CD95-L system

Proffered Papers

genes and for cells with p53 mut or p53 -/- for only 5 or 8 genes, respectively. For selective genes (Bcl2, Caspl, CCND2, DDB2, XPC, Rad51C) these changes were confirmed by qRTPCR. These results demonstrate that even for isogenetic cell lines differing in p53 status huge differences in gene expression were already found prior to irradiation and only few genes were altered after irradiation. Conclusions: The gene expression profile measured for non-irradiated cells appears to be a more informative marker of individual radiosensitivity than to record the changes in gene expression only occurring after irradiation. 132 THE M I C R O - E N V I R O N M E N T TRIGGERED FIBROGENIC D I F F E R E N T I A T I O N I N HUMAN MESENCHYMAL STEM CELLS.

C. Bourgier, M. Mouiseddine, V. Haydont, J. Bourhis, A. Chapel, M.C. Vozenin-Brotons 1UPRES EA 27-10, Radiosensibilit~ des tumeurs et tissus sains, IGR/IRSN. Villejuif, France ;- 2LRPAT, SRBE/DRPH. IRSN. Fontenay-aux-Roses, Fr. 3- LTCRA, SRBE/DRPH, IRSN. Fontenay aux Roses, France Objective: Mesenchymal stem cells (MSC) transplantation may be a promising approach for the development of regenerative therapy after radiotherapy. Recent studies showed that MSC derived from the bone marrow haematopoietic microenvironment are able to home into injured irradiated organs (1) and could improve the acute wound healing response. However, the impact of MSC therapy into delayed radiation injury development has never been investigated so far. Furthermore, Epperly et al. (2) suggested a bone marrow origin of myofibroblasts in irradiation pulmonary fibrosis. In the present study, we hypothesized that a dynamic and reciprocal relationship between cells and extracellular matrix did exist, and investigated whether microenvironmental modifications linked to radio-induced injury could alter MSC phenotype and trigger the fibrogenic differentiation. Material and methods: To answer this question, we developed an original in vitro model of normal and fibrosis extracellular matrix coatings. These coatings were obtained by culturing in SmGM (Cambrex) human primary intestinal smooth muscle cells derived from either normal ileum or radiation enteritis at confluency. Then, cells were solubilized in Triton-NaOH solution to produce normal and fibrosis extracellular matrix (ECM) monolayers respectively. Human mesenchymal stem cells (hMSC) were seeded onto plastic, normal and fibrosis extracellular matrix coating. Cell adhesion and spreading along with CTGF and TGF-bl expression were studied. Results: As compared with plastic, normal ECM coating transiently induced the mRNA level of the two well known fibrogenic growth factor, CTGF and TGF-bl, at 6h (x2) but the mRNA level returned to control level at 24h. This acute mRNA increase was associated with a timedependent induction of secreted CTGF protein. Fibrosis ECM coating did not affect acute expression (6h) of the fibrogenic gene, whereas a significant induction was observed at the later time point (24h). Conclusions: These results showed that ECM modify MSC phenotype and suggested that normal ECM induced a transient wound healing response whereas fibrosis ECM seemed to trigger a sustained fibrogenic response. Later time point kinetics are currently performed to confirm these results and the molecular pathways involved are under investigation. (1) Chapel A et al. Journal of Gene Medecine. 1028-1038, 2003. (2) Epperly et al. 2004. Acknowledgments: CB is fellow of ARC "Association recherche SUE le cancer".

133 R A D I A T I O N - I N D U C E D DAMAGE TO MOUSE UROTHELIAL BARRIER

J..laal 1, W. D6rt~'2 iRadiobiology Laboratory, and 2Experimental Centre, Medical Faculty Carl Gustav Carus, University of Technology Dresden, Germany Objective: Therpeutic irradiation of pelvic tumours results often in unavoidable side effects in a variety of organs. One of

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these - radiation-induced urinary bladder dysfunction- occurs in three phases: An early, reversible reaction, a dosedependent, symptom-free latent time, and a progressive and irreversible late response phase. Since the cellular mechanisms underlying these early and late effects are not fully understood, studies in experimental animal models are required. The present investigation addressed the question whether impairment of the urothelial barrier contributes to radiation effects in the mouse urinary bladder. For this, changes in uroplakin III (UP-III) - a component of the apical urothelial plaque - were examined during the early, as well as the late response phase. Additionally, cell numbers in the urothelial layers were assessed. Material and methods: Female C3H mice were subjected to single dose irradiation with 20 Gy. Groups of mice (3-10) were sacrificed in 2-3 days intervals between days 0 and 31 to study early effects, and at days 90, 120, 180, 240, 360 after irradiation to study late reactions. An age-matched control group (n=3-8) was included at each time point. The bladders were excised, fixed, and processed for histology and UP-III immunohistochemistry. Cell numbers in the urothelial layers were counted per unit length of urothelium. The fractional area with a positive immunohistochemical signal for UP-III on the apical membrane of the superficial umbrella cells was defined. Furthermore, the cytoplasmatic UP-III staining signal was quantified using an arbitrary score ranging from 0 (no staining) to 3 (strong staining signal). Results: Irradiation with 20 Gy resulted in a progressive decrease in cell numbers in all urothelial layers over the entire acute phase, as well as during the initial steps of the late phase. The most pronounced decrease was seen in the number of cells in the superficial layer, i.e. in umbrella cells, with a decrease from 4,11+0,38 cells (mean=l=SD) in controls to 0,27+0,12 cells at day 31 (p<0,0001), 1,1±0,4 at day 90 (p<0,0001), and 1,6+1,6 at day 120 (p=0,005) after the treatment. Subsequently, the number of superficial cells returned to control values by day 180. In un-irradiated bladders, an intensely stained superficial layer of UP-III was found on the luminal membrane of the umbrella cells. Additionally, a weak cytoplasmatic UP-III staining was seen in the cytoplasm of umbrella and intermediate cells, while the basal cell layer was completely negative for UP-III. During the acute phase, a progressive loss of the UP-III layer on top of the umbrella cells was observed, resulting in a total lack of signal at day 31. A significant decrease in UP-III layer was still detectable at days 90 (p<0,0001) and 120 (p=0,029) after the treatment. In both response phases, the loss of apical UP-III correlated with the decrease in the number of umbrella cells (p=0,002). In contrast, a progressive increase in the cytoplasmic staining of UP-III in the upper intermediate layers of the urothelium was seen. The decrease in supe~cial UP-III was negatively correlated with the cytoplasmic UP-III staining intensity (p<0,0001), suggesting that the loss of the superficial, protective layer of UP-III was the trigger for synthesis of cytoplasmic UP-III in the underlying cells. Conclusions: Irradiation of the urinary bladder results in an impairment of the urothelial barrier that is associated with a loss of superficial umbrella cells. However, barrier integrity is restored by day 180 after irradiation, indicating that changes in urothelial permeability might be involved in early radiation effects, and subsequently during the initial steps of the late radiation reaction. 134 I N D U C T I O N OF R A D I A T I O N - I N D U C E D RELIES ON THE C D 9 5 / C D 9 5 - L SYSTEM

PNEUMONITIS

F. Heinzelmann, V. Jendrossek, K. Lauber, R. Boras, 7-. EIdh, K. Nowak, R. Handrick, M. Henkel, W. Budach, C. Belka University of Tuebingen, Department of Radiation Oncology, Germany Objective: Pneumonitis is a dose-limiting side effect of total body irradiation and the main cause for dose restrictions during radiotherapy for any thorax associated neoplasm. Clinically, it may cause an increase in breathing frequency due to respiratory insufficiency and results in a lethality rate up to 10%. However, the underlying mechanisms of irradiationinduced pneumonitis are unclear. Although pneumonitis mostly

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P r o f f e r e d Papers

occurs within irradiated areas of the lung, it may spread to non irradiated areas, indicating that humoral factors might be involved. The current hypothesis suggests that complex alterations concerning epithelial/ endothelial cells, macrophages/ monocytes and a perpetual cascade of cytokine expression patterns are of major importance for the induction of pneumonitis. In this scenario several observations suggest an involvement of the CD9S/CD95-1igand (CD95-L) system in proinflammatory cytokine responses and attraction of neutrophils. Moreover the expression of CD95 and CD95-L is induced by ionizing radiation. The aim of the present study was to gain insight into a putative involvement of the CD95/CD95-L system in radiation*induced pneumonitis. Mice with a genetically defined deficiency of the CD95 receptor (Ipr) or the CD95 ligand (gld) and control mice with an intact CD95/CD95-L system (C57BL6/J) were analysed for their susceptibility to develop radiation-induced pneumonitis. Material and methods: To this end we examined the development of pneumonitis after single irradiation with 0/12,5 Gy to the right hemithorax in CD95 (Ipr) or CD95L deficient (gld) and control mice (C57BL/6J) regarding breathing frequency and histopathological alterations. Results: A highly significant increase in breathing frequency occurred in irradiated contro~ mice between days 5 and 70. Furthermore, a clear inflammatory response with increased alveolar wall thickness, interstitial edema and enhanced number of inflammatory cells in the interstitial and peribronchial space was observed at days 21, 42 and 84 post irradiation (right lung > left lung). In contrast, no increase in breathing frequency and no inflammatory response was detectable in irradiated gld and Ipr mice. Conclusions: These results suggest that the CD95/CD95 system plays an essential role in the induction of morphological and functional alterations in the lung characteristic for radiation-induced pneumonitis. This report demonstrates for the first time that the CD95CD95L system is of major importance for the development of irradiation induced pneumonitis, implicating new perspectives for future therapeutic and preventive strategies. 135 AN HEPARAN-MIMETIC PROTECTS RADIATION-INDUCED MUCOSITIS

MICE

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Results: Radiation-induced mucositis scored after IR plus RGTA®-OTR4131 spray followed by IP injections 3 h after, at day 1 and every 3 days, was significantly lower than after IR alone (p<0.001). A marked decrease of severity and duration of mucositis was observed with administration of RGTA®OTR4131 3 h after IR (p=0.0006) and 24 h after IR (p=0.001) compared to IR alone. Association of amifostine 10 min before IR with RGTA®-OTR4131 24 h after IR evidenced a major and better protection than RGTA®-OTR4131 3 h or 24 h after IR (p=0.002; p=0.001) and than amifostine 10 min before IR (p=0.005). Tumor growth: administration of RGTA®-OTR4131 IP 3h after IR, then at day 1 and every 3 days didn't evidence significant interference on tumor growth associated (p=NS) or not (p=NS) to IR. CIonogenic survival: the assay of radiosensitivity in vitro d i d n ' t show a significative activity of RGTA®-OTR4131 on H E P 2 cells clonogenicity (p=NS). Conclusions: RGTA®-OTR4131 demonstrated a protective activity in radiation-induced mucositis in mice, without interference on tumor growth; RGTA®-OTR4131 associated with amifostine gave an almost total protection. Further investigations are need to understand the selective protective activity on healthy tissues. Mucositis 3,5 3 ~--IR+RGTA®(3h) o

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136 S I M U L T A N E O U S NORMAL TISSUE PROTECTION AND INCREASED TUMOR R A D I O S E N S I T I V I T Y : A NEW GOAL FOR PRAVASTATZN ?

M. Mangoni 1,3, D. ViolotI, V. Frascogna I, C. Morin 2, ].P. Caruelle2, D. Barritau/t~, G. Biti 3, 9. Bourhis I ZInstitut Gustave Roussy, 94805 Villejuif, France ; 2Universit4 Paris-12, 94010 Cr4teil Codex, France; 3Universit~ di Firenze, Florence, Italy; 40TR3 SAS, 4 Rue Frangaise 75001, Paris, France

V. Haydont, O. Gilliot, C. Bourgier, J. Legrand, A. Francois, V. Frascogna, J. Bourhis, M.C. Vozenin-Brotons ~UPRES EA 27-10, Radiosensibilit~ des tumeurs et tissus sains, IGR/IRSN. Villejuif, France ; 2LRPAT, SRBE/DRPH. IRSN. Fontenay-aux-Roses, France

Objective: The purpose was to evaluate the effect of RGTA®OTR4131 (ReGeneraTing Agent), a synthetic heparan-mimetic, on radiation-induced mucositis in a murine model and on tumor growth in vivo and in vitro. Material and methods: Reagents: RGTA®-OTR4131 l m g / k g for intra-peritoneal (IP) injection; 10pg/ml in spray solution; 10pg/ml for in vitro assay. Amifostine (Ethyol) 200mg/kg for IP injection. Radiation-induced mucositis: the oral region of C57 black female mice was selectively irradiated with a single dose of 16.5 Gy. Irradiation (IR) alone was compared with IR plus RGTA®-OTR4131 spray followed by IP injections 3 h after, at day 1 and every 3 days and with IR plus RGTA®-OTR4131 3 h or 24 h after IR, amifostine 10 min before IR, amifostine 10 min before IR with RGTA®-OTR4131 24 h after IR. Mucosal reactions were evaluated daily for 21 days and scored by the Parkins scoring system (Parkins et al, 1983). Tumor growth: 3x106 HEP2 cells (human pharynx cancer cell line) were implanted subcutaneously into nude Balb/c mice. Animals were irradiated with a single dose of 15 Gy selectively to the tumor when tumor diameter reached a mean of 5mm. IR alone was compared with IR plus RGTA®-OTR4131 IP 3h after IR, then at day 1 and every 3 days and with RGTA®-OTR4131 IP alone at day 0, 1 and every 3 days. The tumor volume was evaluated for 21 days by the formula: length x width2/2 and tumoral response was estimated versus initial volume. Clonogenic survival: survival fraction of HEP2 cells was evaluated using the Park model; cells were exposed to IR at 0, 2, 4, 6 Gy and to IR plus RGTA®-OTR4131 2 h after IR.

Objective: Increasing normal tissue tolerance to irradiation is an important goal in radiation therapy. Recent data from our laboratory showed that activation of the Rho/ROCK pathway was involved in radiation induced fibrosis development. Here, we investigated whether a well known inhibitor of Rho isoprenylation: the pravastatin, could prevent fibrosis development. Furthermore, the effect of [Pravastatin+ irradiation] combination on tumour control was assessed in vitro and in-vivo. Material and methods: In the first part, modulation of fibrogenesis by pravastatin was investigated: 1in-vitro, in human primary smooth muscle cells isolated from normal ileum exposed to radiation (X-ray, 19 Gy) cultured in SmGM (Cambrex). Then, modulation of fibrogenic growth factor (TGF-bl and CTGF) expression were studied. 2- in-vivo, in a pro-clinical model of delayed radiation enteritis obtained in Wistar rats locally irradiated (X-rays, 19Gy) after ileum surgical exteriorisation. Pravastatin was administered in drinking water (30mg/Kg/day) 3 days prior to irradiation and 14 days after. Histological follow up of intestinal fibrosis development was performed 6 hours, 3 days, 2, 15 and 26 weeks after irradiation. In a second part, modulation of in vitro radiation sensitivity was assessed in HeLa cells by clonogenic cell survival assay using increasing doses of pravastatin (100500 IJM). In addition, tumour growth delay was studied invivo, using HeLa xenograft in nude mice and treated with pravastatin (30mg/Kg/day), irradiation (2 x 7.5 Gy) or combination. Results: In-vitro, Pravastatin lead to inhibition of the acute radiation-induced TGF-bl peack, whereas no modulation of its target gone, CTGF, was detected. In-vivo,