Posters Friday/Saturday, 20-21 September 2002 $307

our thesis that reactive nitrogen oxide species (RNOS) are responsible for dine on Chinese hamster cell culture. These doses induced: chromatin radioprotective action of L-NAME. decondencation, measured by flow cytometry, chromosomal aberrations,

manifested as bridges and fragments in anaphase and accumulation of 1050 Poster abnormal metaphases (coIchicin-like metaphase) in the cells which after Caffeine-increased radiosensitivity is dependent on a loss of some time died by apoptosis. G2/M arrest in human promyelocyte HL-60 cel l l ine We found also that pre-exposure to low level of e-rays protected actively

growing cells against the additional induction of chromosomal damage by J. Vavrova 1, M. Marekov22, S. Szkanderova 1, D. Vokurkova 3, J. Stullk 1 subsequent exposure to higher acute dose of 137Cs gamma-rays. It was 1purkyne Military Medical Academy Hradec Kr~love, Institute of Radiobiol- shown that activation of signal transduction involved in development of ogy and Immunology, Hradec Kralove, Czech Republic adaptive response is induced by low doses of ionizing irradiation. We 2Faculty of Medicine Hradec Kr~love, Charles University Prague, Depart- showed that the adaptive response measured by the reduction of radiation- ment of Medical Biochemistry, Hradec Kr~love, Czech Repubfic induced chromosome aberrations was completely blocked in the presence 3University Hospital Hradec Kralove, Institute of Clinical Immunology and staurosporin as well as L-NAME (omega- N -nitro - L - arginine-methyl Allergology, Hradec Kr&lov6, Czech Republic ester). These results confirm the participation of protein kinase C in induc-

tion of the adaptive response and first denote on the participation of NO-

In our work we proved that ionizing radiation induces two different types of synthase in this response. apoptesis in HL-60 cells: premitotic and postmitotic. Doses higher than 20 We found that the descendants of the chronic exposure cells were less sen- Gy lead to quick DNA fragmentation, starting up 4-6 hours after irradiation sitive to high doses of gamma-rays than their parents. However, several - premitotic apoptosis (interphase death). In contrast after doses lower than distinct abnormal phenotypic characteristics were found in the surviving 10 Gy cell death with DNA fragmentation was apparent only after G2 phase progeny of irradiated cells.These include the high level of postmitotic apop- cell cycle arrest. After irradiation by dose 6 Gy maximum of apoptosis induc- tosis, decondensed chromatin and anomalous small colonies. These fea- tion was observed after 48-72 hours - postmitotic apoptosis (mitotic death), tures are persisting during 4 years. Radiation-induced apoptosis seems the Four methods were used for apoptosis detection: morphologic (evaluation most interesting characterization of descendants. of nucleus fragmentation in Giemza-Romanovski stained cytospin prepara- Apoptotic form of cell death takes place when level of damages in cell is too tions), flow-cytometric detection of APe2.7 and DNA content analysis high. It is known that high doses of radiation induce cellular interphase (subG1 peak) and western-blot detection of lamin B cleavage to 46 kDa apeptosis. We found that low doses of radiation induce another kind of fragments. 48 hours after irradiation these fragments were detected in both, apoptosis, postmitotic apoptosis. We suggest that induction of postmitotic nucleus and cytoplasma, apoptosis by low doses can be applied for radiotherapy of tumors.

Incubation of cells with 2mM caffeine 30 minutes prior irradiation by 6 Gy prevents cell cycle arrest in G2 phase caused by irradiation. This was con- 1053 Poster nected with radiosensitization and second wave of apoptosis, which was The role of ubiquitin in DNA repair and UV hypersensitive dis- observed (using methods mentioned above) 144-192 hours after irradiation, orders The data suggest that radiosensitization induced by caffeine is in HL-60 R.K. Chiu 1, J. Brun 2, D.A. Gray 2-, B.G. Wouters 1 cells dependent on abrogation of G2 arrest and on induction of apoptosis. 1University Maastricht, Radiation Oncology, Maastricht, The Netherlands

1051 Poster 2Ottawa Regional Cancer Centre, Centre for Cancer Therapeutics, Ottawa, Correlation of HL-60 cell l ine sensitivity to low-dose-rate irra- Canada

diation wlth G2/m-phase block and death by apoptosis The highly conserved protein ubiquitin (Ub) has been implicated in a variety M. Marekovd J. Vavrova, D. Vokurkovd of diverse cellular processes involving the elimination of short-lived or 1Faculty of Medicine Hradec Krdlov~, CU Prague, Department of Med- abnormal proteins. This proteolytic pathway is critical for cellular viability as ical Biochemistry, Hradec Kr~lov6, Czech Repubfic defects are observed in certain cancers and several human diseases. Ub 2purkyne Mifitary Medical Academy Hradec Krdlov~, Institute of Radiobiol- acts as a molecular tag by covalently bonding to specific proteins and sub- ogy and Immunology, Hradec Kr&lov6, Czech Republic sequently to itself to form a linear Ub polymer, thus targeting the substrate 3University Hospital Hradec Kr~lov6, Institute of Cfinical Immunology and for destruction by the 26S proteasome. To add further complexity, these Allergology, Hradec Krdlove, Czech Republic chains communicate different signals depending on the lysine residue used

for elongation. While polymers linked by lysine 48 are recognized as a sig- Low dose rate irradiation is used in brachytherapy and immunotherapy of hal for degradation, some Ub-conjugating enzymes (UBCs) can assemble malignant tumors. This type of irradiation has a protective effect to normal multiUb chains via other linkages, such as lysine 29 or 63. While the bio- tissues. It is generally accepted that accumulation of cells in G1 a0d G2 logical consequence of these alternative chains is not well understood, in phase of cell cycle, which occurs after irradiation by high dose rate gamma yeast the K63-1inked polyUb chains appear to play a role in DNA repair as radiation, prevents the cells from replicating damaged DNA and provides yeast expressing a K63R mutant form of ubiquitin show extreme sensitivity time for reparation of DNA or for apoptosis induction in damaged cells. Cell towards UV light and chemotherapeutic agents such as cisplatin and tira- cycle arrest in G2 phase could be important in developing of tumor cells pazamine. The UBC13/MMS2 complex, which catalyzes these K63 Iink- radioresistance, in contrary irradiation of cells in G2 phase could have ages, belongs to the error-free bypass branch of postreplication repair and radiosensitizing effect. We proved that irradiation of TP53 negative human thus mediates the avoidance of mutagenic repair, a major factor contribut- promyelocyte leukemia cells HL-60 by low-dose rate (3.9 mGy / 1 min) ing to the carcinogenic effects of UV. Interestingly, Xeroderma pigmento- cause prolonged irradiation of cells in G2 phase. Already irradiation by dose sum variant (XPV), has been shown to be caused by a defect in postrepli- 2.5 Gy induced accumulation of 55% of cells in G2 phase. However, this cation repair. In fact, XPV, which accounts for 23% of XP, encodes DNA type of irradiation has lower effect on clonogenic survival compared to sin- polymerase eta, which is responsible for error-free repair. The relevance of

UbK63 in mammalian cells as well as the substrate proteins for this linkage gle high-dose rate irradiation. We studied enhancement of low-dose rate irradiation effect by caffeine. Caffeine prevents G2 cell cycle arrest during are not well defined. To this end, by over expressing dominant negative Ub low dose rate irradiation. This effect is related to induction of second wave mutants in mammalian cells, we show that K63 is necessary for the repair of apoptosis 144-192 hours after beginning of irradiation, of DNA damage induced by cisplatin but not UV. However, K63 is neces-

sary for the suppression of spontaneous and UV-induced mutagenesis fur- 1052 Poster ther suggesting a role for this pathway in carcinogenesis. In order to under- D i rec t and delayed effects induced by acute and chronic stand the biological role of Ub conjugation in DNA repair we are now

pursuing the biochemical characterization of the repair complex, the identi- e x p o s u r e with low doses of ~particles emitted from 14c fication of the UbK63 targets, and the elucidation of the signal conveyed by I. Dpni/ova, N. Gi/iano, S. Arutunyan, L. Noskin the Ub chains produced by this complex. In addition we are interested in the Petersburg Nuc/ear Physics/nstitute, Mo/ecu/ar and Radiation Biophysics, function of the UBC13/MMS2 complex in other cellular processes. Gatchina, Russian Federat

1054 Poster Low doses of radiation are widely used in radiotherapy. It has been shown Additive effects for the combintion of interferon ~ and radia- that the exposure by tow doses results in anti-inflammatory, anaesthetizing effects, tion in four out of five human glioblastoma cell lines It has also been shown, that the exposure of cells by low doses modifies H. Schmidberoer, M. Rave-Frank, E. Weiss, L. Ger/, N. Dettmer, their response to subsequent high doses of radiation. S. G/omme, C.F. Hess We studied the effect of low doses (0.1-20cGy) of ]?,-rays from 14C-thymi- University G6ttingen, Radiotherapy and Radiation Oncology, G6ttingen,

$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.