Francesca Antonelli

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.

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.

The Cosmic Silence experiment: on the putative adaptive role of environmental ionizing radiation.

Previously we reported that yeast and Chinese hamster V79 cells cultured under reduced levels of background environmental ionizing radiation show enhanced susceptibility to damage caused by acute doses of genotoxic agents. Reduction of environmental radiation dose rate was achieved by setting up an underground laboratory at Laboratori Nazionali del Gran Sasso, central Italy. We now report on the extension of our studies to a human cell line. Human lymphoblastoid TK6 cells were maintained under identical in vitro culture conditions for six continuous months, at different environmental ionizing radiation levels. Compared to “reference” environmental radiation conditions, we found that cells cultured in the underground laboratories were more sensitive to acute exposures to radiation, as measured both at the level of DNA damage and oxidative metabolism. Our results are compatible with the hypothesis that ultra-low dose rate ionizing radiation, i.e. environmental radiation, may act as a conditioning agent in the radiation-induced adaptive response.

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.

Age-related effects of X-ray irradiation on mouse hippocampus

Therapeutic irradiation of pediatric and adult patients can profoundly affect adult neurogenesis, and cognitive impairment manifests as a deficit in hippocampal-dependent functions. Age plays a major role in susceptibility to radiation, and younger children are at higher risk of cognitive decay when compared to adults. Cranial irradiation affects hippocampal neurogenesis by induction of DNA damage in neural progenitors, through the disruption of the neurogenic microenvironment, and defective integration of newborn neurons into the neuronal network. Our goal here was to assess cellular and molecular alterations induced by cranial X-ray exposure to low/moderate doses (0.1 and 2 Gy) in the hippocampus of mice irradiated at the postnatal ages of day 10 or week 10, as well as the dependency of these phenomena on age at irradiation. To this aim, changes in the cellular composition of the dentate gyrus, mitochondrial functionality, proteomic profile in the hippocampus, as well as cognitive performance were evaluated by a multidisciplinary approach. Our results suggest the induction of specific alterations in hippocampal neurogenesis, microvascular density and mitochondrial functions, depending on age at irradiation. A better understanding of how irradiation impairs hippocampal neurogenesis at low and moderate doses is crucial to minimize adverse effects of therapeutic irradiation, contributing also to radiation safety regulations.

An Alpha-Particle Irradiator for Radiobiological Research and its Implementation for Bystander Effect Studies

Abstract An experimental system based on an improved version of an existing α-particle irradiator has been developed for radiobiological studies, in particular those investigating bystander effects. It consists of a 20-mm-diameter stainless steel chamber that can be equipped alternatively with 244Cm or 241Am sources of different activities. Mylar®-based petri dishes 56 mm in diameter were specially designed to house adaptors for permeable membrane inserts that reproduce the geometry of commercial cell culture insert companion plates. Characterization of the radiation field at the cell level was performed by experimental measurements and calculations. The average incident LET was about 122 keV/μm for 244Cm and about 125 keV/μm for 241Am. Dose rates at the chosen source-sample distance were 2.8 and 88.6 mGy/min, respectively. These low dose rates are suitable for our planned experiments on low-dose effects. For both sources, the uniformity of the α-particle dose was better than ±7%, and the photon dose calculated at the cell entrance was negligible compared to the α-particle dose. The irradiator is small enough to be inserted into a cell incubator for irradiation under physiological conditions or into a refrigerator to prevent metabolic processes during irradiation. Benchmark experiments using the 241Am source to examine DNA double-strand breaks in directly hit and bystander primary human fibroblasts have shown that the irradiator can be used successfully for bystander effect studies.

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.

Synthetic lethal genetic interactions between Rad54 and PARP-1 in mouse development and oncogenesis

Mutations in DNA repair pathways are frequent in human cancers. Hence, gaining insights into the interaction of DNA repair genes is key to development of novel tumor-specific treatment strategies. In this study, we tested the functional relationship in development and oncogenesis between the homologous recombination (HR) factor Rad54 and Parp-1, a nuclear enzyme that plays a multifunctional role in DNA damage signaling and repair. We introduced single or combined Rad54 and Parp-1 inactivating germline mutations in Ptc1 heterozygous mice, a well-characterized model of medulloblastoma, the most common malignant pediatric brain tumor. Our study reveals that combined inactivation of Rad54 and Parp-1 causes a marked growth delay culminating in perinatallethality, providing for the first time evidence of synthetic lethal interactions between Rad54 and Parp-1 in vivo. Although the double mutation hampered investigation of Rad54 and Parp-1 interactions in cerebellum tumorigenesis, insights were gained by showing accumulation of endogenous DNA damage and increased apoptotic rate in granule cell precursors (GCPs). A network-based approach to detect differential expression of DNA repair genes in the cerebellum revealed perturbation of p53 signaling in Rad54-/-/Parp-1-/-/Ptc1+/-, and MEFs from combined Rad54/Parp-1 mutants showed p53/p21-dependent typical senescent features. These findings help elucidate the genetic interplay between Rad54 and Parp-1 by suggesting that p53/p21-mediated apoptosis and/or senescence may be involved in synthetic lethal interactions occurring during development and inhibition of tumor growth.