$308 Friday/Saturday, 20-21 September 2002 Posters

Germany 1056 Poster Evaluation of apoptoUc response of T-lymphocytes for radia-

Purpose: Local failure is the principle clinical problem in malignant glioma, tion therapy in cancer patients Interferon 13 (IFN-b) has reported antineoplastic and radiosensitizing activi- S. Stankeova 1, N.E.A. Crompton 2, H. Blattmann 2, P. Theiler 2, ty in vivo and in vitro, however clinical results are inconsistent. Our study aimed to investigate the potential of IFN-b to enhance the cytotoxic activity G.C. Emery 2, B. Kaser-Hotz 1 of ionising irradiation against glioma cells, and to elucidate the possible 1University of Zurich, Veterinary Clinics, Zurich, Switzerland mechanisms responsible for the conflicting clinical results. 2psi, Radiation Medicine, Villigen PSI, Switzerland Materials and Methods: Five different glioblastoma cell lines (U87MG, U118MG, U373MG, MO59K, MO59J) with different radiosensitivity and Purpose: A prospective study to evaluate radiation-induced apoptosis in T- genetic background as a surrogate for tumor heterogeneity were used. lymphocytes from dogs, who received a full course of curative or palliative Experiments were performed in exponentially growing cultures, and cell sur- radiation therapy was performed. A major goal was to evaluate the useful- vival was measured by a colony-forming assay. Cells were incubated with ness of the apoptosis assay in identifying patients overly sensitive to radia- IFN-b (30 - 3000 I.U./ml) for 24 h followed by single dose irradiation with 1 tion. Another goal was to examine potential changes in sensitivity of T-lym- to 6 Gy of gamma-rays, phocytes to radiation-induced apoptosis associated with a course of Results: IFN-b reduced the colony forming ability of the glioblastoma cell radiation treatment. lines in a concentration-dependent manner, and significant differences in Material and methods: Blood was collected in heparin tubes, diluted 1:10 in their sensitivity to IFN-b were found. The cell lines also differed in their radi- RPMI medium, irradiated with X-rays and incubated for 48 h. T-lymphocytes ation sensitivity, and there was no correlation between the IFN-b and the were labeled using antibodies, and DNA stained with propidium iodide. For radiation sensitivity. The combined effect of I FN-b and radiation was most- cell analysis a flow cytometer was used. Radiation-induced apoptosis in T- ly additive, only for MO59J cells, which are NHEJ-deficient, supraadditivity lymphocytes was quantified. Blood probes from tumour-bearing dogs, were was observed, taken before the first fraction and at the end of radiation therapy. Conclusion: Our results confirm the remarkable heterogeneity, which is Results: The assay can be successfully applied to canine patients to esti- characteristic for malignant glioma. The five cultures tested differed in their mate radiosensitivity. Apoptosis of lymphocytes is dependent on donor age: radiation response as well as in the response to IFN-b treatment. Three out young animals showed considerably higher levels of apoptosis than older of five cultures were remarkably IFN-b sensitive, tn three out of five cell lines dogs. Bigger dogs displayed higher levels of apoptosis than smaller dogs. the interaction of IFN-b and irradiation was additive. In one cell line, it was Tumour-bearing dogs when compared with healthy dogs showed no signif- slightly subadditive. Synergistic interaction might occur in tumor cells that icant differences in levels of induced apoptosis. No significant changes already have acquired repair deficiencies due to their genetic instability, as were seen in the levels of induced apoptosis in blood taken before or after shown for the MO59J cell line. radiation therapy.

Conclusion: The sensitivity of peripheral blood T-lymphocytes to radiation- 1055 Poster induced apoptosis does not change as a result of the trauma of radiothera- DNase 2, in c o m b i n a t i o n w i th Acid Phosphatase, generated py during the course of tumour treatment. T U N E L reactive ends during Radiation-Induced Apoptosis Y. Nakaeami. M. Ito, M. Omura, T. Hara, I. Ogino, S. Matsubara, T. Inoue 1057 Poster Yokohama CityUniversityschoolofmedicine, radiology, Yokohama, Japan Opportunities and l im i t s o f h y p o f r a c t i o n a t i o n in HDR

brachytherapy During apoptosis, endonuc!eases present in cells cleave DNA at sites locat- ed between nucleosomal units, thus resulting in the characteristic ladder of P. Smini~ 1, C.J. Schneider 2-, J.F. Fowler 3 DNA fragments.Several attempts have been made to characterize the I VU University Medical Center, Radiation Oncology, section Radiobiology, nucleas'es that are responsible for this fragmentation. For instance, DNase Amsterdam, The Netherlands 1, Nuc 18 and NUC 70 have been reported to be associated with apoptotic 2The Netherlands Cancer Institute, Radiation Oncology, Amsterdam, The DNA cleavage. DNase gamma and caspase-activated-DNase (CAD) have Netherlands also been discovered. In addition, DNase 2 has also been proposed to be 3University of Wisconsin Hospital, Human Oncology, Madison, Wisconsin, involved in specific cases of apoptosis. However, according to past reports, USA DNase 2 doesn't directly generate TUNEL-positive cells. We studied the participation of acid phosphatase in the generation of Indications on the existence of long repair half times in the order of 2-4 h for TUNEL-positive cells. In our study, the DNase 2 like proteins, whose mole- late responding human normal tissues have been used to explain, on basis cular weights were 62- and 32-kDa, were detected in nuclear extracts of of the Biological Effective Dose, the superiority of fractionated HDR with HL60 cells post gamma-irradiation by SDS-PAGE and immunohistochem- istry. Zymography assays were done for endonuclease activity. And acidic large fraction sizes of 5-7 Gy over continuous CLDR irradiation at 0.5 Gy/h nuclease activity was especially active in 32-kDa bands but not in 62-kDa dose rate. Hypofractionation in HDR in brachytherapy might have its oppor-

tunities for widening the therapeutic window, but definitely has its limits. bands. TUNEL assay was positive post gamma-irradiation. From measure- The therapeutic ratio of HDR and CLDR is largely dependent on the [a] merits of the activity of acid phosphatase, the activity in nuclear extracts number of HDR fractions (hence fraction size), [b] overall treatment duration increased remarkably post gamma-irradiation. (the time interval between fractions in HDR and the dose rate in CLDR) and At the beginning of apoptosis, the translocation of DNase 2 and acid phos- phatase occurred. Also, terminal transferase used for TUNEL reaction [c] repair characteristics of the exposed tissues (aJb ratio and repair half extends 3'-hydroxyl ends of DNA. For instance, CAD cleaves DNA leaving time). While parameters [a] and [b] could be chosen, from [c], the repair half 5'-phosphate and 3'-hydroxyl ends, confirming that CAD is responsible for time could only be estimated from experimental and clinical data. Previous- the cell autonomous generation of TUNEL-reactive DNA fragments. In case ly we showed that, with the assumption of the repair half time of normal tis-

sue being three times longer (3h) than that of the tumour (1 h), hypofrac- of our study, however, the most likely candidate for the acid DNase in lyso- tionation in HDR could relatively spare normal tissue if the optimum fraction somes that causes DNA fragmentation is DNase 2. However, it is unlikely size is selected. Aiming at a constant BED for the tumour, the physical dose that the action of DNase 2 directly generates TUNEL-positive cells, because can be reduced allowing absolute normal tissue sparing. However, the opti- cleavage of DNA by DNase 2 generates 5'-hydroxyl and 3'-phosphate ends mum HDR fractionation scheme, i.e. the combination of fraction size and that cannot be used as substrates for terminal transferase. Lysosomes are time between the fractions most advantageous with regard to normal tissue rich in acid phosphatases that can remove the phosphate group from the 3'- sparing, is largely dependent on the CLDR dose rate. end of DNA fragments. And acid phosphatase activity in the nuclei of HL60 On basis of the BEDNT/TUM ratio of HDR over CLDR, three times 6.7 Gy cells increased time-and dose-dependently after gamma-irradiation, would be the optimal HDR fractionation scheme for replacement of a CLDR These results suggest that acid phosphatase was translocated from lyso- scheme of 20 Gy in 10 to 30 h (dose rate of 2 to 0.7 Gy/h). However, 4 frac- somes into nuclei during gamma-radiation induced apoptosis at the begin- tions of 5 Gy would be preferential for replacement of 20 Gy CLDR in 40 h, ning. Thus, it is possible to assume that DNase 2, in combination with acid still assuming large differences between tumour and normal tissue repair phosphatase in lysosomes, generates TUNEL reactive ends. half times and equal overall treatment time. However, the most advanta-

geous fraction size would be smaller by prolorlgation of the HDR overall treatment time. This dependency on treatment choices and treatment conditions illustrates the opportunities and limits of hypofractionation in HDR brachytherapy.