (Santa Cruz, clone H-12) 1:1000; anti-src Y416 (cell signaling, polyclonal), 1:1000: anti-caveolin 1 (BD Transduction Laboratories, clone 2297) 1:1000; anti-caveolin pY14 (BD Transduction Laboratories, clone 56) 1:1000; antiDNA-PK (PharMingen, clone 4F10C5) 1:500: anti-DNA-PK phospho-Thr No 2609 (Rockland) 1:1000; anti-lamin B1 (Biozol, clone ZL-5) 1:1000. Quantification of binding was achieved by incubation with a secondary peroxidase-conjugated antibody with the ECL system (Amersham). EGFR was immune-precipitated from cytoplasmic and nuclear protein fractions obtained from 20 · 106 cells with antiEGFR antibody clone 13 (BD Transduction Laboratories).
Subcellular fractionation Cytoplasmic and nuclear extracts were prepared according to the instructions of the NE-PER nuclear and cytoplasmic extraction kit (Pierce, Rockford, IL, USA).
Quantification of cH2AX-foci formation Cells were cultivated on CultureSlides (Becton–Dickinson), incubated with BBI (10 lM) for 16 h, irradiated and fixed with 70% ice-cold ethanol after 24 h. For immune-fluorescence analysis cells were incubated with cH2AX antibody (Upstate, clone JBW301) (1:500) for 2 h at room temperature. Positive foci were visualized by incubation with a 1:500 dilution of Alexa488-labelled goat anti-mouse serum (Molecular Probes) for 30 min. Coverslips were mounted in Vectashield/DAPI (Vector Laboratories). For each data point, 300–500 nuclei were evaluated.
Cell culture and irradiation The human bronchial carcinoma cell line, designated A549 (wt. TP53) (ATCC), and transformed fibroblast cell line, HH4dd (mt. TP53) [10], were used. Trypsinized cells were seeded for the colony formation assay at a density of 250 per 9.6 cm2 well and 24 h after plating cells were treated with BBI (10 lM). Subsequently, cells were irradiated with 225-kV photons (Gulmay RS 225) with a dose rate of 1 Gy/min at 37 C. BBI was purchased from Sigma and used at a concentration of 10 lM for 16 h before irradiation.
Confocal microscopy A549 cells were cultivated on CultureSlides (Becton– Dickinson, Franklin Lakes, NJ, USA), irradiated with 4 Gy, and after 20 min fixed with periodate-lysine-paraformaldehyde (PLP). For immunofluorescence analysis, cells were incubated with Anti-EGFR (BD Bioscience, clone 13) (1:20) for 1 h at 4 C. Bound antibodies were visualized by incubation with a 1:500 dilution of a Cy3-donkey anti–mouse serum (Dianova, Hamburg, Germany) for 1 h. DNA was stained with YO-PRO (Molecular Probes, Leiden, The Netherlands). The nuclei were analyzed with a confocal laser scanning microscope (Leica TCS SP, Leica Microsystems, Bensheim, Germany).
Results BBI treatment induced nuclear EGFR accumulation To elucidate the subcellular localization of EGFR after BBI- and radiation-treatment, we performed confocal microscopy. We failed to detect EGFR (red) within the nuclear region (DNA = green) of non-irradiated bronchial carcinoma line A549 (Fig. 1). However, there was a strong signal for EGFR in the peri-nuclear cell compartment in these cells. Twenty min after irradiation with 4 Gy, a clear accumulation of EGFR protein within the nuclear region and a co-localization with DNA was observed (merged colour yellow) (Fig. 1). A 16 h pretreatment with BBI resulted in a nuclear EGFR accumulation even in non-irradiated cells. Subsequent irradiation stimulated nuclear EGFR accumulation further.
BBI treatment activated src kinase and triggered EGFR phosphorylation at residue Y845 To elucidate the molecular mechanism of BBI on nuclear EGFR accumulation, we incubated confluent A549 and
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Fig. 1. Nuclear EGFR transport induced by BBI treatment and irradiation. Confluent A549 cells were either treated with BBI for 16 h and/or irradiated with 4 Gy. Twenty minutes after irradiation, cells were fixed and immune-stained with anti-EGFR antibody. Binding was visualized by a secondary antibody labelled with Cy3 (red). DNA was stained with YO-PRO (green). Analysis of nuclei was performed by confocal microscopy; fluorescence signals of Cy3 and YO-PRO were superimposed (yellow).
HH4dd cells with BBI and immune-precipitated EGFR from a fraction of cytoplasmic proteins for various treatment times (Fig. 2). In A549 cells, BBI treatment resulted in an increase of EGFR protein over the time with a maximum after 6 h. This increase was associated with phosphorylation of EGFR at tyrosine residue 845 (Fig. 2A). Since it was reported, that phosphorylation of EGFR at Y845 results from src kinase activity [23] and is essential for EGFR transactivation, we screened for activated src kinase bound to EGFR. Indeed, we observed, that BBI-induced EGFR phosphorylation at Y845 correlated to complex formation with activated src, characterized by Y416 phosphorylation (Fig. 2A) during the first 6 hours of BBI treatment. However, longer treatment times resulted in a loss of this correlation and EGFR phosphorylation at Y845 decreased, whereas activated src increased within the complex with EGFR. To understand the functional importance of this complex formation, we screened for binding of caveolin 1, which is reported to be involved in EGFR internalization following transactivation [24]. As shown in Fig. 2A, caveolin 1 is part of this BBI-induced protein complex. Moreover, bound caveolin 1 is phosphorylated at tyrosine residue 14, which represents the activated status of caveolin 1. This phosphorylation is also performed by src kinase [24]. For transformed HH4dd cells, comparable reaction patterns were observed (Fig. 2B), but it is noteworthy, that in these cells the src response upon BBI treatment is much more pronounced. This effect is consequently associated with both increased phosphorylation of EGFR at Y845 and increased phosphorylation of caveolin 1 at Y14.
Fig. 2. BBI treatment stimulated src kinase and triggers EGFR phosphorylation at residue Y845. Confluent A549 (A) and HH4dd cells (B) were treated with 10 lM BBI for 16 h. At the time points given, cells were lysed and cytoplasmic proteins were isolated. The EGFR was immune-precipitated from the fraction of cytoplasmic proteins. Proteins were separated by SDS–PAGE and proteins were transferred to nitrocellulose membranes by blotting. The expression of EGFR, EGFR phosphorylated at Y845, src, src phosphorylated at Y416, caveolin 1 and caveolin 1 phosphorylated at Y14 were detected. The experiment was performed three times; representative results are shown.
BBI amplified radiation-induced effects on EGFR Irradiation with 4 Gy resulted in an increase in EGFR protein within the cytoplasmic protein fraction of A549 cells (Fig. 3A). This process correlated with EGFR phosphorylation at Y845, binding of activated src and binding of activated caveolin. As shown above (Fig. 2) BBI treatment alone for 16 h increased the amount of EGFR in the absence of irradiation. In addition, BBI treatment per se increased EGFR phosphorylation at Y845, binding of activated src and binding of activated caveolin. Combined treatment with BBI and irradiation further increased the EGFR amount in the fraction of cytoplasmic proteins and this process correlated with EGFR phosphorylation at Y845, binding of activated src and binding of activated caveolin. For HH4dd cells, combined treatment of BBI and irradiation resulted in the same reaction patterns (Fig. 3B); however, as already described in Fig. 2, the response upon radiation alone and
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Fig. 3. BBI amplified radiation-induced molecular effects upon EGFR. Confluent A549 (A) and HH4dd cells (B) were incubated with BBI (10 lM) for various times and irradiated with a dose of 4 Gy. Cytoplasmic proteins were isolated from 20 · 106 cells, EGFR immune-precipitation was performed and proteins were separated by SDS–PAGE. After transfer to nitrocellulose, amounts of EGFR, EGFR phosphorylated at T654, src, src phosphorylated at Y416, caveolin 1 and caveolin 1 phosphorylated at Y14 were detected with the help of specific antibodies. Experiments were performed three times; representative results are shown.
also for combined treatment with BBI was much more pronounced.
BBI- and radiation-treatment resulted in EGFR accumulation within the cell nucleus As reported earlier [21], the EGFR protein was accumulated within the cell nucleus following irradiation. Fig. 4A shows a clear increase of nuclear EGFR 10–20 min after irradiation. As reported recently [20] and also shown herein, this nuclear accumulation was associated with phosphorylation of EGFR at T654 [20]. Nuclear EGFR seems to be involved in regulation of DNA-PK kinase activity [21,25–27]. In agreement with that nuclear EGFR accumulation correlated with the appearance of activated DNA-PK phosphorylated at residue T2609 [28]. As already observed for cytoplasmic proteins (Fig. 3), BBI treatment also increased nuclear EGFR protein (Fig. 4A). This process correlated with increased phosphorylation of EGFR at T654 and phosphorylation of DNA-PK at T2609. In combination with irradiation these accumulation and phosphorylation processes were even further increased. Comparable effects of BBI treatment were observed with HH4dd cells (Fig. 4B).
Fig. 4. BBI mediated modulation of radiation-induced nuclear EGFR accumulation. A549 cells (A) and HH4dd cells (B) were incubated with BBI (10 lM) for 16 h and subsequently irradiated with 4 Gy. At the time points given after irradiation, nuclear proteins were isolated, separated by SDS–PAGE and blotted. With help of specific antibodies the amounts of EGFR, EGFR phosphorylated at T654, DNA-PK and DNA-PK phosphorylated at T2609 were detected. Lamin B1 protein was detected as a loading control. Experiments were performed three times; representative results are shown.
BBI treatment stimulated DNA-double strand break repair in A549 cells, but failed in HH4dd cells Radiation induced a dose dependent increase in residual DNA-double strand breaks in A549 and HH4dd cells (Fig. 5). BBI pre-treatment, however, reduced this residual damage only in A549 cells, but not in HH4dd cells. These data fit the observation, published earlier [10], that BBI acts as a radioprotector for TP53 wt. A549 cells but not for HH4dd cells with mt. TP53.
BBI increased radiation induced complex formation between EGFR, TP53 and MDC1 To elucidate a possible link between EGFR and TP53, which may explain the different biological effects of BBI upon A549 and HH4dd cells reported earlier [10], we performed an EGFR immune-precipitation and tested for binding of TP53 to EGFR. As presented in Fig. 6A, TP53 is bound to nuclear EGFR upon incubation with BBI (Fig. 6). This complex formation increased in A549 with prolonged BBI incubation time. Complex formation could be observed for TP53 protein as well as for activated TP53 phosphorylated at residue Ser15. For HH4dd cells, a complex formation between EGFR and TP53 was also observed. However, the mutated TP53 of HH4dd cells [10] and also its Ser15 phosphorylated
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Fig. 5. BBI decreases radiation induced residual DNA damage. Confluent A549 cells (A) and HH4dd cells (B) were treated with BBI (10 lM) for 16 h. Subsequently cells were irradiated with 2, 4 and 6 Gy and after 24 h cells were fixed. Residual damage was visualized by incubation with cH2AX antibody. Each bar represents means ± SE of residual repair foci positive for cH2AX per cell. For each data point 300–500 nuclei were evaluated. Asterisks indicate significant differences (Student’s t-test *p < 0.05).
form were bound in excess to EGFR and BBI treatment failed to increase complex formation. Most interestingly, BBI increased complex formation between EGFR and TP53 in A549 cells was associated with increased binding of MDC1 protein, whereas in HH4dd cells MDC1 binding was weak and not increased by BBI (Fig. 6A). In combination with irradiation, BBI pre-treatment strengthened the radiation induced complex formation between EGFR, TP53 and MDC1 in A549 cells characterized by a wt. TP53 (Fig. 6B). In contrast, HH4dd cells characterized by a mt. TP53, failed to respond upon BBI or irradiation treatment with a formation of the EGFR/MDC1 complex (Fig. 6C).
Discussion Several recent reports describe EGFR accumulation within the cell nucleus [29–31]. These reports link an increased nuclear EGFR with increased treatment resistance and poor prognosis for patients [32–34]. One explanation could be that nuclear accumulation of EGFR is part of the cellular stress response and ensures increased cell survival. This idea is strengthened by the observation that nuclear EGFR
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Fig. 6. BBI and irradiation modulate complex formation of nuclear EGFR, TP53 and MDC1 Confluent A549- and HH4dd cells were treated with 10 lM BBI up to 16 h (A). In different experiments, A549 cells (B) and HH4dd cells (C) were pretreated with BBI for 16 h and subsequently cells were irradiated with 4 Gy. At the time points given, cells were lysed and nuclear proteins were isolated. The EGFR was immune-precipitated from the fraction of nuclear proteins. Proteins were separated by SDS–PAGE and proteins were transferred to nitrocellulose membranes by blotting. The expressions of EGFR, TP53, TP53 phosphorylated at S15 and MDC1 were visualized by means of specific antibodies. Experiments were performed three times; representative results are shown.
accumulation can be stimulated by cellular treatment with stressors, e.g., radiation, heat, radicals [21]. In addition, it could be shown that nuclear EGFR was linked with activation of DNA-PK, which plays a crucial role during DNA repair processes [28]. Nuclear accumulation of EGFR could be correlated to the pool of cytoplasmic and membrane-bound EGFR, suggesting that both compartments can serve as a protein reservoir for nuclear transport [35]. Membranebound EGFR is activated upon treatment with stress factors and is then sorted into caveolae and internalized [24]. Within caveolae, EGFR is stable and accumulates in a peri-nuclear localization [24]. Earlier observations have shown that BBI interacts with EGFR [36] and that BBI is able to induce
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expression of repair relevant genes [14]. In addition, BBI treatment prevented formation of dicentric chromosomes by activation of DNA-PK [15]. All together these data argue for a link between BBI, EGFR, and DNA repair. Herein we demonstrate that BBI treatment induced EGFR nuclear accumulation, which could also be observed following radiation exposure [37]. Thus, as already suggested earlier [10], BBI-triggered cell signalling runs parallel to radiation-induced activation of DNA repair and increased cellular survival [21]. Indeed, as a consequence of BBI treatment alone, we observed internalization of EGFR via caveolae to a similar extent as observed after irradiation. EGFR internalization seems to be src-kinase dependent as shown by us and others [24] and is associated with phosphorylation of caveolin 1 at Y14. Src activity is also thought to be responsible for EGFR phosphorylation at residue Y845 [38], however this phosphorylation seems not to be essential for EGFR internalization following stress exposure [24]. It is suggested that EGFR phosphorylation at Y845 is involved in regulation of tyrosine kinase activity of EGFR [39]. Both irradiation and BBI treatment induced EGFR internalization. However, BBI treatment did not induce DNA-damage or reduced clonogenic survival [10,15], which suggests a regulation of EGFR internalization independent from DNA damage. BBI-induced EGFR internalization was linked to nuclear EGFR accumulation, which could be followed by increased protein amount and phosphorylation at T654 [20]. As reported earlier [21], nuclear EGFR accumulation was associated with increased phosphorylation of DNA-PK at residue T2609, which correlated with enzyme activation. Activation of DNA-PK is essential for the endonuclease Artemis to ensure DNA end-joining [28] during repair of double strand breaks. These data are in agreement with previous reports [15] that BBI stimulated DNA-PK, reduced the amount of radiation-induced dicentric chromosomes and increased post-irradiation clonogenic survival [9]. Dicentric chromosomes are derived from errors during non-homologous end-joining and are linked with increased cell death [40]. Data presented earlier show that the radioprotective effect of BBI depends on the presence of a wt. TP53 [10]. In the present study we show that BBI induced molecular changes linked to EGFR signalling were independent of the TP53 status. One explanation for this observation would be that BBI-triggered EGFR signalling acts upstream of TP53. In agreement with this idea, we observed that nuclear EGFR is in complex with TP53 after BBI treatment independent from TP53 cellular status. Since TP53 can play an essential role during DNA repair [41,42], we assume that TP53 links BBI mediated effects on EGFR with DNA repair processes. In agreement with that idea, data presented in Fig. 6 provide the first mechanistic evidence for BBI-mediated TP53-dependent radioprotection. In cells with wt. TP53, we observed that BBI-induced nuclear EGFR accumulation was linked with stimulated complex formation of EGFR, TP53, and the protein MDC1. MDC1 was reported to translocate to sites of DNA lesions, where it collaborates with other proteins and with phosphorylated histone H2AX to mediate the accumulation of checkpoint and repair factors into nuclear foci [43]. Thus MDC1 can act as a positive regulator of NHEJ-repair [44] and is regarded as a tumor suppressor [45]. Generally, TP53 and MDC1 can be involved
in both non-homologous end-joining [46] and homologous recombination [47]; however, the use of confluent cells – which repair double strand breaks by NHEJ predominantly – argue for an involvement of both factors in NHEJ under the experimental conditions used. Nevertheless, we cannot exclude an effect upon homologous recombination also. Interestingly, both irradiation and BBI modulated nuclear complex formation between EGFR and MDC1 in cells with wt. TP53. Nevertheless, despite comparable protein expression of MDC1 in HH4dd cells and A549 cells (data not shown), the complex formation between EGFR and MDC1 failed in HH4dd cells with both treatments and may explain the lack of enhancement of double strand break repair by BBI following irradiation. This observation may imply that the presence of a wt. TP53 is mandatory for binding of MDC1 to the EGFR/TP53 complex and double strand break repair. The TP53 mutation in HH4dd cells is located within exon 5, which codes for the core domain of the molecule and is involved in sequence-specific DNA-binding [10]. Indeed this mutation is linked with altered DNA-binding and with failure to induce TP53 dependent gene transcription by ionizing radiation [10]. How this mutation is involved in the failure of EGFR/MDC1 complex formation will be the subject of future investigations. In summary, treatment of cells with BBI resulted in an increased internalization of EGFR into the cytoplasm as well as stimulated nuclear EGFR transport. Increased nuclear EGFR is associated with activation of DNA-PK and binding of TP53 and MDC1. In cells with mt. TP53, MDC1 binding is impaired, which may explain the dependence of BBI mediated radioprotection on the presence of functional TP53.
Acknowledgements We thank Nadine Hoffmann for excellent technical assistance. This work was supported by grants from Deutsche Forschungsgemeinschaft (DI 402/9-1) and Deutsche Krebshilfe (No. 106401). * Corresponding author. Klaus Dittmann, Division of Radiobiology and Environmental Research, Department of Radiation Oncology University of Tu ¨ntgenweg 11, 72076 Tu ¨bingen, Ro ¨bingen, Germany. E-mail address: [email protected] Received 31 October 2007; received in revised form 28 December 2007; accepted 3 January 2008; Available online 30 January 2008
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