920: DNA damage and oxidative stress after low doses of X and proton beam irradiation

EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 characterized by cell dispersion and membrane protrusions, of the irradiated survivor’s progeny cells was partially reverted by the ERK1/2 inhibitor. The inhibition of PI3K/AKT was able to decrease the high migratory potential induced by the radiation as compared to control progeny. On the other hand, ERK1/2 inhibitor was more effective in reducing the invasiveness potential of the irradiated survivor’s progeny. Conclusions: Our results showed that radiation survivor cells could generate highly aggressive progeny and that PI3K/AKT and ERK1/2 signaling pathways play an crucial role in this event. Therefore, the components of these signaling pathways may constitute important targets for new adjuvant treatment schedules with radiotherapy in the treatment of CRC. No conflict of interest. 918 Is single amino acid arginine deprivation a strategy to treat non-auxotrophic glioblastoma? M. Ingargiola1,2 , L. Radon1,2 , N. Hinrichs1,2 , A. Kohn ¨ Luque3 , R. Wiedemuth4 , A. Temme4 , A. Deutsch3 , L. Kunz-Schughart1,2 . 1 OncoRay − National Center for Radiation Research in Oncology, Faculty of Medicine Carl Gustav Carus, TU Dresden, Germany, 2 Institute of Radiooncology, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany, 3 Innovative Methods of Computing, Center for Information Services and High Performance Computing, TU Dresden, Germany, 4 Experimental Neurosurgery/Tumorimmunology, University Hospital Carl Gustav Carus, TU Dresden, Germany Introduction: Arginine deprivation is a promising strategy in metabolic anticancer therapy. It was shown to inhibit tumor growth of arginine-auxotrophic cancers in vitro and in vivo. We previously reported that acute arginine withdrawal sensitizes cancer cells in a multicellular spheroid (MCTS) assay to radiotherapy even in the absence of complete arginine auxotrophy. Because of its systemic applicability enzymatic arginine deprivation could thus be of interest for the treatment of therapeutically challenging glioblastomas. In the present study, we evaluated the impact of arginine withdrawal on 3-D growth and invasive capacity of glioblastoma cells with wildtype and mutated/knockdown p53 status. Material and Methods: U87-MG, U87-MG-shp53 (p53-knockdown) and U251MG (p53-mutant) transduced with eGFP were applied. Spheroids were cultured and analysed in liquid overlay using a standardized semi-automated set-up. Arginine deprivation was realized by transfer of MCTS into argininefree medium containing dialysed serum. Upon treatment, spheroid volume was monitored by phase contrast imaging, viable cell numbers were determined, and invasion distance of spheroid cells was evaluated in a 3-D collagen-I gel by fluorescence microscopy. Results and Discussion: All three glioblastoma cell lines form MCTS but only two of them (U87-MG-wt, U87-MG-shp53) show spheroid volume growth. In contrast, all spheroid types clearly invade into collagen-I gel. Interestingly, the p53-knockdown U87-MG-shp53 cells exhibit a slightly enhanced growth in both monolayer and spheroid culture but appeared to be less invasive in the collagen-I gel as compared to their U87-MG-wt counterpart. Acute arginine deprivation results in a complete loss of cell growth in monolayer and MCTS culture independent of the p53-status. In parallel, acute arginine starvation affects the invasion capacity of U87-MG-wt but neither of the p53knockdown descendant nor of the p53-mutated U251-MG spheroids. Both invasion capacity and MCTS volume growth are restored after completing an arginine-free diet even after long-term exposure. Conclusions: Arginine deprivation effectively inhibits 3-D volume growth independent of the p53 status and appears to reduce the invasion of at least p53-wt glioblastoma cells. The strategy has a clear potential and shall thus be further tested in other models and in combination with standard treatments for glioblastoma patients in particular radiotherapy. This project is funded by the European Social Fund (ESF) and the Free State of Saxony. No conflict of interest. 919 Arginine deprivation and radiation as combination therapy: A systematic study in HNSCC 2-D vs. 3-D culture models F. Manig1,2 , L. Lehmann1,2 , M. Huether1,2 , M. Baumann1,2 , O. Stasyk3 , L.A. Kunz-Schughart1,2 . 1 OncoRay − National Center for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, TU Dresden, Germany, 2 Institute of Radiooncology, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany, 3 Institute of Cell Biology, NASU, Lviv, Ukraine Background: Arginine auxotrophic tumors lacking the enzyme argininosuccinatesynthase (ASS) to convert citrulline to arginine are highly susceptible to arginine starvation. As a consequence, arginine deprivation as monotherapy is in clinical trial for such tumor entities. A promising approach for ASS-positive tumors was recently indicated in an in vitro study when treating 3-D cultures of colorectal cancer cells with irradiation under arginine-deficient conditions. In the current study we therefore evaluated ASS-1 protein pattern and the impact of this treatment strategy in head and neck squamous cell carcinoma (HNSCC) in vitro models.

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Material and Methods: A panel of HNSCC cell lines was applied including SAS and FaDu which also grow as spheroids. ASS-1 protein expression was determined by western blot analysis. Arginine starvation was achieved in arginine-free media supplemented with dialyzed serum or by rh-arginase. Monolayer cell growth was assessed by cell counting and spheroid volume growth was monitored semi-automatically via phase contrast imaging. Arginine deprivation was combined with single-dose X-ray and assessed in colony formation (CFA) and spheroid control probability (SCP) assays. Results: All HNSCC cell lines express ASS-1 protein but show different intrinsic levels. Independent of that, arginine deprivation leads to an acute cell growth arrest in all 2-D cultures and reduced regrowth capacity upon completion of the diet. Similarly, no volume growth during starvation was observed for the two 3-D culture models studied. However, spheroid volume growth was entirely restored even after long-term (15 days) arginine starvation. Arginine withdrawal resulted in a strong radiosensitization in one of the two models (SAS) both in classical CFA and sophisticated SCP experiments. This effect was not abolished by the addition of physiological concentrations of the arginine-precursor citrulline. Conclusions and Perspectives: Cell growth of HNSCC cells in monolayer and spheroid culture requires arginine, but an impact of arginine withdrawal on cell survival and regrowth capacity is only seen in 2-D culture. In spite of this loss of susceptibility in the 3-D environment, the radiosensitizing effect is preserved in SAS spheroids. Experiments are ongoing to gain deeper insight into the underling mechanism and to discriminate responder and nonresponder HNSCC cells with respect to radiosensitization. This work is supported by the European Social Fund, grant no. 100147786. No conflict of interest. 920 DNA damage and oxidative stress after low doses of X and proton beam irradiation K. Jasinska1 , A. Cierniak2 , A. Borkowska1 , J. Jura2 , P. Olko3 , B. Romanowska-Dixon4 , M. Elas1 , K. Urbanska1 . 1 Biochemistry Biophysics and Biotechnology, Biophysics, Krakow, Poland, 2 Biochemistry Biophysics and Biotechnology, General Biochemistry, Krakow, Poland, 3 Institute of Nuclear Physics PAS, Radiation Physics and Dosimetry, Krakow, Poland, 4 Jagiellonian University Medical College, Ophthalmology and Ocular Oncology, Krakow, Poland Background: Ionizing radiation induces damage in DNA by direct ionization and through generation of radicals which attack DNA. Recent studies argue that relative biological effectiveness of proton irradiation might be higher than usually accepted 1.1 [1]. Proton beam might also induce slightly different DNA damages than photon irradiation [2]. The goal of this study was to compare the DNA damage and oxidative stress after a low dose of X versus proton beam irradiation. Methods: BLM human melanoma and E0771 murine medullary breast adenocarcinoma were irradiated with 1−5 Gy of X ray (300 kV Philips, 1 Gy/min) or proton beam (58 MeV) from AIC-144 cyclotron. Immediately after cells were tested for survival using MTT test, DNA damage using comet assay and micronucleus assay, and TBARs for oxidative damage. Results: Proliferation of cells were slightly inhibited after 1−3 Gy, and strongly inhibited after 5 Gy. Comet assay showed increase in DNA damage in time. TBARS demonstrated the presence of oxidative damage in cells after 5 Gy, but not after lower doses. Conclusions: E0771 cells are more sensitive to radiation than BLM cells. X-ray and proton beam irradiation cause DNA damage in a dose-dependent manner. Reference(s) [1] Britten RA. et al. (2013) Variations in the RBE for cell killing along the depth-dose profile of a modulated proton therapy beam. Radiation Res., 179(1): 21−8. [2] Girdhani, S. et al. (2013) Biological Effects of Proton Radiation: What We Know and Don’t Know. Radiation Res., 279, 257–272. No conflict of interest. 921 Importance of Mcl-1 and the Mcl-1-modulating enzyme USP9x for radiosensitivity in prostate cancer cells J. Rudner1 , S.A. Hogh-Binder2 , F. Wolfsperger2 , S. Huber2 , V. Jendrossek3 . 1 University Hospital Essen, Essen, Germany, 2 University Hospital Tuebingen, Radiation Oncology, Tuebingen, Germany, 3 University Hospital Essen, Insitute for Cell Biology (Tumor Research), Essen, Germany Background: Myeloid cell leukemia sequence 1 (Mcl-1) is a short-lived antiapoptotic member of the Bcl-2 protein family prolonging survival in response to genotoxic and cytotoxic stress and limiting the therapeutic success of cancer patients. The stability of this protein is tightly regulated by ubiquitylation and deubiquitylation. While ubiquitin ligases attach ubiquitin moieties to the proteins targeting it for proteasomal degradation, the deubiquitylating enzymes (like the Mcl-1 recognizing deubiquitinase USP9x) can remove the polyubiquitin chains thereby stabilizing the instable protein. Usually, Mcl-1 levels