Valentina Dini

DNA DSB induction and rejoining in V79 cells irradiated with light ions: a constant field gel electrophoresis study.

Purpose : To study the induction and the time-course of rejoining of DNA double strand breaks (DSB) in V79 cells irradiated with light ions with different linear energy transfer (LET). Materials and methods : V79 cells were irradiated in monolayer with monoenergetic proton, deuteron, helium-3 or helium-4 ion beams, each at two different energy values. Gamma rays were used as reference radiation. DSB have been measured by constant field gel electrophoresis (CFGE). Results : The initial yield depended little on the particle type and LET. The amount of DSB left unrejoined for up to 2 h incubation time could be roughly described by a decreasing exponential function with a final plateau, although more complex functions cannot be excluded. Radiation quality had little effect on the rejoining rate but affected the plateau. The amount of residual DSB after 2h was higher for densely than for sparsely ionizing radiation, and for the same particle was dependent on LET. The corresponding RBE ranged from 1.8 to 6.0. Conclusions : The results support the hypothesis that complex, less reparable DSB are induced in higher proportion by light ions with respect to gamma-rays and that, for the same ion, increasing LET leads to an increase in this proportion.PURPOSE To study the induction and the time-course of rejoining of DNA double strand breaks (DSB) in V79 cells irradiated with light ions with different linear energy transfer (LET). MATERIALS AND METHODS V79 cells were irradiated in monolayer with monoenergetic proton, deuteron, helium-3 or helium-4 ion beams, each at two different energy values. Gamma rays were used as reference radiation. DSB have been measured by constant field gel electrophoresis (CFGE). RESULTS The initial yield depended little on the particle type and LET. The amount of DSB left unrejoined for up to 2 h incubation time could be roughly described by a decreasing exponential function with a final plateau, although more complex functions cannot be excluded. Radiation quality had little effect on the rejoining rate but affected the plateau. The amount of residual DSB after 2 h was higher for densely than for sparsely ionizing radiation, and for the same particle was dependent on LET. The corresponding RBE ranged from 1.8 to 6.0. CONCLUSIONS The results support the hypothesis that complex, less reparable DSB are induced in higher proportion by light ions with respect to gamma-rays and that, for the same ion, increasing LET leads to an increase in this proportion.

Induction and Repair of DNA Double-Strand Breaks in Human Cells: Dephosphorylation of Histone H2AX and its Inhibition by Calyculin A

Abstract Antonelli, F., Belli, M., Cuttone, G., Dini, V., Esposito, G., Simone, G., Sorrentino, E. and Tabocchini, M. A. Induction and Repair of DNA Double-Strand Breaks in Human Cells: Dephosphorylation of Histone H2AX and its Inhibition by Calyculin A. Radiat. Res. 164, 514–517 (2005). Phosphorylation of histone H2AX at serine 139 (γ-H2AX) represents one of the earliest steps in DNA DSB signaling and repair, but the mechanisms of coupling this histone modification to DSB processing remain to be established. In this work, H2AX phosphorylation-dephosphorylation kinetics induced by low doses of γ rays in MRC-5 human fibroblasts was studied. The number of γ-H2AX foci increased rapidly, with the maximum reached 20 min after irradiation. Using calyculin A, a protein phosphatase inhibitor, no significant dephosphorylation was found in this time. At longer times, no further induction of γ-H2AX foci occurred. This indicates that the number of γ-H2AX foci scored at 20 min can be used as representative of the initial number of DSBs. Pulsed-field gel electrophoresis (PFGE) was also used to determine whether calyculin A-mediated inhibition of γ-H2AX dephosphorylation and DSB rejoining are independent phenomena. We found that the maintenance of the phosphate group at Ser 139 in γ-H2AX does not represent an obstacle for DSB rejoining. Preliminary experiments performed with 62 MeV/nucleon carbon ions have shown a longer persistence of γ-H2AX foci with respect to γ rays, consistent with the induction of damage that is more severe and difficult to repair.

DNA DSB induced in human cells by charged particles and gamma rays: Experimental results and theoretical approaches

Purpose:To quantify the role played by radiation track structure and background fragments in modulating DNA fragmentation in human cells exposed to γ-rays and light ions. Materials and methods: Human fibroblasts were exposed in vitro to different doses (in the range from 40 – 200 Gy) of 60Co γ-rays and 0.84 MeV protons (Linear Energy Transfer, LET, in tissue 28.5 keV/μm). The resulting DNA fragments were scored under two electrophoretic conditions, in order to optimize separation in the size ranges 0.023 – 1.0 Mbp and 1.0 – 5.7 Mbp. In parallel, DNA fragmentation was simulated both with a phenomenological approach based on the “generalized broken-stick” model, and with a mechanistic approach based on the PARTRAC (acronym of PARticle TRACk) Monte Carlo code (1.32 MeV photons were used for the simulation of 60Co γ-rays). Results: For both γ-rays and protons, the experimental dose response in the range 0.023 – 5.7 Mbp could be approximated as a straight line, the slope of which provided a yield of (5.3 ± 0.4) • 10−9 Gy−1 bp−1 for γ-rays and (7.1 ± 0.6) • 10−9 Gy−1 bp−1 for protons, leading to a Relative Biological Effectiveness (RBE) of 1.3 ± 0.2. From both theoretical analyses it appeared that, while γ-ray data were consistent with double-strand breaks (DSB) random induction, protons at low doses showed significant deviation from randomness, implying enhanced production of small fragments in the low molecular weight part of the experimental range. The theoretical analysis of fragment production was then extended to ranges where data were not available, i.e. to fragments larger than 5.7 Mbp and smaller than 23 kbp. The main outcome was that small fragments (<23 kbp) are produced almost exclusively via non-random processes, since their number is considerably higher than that produced by a random insertion of DSB. Furthermore, for protons the number of these small fragments is a significant fraction (about 20%) of the total number of fragments; these fragments remain undetected in these experiments. Calculations for 3.3 MeV alpha particle irradiation (for which no experimental data were available) were performed to further investigate the role of fragments smaller than 23 kbp; in this case, besides the non-random character of their production, their number resulted to be at least as much as half of the total number of fragments. Conclusion: Comparison between experimental data and two different theoretical approaches provided further support to the hypothesis of an important role of track structure in modulating DNA damage. According to the theoretical approaches, non-randomness of fragment production was found for proton irradiation for the smaller fragments in the experimental size range and, in a significantly larger extent, for fragments of size less than 23 kbp, both for protons and alpha particles.

DNA fragmentation in V79 cells irradiated with light ions as measured by pulsed-field gel electrophoresis. I. Experimental results.

Purpose : To compare the results on DNA fragmentation induced in Chinese hamster V79 cells by various doses of γ-rays and low-energy protons and helium-4 ions. Materials and methods : V79 cells were irradiated as monolayers with monoenergetic protons and helium-4 ions; γ-rays were used as the reference radiation. DNA double-strand breaks were evaluated by calibrated pulsed-field gel electrophoresis using conditions covering the range 5.7 Mbp-23.1 kbp. Results : The fragment-counting method gave double-strand breaks yields and the relative biological effectiveness higher than those obtained by the fraction of activity released method. The frequency distribution of fragments showed that protons and helium ions induced more fragments below the Mbp region than did γ-rays at the same dose. The distributions for both the irradiated and non-irradiated samples clearly appeared to be non-random. Conclusion : Differences were observed in the yield and spatial correlation, at a molecular size scale characteristic of loop dimensions, of the double-strand breaks induced by γ-rays and by light ions. These effects may have a role in the observed different cell response to these radiations.

Induction and Repair of DNA DSB as Revealed by H2AX Phosphorylation Foci in Human Fibroblasts Exposed to Low- and High-LET Radiation: Relationship with Early and Delayed Reproductive Cell Death.

The spatial distribution of radiation-induced DNA breaks within the cell nucleus depends on radiation quality in terms of energy deposition pattern. It is generally assumed that the higher the radiation linear energy transfer (LET), the greater the DNA damage complexity. Using a combined experimental and theoretical approach, we examined the phosphorylation-dephosphorylation kinetics of radiation-induced γ-H2AX foci, size distribution and 3D focus morphology, and the relationship between DNA damage and cellular end points (i.e., cell killing and lethal mutations) after exposure to gamma rays, protons, carbon ions and alpha particles. Our results showed that the maximum number of foci are reached 30 min postirradiation for all radiation types. However, the number of foci after 0.5 Gy of each radiation type was different with gamma rays, protons, carbon ions and alpha particles inducing 12.64 ± 0.25, 10.11 ± 0.40, 8.84 ± 0.56 and 4.80 ± 0.35 foci, respectively, which indicated a clear influence of the track structure and fluence on the numbers of foci induced after a dose of 0.5 Gy for each radiation type. The γ-H2AX foci persistence was also dependent on radiation quality, i.e., the higher the LET, the longer the foci persisted in the cell nucleus. The γ-H2AX time course was compared with cell killing and lethal mutation and the results highlighted a correlation between cellular end points and the duration of γ-H2AX foci persistence. A model was developed to evaluate the probability that multiple DSBs reside in the same gamma-ray focus and such probability was found to be negligible for doses lower than 1 Gy. Our model provides evidence that the DSBs inside complex foci, such as those induced by alpha particles, are not processed independently or with the same time constant. The combination of experimental, theoretical and simulation data supports the hypothesis of an interdependent processing of closely associated DSBs, possibly associated with a diminished correct repair capability, which affects cell killing and lethal mutation.

DNA fragmentation induced in human fibroblasts by 56Fe ions: Experimental data and monte carlo simulations

Abstract Campa, A., Alloni, D., Antonelli, F., Ballarini, F., Belli, M., Dini, V., Esposito, G., Facoetti, A., Friedland, W., Furusawa, Y., Liotta, M., Ottolenghi, A., Paretzke, H. G., Simone, G., Sorrentino, E. and Tabocchini, M. A. DNA Fragmentation Induced in Human Fibroblasts by 56Fe Ions: Experimental Data and Monte Carlo Simulations. Radiat. Res. 171, 438–445 (2009). We studied the DNA fragmentation induced in human fibroblasts by iron-ion beams of two different energies: 115 MeV/nucleon and 414 MeV/nucleon. Experimental data were obtained in the fragment size range 1–5700 kbp; Monte Carlo simulations were performed with the PARTRAC code; data analysis was also performed through the Generalized Broken Stick (GBS) model. The comparison between experimental and simulated data for the number of fragments produced in two different size ranges, 1–23 kbp and 23–5700 kbp, gives a satisfactory agreement for both radiation qualities. The Monte Carlo simulations also allow the counting of fragments outside the experimental range: The number of fragments smaller than 1 kbp is large for both beams, although with a strong difference between the two cases. As a consequence, we can compute different RBEs depending on the size range considered for the fragment counting. The PARTRAC evaluation takes into account fragments of all sizes, while the evaluation from the experimental data considers only the fragments in the range of 1–5700 kbp. When the PARTRAC evaluation is restricted to this range, the agreement between experimental and computed RBE values is again good. When fragments smaller than 1 kbp are also considered, the RBE increases considerably, since γ rays produce a small number of such fragments. The analysis performed with the GBS model proved to be quite sensitive to showing, with a phenomenological single parameter, variations in double-strand break (DSB) correlation.

DNA Fragmentation Induced in Human Fibroblasts by Accelerated 56Fe Ions of Differing Energies

Abstract Belli, M., Campa, A., Dini, V., Esposito, G., Furusawa, Y., Simone, G., Sorrentino, E. and Tabocchini, M. A. DNA Fragmentation Induced in Human Fibroblasts by Accelerated 56Fe Ions of Differing Energies. Radiat. Res. 165, 713–720 (2006). DNA fragmentation was studied in the fragment size range 0.023–5.7 Mbp after irradiation of human fibroblasts with iron-ion beams of four different energies, i.e., 200 MeV/nucleon, 500 MeV/nucleon, 1 GeV/nucleon and 5 GeV/nucleon, with γ rays used as the reference radiation. The double-strand break (DSB) yield (and thus the RBE for DNA DSB induction) of the four iron-ion beams, which have LETs ranging from 135 to 442 keV/μm, does not vary greatly as a function of LET. As a consequence, the variation of the cross section for DSB induction mainly reflects the variation in LET. However, when the fragmentation spectra were analyzed with a simple theoretical tool that we recently introduced, the results showed that spatially correlated DSBs, which are absent after γ irradiation, increased markedly with LET for the iron-ion beams. This occurred because iron ions produce DNA fragments smaller than 0.75 Mbp with a higher probability than γ rays (a probability that increases with LET). These sizes include those expected from fragmentation of the chromatin loops with Mbp dimensions. This result does not exclude a correlation at distances smaller than the lower size analyzed here, i.e. 23 kbp. Moreover, the DSB correlation is dependent on dose, decreasing when dose increases; this can be explained with the argument that at increasing dose there is an increasing fraction of fragments produced by DSBs caused by separate, uncorrelated tracks.

Influence of PMMA Shielding on DNA Fragmentation Induced in Human Fibroblasts by Iron and Titanium Ions

Abstract Dini, V., Antonelli, F., Belli, M., Campa, A., Esposito, G., Simone, G., Sorrentino, E. and Tabocchini, M. A. Influence of PMMA Shielding on DNA Fragmentation Induced in Human Fibroblasts by Iron and Titanium Ions. Radiat. Res. 164, 577–581 (2005). In the framework of a collaborative project on the influence of the shielding on the biological effectiveness of space radiation, we studied DNA fragmentation induced by 1 GeV/nucleon iron ions and titanium ions with and without a 197-mm-thick polymethylmethacrylate (PMMA) shield in AG1522 human fibroblasts. Pulsed- and constant-field gel electrophoresis were used to analyze DNA fragmentation in the size range 1– 5700 kbp. The results show that, mainly owing to a higher production of small fragments (1–23 kbp), titanium ions are more effective than iron ions at inducing DNA double-strand breaks (DSBs), their RBE being 2.4 and 1.5, respectively. The insertion of a PMMA shield decreases DNA breakage, with shielding protection factors (ratio of the unshielded/shielded cross sections for DSB production) of about 1.6 for iron ions and 2.1 for titanium ions. However, the DSB yield (no. of DSBs per unit mass per unit dose) is almost unaffected by the presence of the shield, and the relative contributions of the fragments in the different size ranges are almost the same with or without shielding. This indicates that, under our conditions, the effect of shielding is mainly to reduce the dose per unit incident fluence, leaving radiation quality practically unaffected.

A laboratory inter-comparison of the importance of serum serotonin levels in the measurement of a range of radiation-induced bystander effects: Overview of study and results presentation

Abstract Purpose: Recent research has suggested that serotonin may play an important role in the expression of radiation-induced bystander effects. Serotonin levels in serum were reported to range from 6–22 μM and to correlate inversely with the magnitude of cellular colony-forming ability in medium transfer bystander assays. That is, high serotonin concentration correlated with a low cloning efficiency in cultures receiving medium derived from irradiated cells. Methods: Because of the potential importance of this observation, the European Unions Non-targeted Effects Integrated Project (NOTE) performed an inter-comparison exercise where serum samples with high and low serotonin levels were distributed to seven laboratories which then performed their own assay to determine the magnitude of the bystander effect. Results: The results provided some support for a role for serotonin in four of the laboratories. Two saw no difference between the samples and one gave inconclusive results. In this summary paper, full data sets are presented from laboratories whose data was inconclusive or insufficient for a full paper. Other data are published in full in the special issue. Conclusion: The data suggest that there may be multiple bystander effects and that the underlying mechanisms may be modulated by both the culture conditions and the intrinsic properties of the cells used in the assay.

Glioblastoma stem cells: radiobiological response to ionising radiations of different qualities

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumour, with very poor prognosis. The high recurrence rate and failure of conventional treatments are expected to be related to the presence of radio-resistant cancer stem cells (CSCs) inside the tumour mass. CSCs can both self-renew and differentiate into the heterogeneous lineages of cancer cells. Recent evidence showed a higher effectiveness of C-ions and protons in inactivating CSCs, suggesting a potential advantage of Hadrontherapy compared with conventional radiotherapy for GBM treatment. To investigate the mechanisms involved in the molecular and cellular responses of CSCs to ionising radiations, two GBM stem cell (GSC) lines, named lines 1 and 83, which were derived from patients with different clinical outcomes and having different metabolic profiles (as shown by NMR spectroscopy), were irradiated with (137)Cs photons and with protons or C-ions of 62 MeV u(-1) in the dose range of 5-40 Gy. The biological effects investigated were: cell death, cell cycle progression, and DNA damage induction and repair. Preliminary results show a different response to ionising radiation between the two GSC lines for the different end points investigated. Further experiments are in progress to consolidate the data and to get more insights on the influence of radiation quality.