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curves and clonogenic assay were performed in order to determine celt same medium for various times. For TRF2 and human neutrophil elastase growth inhibition, detection, samples were boiled at 95 degrees centigrade for 5 minutes with Results and conclusions: exposition to IIF resulted in a strong inhibition of equal volumes of sample buffer. After this treatment, 15 microliter aliquots cell proliferation and in a marked induction of neuronal differentiation as were fractionated by SDS-PAGE using 10-20 percent gradient polyacry- revealed by neurite extensioin, increase of acetylcholinesterase specific lamide gels. TRF2 and human neutrophil elastase proteins were detected activity and tyrosine hydroxylase mRNA expression. When the effect on using a 1/500-t/2000 dilution of anti-TRF2 and anti-human neutrophil elas- cell growth induced by IIF or by ATRA, or by 9cRA, or by NaB were corn- tase antibodies. TOYOBO-ABC high-HRP Immunostaining Kit was used for pared, it resulted that the inhibitory effect of IIF was higher than the one by immunohistochemistry. A DNA fragmentation assay was performed for the ATRA, 9cRA or by NaB. The extensive neudte outgrowth induced by IIF detection of apoptosis. In order to investigate interaction between elastase resulted of the same extent when compared with the one induced by 9cRA and TRF2, in vitro test was also performed. and the marked increase of acetylcholinesterase specific activity induced by Results: Western blot analyses and immunohistochemistry showed that IIF resulted quite higher than that induced by ATRA. gannma-rays induce the temporary accumulation and subsequent loss of These results demonstrate the effectiveness of the new derivative of TRF2 protein in the nuclei of irradiated HL60 cells. Following DNA frag- retinoic acid IIF on differentiation and proliferation in TS12 neuroblastoma mentation, the loss of TRF2 could be detected. TRF2 was broken down by cell line. elastase, which was translocated into the nucleus before the toss of TRF2.

Conclusions: The results of the study showed that irradiation first induces 1067 Poster activation of TRF2, consequently protecting the end of the chromosome. A novel three-dimensional tumour cell culture system for Subsequently, translocation of elastase into the nucleus results in the improved therapeutically orientated investigations in vitro breakdown of TRF2 after DNA fragmentation has occurred.

B. Forthuber 1, T. Seppi 1, K. Pfallet 2, L Skvortsova 1, P. Lukas 1 1069 Poster 1University Clinics of Innsbruck, Dept. of Radiotherapy-Radio- Effects of digitoxin on cell growth and radiosensitivity in oncology, Innsbruck, Austria g l i ob las toma cel l lines

'2University of Innsbruck, Institute of Histology, Innsbruck, Austria A.B.L. Marthinsen. 72 Strickert, K.M. Jensen, J. Haux St. Olavs Hospital, Cancer Department, Trondheim, Norway

Purpose: Substantial differences in response to radio- and chemotherapy have been observed between cells grown in monolayer cultures and in vivo Digitoxin is lipid soluble and crosses the blood-brain barrier and accumu- tumour cells. Many of these differences seem to be directly related to the lates in brain tissue. We have previously shown that digitalis, in non toxic differences in spatial organisation and in cell-cell- and cell-matrix interac- consentrations, induces apoptosis in several different human malignant cell tions. 3D cultures may preserve specific biochemical and morphological lines, and have also effect on radiosensitivity. This indicate that cardiac gly- features similar to the corresponding tissues in vivo and can stay in a dif- cosides have a possible role in cancer treatment. ferentiated, active functional state for many weeks thereby providing a more We have studied three different glioblastoma cell lines (U-373 MG, U-118 reliable in vitro model for exploring tumour cell response to various treat- MG and U-87 MG) exposed to digitoxin (in concentrations up to 100 ng/ml). ment modalities. Up to the current point of time, 3D .culture models such as Growth rates (cell counting at different times after seeding), viability (MTT multicellular tumour spheroids cannot completely represent the in vivo situ- assay), proliferation (3H thymidine incorporation) and DNA histograms ation, especially in regard of varying oxygen and nutrient supply to different (flowcytometry) were assessed. Cells exposed to digitoxin (3 days) were tumour regions, irradiated (0 - 8 Gy) and radiosensitivity were determined by a clonogenic Considerable progress has been made more recently in generating bio-arfi- assay. Digitoxin decreased viability, proliferation and plating efficiency of all ficial tissues in vitro. Unfortunately, the development of a simple assay for the three cell lines in a dose dependent manner, the U-373 MG line being vascularisation of celt aggregates in vitro still represents one of the main most resistant. DNA histogram analysis revealed accumulation of the digi- problems. The presented work focuses on the development of a scaffold toxin treated cells in the G2M phase as well as increased DNA fragmenta- based 3D tumour model equipped with thin semiartificial vessels, thus tion. Morphological changes together with other results suggest cell death enabling oxygen and nutrient supply or drug delivery, through apoptosis. The U-373 MG cell line was more radioresistant and had Procedure: A patented incubation device consisting of an artificial fibrous higher plating efficiency than the other two cell lines. Digitoxin increased the support and a vessel network has been constructed to provide a constant radiosensitivity of the cell lines in an additive dose dependent response microenvironment with continuous nutrient supply and removal of catabolic (isobologram analysis). waste products. 3D cultures can be maintained over several weeks and Digitoxin induces growth repression and additively increases radiosensitivi- used for various morphological, physiological and toxicological investiga- ty in glioblastoma cell lines in vitro. The effects occur at concentrations tions. So far, cell cycle distributions and apoptotic fractions under varying achieved in brain tissue by therapeutic plasma concentrations of digitoxin. perfusion rates have been assessed to standardise the novel system. Digitoxin may be of benefit in the treatment of gliobiastoma. Results and Conclusion: Cells in this scaffold cultured system exhibit a nor- mal cell cycle distribution accompanied by low levels of apoptosis and sim- 1070 Poster ilar morphology to the in vivo situation. The most striking argument for the Low-dose hypersensitivity might be dependent on cel l s ize use of such systems seems to be the fact that, in contrast to tumour spher- selection criteria oids, proliferation, death and repopulation of cells are not limited to restrict- L.M. Persson, B.K. Lind, L Hedlof, M.R. Edgren, A. Brahme ed areas but irregularly distributed over the whole artificial tissue. This Medical Radiation Physics, Oncology-Phatology, Stockholm, Sweden model simulates the complex microenvironmental situation of solid tumours in vivo much better than the tumour spheroid system, and may therefore Different selection criteria using a fluorescence activated cell sorter (FACS) represent a mandatory test system in therapeutic screening programs or in have been applied on human colon cancer (HT-29) and a small cell lung radiobiological research, respectively, cancer (U-1690) cells in order to resolve low-dose hypersensitivity with

1068 Poster clonogenic survival. Clonogenic cell survival after irradiation with a Co -60 beam was measured

Telomere repeat binding factor 2 (TRF2) degenerated by radi- for cells counted with the FACS with no selection criterion and cells select- a t i on - induced apoptosis ed with medium cell size. A clear influence of the selection criterion is M./to, Y. Nakagami, M. Omura, 7". Hara,/. Ogino, S. Matsubara, T./noue shown in the cell survival results. Cell population with medium cell size Yokohama City University School of Medicine, radio/ogy, Yokohama, Japan shows smaller amount of low-dose hypersensitivity than cells with no selec-

tion criterion. Plating efficiency and microscopic examination show that Purpose: A telomere consists of the short tandem DNA repeats of small cells are not viable cells and that the large cells are cells in late G2- (T2AG3)n localized to the distal ends of chromosomes. Telomere repeat and M- phase. The present investigation indicates that the expression of binding factor 2 (TRF2) has been implicated in the protection of chromo- low-dose some ends. Recently, it has been reported that the loss of TRF2 induces hypersensitivity is strongly dependent on sorting criteria that may depend apoptosis by various stimuli or genetic technique, however, the effects of on cell cycle selection. radiation are not known. Therefore, this study investigated the interaction between TRF2 and radiation. Materials and Methods: HL60 and HeLa $3 cells were used in this study, since apoptosis is known to be induced by gamma-irradiation in HL60 cells but not HeLa $3 cells. In order to induce apoptosis, both cells were treated with 1, 5, 10, 20, and 30 Gy of 137Cs radiation, and then incubated in the