Perspectives in linear accelerator for FLASH VHEE: Study of a compact C-band system.

PURPOSE: In order to translate the FLASH effect in clinical use and to treat deep tumors, Very High Electron Energy irradiations could represent a valid technique. Here, we address the main issues in the design of a VHEE FLASH machine. We present preliminary results for a compact C-band system aiming to reach a high accelerating gradient and high current necessary to deliver a Ultra High Dose Rate with a beam pulse duration of 3µs. METHODS: The proposed system is composed by low energy high current injector linac followed by a high acceleration gradient structure able to reach 60-160 MeV energy range. To obtain the maximum energy, an energy pulse compressor options is considered. CST code was used to define the specifications RF parameters of the linac. To optimize the accelerated current and therefore the delivered dose, beam dynamics simulations was performed using TSTEP and ASTRA codes. RESULTS: The VHEE parameters Linac suitable to satisfy FLASH criteria were simulated. Preliminary results allow to obtain a maximum energy of 160 MeV, with a peak current of 200 mA, which corresponds to a charge of 600 nC. CONCLUSIONS: A promising preliminary design of VHEE linac for FLASH RT has been performed. Supplementary studies are on going to complete the characterization of the machine and to manufacture and test the RF prototypes.

[Flash radiotheray at very high dose-rate: A brief account of the current situation]. / Radiothérapie flash à très haut débit de dose : point sur les avancées récentes.

In the last decade, major advances in high precision treatment delivery and multimodal imaging allowed radiotherapy to be more efficient and better tolerated. However, the technology of the accelerators used to generate X-ray beams is outdated and does not allow to explore the tolerance to novel approaches in terms of dose-rate. We have been the first to propose a completely novel modality of irradiation, named Flash radiotherapy, in which the dose per pulse and the instant dose-rate during the pulses is 103 to 104 higher than those used in conventional facilities. Flash has been shown to spare mouse lung from radio-induced fibrosis, whilst leaving unchanged the antitumor potential. Other teams have shown that the advantage of Flash in terms of reduced complications extends to normal brain and intestinal crypts. The goal of this paper is to review the progress of studies dealing with very high dose-rate "Flash" irradiation, describe the theoretical models proposed to explain the underlying mechanisms, and discuss the prospects for clinical applications of this emerging technique.

Additive and supraadditive interaction between ionizing radiation and pazelliptine, a DNA topoisomerase inhibitor, in Chinese hamster V-79 fibroblasts.

The cytotoxic effect of the 9-azaellipticine derivative pazelliptine in combination with gamma-ray irradiation was investigated using Chinese hamster V-79 cells in culture. gamma-ray irradiation and drug treatment (1-h drug exposure) were applied at 1-h intervals for partial DNA damage recovery in growth medium. Isobologram analysis of the clonogenic potential gave evidence of supraadditive interaction in the radiation----drug sequence with 10% survival as an endpoint. No synergistic potentiation was observed at higher survival or as pazelliptine was applied first. Pazelliptine abolished the low-dose shoulder characteristic of asynchronous cell response to gamma-rays. Although rejoining of radiation-induced DNA strand breaks was completed at the time of drug exposure, pazelliptine brought about a larger amount of DNA strand breaks in preirradiated than in nonirradiated cells. The time and dose dependencies of DNA strand break formation and repair with radiation and/or pazelliptine were analyzed by neutral and alkaline filter elution. Pazelliptine in the micromolar range showed the same pattern of double-stranded cleavable complex formation as expected of a DNA topoisomerase II-targeting agent. At a low concentration of pazelliptine, however, protein-concealed breaks were mostly in the form of single-stranded adducts. Such single-stranded complexes have been reported to occur with some topoisomerase II-targeting drugs; their properties are also reminiscent of those induced by the topoisomerase I poison, camptothecin. It is proposed that topoisomerase poisoning interacts with the repair of radiation-induced lesions.

Interaction of ionizing radiation with paclitaxel (Taxol) and docetaxel (Taxotere) in HeLa and SQ20B cells.

Altered gamma-ray response by brief (1 h), concomitant exposure to paclitaxel (Taxol) or docetaxel (Taxotere) was investigated in growing HeLa and SQ20B human tumor cells in vitro. For both cell lines, both taxoids were able to reduce or enhance radiation cell killing, depending on the drug concentration. Large reduction of radiosensitivity (up to 3.3-fold reduction relative to radiation alone) was observed in HeLa cells over a wide range of drug concentrations, extending to 1.5- (paclitaxel) or 3.3- fold (docetaxel) the IC50s determined for drug alone. This antagonistic effect was also observed with SQ20B cells. It disappeared for drug concentrations exceeding 0.9 (SQ20B), 1.6 (HeLa; paclitaxel), and 3.4 (HeLa; docetaxel) IC50 equivalents, above which a drug dose-dependent, supra-additive radiation-drug interaction was observed. Reduction of radiation susceptibility in the low-drug dose range also held for mid-G1 synchronized HeLa cells, i.e., in the cell cycle compartment characterized as the most resistant one to docetaxel (C. Hennequin et al., Br. J. Cancer, 71: 1194-1198, 1995). In the case of SQ20B cells, the cytotoxicity of either drug or radiation alone was primarily dependent on the state of growth, with quiescent (G(0)) cells showing increased radiosensitivity and reduced drug toxicity compared to the growing fraction. The effect of taxoids (1-h contact) was finally investigated in sequential treatment as a function of the time elapsed between radiation and exposure to drugs. In HeLa cells, the postirradiation time-dependence of the response to combined treatment was biphasic. The radioprotecting potential of either taxoid disappeared in approximately 1.5 h following radiation. At longer postirradiation delays, radiation-induced redistribution in the cell cycle appeared to be the major determinant of HeLa cell survival, in relation to the differential cell cycle phase specificity of each drug. Pronounced paclitaxel recovery versus increased sensitivity to docetaxel occurred over 8 h after irradiation. SQ20B cells showed monophasic radiation recovery with both drugs over the same time range.

Likelihood of the new antitumoral drug 10-[gamma-diethylaminopropylamino]-6-methyl-5H-pyrido[3',4':4,5]pyrrolo [2,3-g]isoquinoline (BD-40), a pyridopyrroloisoquinoline derivative, to induce DNA strand breaks in vivo and its nonmutagenicity in yeast.

BD-40, a pyridopyrroloisoquinoline analogue of ellipticines, has dose-dependent cytostatic and cytotoxic effects on cultures of Saccharomyces cerevisiae. These inhibitory effects take place only in growing cells and are enhanced in the presence of oxygen. Among the different repair-deficient mutants examined, a mutant defective in DNA strand break repair (rad52-1) was found to be the most sensitive to such a toxic effect. A triple mutant blocked in the excision (rad2), the mutagenic (rad6), and the recombinogenic (rad52) repair pathways demonstrated the same sensitivity as the single rad52 mutant. Nuclear reversion and forward mutations as well as mitochondrial "petite" mutation were not induced by BD-40. These results indicate that: (a) the lesions induced in vivo by BD-40 are likely to be DNA strand breaks; (b) such damage is repairable in the wild type and is not of the mutagenic type; and (c) the excision pathway is not involved in such a repair of BD-40-induced lesions, and the mutagenic pathway plays a minor role. Since DNA strand breaks were not detected in vitro whether exposure of DNA to BD-40 was achieved in the presence or the absence of microsomal S-9 mix, it is suggested that an oxygen-dependent enzymatic processing, not linked to the microsomal monooxygenase complex, is required for the development of the cytotoxic activity of BD-40.

Interaction of ionizing radiation with the topoisomerase I poison camptothecin in growing V-79 and HeLa cells.

Interaction between gamma-rays and camptothecin (CPT) was investigated in vitro, using log phase HeLa-S3 cells and Chinese hamster V-79 fibroblasts. A plateau of toxicity was rapidly reached for both cell lines upon exposure to CPT alone, consistent with S-phase specificity of CPT. With synchronized HeLa cells, however, CPT proved cytotoxic after the middle of G1 phase. This effect was abolished by cotreatment with the DNA polymerase alpha inhibitor aphidicolin. CPT enhanced the initial slope of the radiation survival curves for asynchronous cells by a factor of 1.1 (HeLa) to 2.3 (V-79). This apparent radiation sensitization correlated with the intrinsic radiosensitivities of the drug-surviving fractions within the different compartments of the cell cycle. There was no evidence of mutual potentiation of CPT and radiation in terms of survival as well as with regard to the formation and rejoining of DNA double-strand breaks. V-79 cells exhibited pronounced postirradiation recovery. In contrast, the response of HeLa cells to drug did not vary appreciably for over 8-h following radiation. This difference proceeded from differential cell cycle redistribution. In both cell lines, acute irradiation produced depletion of the G1 compartment, accumulation at the S-G2 junction, and G2 arrest in proportion to the gamma-ray dose. However, while radiation brought about rapid depletion of the S-phase compartment in V-79 cells, it induced accumulation of HeLa cells into S phase. As a result, the CPT-sensitive HeLa cell population, consisting of the early-S, mid-S, and late G1 fractions, did not vary very much after irradiation. Exposure to CPT under conditions of low dose rate irradiation (1 Gy/h) selectively reversed the radiation effect on S-phase progression in V-79 cells; i.e., it induced accumulation of cells in S phase in the same way as found with HeLa cells. Isobolic analysis of survival data consistently showed supraadditivity of cell killing in both cell lines upon concomitant exposure to CPT and low dose rate irradiation. Cytokinetic cooperation appears to be the major determinant of cell survival in treatments associating CPT and radiation in growing cells. Attempts to predict the outcome of such a combined modality thus should take into consideration the response of the growing fraction of each cell line in terms of cell cycle-regulatory processes and redistribution.

DNA repair and cell cycle interactions in radiation sensitization by the topoisomerase II poison etoposide.

The interactions between ionizing radiation and etoposide were probed using asynchronous growing V-79 fibroblasts. Synergistic cell kill was observed as gamma-rays were applied prior to or concomitantly with the drug. Three major determinants of enhanced cytotoxicity in combined treatment were identified. The kinetic analysis of radiation recovery bore evidence of two repair interaction mechanisms. First, rapidly repairable radiation-induced DNA damage was fixed into lethal lesions by etoposide, thus giving rise to marked supra-additive interaction under concomitant radiation-drug exposure. Second, cells arrested in G2 phase following radiation proved hypersensitive to the cytotoxic effect of etoposide. It is proposed that either topoisomerase II alpha is closely involved in some rapid DNA repair pathway operating during all phases of the cell cycle, and even further involved in DNA repair acting within the radiation-induced G2 block, or that the lesions induced by etoposide are able to impair these processes. The shoulder of the radiation survival curve was abolished as gamma-rays and drug were applied at 1-h intervals. This effect, corresponding to mode II additivity from isobologram determinations, appeared to be correlated with a differential sensitivity of the various phases of the cell cycle to drug and radiation.

One-electron photo-oxidation of reduced Desulfovibrio vulgaris flavodoxin on laser excitation at 355 nm.

Electron ejection from the reduced flavin in flavodoxin from Desulfovibrio vulgaris was obtained on exposure of the protein to the third harmonic radiation (354.7 nm) generated from a pulsed Nd/YAG laser. The results indicate that the reaction is due to stepwise two-photon excitation of the reduced flavin via the excited singlet state. The absorption spectrum of the neutral flavosemiquinone radical formed in this process was obtained. This spectrum remains stable over the time of study (0.2 ms) in the pH range studied, except for a slight evolution during the first microseconds, attributed to conformational readjustments of the active site. This two-photon excitation method provides a convenient means of generating the flavosemiquinone for ultrafast kinetic studies.

Physicochemical properties of flavodoxin from Desulfovibrio vulgaris.

Reductive titration curves of flavodoxin from Desulfovibrio vulgaris displayed two one-electron steps. The redox potential E-2 for the couple oxidized flavodoxin/flavodoxin semiquinone was determined by direct titration with dithionite. E-2 was -149 plus or minus 3 mV (pH 7.78, 25 degrees C). The redox potential E-1 for the couple flavodoxin semiquinone/fully reduced flavodoxin was deduced from the equilibrium concentration of these species in the presence of hydrogenase and H-2. E-1 was -438 plus or minus 8 mV (pH 7.78, 25 degrees C). Light-absorption and fluorescence spectra of flavodoxin in its three redox states have been recorded. Both the rate and extent of reduction of flavodoxin semiguinone with dithionite were found to depend on pH. An equilibrium between the semiquinone and hydroquinone forms occurred at pH values close to the neutrality, even in the presence of a large excess of dithionite, suggesting an ionization in fully reduced flavodoxin with a pK-a = 6.6. The association constants K for the three FMN redox forms with the apoprotein were deduced from the value of K (K = 8 times 10-7 M-1) measured with oxidized EMN at pH 7.0. Oxidized flavodoxin was found to comproportionate with the fully reduced protein (k-comp = 4.3 times 10-3 M-1 times s-1, pH 9.0, 22 degrees C) and with reduced free FMN (K-comp = 44 M-1 times s-1, pH 8.1, 20 degrees C). Fast oxidation of reduced flavodoxin occurred in the presence of O-2. Slower oxidation of semiquinone was dependent on pH in a drastic way.

[Ultrahigh dose-rate, "flash" irradiation minimizes the side-effects of radiotherapy]. / Radiothérapie « flash ¼ à très haut débit de dose : un moyen d'augmenter l'indice thérapeutique par minimisation des dommages aux tissus sains ?

PURPOSE: Pencil beam scanning and filter free techniques may involve dose-rates considerably higher than those used in conventional external-beam radiotherapy. Our purpose was to investigate normal tissue and tumour responses in vivo to short pulses of radiation. MATERIAL AND METHODS: C57BL/6J mice were exposed to bilateral thorax irradiation using pulsed (at least 40 Gy/s, flash) or conventional dose-rate irradiation (0.03 Gy/s or less) in single dose. Immunohistochemical and histological methods were used to compare early radio-induced apoptosis and the development of lung fibrosis in the two situations. The response of two human (HBCx-12A, HEp-2) tumour xenografts in nude mice and one syngeneic, orthotopic lung carcinoma in C57BL/6J mice (TC-1 Luc+), was monitored in both radiation modes. RESULTS: A 17 Gy conventional irradiation induced pulmonary fibrosis and activation of the TGF-beta cascade in 100% of the animals 24-36 weeks post-treatment, as expected, whereas no animal developed complications below 23 Gy flash irradiation, and a 30 Gy flash irradiation was required to induce the same extent of fibrosis as 17 Gy conventional irradiation. Cutaneous lesions were also reduced in severity. Flash irradiation protected vascular and bronchial smooth muscle cells as well as epithelial cells of bronchi against acute apoptosis as shown by analysis of caspase-3 activation and TUNEL staining. In contrast, the antitumour effectiveness of flash irradiation was maintained and not different from that of conventional irradiation. CONCLUSION: Flash irradiation shifted by a large factor the threshold dose required to initiate lung fibrosis without loss of the antitumour efficiency, suggesting that the method might be used to advantage to minimize the complications of radiotherapy.

Covalent and non-covalent interaction of chymotrypsin with alpha 2-macroglobulin.

The pattern of covalent crosslinking between human alpha 2-macroglobulin (alpha 2M) and chymotrypsin has been investigated by chromatography and polyacrylamide gel electrophoresis in denaturing medium. Reaction with a single mol of chymotrypsin per mol alpha 2M results in the formation of a 95% covalent 1:1 chymotrypsin-alpha 2M complex and in the proteolytic cleavage of both 180 kDa monomers in one alpha 2M subunit. Proteolytic cleavage in the other alpha 2M subunit requires the presence of a second mol of chymotrypsin; part (20%) of the protease in the 2:1 chymotrypsin-alpha 2M complex thus formed appears to be non-covalently bound to the alpha 2M chains. Covalent binding is abolished when the reaction of alpha 2M with the protease is carried out in the presence of hydroxylamine. A single mol of the protease is then able to cleave all four 180 kDa monomers in alpha 2M.

Biological basis for chemo-radiotherapy interactions.

For over 10 years, chemo-radiotherapeutic combinations have been used to treat locally advanced epithelial tumours. The rationale for these combinations relies on spatial cooperation or interaction between modalities. Interactions may take place (i) at the molecular level, with altered DNA repair or modification of the lesions induced by drugs or radiation, (ii) at the cellular level, notably through cytokinetic cooperation arising from differential sensitivity of the various compartments of the cell cycle to the drug or radiation, and (iii) at the tissue level, including reoxygenation, increased drug uptake or inhibition of repopulation or angiogenesis. Some mechanisms underlying interaction of radiation with cis-diammino-platinum (II) (cis-Pt), 5-fluoro-2'-deoxyuridine (5-FU), taxanes and gemcitabine are described. It is shown how various mechanisms including cell synchronisation and reoxygenation concur to paclitaxel-induced radiosensitisation. In the future, specific targeting of tumours, for example, with the epidermal growth factor receptor (EGFR) or angiogenesis inhibitors, should be achieved in order to increase the therapeutic index.

Gamma-radiolysis study of the reductive activation of neocarzinostatin by the carboxyl radical.

The activation of the antitumor protein antibiotic neocarzinostatin (NCS) by the carboxyl radical CO-2, a one-electron donor obtained selectively from gamma-ray irradiation of nitrous oxide-saturated formate buffer, has been investigated in the presence and in the absence of DNA at pH 4.7 and pH 7.0. The reaction of NCS with CO-2 in the absence of DNA is followed by a marked red shift (420----441 nm) and a pronounced increase (X 8.8) of the fluorescence emission corresponding to the naphthalene moiety of the NCS chromophore. The light absorption spectrum shows in parallel a hypochromic change with considerable fine structure throughout the 250-400 nm wavelength range. When DNA is present, the fluorescence intensity at completion of the reaction is slightly reduced (by 5 to 15 per cent) and the maximum emission wavelength shifted to 436-438 nm. However, the bulk rate of reaction is not altered by DNA and is independent of the pH, of the temperature and of the concentration of NCS. The NCS concentration-independence of the reaction rate is consistent with a high intrinsic rate (k greater than 10(8)M-1 . s-1) for the reaction of CO-2 with the NCS chromophore. Complete reduction of the NCS chromophore involves a total of three electron-equivalents. The final product does not react with oxygen, shows no odd electron spin, and is unable to induce DNA strand scission. Its molecular state, however, is fundamentally different when gamma-ray irradiations are performed with DNA. This bears evidence of short-lived one electron or two-electrons reduced intermediates decaying via non-identical routes depending on the presence of the acceptor DNA. Actually, dose-related strand breaks appear in DNA exposed to the action of NCS and CO-2. Some NCS chromophore-DNA covalent adducts are also found. DNA strand breakage by CO-2-activated NCS is correlated with thymine release and is inhibited by a redox-stable intercalating agent. The DNA-nicking process thus bears resemblance to that reported by other authors using mercaptans to initiate reductive activation of the NCS chromophore. However, some spectral differences are observed between the CO-2-reacted and the thiol-treated chromophores. Moreover, thymine release and strand scission in DNA incubated with CO-2 and NCS proceed under anaerobic conditions. It is proposed that the strict oxygen requirement for DNA damage by NCS in the presence of mercaptans is due, at least partly, to competition between oxygen and thiols for reaction with the same primary deoxyribose radical resulting from DNA attack by the reductively activated NCS chromophore.

On the mechanism of reductive activation in the mode of action of some anticancer drugs.

Bioactivation of a number of DNA-specific antitumor drugs depends on oxidoreduction. Bleomycin, neocarzinostatin and anthracycline glycosides are the best known among such drugs in terms of reductive activation processes. Their reduction results in short-lived radical or electrophilic intermediates attacking DNA stereospecifically. The physico-chemical properties of these drugs and the nature of DNA damage are reviewed. Models for DNA-intercalation, electron-donor systems involved in drug metabolisation, and the role of oxygen in radical reactions, are discussed in the light of recent reports.

The seven-stranded beta-barrel structure of apo-neocarzinostatin as compared to the immunoglobulin domain.

The three-dimensional structure of apo-NCS, as revealed by proton NMR, is based on an antiparallel seven-stranded beta-barrel. This fold is frequently encountered in protein structures, especially for immunoglobulin domains. The strands forming the barrel are joined by flexible loops of which three are implicated in the ligand binding site of these proteins. In this paper a preliminary comparison is given with respect to the static and dynamic properties of both the constant beta-barrel and the active loops for apo-NCS and the variable VH domain of an immunoglobulin Fab' fragment.

Visualization of the parietal mass transfer by using immobilized peroxidase.

A new method is developed for direct visualization of the local mass transfer at solid liquid interfaces involving the chemiluminescent oxidation of luminol by H2O2 catalyzed by immobilized peroxidase. At low concentration of H2O2 (C less than 5 x 10(-5) M) this reaction is controlled by diffusion and it is possible to characterize the diffusion convection of H2O2 at each point of the tube as a function of the local hydrodynamic properties. These properties are characterized by pulsed Doppler ultrasound velocimetry. 1) The rate of light emission depends on Re1/3 for the laminar case and decreases when going downstream in accordance with the known theories of diffusion convection along tubes. 2)Downstream to a stenosis, a maximum of light appears which depends on the input Reynolds number in a manner similar to the reattachment point of the flow. This constitutes the first experimental confirmation of calculations on diffusion convection downstream to stenoses. These first experiments show the capacity of the method to detect the local properties of the parietal mass transfer phenomenon as a function of the geometry of the wall and the hydrodynamic properties of the flow.

Radiation-induced crosslinking between poly(deoxyadenylic-deoxythymidylic acid) and tripeptides containing aromatic residues.

OH.-induced covalent peptide-nucleotide adducts have been isolated by reverse-phase chromatography from the enzymic hydrolyzates of gamma-ray irradiated solutions containing double-stranded poly(deoxyadenylic-deoxythymidylic acid) and one of the tripeptides, lysyl-tryptophyl-lysine or lysyl-tyrosyl-lysine. Numerous compounds were formed, resulting presumably from different modes of radical addition. All isomers appeared to have the same general structure peptide-d(ApTpA), based mostly on double-labelling experiments of bases and phosphate groups in DNA. The major adduct fraction obtained from Lys-Trp-Lys and poly(dA-dT) was purified to homogeneity by sequential reverse-phase and ion-exchange chromatography, and characterized spectrally. The pattern of acid and alkaline hydrolysis suggests that thymine is the site of peptide-nucleotide binding in this particular adduct fraction.

Radiation-induced arrest of cells in G2 phase elicits hypersensitivity to DNA double-strand break inducers and an altered pattern of DNA cleavage upon re-irradiation.

PURPOSE: To determine how radiation-induced arrest in G2 affects the response of mammalian cells to a challenging dose of radiation or to antitumour drugs producing DNA double-strand breaks. MATERIALS AND METHODS: V79 fibroblast survival to 5 Gy gamma-rays followed at intervals by 3 Gy irradiation or by contact with an equitoxic dose of neocarzinostatin or etoposide, was measured by clonogenic assays. The pattern of radiation-induced DNA double-strand breaks was determined by filter elution and CFGE (continuous field gel electrophoresis) or PFGE (pulsed-field gel electrophoresis) in G2-arrested cells as well as in nonpre-irradiated asynchronous or synchronized cells. The cell-cycle phase specificity of drug susceptibility was determined in synchronized HeLa cells. RESULTS: Cell kill by radiation-drug combined treatment varied markedly with the time elapsed after priming irradiation. Pre-irradiated, G2-arrested V79 fibroblasts demonstrated excess double-stranded DNA cleavage upon re-irradiation and hypersensitivity to drugs and radiation, although maximum resistance to both neocarzinostatin and etoposide in synchronized HeLa cells was in G2. This effect occurred in the megabase range only, with a peak around 4 Mbp; no change in the electrophoretic migration profile of DNA was observed below 1 Mbp. Moreover, the DNA migration profile and the yield of DNA cleavage in G2-arrested cells were close to those expected from S-phase cells. CONCLUSION: The available data suggest that mechanisms operating within the radiation-induced G2 block promote susceptibility to DNA double-strand break inducers at this stage. It is also proposed that the conformation of DNA in cells accumulated in G2 following irradiation bears resemblance to that for cells in S phase, due either to active repair mechanisms or to inhibition of chromosome disentanglement at the S-G2 transition.

Hyperfast, early cell response to ionizing radiation.

PURPOSE: To determine whether the oscillatory changes of radio-sensitivity which occur within fractions of a second to a few minutes following flash irradiation correlate with an altered incidence of apoptosis, DNA strand breaks or lipid-coupled signalling. MATERIALS AND METHODS: Human tumor cells (SQ-20B, LoVo) or Chinese hamster V79 fibroblasts were exposed to split-dose, pulse irradiation with 3.5 MeV electrons at high dose-rate (12 or 120 Gy x s(-1)) and the effects assessed by clonogenic assays, analysis of DNA cleavage and microscopic observation. RESULTS: The processes underlying oscillatory radiation response were saturable, but did not correlate with an increased incidence of DNA single- or double-strand breaks or apoptosis. N-acetylcysteine and inhibitors of lipid-derived signalling also failed to alter oscillatory response. However, this response did correlate with phenotypic alterations evoking mitotic or delayed cell death. Furthermore, high dose-rate irradiation provided a lower level of instability than protracted gamma-ray irradiation. CONCLUSIONS: It is proposed that the early steps of DNA damage recognition and repair following priming radiation exposure bring about rapid, synchronous remodeling of chromatin, evoking enhanced chromosome damage upon re-irradiation.

Poly(ADP-ribose) polymerase, a major determinant of early cell response to ionizing radiation.

PURPOSE: To determine whether DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase (PARP-1) are involved in eliciting the rapid fluctuations of radiosensitivity that have been observed when cells are exposed to short pulses of ionizing radiation. MATERIALS AND METHODS: The effect of DNA-PK and PARP-1 inhibitors on the survival of cells to split-dose irradiation was investigated using Chinese hamster V79 fibroblasts and human carcinoma SQ-20B cells. The responses of PARP-1 proficient and PARP-1 knockout mouse 3T3 fibroblasts were compared in a similar split-dose assay. RESULTS: Inactivation of DNA-PK by wortmannin potentiated radiation-induced cell kill but it did not alter the oscillatory, W-shaped pattern of early radiation response. In contrast, oscillatory radiation response was abolished by 3-aminobenzamide, a reversible inhibitor of enzymes containing a PARP catalytic domain. The oscillatory response was also lacking in PARP-1 knockout mouse 3T3 fibroblasts. CONCLUSION: The results show that PARP-1 plays a key role in the earliest steps of cell response to ionizing radiation with clonogenic ability or growth as endpoint. It is hypothesized that rapid poly(ADP-ribosylation) of target proteins, or recruitment of repair proteins by activated PARP-1 at the sites of DNA damage, bring about rapid chromatin remodelling that may affect the incidence of chromosomal damage upon re-irradiation.