Cellular and molecular effects of protons: apoptosis induction and potential implications for cancer therapy.

Due to their ballistic precision, apoptosis induction by protons could be a strategy to specifically eliminate neoplastic cells. To characterize the cellular and molecular effects of these hadrons, we performed dose-response and time-course experiments by exposing different cell lines (PC3, Ca301D, MCF7) to increasing doses of protons and examining them with FACS, RT-PCR, and electron spin resonance (ESR). Irradiation with a dose of 10 Gy of a 26,7 Mev proton beam altered cell structures such as membranes, caused DNA double strand breaks, and significantly increased intracellular levels of hydroxyl ions, are active oxygen species (ROS). This modified the transcriptome of irradiated cells, activated the mitochondrial (intrinsic) pathway of apoptosis, and resulted in cycle arrest at the G2/M boundary. The number of necrotic cells within the irradiated cell population did not significantly increase with respect to the controls. The effects of irradiation with 20 Gy were qualitatively as well as quantitatively similar, but exposure to 40 Gy caused massive necrosis. Similar experiments with photons demonstrated that they induce apoptosis in a significantly lower number of cells and in a temporally delayed manner. These data advance our knowledge on the cellular and molecular effects of proton irradiation and could be useful for improving current hadrontherapy protocols.

Modelled microgravity does not modify the yield of chromosome aberrations induced by high-energy protons in human lymphocytes.

The aim was to evaluate the effect of modelled microgravity on radiation-induced chromosome aberrations (CAs). G0 peripheral blood lymphocytes were exposed to 60 MeV protons or 250 kVp X-rays in the dose range 0-6 Gy, and allowed to repair DNA damage for 24 h under either normal gravity or microgravity modelled by the NASA-designed rotating-wall bioreactor. Cells were then stimulated to proliferate by phytohaemagglutinin (PHA) under normal gravity conditions and prematurely condensed chromosomes were harvested after 48 h. CAs were scored in chromosomes 1 and 2 by fluorescence in-situ hybridization. Proliferation gravisensitivity was examined by cell growth curves and by morphological evaluation of mitogen-induced activation. Cell replication rounds were monitored by bromodeoxyuridine labelling. Modelled microgravity markedly reduced PHA-mediated lymphocyte blastogenesis and cell growth. However, no significant differences between normal gravity and modelled microgravity were found in the dose-response curves for the induction of aberrant cells or total interchromosomal exchange frequency. Rotating-wall bioreactor-based microgravity reproduced space-related alterations of mitogen stimulation in human lymphocytes but did not affect the yield of CAs induced by low-linear energy transfer radiation.

The use of thermoluminescent detectors for measurements of proton dose distribution.

The aim of this work was to investigate the use of TLD-100 detectors in the field of relative dosimetry in proton eye facilities. These dosemeters, of different sizes, were used to measure transverse and longitudinal distributions of 62 MeV unmodulated proton beams at INFN-LNS in Catania. Comparison with other detectors, such as ionisation chambers, GAF and radiographic film, is extensively discussed.

Preliminary results on a dedicated silicon diode detector for proton dosimetry.

The present work reports preliminary measurements on the behaviour of a new p-type stereotactic silicon diode. Hi-pSi, produced by Scanditronix and dedicated to proton dosimetry. Diode response was investigated in low-energy proton beams (26.7 MeV and 12 MeV nominal energy), mainly with attention to stability, linearity, dose rate and energy dependence of the detector response. Three different Hi-pSi diodes of the same type were investigated. The diode response was linear with dose and the standard deviation of repeated readings was less than 2.5%. A marked dependence on dose rate was observed for one of the diodes (a response increase of 47% in the 0.7-11 Gy x min(-1) range). After the dose rate and water to silicon mass collision stopping power ratio correction of the diode response in the depth dose measurements, the difference, at the Bragg peak, with respect to the reference chamber was about 4%, ascribed to poor knowledge of the materials in front of the sensitive volume. The diode response was also nearly independent of linear energy transfer (LET) in the 9.6-21.5 MeV effective energy range.

The late effects of proton irradiation on cell growth, cell cycle arrest and apoptosis in a human melanoma cell line.

The aim of this work is the in vitro study of the late effects of single proton irradiation on HTB63 human melanoma cell growth, cell cycle and cell death. The experimental conditions were focused on analyzing the effects of irradiation on the periphery of tumour that can be, in clinical practice, close to critical organs. Confluent cell monolayers were irradiated with single doses ranging from 1 - 20 Gy, using proton beams having an energy of 22.6 MeV at the target. Antiproliferative effect of protons, cell cycle analysis and initiation of cell death, were followed 48 hours after irradiation. The inhibition of melanoma cell growth was observed, especially after single application of 12 and 16 Gy. Cell cycle analysis and cell viability have shown the G2/M and G1/G0 arrest of irradiated cells correlating with the increase of the applied dose. The flow cytometric analysis has shown presence of apoptotic nuclei. These data demonstrate that irradiation with protons, under the chosen experimental conditions, have significant effects on melanoma cell growth inhibition being dose dependent, G2/M cell cycle arrest and appearance of apoptotic nuclei, even 48 hours after irradiation. The results obtained may help the understanding of the relationship between cell proliferation, death and cell cycle regulation of melanomas after proton irradiation.

Radiochromic film dosimetry of a low energy proton beam.

In this work some dosimetric characteristics of MD-55-2 GafChromic films were studied in a low energy proton beam (21.5 MeV) directly in a water phantom. The nonlinearity of the optical density was quantified by a factor P(lin). A correction factor P(en), that accounts for optical density dependence on the energy, was empirically determined. The effects of detector thickness in depth dose measurements and of the film orientation with respect to beam direction were investigated. The results show that the MD-55-2 films provide dose measurements with the films positioned perpendicularly to the proton beam. A dosimetric formalizm is proposed to determine the dose to water at depth d, with films oriented perpendicularly to the beam axis. This formalism uses a calibration factor of the radiochromic film determined directly on the proton beam at a reference depth in water, and the P(lin) factor, that takes into account the nonlinearity of the calibration curve and the P(en) factor that, in turn takes into account the change of proton beam energy in water. The MD-55-2 films with their high spatial resolution and the quasiwater equivalent material are attractive, positioned perpendicularly along the beam axis, for the absolute dose determination of very small beam sizes and modulated proton beams.