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EP-1585 EVALUATION OF AN INDUCIBLE P21 PROMOTER FOR RADIATION INDUCED TRANSCRIPTIONAL TARGETING
S608
dose lowers cell survival in both cell lines. The hyperfractionation regime applied alone without any pre-irradiation decreases cell survival significantly comparing to control and 0.03Gy in HT29 cell line indicating protective effect of the dose. The latter may indicate that applied dose induced activity of cell protective mechanisms. The obtained results may be introduced into optimization the balance between tumour treatment and normal tissue spearing in radiotherapy process. EP-1585 EVALUATION OF AN INDUCIBLE P21 PROMOTER FOR RADIATION INDUCED TRANSCRIPTIONAL TARGETING U. Kamensek1, G. Sersa1, S. Kranjc1, M. Cemazar1 1 Institute of Oncology Ljubljana, Department of experimental oncology, Ljubljana, Slovenia Purpose/Objective: Radiation induced transcriptional targeting is a gene therapy approach that takes advantage of targeting abilities of radiotherapy using promoters of genes that are activated by irradiation to spatially and temporally limit expression of the transgene to the irradiated tumor volume. One of the promoters used for this purpose, is that of the cyclin dependent kinase inhibitor 1 (CDKN1A) gene, more commonly known as p21 gene or also as WAF1, Cip1, Sdi1. Gene p21 is an early response gene, mediating cell cycle G1 phase arrest in response to a variety of stress stimuli including DNA damaging agents like irradiation. Materials and Methods: The suitability of the p21 promoter for inducible therapy was tested using three in vitro and in vivo reporter gene experimental models: a model of stably transfected cell lines, a model of stably transfected tumors and a model of transiently transfected mouse muscles. To prepare stably transfected cell lines murine fibrosarcoma LPB and mammary carcinoma TS/A cells were stably transfected with plasmids encoding p21 promoter-driven green fluorescent protein (GFP) gene using electroporation; stably transfected fluorescent TS/A tumor cells were then used for the induction of subcutaneous tumors; and for the preparation of the transiently transfected muscles model, mouse rear leg muscles were transiently transfected with plasmid DNA, also using electroporation. To test the inducibility of p21 promoter, stably transfected cells were irradiated with single dose of 6 Gy; model of stably transfected tumors was used to test dose response of the promoter using irradiation with 2, 6 and 10 Gy; and model of stably transfected muscles was used to test if promoter can be re-induced 8 days after first induction with dose of 6 Gy. Reporter gene expression was determined using a fluorescence microplate reader in vitro and by non-invasive fluorescence imaging using fluorescence stereomicroscope and ImageJ computer software in vivo. Results: Irradiation of cells with 6 Gy caused statistically relevant induction in the expression of reporter gene. Using the stably transfected tumor model, we showed that the induction was not dose dependent, since the dose of 6 Gy induced higher reporter gene expression than dose of 10 Gy. Radiation with 6 Gy also activated the reporter gene expression in the model of transiently transfected muscles, although the induction was not statistically relevant. In the latter model re-inducibility of promoter was proven. Conclusions: In reporter gene experimental models, the p21 promoter was proven to be inducible with irradiation (6 Gy) which makes it a suitable promoter to control the expression of therapeutic genes, such as IL12, in radio-gene therapy. Further optimization and improvements to this combined therapy could lead to better controlled gene therapy, temporally as well as spatially, which is crucial for translation of this approach into the clinical setting. EP-1586 RAT MODEL FOR INVESTIGATION ON THE POTENTIAL MODIFIERS OF IONIZING RADIATION INDUCED BRAIN INJURY I. Plangár1, I. Mán2, E.R. Szabó2, G. Volford3, T. Tokés4, Z.K. Varga5, I. Németh6, R. Weiczner7, K. Hideghety2 1 Albert Szent-Györgyi Univ.Med.Sch., Department of Neurology, Szeged, Hungary 2 Albert Szent-Györgyi Univ.Med.Sch., Department of Oncotherapy, Szeged, Hungary 3 Albert Szent-Györgyi Univ.Med.Sch., Department of Radiology, Szeged, Hungary 4 Albert Szent-Györgyi Univ.Med.Sch., Institute of Surgical Research, Szeged, Hungary 5 University of Pécs, Biotecont Ltd, Szeged, Hungary 6 Albert Szent-Györgyi Univ.Med.Sch., Department of Dermatology,
ESTRO 31
Szeged, Hungary University of Szeged, Department of Anatomy, Szeged, Hungary
7
Purpose/Objective: The aim of this study was to establish a rat model to study the radiation dose related cognitive dysfunction in correlation to MR-morphologic and histopathologic changes in order to test the supposed radioprotective effect of certain molecules and drugs. Materials and Methods: Behavioural dysfunction, magnetic resonance imaging (MRI) changes and histological abnormalities in the brain were investigated in 38 Sprague-Dawley male rats aged 6 weeks treated with local brain irradiation using small field (10mm of diameter), 6 MeV energy electron beam directed to the corpus callosum of one hemisphere of the brain. Dose were escalated by 10 Gy from 40 to 120 Gy and compared with 12 sham-irradiated rats as controls. Performance of the open-field task was examined weekly and the Morris water maze test at the beginning four times a week, after that once a month. Additionally T2 weighed 3D Cube saggital and T1 weighed 3D IR-FSPGR axial scans of MRI were performed using the human head and skull coil supplemented with special small animal positioning device. Histological changes were examined at 1,2 and 3 months post irradiation (PI) using standard, furthermore axon and myelin specific staining. Results: The robust macroscopic damages that we could detect were dose-dependent, which could be localized in vivo by means of MRI. In the open field test we did not find significant differences between the treated and control groups during 3 months PI. The damaging effects detected by the water maze test appeared in the periods between 30120 days after the irradiation depending on the dose delivered. The structural damage in the rat brain, detected by MRI correlated well with the histological findings. The microscopic examination of the brain sections showed several types of brain damage, principally necrosis with reactive astrogliosis. Conclusions: On the basis of our results so far, we defined the 40 Gy dose level with marked behavioural, macroscopic and histological changes at 3 months PI for future investigation on neuroradioprotective agents.
ELECTRONIC POSTER: RADIOBIOLOGY TRACK: NOVEL RADIATION MODALITIES - PARTICLES, HIGH LET EP-1587 CELL SURVIVAL RESPONSES TO MODULATED PROTON BEAMS DELIVERED BY PASSIVE SCATTERING AND PENCIL BEAM SCANNING C.K. Mc Garry1, K.T. Butterworth2, T.P.G. : B. Clasie J. Schumann A Carabe-Fernandez N. Depauw H. Kooy H.S. Ming S. Tang3, J.M. O'Sullivan2, G. Schettino2, S.J. McMahon2, H. Paganetti3, K. Held3, K.M. Prise2, A.R. Hounsell1 1 Northern Ireland Cancer Centre, Belfast Health and Social Care Trust, Belfast, United Kingdom 2 Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, United Kingdom 3 Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, USA Purpose/Objective:To evaluate the relative biological effectiveness of passive scattering and pencil beam scanning proton beams and determine the radiobiological impact of modulated beam deliveries on cell survival occurring in- and out-of-field with intercellular communication intact or physically inhibited. Materials and Methods: Cell survival was determined by clonogenic assay in the human prostate cancer cell line (DU145) following exposure to uniform and non-uniform proton fields delivered by passive scattering or active scanning and compared to survival responses observed using a 6 MV photon beam produced with a Varian linear accelerator. Results: The survival response of DU-145 cells exposed to proton beams using passive scattering or active scanning showed no significant difference from the observed response for 6 MV photons taking into account a relative biological effectiveness (RBE) of 1.1 for protons. Non-uniform dose distributions delivered by passive scattering or active scanning used a brass aperture to shield half of the cell population. Clonogenic survival in the shielded region was significantly lower than that predicted from the linear quadratic model with 50 – 60 % survival being observed at scattered doses of 0.05 Gy and 0.03 Gy for passive scattering and active scanning beams respectively. The observed out-of-field responses were shown to be
dose lowers cell survival in both cell lines. The hyperfractionation regime applied alone without any pre-irradiation decreases cell survival significantly comparing to control and 0.03Gy in HT29 cell line indicating protective effect of the dose. The latter may indicate that applied dose induced activity of cell protective mechanisms. The obtained results may be introduced into optimization the balance between tumour treatment and normal tissue spearing in radiotherapy process. EP-1585 EVALUATION OF AN INDUCIBLE P21 PROMOTER FOR RADIATION INDUCED TRANSCRIPTIONAL TARGETING U. Kamensek1, G. Sersa1, S. Kranjc1, M. Cemazar1 1 Institute of Oncology Ljubljana, Department of experimental oncology, Ljubljana, Slovenia Purpose/Objective: Radiation induced transcriptional targeting is a gene therapy approach that takes advantage of targeting abilities of radiotherapy using promoters of genes that are activated by irradiation to spatially and temporally limit expression of the transgene to the irradiated tumor volume. One of the promoters used for this purpose, is that of the cyclin dependent kinase inhibitor 1 (CDKN1A) gene, more commonly known as p21 gene or also as WAF1, Cip1, Sdi1. Gene p21 is an early response gene, mediating cell cycle G1 phase arrest in response to a variety of stress stimuli including DNA damaging agents like irradiation. Materials and Methods: The suitability of the p21 promoter for inducible therapy was tested using three in vitro and in vivo reporter gene experimental models: a model of stably transfected cell lines, a model of stably transfected tumors and a model of transiently transfected mouse muscles. To prepare stably transfected cell lines murine fibrosarcoma LPB and mammary carcinoma TS/A cells were stably transfected with plasmids encoding p21 promoter-driven green fluorescent protein (GFP) gene using electroporation; stably transfected fluorescent TS/A tumor cells were then used for the induction of subcutaneous tumors; and for the preparation of the transiently transfected muscles model, mouse rear leg muscles were transiently transfected with plasmid DNA, also using electroporation. To test the inducibility of p21 promoter, stably transfected cells were irradiated with single dose of 6 Gy; model of stably transfected tumors was used to test dose response of the promoter using irradiation with 2, 6 and 10 Gy; and model of stably transfected muscles was used to test if promoter can be re-induced 8 days after first induction with dose of 6 Gy. Reporter gene expression was determined using a fluorescence microplate reader in vitro and by non-invasive fluorescence imaging using fluorescence stereomicroscope and ImageJ computer software in vivo. Results: Irradiation of cells with 6 Gy caused statistically relevant induction in the expression of reporter gene. Using the stably transfected tumor model, we showed that the induction was not dose dependent, since the dose of 6 Gy induced higher reporter gene expression than dose of 10 Gy. Radiation with 6 Gy also activated the reporter gene expression in the model of transiently transfected muscles, although the induction was not statistically relevant. In the latter model re-inducibility of promoter was proven. Conclusions: In reporter gene experimental models, the p21 promoter was proven to be inducible with irradiation (6 Gy) which makes it a suitable promoter to control the expression of therapeutic genes, such as IL12, in radio-gene therapy. Further optimization and improvements to this combined therapy could lead to better controlled gene therapy, temporally as well as spatially, which is crucial for translation of this approach into the clinical setting. EP-1586 RAT MODEL FOR INVESTIGATION ON THE POTENTIAL MODIFIERS OF IONIZING RADIATION INDUCED BRAIN INJURY I. Plangár1, I. Mán2, E.R. Szabó2, G. Volford3, T. Tokés4, Z.K. Varga5, I. Németh6, R. Weiczner7, K. Hideghety2 1 Albert Szent-Györgyi Univ.Med.Sch., Department of Neurology, Szeged, Hungary 2 Albert Szent-Györgyi Univ.Med.Sch., Department of Oncotherapy, Szeged, Hungary 3 Albert Szent-Györgyi Univ.Med.Sch., Department of Radiology, Szeged, Hungary 4 Albert Szent-Györgyi Univ.Med.Sch., Institute of Surgical Research, Szeged, Hungary 5 University of Pécs, Biotecont Ltd, Szeged, Hungary 6 Albert Szent-Györgyi Univ.Med.Sch., Department of Dermatology,
ESTRO 31
Szeged, Hungary University of Szeged, Department of Anatomy, Szeged, Hungary
7
Purpose/Objective: The aim of this study was to establish a rat model to study the radiation dose related cognitive dysfunction in correlation to MR-morphologic and histopathologic changes in order to test the supposed radioprotective effect of certain molecules and drugs. Materials and Methods: Behavioural dysfunction, magnetic resonance imaging (MRI) changes and histological abnormalities in the brain were investigated in 38 Sprague-Dawley male rats aged 6 weeks treated with local brain irradiation using small field (10mm of diameter), 6 MeV energy electron beam directed to the corpus callosum of one hemisphere of the brain. Dose were escalated by 10 Gy from 40 to 120 Gy and compared with 12 sham-irradiated rats as controls. Performance of the open-field task was examined weekly and the Morris water maze test at the beginning four times a week, after that once a month. Additionally T2 weighed 3D Cube saggital and T1 weighed 3D IR-FSPGR axial scans of MRI were performed using the human head and skull coil supplemented with special small animal positioning device. Histological changes were examined at 1,2 and 3 months post irradiation (PI) using standard, furthermore axon and myelin specific staining. Results: The robust macroscopic damages that we could detect were dose-dependent, which could be localized in vivo by means of MRI. In the open field test we did not find significant differences between the treated and control groups during 3 months PI. The damaging effects detected by the water maze test appeared in the periods between 30120 days after the irradiation depending on the dose delivered. The structural damage in the rat brain, detected by MRI correlated well with the histological findings. The microscopic examination of the brain sections showed several types of brain damage, principally necrosis with reactive astrogliosis. Conclusions: On the basis of our results so far, we defined the 40 Gy dose level with marked behavioural, macroscopic and histological changes at 3 months PI for future investigation on neuroradioprotective agents.
ELECTRONIC POSTER: RADIOBIOLOGY TRACK: NOVEL RADIATION MODALITIES - PARTICLES, HIGH LET EP-1587 CELL SURVIVAL RESPONSES TO MODULATED PROTON BEAMS DELIVERED BY PASSIVE SCATTERING AND PENCIL BEAM SCANNING C.K. Mc Garry1, K.T. Butterworth2, T.P.G. : B. Clasie J. Schumann A Carabe-Fernandez N. Depauw H. Kooy H.S. Ming S. Tang3, J.M. O'Sullivan2, G. Schettino2, S.J. McMahon2, H. Paganetti3, K. Held3, K.M. Prise2, A.R. Hounsell1 1 Northern Ireland Cancer Centre, Belfast Health and Social Care Trust, Belfast, United Kingdom 2 Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, United Kingdom 3 Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, USA Purpose/Objective:To evaluate the relative biological effectiveness of passive scattering and pencil beam scanning proton beams and determine the radiobiological impact of modulated beam deliveries on cell survival occurring in- and out-of-field with intercellular communication intact or physically inhibited. Materials and Methods: Cell survival was determined by clonogenic assay in the human prostate cancer cell line (DU145) following exposure to uniform and non-uniform proton fields delivered by passive scattering or active scanning and compared to survival responses observed using a 6 MV photon beam produced with a Varian linear accelerator. Results: The survival response of DU-145 cells exposed to proton beams using passive scattering or active scanning showed no significant difference from the observed response for 6 MV photons taking into account a relative biological effectiveness (RBE) of 1.1 for protons. Non-uniform dose distributions delivered by passive scattering or active scanning used a brass aperture to shield half of the cell population. Clonogenic survival in the shielded region was significantly lower than that predicted from the linear quadratic model with 50 – 60 % survival being observed at scattered doses of 0.05 Gy and 0.03 Gy for passive scattering and active scanning beams respectively. The observed out-of-field responses were shown to be