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 However, the technology of the accelerators used to generate X-ray beams is outdated and does not allow to explore the tolerance to novel approaches in terms of dose-rate.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.

[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.

The CEACAM1 tumor suppressor is an ATM and p53-regulated gene required for the induction of cellular senescence by DNA damage.

The p53 tumor-suppressor protein has a key role in the induction of cellular senescence, an important barrier to cancer development. However, very little is known about the physiological mediators of cellular senescence induced by p53. CEACAM1 is an immunoglobulin superfamily member whose expression is frequently lost in human tumors and exhibits tumor-suppressor features in several experimental systems, including Ceacam1 knockout mice. There is currently little understanding of the pathways and mechanisms by which CEACAM1 exerts its tumor-suppressor function. Here we report that CEACAM1 is strongly upregulated during the cellular response to DNA double-strand breaks (DSBs) starting from the lowest doses of DSB inducers used, and that upregulation is mediated by the ataxia telangiectasia mutated (ATM)/p53 pathway. Stable silencing of CEACAM1 showed that CEACAM1 is required for p53-mediated cellular senescence, but not initial cell growth arrest, in response to DNA damage. These findings identify CEACAM1 as a key component of the ATM/p53-mediated cellular response to DNA damage, and as a tumor suppressor mediating cellular senescence downstream of p53.

[New perspectives for radiosensitization in pancreatic carcinoma: a review of mechanisms involved in pancreatic tumorigenesis]. / Mécanismes de carcinogenèse des cancers du pancréas : quelles pistes pour la radiosensibilisation ?

Pancreatic carcinoma is the fifth leading cause of cancer-related mortality. The 5-year overall survival is less than 5 %. This very poor prognosis can be explained both by late diagnosis and by treatment resistance, including resistance to radiation therapy. A better understanding of the pancreatic tumorigenesis and knowledge of the most frequent mutations in pancreatic adenocarcinoma (KRAS, p16, TP53, Smad4) open new perspectives for the development of more effective treatments. This review presents the major genetic and molecular alterations in pancreatic cancer that could be targeted to improve radiosensitization.

[Biological basis of chemo-radiotherapy associations]. / Bases biologiques des associations chimio-radiothérapies.

For many tumour types, chemoradiotherapy is now the standard of care. Concomitant administration seems to improve the results over sequential schedules in many clinical situations. A lot of mechanisms of interaction between drugs and radiation have been described, according to the mechanism of cytotoxicity of the compound. Modifications of radio-induced DNA damages or of their repair have been involved for cisplatin or 5-fluoro-uracil. Synchronisation or cytokinetic cooperation is also usually described, particularly for taxanes. At the tissular level, reoxygenation of the tumour or inhibition of the proliferation could be obtained with different drugs, and today with targeted therapies. The study of these interactions in vitro or in vivo could help the physician in selecting the best drugs to use in conjunction with irradiation and in designing his clinical protocols.

Additive interaction of gefitinib ('Iressa', ZD1839) and ionising radiation in human tumour cells in vitro.

Cultures of human carcinoma A-431, A-549 and HeLa cells were challenged with gamma-rays without or with concomitant exposure to gefitinib, a potent inhibitor of the tyrosine kinase activity of epidermal growth factor receptor (EGFR). The outcome of treatment was determined from cell and colony count, cell cycle progression and DNA double-strand break formation and rejoining. Apoptosis was measured in parallel from hypodiploid DNA and using an annexin V assay. Gefitinib developed a cytostatic effect in all cell lines, with drug sensitivity correlating the level of EGFR expression. A weak cytotoxicity of gefitinib was observed in HeLa cells only, although the drug was unable to induce significant cell cycle redistribution in this cell line. In contrast, substantial G1 block and S-phase depletion was observed in A-431 and A-549 cells exposed to gefitinib. The drug brought about additive to subadditive interaction with radiation with regard to growth inhibition, clonogenic death and induction of apoptosis. Consistently, gefitinib did not hinder the rejoining of radiation-induced DNA double-strand breaks in any cell line. The results demonstrate that gefitinib may elicit cytotoxicity at high concentration, but does not act as a radiosensitiser in vitro in concomitant association with radiation.

[Targeted drugs in radiation therapy]. / Thérapeutiques ciblées et radiotherapie.

New drugs aiming at the development of targeted therapies have been assayed in combination with ionizing radiation over the past few years. The rationale of this concept comes from the fact that the cytotoxic potential of targeted drugs is limited, thus requiring concomitant association with a cytotoxic agent for the eradication of tumor cells. Conversely a low level of cumulative toxicity is expected from targeted drugs. Most targeted drugs act through inhibition of post-translational modifications of proteins, such as dimerization of growth factor receptors, prenylation reactions, or phosphorylation of tyrosine or serine-threonine residues. Many systems involving the proteasome, neoangiogenesis promoters, TGF-beta, cyclooxygenase or the transcription factor NF-kappaB, are currently under investigation in hopes they will allow a control of cell proliferation, apoptosis, cell cycle progression, tumor angiogenesis and inflammation. A few drugs have demonstrated an antitumor potential in particular phenotypes. In most instances, however, radiation-drug interactions proved to be strictly additive in terms of cell growth inhibition or induced cell death. Strong potentiation of the response to radiotherapy is expected to require interaction with DNA repair mechanisms.

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.

[Regulation of cell cycle and radiation-induced cell death]. / Régulation du cycle cellulaire et de la mort cellulaire radio-induite.

Tight control of cell proliferation is mandatory to prevent cancer formation as well as to normal organ development and homeostasis. This occurs through checkpoints that operate in both time and space and are involved in the control of numerous pathways including DNA replication and transcription, cell cycle progression, signal transduction and differentiation. Moreover, evidence has accumulated to show that apoptosis is tightly connected with the regulation of cell cycle progression. In this paper we describe the main pathways that determine checkpoints in the cell cycle and apoptosis. It is also recalled that in solid tumors radiation-induced cell death occurs most frequently through non-apoptotic mechanisms involving oncosis, and mitotic or delayed cell death.

[Clinical aspects of research in radiobiology. Past and future directions]. / Applications cliniques des recherches en radiobiologie. Etat des lieux et perspectives.

Over the last ten years the impact of fundamental radiation biology into daily radiotherapy has been of concern chiefly to fractionation, prediction of radiation response, tumour oxygenation, intrinsic radiosensitivity including genetic approaches, and the determinants of the outcome of chemoradiotherapy combinations. Future goals will rely on sophisticated approaches, based on the progress of molecular and cellular biology and the characterisation of new targets for radiation. Some of these novel advances will be discussed.

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.

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.

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.

[DNA-dependent protein kinase (DNA-PK), a key enzyme in the re-ligation of double-stranded DNA breaks]. / La protéine kinase dépendante de l'ADN (DNA-PK), une enzyme clé de la religation des cassures double-brin de l'ADN.

Repair pathways of DNA are now better defined, and some important findings have been discovered in the last few years. DNA non-homologous end-joining (NEHJ) is a crucial process in the repair of radiation-induced double-strand breaks (DSBs). NHEJ implies at least three steps: the DNA free-ends must get closer, preparation of the free-ends by exonucleases and then a transient hybridisation in a region of DNA with weak homology. DNA-dependent protein kinase (DNA-PK) is the key enzyme in this process. DNA-PK is a nuclear serine/threonine kinase that comprises three components: a catlytic subunit (DNA-PKCS) and two regulatory subunits, DNA-binding proteins, Ku80 and Ku70. The severe combined immunodeficient (scid) mice are deficient in DNA-PKCS: this protein is involved both in DNA repair and in the V(D)J recombination of immunoglobulin and T-cell receptor genes. It is a protein-kinase of the P13-kinase family and which can phosphorylates Ku proteins, p53 and probably some other proteins still unknown. DNA-PK is an important actor of DSBs repair (induced by ionising radiations or by drugs like etoposide), but obviously it is not the only mechanism existing in the cell for this function. Some others, like homologous recombination, seem also to have a great importance for cell survival.

Synthesis and in vitro evaluation of novel 2,6,9-trisubstituted purines acting as cyclin-dependent kinase inhibitors.

Novel C-2, C-6, N-9 trisubstituted purines derived from the olomoucine/roscovitine lead structure were synthesized and evaluated for their ability to inhibit starfish oocyte CDK1/cyclin B, neuronal CDK5/p35 and erk1 kinases in purified extracts. Structure activity relationship studies showed that increased steric bulk at N-9 reduces the inhibitory potential whereas substitution of the aminoethanol C-2 side chain by various groups of different size (methyl, propyl, butyl, phenyl, benzyl) only slightly decreases the activity when compared to (R)-roscovitine. Optimal inhibitory activity against CDK5, CDK1 and CDK2, with IC50 values of 0.16, 0.45 and 0.65 microM, respectively, was obtained with compound 21 containing a (2R)-pyrrolidin-2-yl-methanol substituent at the C-2 and a 3-iodobenzylamino group at the C-6 of the purine. Compound 21 proved cytotoxic against human tumor HeLa cells (LD50-6.7 microM versus 42.7 microM for olomoucine, 24-h contact). Furthermore, unlike olomoucine, compound 21 was effective upon short exposure (LD50= 25.3 microM, 2-h contact). The available data suggest that the affinity for CDKs and the cytotoxic potential of the drugs are inter-related. However, no straightforward cell cycle phase specificity of the cytotoxic response to 21 was observed in synchronized HeLa cells. With the noticeable exception of pronounced lengthening of the S-phase transit by 21 applied during early-S in synchronized HeLa cells, and in striking contrast with earlier reports on studies using plant or echinoderm cells. olomoucilnc and compound 21 were unable to reversibly arrest cell cycle progression in asynchronous growing HeLa cells. Some irreversible hlock in GI and G2 phase occurred at high olomoucine concentration, correlated with induced cell death. Moreover, chmronic exposure to lethal doses of compound 21 resulted in massive nuclear fragmentation, evocative of mitotic catastrophe with minour amounts of apoptosis only. It was also found that olomoucine and compound 21 reversibly block the intracellular uptake of nuicleosides with high efficiency.

Pulse treatment of interphasic HeLa cells with nanomolar doses of docetaxel affects centrosome organization and leads to catastrophic exit of mitosis.

In order to investigate the role of centrosome duplication in mitotic spindle morphogenesis, we designed a 1 hour pulse treatment protocol on synchronized HeLa cells with nanomolar doses of taxoids that might impair centrosome biogenesis but would allow the recovery of normal microtubule (Mt) dynamics before mitosis. We were prompted to use this approach as docetaxel (DOC; taxotereTM), a taxoid known to promote Mt polymerization, was shown to be more cytotoxic when applied during S phase. We show that pulse drug exposure is most efficient in late S and in G2 and results in a marked disorganization of the centrosome in G2, the pericentriolar material (PCM) being dissociated from centrioles. Separation of centrosomes at the G2-M transition is also impaired and mitotic spindle morphogenesis is grossly abnormal: although in most spindles chromosomes align in a metaphase plate, the two centrosomes stay most often unseparated at one pole and most of the NuMA protein accumulates at the other. Interestingly, we find that the centrosomes' ability to duplicate is not abolished as they are still able to trigger parthenogenetic development of frog eggs. Despite spindle asymmetry, the progression through mitosis is not blocked. This results in a catastrophic exit from mitosis, each mitotic cell generating several micronucleated cells linked together by multiple midbodies. Lack of mitotic block appears therefore as the prime cause of cell lethality. These experiments suggest that NuMA redistribution at the onset of mitosis depends upon the correct redistribution of PCM between centriole pairs. They also indicate that the presence of aberrant spindle poles does not alert the surveillance mechanism controlling the exit of mitosis.

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.