120 ROLE OF THE MAJOR VAULT PROTEIN IN TUMOR CELLS AND IN RESPONSE TO IONIZING RADIATION

S49 parameters (V10-V40). Correlation between Jacmean and these parameters was assessed through KruskallWallis or Spearman tests. An univariate and a stepwise logistic multivariate analysis (MVA, selecting variables with p-value<0.1) were performed by considering as the end point the Jacmean smaller than the first quartile value (Q1). Parotid glands DVHs were stratified according to their degree of deformation, trying to assess the most predictive dose-volume combination in the low and medium dose region. A logistic MVA analysis including the shape of DVHs was also performed.

ICTR-PHE 2012 more likely to experience a clinically relevant toxicity. Quantitative assessment of parotid deformation during RT may be well represented in terms of Jacmean. Our analysis shows that the pattern of deformation may be well predicted by the shape of the planned dose distribution. In particular, a bad DVH (high value of V10 and V40) seems the most important predictor of parotid shrinkage. Interestingly, our results suggest the dose bath at low dose (10-15 Gy) is more predictive of the fraction of parotid receiving the “high” dose. References: [1] Fiorino et al., Introducing the Jacobian-volumehistogram of deforming organs: application to parotid shrinkage evaluation. Phys Med Biol. 2011 Jun 7;56(11):3301-12 [2] Broggi et al., A two-variable linear model of parotid shrinkage during IMRT for head and neck cancer. Radiother. Oncol. 2010 Feb; 94(2):206-12.

Figure 1: The risk to have a large shrinkage (Jacmean
120 ROLE OF THE MAJOR VAULT PROTEIN IN TUMOR CELLS AND IN RESPONSE TO IONIZING RADIATION M. Zimmermann1, A. Hollenstein2, M. Pruschy1, T. Codilupi1, A. Sartori1 1 Laboratory for Molecular Radiobiology, University Hospital Zurich, Switzerland 2 The Netherlands Cancer Institute, Amsterdam, The Netherlands

Results. For the enrolled patients an average Jacmean equal to 0.76 was estimated, with the first quartile (Q1) equal to 0.67. A low value of Jacmean corresponds to a large parotid shrinkage effect. Based on correlation tests, OVPPTV1 (p=0.0045), OVPPTV2 (p= 0.0026), age (p= 0.0209) were found as the pre-treatment anatomical/clinical/ geometrical variables correlated with Jacmean; Dmean (p= 0.0005) and most of the DVH parameters, V10 (p<0.0001), V15 (p=0.0001), V20 (p=0.0015), V40 (p=0.0003), were also found significantly correlated. Similar results were found for the univariate analysis by considering JacmeanQ1, V10 and V40 were assessed as the most predictive dosimetric parameters. Based on a ROC analysis, V10=93% (sensitivity:41.4%; specificity:82.5%) and V40= 36% (sensitivity:68%; specificity:52.5%) were estimated as the best cut-off values. Based on DVH shape, parotid glands were then separated in three different groups: bad-DVH (V10>93% and V40>36%), medium-DVH (V10>93% and V40<36%), good-DVH (V10<93%). The risk to have Jacmean< Q1 was 39.6 % vs 19.6% vs 11.3% in the three groups respectively (Figure1). By including in the MVA analysis this “DVH grouping” parameter bad-DVH (p=0.001026, OR=0.29; 95%CI:0.138-0.607) was found as the most predictive parameter for risk of large shrinkage (Jacmean
With a mass of 13 MDa, vaults are the largest ribonucleoprotein complexes in the cell, but their functions are hardly investigated. Overexpression of the major vault protein (MVP), the main component of vaults, correlates with enhanced drug resistance in tumor cells, supposedly by sequestering the pharmacologically active substances to lysosomes. Our laboratory initially focused on a putative link between the MVP and the DNA damage response induced by ionizing radiation. We used an siRNA-based approach to downregulate MVP in tumor cells and could successfully demonstrate enhanced radiosensitivity in A549 lung adenocarcinoma cells and HCT116 colon adenocarcinoma cells. Enhanced radiosensitivity also correlated with the increased amount of residual DNAdamage in MVP-depleted cells 24 hours after irradiation. However the proposed drug transporter activity of MVP leading to enhanced drug resistance is not applicable in the case of IR. Interestingly, cell proliferation on MVPdownregulation alone also was drastically reduced, which correlated with a strong reduction of DNAsynthesis (BrdU-incorporation) and a strong cell cycle redistribution in MVP-downregulated cells. In parallel cleavage of PARP (as readout for apoptosis induction) and strongly reduced levels of the Rad51 protein could also be detected on MVP-downregulation. Rad51 is a key player of homologous recombination and was also shown to be a caspase substrate. However as opposed to PARP-cleavage, reduction of Rad51 protein level upon MVP downregulation could not be reversed by cellular pretreatment with a pan-caspase inhibitor. Furthermore we could not discern so far whether MVP has a direct function in cell cycle progression, or whether cell cycle redistribution upon MVP downregulation might be the consequence of impaired

S50 homologous recombination by downregulation of Rad51. Interestingly, MVP knockout mice do not show any impairment in development and viability and thus, our results suggest that the status of MVP might play an important role for the cellular phenotype and stress response in particular in the background of oncogenic transformation. Most of our experiments were performed with the lung adenocarcinoma cell line A549, which is p53-wildtype. We therefore extended our experiments to p53wildtype and p53-deficient colon adenocarcinoma cell HCT116 (p53+/+; p53-/-). Intriguingly, MVPdownregulation induced a much stronger antiproliferative effect in the p53-wildtype than in the p53-deficient cells and interestingly the MVP status also determines IR-induced activation of p53dependent transcriptional activity in HCT116 cells, as determined by reporter gene-expression under control of p53-responsive elements. A similar p53-dependent antiproliferative effect was also observed in E1A/ras transformed mouse embryo fibroblasts on MVPdownregulation. Overall, these results suggest that the status of MVP/vaults not only co-determines the radiosensitivity of tumor cells, but that MVP might play a much broader role for the integrity and stress threshold in tumor cells with still intact p53. 121 DELAYED CELL DEATH ASSOCIATED WITH MITOTIC CATASTROPHE IN GAMMA-IRRADIATED STEM-LIKE GLIOMA CELLS G. Niedermann, E. Firat University Hospital, Freiburg, Germany Background and Purpose: Stem-like tumour cells are regarded as highly resistant to ionizing radiation (IR). Previous studies have focused on apoptosis early after irradiation, and the apoptosis resistance observed has been attributed to reduced DNA damage or enhanced DNA repair compared to non-stem tumour cells. Here, early and late radioresponse of patient-derived stemlike glioma cells (SLGCs) and differentiated cells directly derived from them were examined for cell death mode and the influence of stem cell-specific growth factors. Materials and Methods: Primary SLGCs were propagated in serum-free medium with the stem-cell mitogens epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). Differentiation was induced by medium containing serum without EGF and FGF. Radiation sensitivity was evaluated by assessing proliferation, clonogenic survival, apoptosis, and mitotic catastrophe. DNA damage-associated γH2AX as well as p53 and p21 expression were determined by Western blots. Results: SLGCs failed to apoptose in the first 4 days after irradiation even at high single doses up to 10Gy, but we observed substantial cell death later than 4 days postirradiation in 3 of 6 SLGC lines treated with 5 or 10Gy. This delayed cell death was observed in 3 of the 4 SLGC lines with nonfunctional p53, was associated with mitotic catastrophe and occurred via apoptosis. The early apoptosis resistance of the SLGCs was associated with lower γH2AX compared to differentiated cells, but we found that the stem-cell culture cytokines EGF plus FGF-2 strongly reduce

ICTR-PHE 2012 γH2AX levels. Nonetheless, γIR-induced apoptosis even correlated with EGF/FGF-induced proliferation and mitotic catastrophe in two p53-deficient SLGC lines examined. In a line containing CD133-positive and negative stem-like cells, the CD133-positive cells proliferated faster and underwent more γIR-induced mitotic catastrophe. Conclusions: Our results suggest the importance of delayed apoptosis, associated mitotic catastrophe, and cellular proliferation for γIR-induced death of p53deficient SLGCs. This may have therapeutic implications. We further show that the stem-cell culture cytokines EGF plus FGF-2 activate DNA repair and thus confound in vitro comparisons of DNA damage repair between stem-like and more differentiated tumour cells. 122 CONCEPTUAL DESIGN OF THE PROTON BEAM USER LABORATORIES AT THE ESS-BILBAO ACCELERATOR: RADIOBIOLOGY APPLICATIONS M. Huerta, R. Martínez, E. Abad, F.J. Bermejo ESS-Bilbao, Universidad del Pais Vasco, 48940 Leioa (Vizcaya), Spain. The preliminary design of the ESS-Bilbao light ion linear accelerator has been completed. The first phase of the project up to 50 MeV is currently under construction. In addition, the conceptual design of the user laboratories is being carried out. One of the foreseen laboratories will be dedicated to radiation biology research oriented to proton beam therapy. 1. Introduction The ESS-Bilbao light ion linear accelerator has been conceived as a modular and multi- purpose machine, useful as the core of a new standalone accelerator facility in southern Europe, giving support to local beam users and accelerator physicists, as well as fulfilling specifications to serve as a benchmark for components and systems relevant for the European Spallation Source (ESS) project. At first stage, the light ion linear accelerator is expected to generate proton beams with energies up to 50 MeV. These energetic beams will be used by a local community of users who have shown growing interest in using charged particle beams in physical and biomedical studies. 2. Current project status Within the first stage of the ESS-Bilbao project, a light ion linear accelerator has been designed with the parameters shown in Table 1.

Table 1. Basic parameters of the ESS-Bilbao proton beam accelerator for the firsBasic parameters of the ESS-Bilbao proton beam accelerator for the first stage. The 50 MeV proton beam of the ESS-Bilbao accelerator will be used in different laboratories; a low intensity neutron source (LAN) with three experimental lines, and three proton irradiation facilities: - Protons for Materials (P4M): To study the radiation