EP-1581 IRRADIATION UPREGULATES CMV PROMOTER OF STABLY TRANSFECTED CELLS AND TUMORS IN MICE

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Conclusions: Cu, Zn, Fe, and Se are known as trace elements. These elements are important in enzyme and protein function such as glutathione peroksidase, catalase, superokside dismutase (SODs), ceruloplasmin, metallothionein. Radioprotective effect of these trace elements’ supplementation before and in early period after radiotherapy has been showed in many experimental studies. Also, it has been demonstrated in different studies that to reach normal levels within six to eight weeks after irradiation and our study confirmed this result. EP-1579 STUDY ON IONIZING RADIATION ON VERTEBRATE MODEL USING WILD TYPE AND TRANSGENIC ZEBRAFISH EMBRYOS E.R. Szabo1, I. Man1, I. Plangar2, Z.K. Varga3, T. Tokes4, Z. Szabo1, G. Fekete1, Z. Nagy1, K. Hideghety1 1 Albert Szent-Györgyi Univ.Med.Sch., Department of Oncotherapy, Szeged, Hungary 2 Albert Szent-Györgyi Univ.Med.Sch., Department of Neurology, Szeged, Hungary 3 University of Pecs, Biotecont Ltd, Pecs, Hungary 4 Albert Szent-Györgyi Univ. Med. Sch., Institute of Surgical Research, Szeged, Hungary Purpose/Objective: The aim of our investigation was to study the effects of ionizing radiation (IR) on wild type zebrafish (Danio rerio) and on transgenic endothel and neuron labelled fish larvae. Materials and Methods: Wild type, FLI-1 (blood vessels labelled) and neuronal expression of green fluorescent protein (EGFP) transgenic zebrafish embryos and larvae were used. The zebrafish adults were mated and viable embryos were exposed to IR using 60Co facility at 3 and 24 hours post fertilization (hpf) with escalated single fraction doses (0, 5, 10, 15, 20 Gy). Digital images of both wild type and transgenic fish were taken to detect the morphological abnormalities of the treated groups in comparison to the control. Mortality and malformation rates of all types were evaluated each day in embryonic and early larval stage (24-168 hpf) furthermore the development of vascular and nervous system were studied by confocal/fluorescense microscope. Results: The IR at 3 hpf has caused significant changes in the survival at 15 Gy on the 7th day. The lethal dose 50% (LD50) was detected on this dose level and total lethality at 20 Gy on the 5th day. We have observed significant deterioration in the developing wild type embryos. In the transgenic and wild type lines we found the LD50 of IR at 20 Gy in the stage of embryos (24 hpf). Further we have detected significant morphological deterioration in the development of the vascular and neural system during the embryogenesis. The degree of the deterioration was dose-dependent at 24 hpf. Conclusions: Our preliminary results show that the zebradanio lines provide a useful in vivo model for investigation on the dose-and timedependent effects of ionizing radiation and to validate modifiers of the radiation response in the future. EP-1580 DATA BASE IMPLEMENTATION TO IMPROVE THE MANAGEMENT OF THE INTERRUPTIONS IN RADIOTHERAPY J. de la Vega Fernandez1, S. Ruiz-Arrebola1, F. Simancas1, A. TorneroLópez1, M. Vilches1, R. Guerrero1, D. Guirado1 1 Hospital Universitario S. Cecilio, Servicio de Radiofísica Hospitalaria, Granada, Spain Purpose/Objective: The prolongation of the overall treatment time has influence in the control probability for certain type of tumours, such as head and neck squamous cell carcinoma (HNSCC). It is necessary to adopt the appropriate measures to compensate the adverse interruption effects; however, the absence of a register of the patient interruptions difficult the application and the obtaining of statistics to improve the management of these measures. We develop a data base to facilitate the administration of compensatory measures to each patient and to have faster and more effective access to a statistic. Materials and Methods: We use Microsoft Access 2007 © software to create a data base of the treated patient in two Siemens Linear Accelerators: Mevatron KDS and Artiste ©. We use the combination of the medical history number and the date of the treatment beginning as the indentifying code because some patient may have more than one treatment. In each register there are several fields to be filled in when a patient starts the treatment. The more important fields are: demographic

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data (medical history number, name, surname…), the ICD code of the disease, treatment unit, type and quality of the beams, number of fractions and the dose per fraction, starting treatment date and the date in which the treatment should finish. In addition, each week a medical physicist reviews the treatment sheet of all patients and fills in the following fields if an interruption occurred: the reason, the starting date and the ending date. The data base generates a list of the patients in treatment that have had an interruption, on Friday it is decided the compensatory measures in a technical session (with medical physicists, physicians, nurses and RTT) and they are introduced in the data base. Finally when a patient finishes the treatment the physicist fills in the field: ending date of the real treatment. We started the register on March of 2011, so far 413 patients were registered and compared with a previous work [1] to quantify the improvement in the management. Results: We have found that 80.1% of the patients interrupt their treatment. Panel A of the figure shows the causes of the interruption, breaks with unknown reason were only 1.78%, in the previous work the percentage was 9.5%. The average prolongation in all the treatments was 3.5 days (SD = 3.5 days), the panel B shows the histogram, and in the previous work was 5.6 days (SD = 5.6 days). The percentage of patients whose interruptions were not evaluated was 7.06%. An internal audit showed that before the data base started only the 50% of the patients that are more affected by interruptions received an evaluation. In the HNSCC the average prolongation was 5.0 days (SD = 5.4 days), in the previous work was 7.5 days (SD = 6.0).

Conclusions: The implementation of data base shows an improvement in the application of the interruption protocols for it obliges to control every patient every week and automates the process. Moreover the obtaining of statistics of the compensatory measures is easier and permits to take decisions about the system weakness. References. [1] M.B. Ríos Pozo et al. Radiother Oncol 2010;91(s 1): S618. EP-1581 IRRADIATION UPREGULATES CMV PROMOTER OF STABLY TRANSFECTED CELLS AND TUMORS IN MICE M. Cemazar1, U. Kamensek1, S. Kranjc1, G. Sersa1 1 Institute of Oncology, department of experimental oncology, Ljubljana, Slovenia Purpose/Objective: The immediate/early promoter/enhancer of cytomegalovirus (CMV promoter) is one of the most commonly used promoters for expression of transgenes in mammalian cells. Although is often thought of as a strong constitutive and unregulated promoter, it was shown that its activity depends on host-cell transcriptional environment which is altered in a stressed cell. The aim of this study was to examine the influence of stresses, like irradiation (IR) and treatment with chemotherapeutic agent cisplatin (CDDP), on the activity of the CMV promoter in stably transfected tumor cells and tumors in mice as well as in transiently transfected mouse muscle. Materials and Methods: Two cell lines fibrosarcoma LPB and mammary carcinoma TS/A were transfected with plasmid DNA containing CMV promoter driven green fluorescent protein (GFP) reporter gene (pCMV-EGFP) using electroporation. After 2 months of culturing under increasing concentrations of selection agent

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geneticin, stably transfected cells were obtained. To test stressinduced regulation of the CMV promoter, cells were exposed to IR (6 Gy) and CDDP (3microg/ml). Expression of reporter gene was determined by fluorescence microplate reader and fluorescence microscopy at day 3 post-treatment. Solid TS/A tumors were induced by subcutaneous injection of stably transfected GFP-expressing tumor cells. Muscles of rear leg in mice were transiently transfected with the plasmid pCMV-EGFP using electroporation. Mice were treated by tumor/leg IR (6 Gy) or intravenous injection of CDDP (4mg/kg). GFP expression was followed by non-invasive fluorescence imaging using stereomicroscope for 7 days. Results: Exposure of cells to IR or CDDP resulted in significant upregulation of the expression in both cell lines, although the effects were more evident in TSA cell line. Treatment with IR or CDDP resulted in significant up-regulation of GFP expression in both in vivo models. After IR, the fluorescence intensity of tumors was significantly increased by a factor of 1.26 already on the third day after treatment. Similarly, the increase in fluorescence intensity of transiently transfected muscles following IR was by a factor of 1.17 on the second day. CDDP up-regulated the CMV promoter, resulting in significantly increased fluorescence intensity in both, tumors and transiently transfected muscles. Up-regulation was the most pronounced on the fourth day post-treatment for tumors and on the fifth day for muscles. Conclusions: Observed upregulation of the CMV promoter limits its usefulness of this widely used promoter as a constitutive promoter. The choice of promoter linked to the gene of interest is crucial for success of gene therapy as well as basic research. On the other hand, inducibility of CMV promoters can be beneficially used in gene therapy when combined with standard cancer treatment, such as radiotherapy and chemotherapy. EP-1582 EXPERIMENTAL INVESTIGATION OF CELL KILLING THROUGH MEDIUMBORNE SIGNALS IN HUMAN PROSTATE CANCER CELL LINE S. Sjostedt1, E. Bezak1 1 RAH, Medical Physics, Adelaide, Australia Purpose/Objective: It has been demonstrated that exposure of nonirradiated cells to Irradiated Cell Conditioned Medium (ICCM) can cause a cascade of events leading to effects similar to those resulting from direct radiation damage. The stochastic model, based on microdosimetric principles, relates absorbed dose to the emission and processing of cell death signals by non-irradiated cells. According to the model the fraction of non-irradiated recipient cells, surviving transfer of ICCM, decreases exponentially with increase in the signal intensity or the number of irradiated donor cells. This study attempts to validate this model in vitro in PC3 human prostate cancer cell line by investigating whether the toxicity of ICCM depends on the absorbed dose received by donor cells and on the cellular concentration of donor cells releasing toxic medium-borne signals due to radiation hits. Materials and Methods: The toxicity of ICCM was determined by measuring the recipient cell survival after exposure to irradiated cell conditioned medium which was derived from donor cells exposed to various doses. Additionally, recipient cell survival was measured after exposure to ICCM derived from donor cells of various concentrations irradiated with 2 Gy. Results: Exposure to 2 Gy, 4 Gy, 6 Gy and 8 Gy of ICCM resulted in a significant (P<0.001) decrease in clonogenic survival in non-irradiated recipient cells compared to the control group. However no dependency above 2 Gy dose was observed. Additionally, a significant (P<0.001) decrease in cell survival was found in the recipient cells exposed to the ICCM, derived from different concentrations of donor cells, exposed to 2 Gy compared to the control that received cell-free sham-irradiated media. A recipient cell survival, after receiving ICCM obtained from 2x102 cells, was found to be significantly higher compared to the rest of donor cell concentrations. This indicates that the toxicity of ICCM was dependent on the cellular concentration of donor cells. Conclusions: Data fitting provided reasonable agreement with the microdosimetric model for the induction of cell killing through medium-borne signals. EP-1583 IMPLICATIONS OF THE DIFFERENT BYSTANDER EFFECT IN MELANOMA SKIN-CANCER CELLS AND UMBILICAL-CORD STROMAL STEM CELLS J. Gomez-Millan Barrachina1, I.S. Santos Katz2, V. De Araujo Farias3, J.L. Linares Fernandez3, J. Lopez Peñalver3, G. Ortiz Ferrón3, C. Ruiz Ruiz3, F.J. Oliver4, J.M. Ruiz de Almodovar3

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Hospital Universitario Virgen de la Victoria, Radiation Oncology Department, Malaga, Spain Instituto Butantan, Centro de Pesquisa e Formacao em Imunologia., Sao Paulo, Brazil 3 Instituto de Biopatología y Medicina Regenerativa, Biopatologia, Granada, Spain 4 Instituto de Parasitología y Biomedicina Lopez-Neyra, Biomedicina, Granada, Spain 2

Purpose/Objective: Radiation induces direct DNA damage over irradiated cells. In the bystander effect the irradiated tumour cells send signals to neighbours that elicit effects that exceed radiation induced cell death. To understand the molecular mechanisms involved in the bystander effect we have studied the radiation-induced effects in normal umbilical-cord stromal stem cell (HCSSC) lines and in human cancer cells Materials and Methods: Two cell lines (UCSSC 35 and UCSSC 37) and two human melanoma skin-cancer cells (A375 and G361) were exposed to ionizing radiation to measure acute radiation-dosage cell-survival curves and radiation-induced bystander cell-death response. Survival was measured with colony forming assay. Apoptosis was measured by Flow cytometry. DNA repair was measured with comet assay Results: Clonogenic dose response curves showed that normal cells were resistant to the bystander effect whilst tumor cells were sensitive to irradiated cell-conditioned media, showing a dose– response relationship that became saturated at relatively low doses. Flow cytometry showed that cell death mechanism was mediated through apoptosis. We applied a biophysical model to describe bystander cell-death through the binding of a ligand to the cells. This model allowed us to calculate the maximum cell death (vmax) produced by the bystander effect together with its association constant (KBy) in terms of dose equivalence (Gy). The values obtained for KBy in A375 and G361 cells were 0.23 and 0.29 Gy, respectively Conclusions: Our findings help to understand how anticancer therapy could have an additional decisive effect in that the response of sublethally hit tumor cells to damage might be required for therapy to be successful because the survival of cells communicating with irradiated cells is reduced. EP-1584 LOW DOSE PRE-IRRADIATION EFFECT ON CELL SURVIVAL IN HT29 AND MRC5 CELL LINES I. Djan1, B. Petrovic1, S. Solajic2, M. Djan3, G. Bogdanovic2, M. Erak1 1 Institute of Oncology, Radiotherapy Department, Sremska Kamenica, Serbia 2 Institute of Oncology, Experimantal Oncology Department, Sremska Kamenica, Serbia 3 University of Novi Sad Faculty of Sciences, Department of Biology and Ecology, Novi Sad, Serbia Purpose/Objective: Numerous researches focus on a radio-adaptive response of different cell lines. Also, different pre-irradiation doses and different challenging doses have been investigated. The radioadaptive dose is most probably associated with the regulation of DNA repair mechanisms, stress response genes, cell cycle genes and apoptosis. The aim of this paper is to evaluate effect of low dose preirradiation followed by hyperfractionation regime (1.3Gy twice per day, in four days). Materials and Methods: Low dose pre-irradiation effect was studied on two cell lines HT29 (human colorectal cancer cells) and MRC5 (human pulmonary fibroblasts cells). The following pre-irradiation doses were applied: 0.03Gy, 0.05Gy and 0.07Gy. Two hours after preirradiation cells were further irradiated with 1.3Gy dose twice per day, with four hours period between them. Described regime was repeated in next three days without pre-irradiation doses. Results: The mean survival of HT29 cells in a described regime was significantly lower in applied pre-irradiation doses 0.05Gy and 0.07Gy, as well as without pre-irradiation but with hyperfractionation alone comparing to control, while 0.03Gy pre-irradiation dose showed no significant effect on cell survival. The mean survival of MRC5 cells was significantly lower in all applied pre-irradiation doses and without pre-irradiation but with hyperfractionation alone comparing to control. It seems that in HT29 cell line the pre-irradiation dose of 0.03Gy induced significant radioprotective effect, while the same effect was not detected in fibroblasts (cell line MRC5). Conclusions: The presented results showed that low-dose preirradiation has significant effect in cell survival in both cell lines, HT29 and MRC5, except that 0.03Gy pre-irradiation dose has no effect in HT29 cell line. It was also proved that increasing of pre-irradiation