M. Ugolini 1,2, G. Babini 1,2, G. Baiocco 1,2, D. Cappelletti 1,2, L. Mariotti 1,2, J. Morini 1,2,3 and A. Ottolenghi 1,2

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1 M. Ugolini 1,2, G. Babini 1,2, G. Baiocco 1,2, D. Cappelletti 1,2, L. Mariotti 1,2, J. Morini 1,2,3 and A. Ottolenghi 1,2 1 Department of Physics, University of Pavia, Pavia, Italy 2 Istituto Nazionale di Fisica Nucleare (INFN), Sezione di Pavia, Pavia, Italy 3 Department of Molecular Medicine, University of Pavia, Pavia, Italy

2 Outline General introduction and objectives of the work: ionizing radiation perturbed extracellular signaling the role of ERK1/2 and AKT pathways Experimental study: ERK1/2 and AKT spatial localization: Immunocytochemistry assay ImageJ Analysis and Results Summary and future developments

3 ERK1/2 and AKT pathways ERK1/2 and AKT are involved in a variety of different biological responses, such as apoptosis, differentiation and proliferation Proteins activation due to: Extracellular signals: cytokines, interleukins, growth and stress factors; 0,25 Gy 0-0,1 Gy 1 Gy Environmental stress: medium change and ionizing radiation Negative feedback loop L. Mariotti et al, Radiation Research,

4 ERK1/2 and AKT pathways Why are we interested in studying these pathways activation? Disregulation in many diseases (cancer & noncancer) J. A. McCubrey et al., Roles of the RAF/MEK/ERK and PI3K/PTEN/AKT pathways in malignant transformation and drug resistance, Advances in enzyme regulation, 46:249-79, 2006 S. Schubbert et al., Hyperactive Ras in dedevelopmental disorders and cancers, Nature Publishing Group, 7(4): , 2007 M. Osaki et al., PI3K-AKT pathway: its functions and alterations in human cancers, Apoptosis, ,2004 Possible targets of antitumor therapy and M. Drigotas et al., Reactive Oxygen Spercie activation of MAPK pathway results in VEGF upregulation as an undesired irradiation response, Journal of Oral Pathology & Medicine, 42(8):612-9, 2013 HF. Li et al., Radiation-induced AKT activation modulates radioresistance in human glioblastoma cells, Radiation Oncology, 4:43, 2009 JA: Vara Fresno et al, PI3K/AKT signaling pathway and cancer, Cancer Treat Rev., 30(2): , 2004

5 Experimental setup Irradiation facility: CLINAC 2100 (IRCCS S. Maugeri, Pavia) Radiation field 20x20 cm Flatness and symmetry guaranteed at a 3% level Dose Rate 3 Gy/min Adopted cell line: AG01522, Normal Human Skin Fibroblasts Samples

6 Immunocytochemistry assay 3 doses (0-0,5-2 Gy) Immunocytochemistry (ICC) x is a technique used to detect 5 times (30min, the presence 2h, 4h, 8h, 6h) of a particular protein or x antigen in cells through 10x3 images a specific (nucleus antibody identification that binds + 2 (one to per it. protein: Such antibody ERK1/2 is coupled and AKT)) with a x fluorescent chemical compound that can 2 (biological duplicate ) re-emit light upon light excitation. = 900 images p-erk1/2 NUCLEI p-akt

7 ImageJ analysis ImageJ: Purposely developed semi-automatic procedure p-erk1/2 p-akt ROI s identification -1 (fully cytoplasmatic ) < R = (N-C)/(N+C) < +1 (fully nuclear)

8 Results Dose dependence at 30 minutes after irradiation A preliminary data analysis indicates no significant dose dependence at later times: 2h, 4h, 6h and 8h. Fast activation

9 Summary and future developments Both ERK1/2 and AKT pathways are activated by ionizing radiation To measure the activation of these pathways the migration of these proteins from the cytoplasm to the nucleus has been investigated A dose dependence was observed at 30 minutes while no significant dose dependence was found at later times Their activation seems to be very quick, so further study at shorter time are needed; The response of other important pathways to ionizing radiation will be considered, both intracellular (XIAP, FLIP and p38) and extracellular (TGFβ and TNFα); In order to evaluate the role of the quality of radiation further study will be performed using both carbon ions and protons;