Inhibiting Survivin Expression Increases The Radiosensitivity of Human Hepatoma HepG2 Cells to High-LET Radiation

The influence of survivin expression on the radiosensitivity of tumor cells to high linear energy transfer (LET) radiation is investigated. Survivin-specific short-interfering RNA (siRNA) oligonucleotides were synthesized based on the survivin sequence provided by GenBank. Human hepatoma HepG2 cells were transfected with survivin-specific siRNA to inhibit its expressions. It was found that the transfection with surviving-specific siRNA increased the levels of G2/M arrest and the apoptotic rates induced by radiation in HepG2 cells. After exposure to high-LET carbon ions, a reduced clonogenic survival effect was observed in the cells treated with siRNA. These results show that survivin plays a key role in mediating the radioresistance of cells to high-LET radiation.

Early effects of low dose C-12(6+) ion or X-ray irradiation on human peripheral blood lymphocytes

The aim of this study was to estimate the acute effects of low dose C-12(6+) ions or X-ray radiation on human immune function. The human peripheral blood lymphocytes (HPBL) of seven healthy donors were exposed to 0.05 Gy C-12(6+) ions or X-ray radiation and cell responses were measured at 24 h after exposure. The cytotoxic activities of HPBL were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT); the percentages of T and NK cells subsets were detected by flow cytometry; mRNA expression of interleukin (IL)-2, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were examined by real time quantitative RT-PCR (qRT-PCR); and these cytokines protein levels in supematant of cultured cells were assayed by enzyme-linked immunosorbent assays (ELISA). The results showed that the cytotoxic activity of HPBL, mRNA expression of IL-2, IFN-gamma and TNF-alpha in HPBL and their protein levels in supernatant were significantly increased at 24 h after exposure to 0.05 Gy C-12(6+) ions radiation and the effects were stronger than observed for X-ray exposure. However, there was no significant change in the percentage of T and NK cells subsets of HPBL. These results suggested that 0.05 Gy high linear energy transfer (LET) C-12(6+) radiation was a more effective approach to host immune enhancement than that of low LET X-ray. We conclude that cytokines production might be used as sensitive indicators of acute response to LDL (C) 2009 COSPAR. Published by Elsevier Ltd. All rights reserved.

Effects of Ionizing Radiation on Three-Dimensional Human Vessel Models: Differential Effects According to Radiation Quality and Cellular Development

Little is known about the effects of space radiation on the human body. There are a number of potential chronic and acute effects, and one major target for noncarcinogenic effects is the human vasculature. Cellular stress, inflammatory response, and other radiation effects on endothelial cells may affect vascular function. This study was aimed at understanding the effects of space ionizing radiation on the formation and maintenance of capillary-like blood vessels. We used a 3D human vessel model created with human endothelial cells in a gel matrix to assess the effects of low-LET protons and high-LET iron ions. Iron ions were more damaging and caused significant reduction in the length of intact vessels in both developing and mature vessels at a dose of 80 cGy. Protons had no effect on mature vessels up to a dose of 3.2 Gy but did inhibit vessel formation at 80 cGy. Comparison with gamma radiation showed that photons had even less effect, although, as with protons, developing vessels were more sensitive. Apoptosis assays showed that inhibition of vessel development or deterioration of mature vessels was not due to cell death by apoptosis even in the case of iron ions. These are the first data to show the effects of radiation with varying linear energy transfer on a human vessel model. (C) 2011 In Radiation Research Society

Loss of p15/Ink4b accompanies tumorigenesis triggered by complex DNA double-strand breaks

DNA double-strand breaks (DSBs) are the most deleterious lesion inflicted by ionizing radiation. Although DSBs are potentially carcinogenic, it is not clear whether complex DSBs that are refractory to repair are more potently tumorigenic compared with simple breaks that can be rapidly repaired, correctly or incorrectly, by mammalian cells. We previously demonstrated that complex DSBs induced by high-linear energy transfer (LET) Fe ions are repaired slowly and incompletely, whereas those induced by low-LET gamma rays are repaired efficiently by mammalian cells. To determine whether Fe-induced DSBs are more potently tumorigenic than gamma ray-induced breaks, we irradiated 'sensitized' murine astrocytes that were deficient in Ink4a and Arf tumor suppressors and injected the surviving cells subcutaneously into nude mice. Using this model system, we find that Fe ions are potently tumorigenic, generating tumors with significantly higher frequency and shorter latency compared with tumors generated by gamma rays. Tumor formation by Fe-irradiated cells is accompanied by rampant genomic instability and multiple genomic changes, the most interesting of which is loss of the p15/Ink4b tumor suppressor due to deletion of a chromosomal region harboring the CDKN2A and CDKN2B loci. The additional loss of p15/Ink4b in tumors derived from cells that are already deficient in p16/Ink4a bolsters the hypothesis that p15 plays an important role in tumor suppression, especially in the absence of p16. Indeed, we find that reexpression of p15 in tumor-derived cells significantly attenuates the tumorigenic potential of these cells, indicating that p15 loss may be a critical event in tumorigenesis triggered by complex DSBs.

低剂量12C6+辐射对小鼠免疫系统损伤和抗氧化剂氮乙酰半胱氨酸的保护作用研究

实验目的：随着科技的发展，人类活动范围已经逐渐向外太空扩展，对于人类太空探索的最大威胁是太空中的各种粒子辐射。这些辐射包括太阳辐射（质子和电子）和银河辐射（质子占85%，氦离子占14%，重离子占1%）。众所周知，重离子与常规X和γ射线相比有较高的传能线密度（linear energy transfer, LET）和相对生物学效应（relative biological effectiveness, RBE），对机体组织和器官有较强的影响。放射治疗是肿瘤治疗的重要手段之一，由于肿瘤细胞的异质性，其对放、化疗的反应相差悬殊。本研究的目的是： 1评估辐射对健康机体产生的生物学风险； 2研究抗氧化剂氮乙酰半胱氨酸（NAC）对机体辐射损伤的保护作用 3不同肿瘤细胞辐射敏感性的差异。实验方法： 1 X射线或12C6+离子对小鼠进行不同剂量的全身辐射。NAC处理组小鼠在照射前1小时腹腔注射200mg/kg的NAC，对照组注射等体积的生理盐水。照射后不同时间点取样，利用流式细胞仪检测小鼠免疫细胞周期和凋亡情况，单细胞电泳检测淋巴细胞DNA损伤，MTT法（3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide）检测脾脏NK（natural killer，NK）细胞活性，微核法检测淋巴细胞染色体损伤情况，小鼠体内干扰素－γ（Interferon-γ，IFN-γ）由ELISA方法得到，小鼠血清中超氧化物岐化酶(Surperoxide dismutase SOD）由分光光度法测定，并观察胸腺和脾脏指数变化。 2 不同剂量X射线和12C6+离子辐射人肺腺癌细胞H1299和A549，用细胞克隆法检测照射后细胞存活曲线，流式细胞仪检测细胞周期和凋亡，Western-blot 检测A549 细胞P53蛋白表达。 结果： 1小鼠外周血淋巴细胞、胸腺细胞和脾脏淋巴细胞周期随着X射线照射剂量的增大而被阻滞在了G0/G1期，相同剂量的12C6+离子辐射时外周血淋巴细胞周期被阻滞在S期，分次连续X射线照射时，外周血淋巴细胞周期随着累积剂量的增加被阻滞在G2/M期；细胞凋亡比例随着照射剂量的增加而增加。小鼠血清中IFN-γ水平和脾脏中NK细胞活性在重离子照射剂量为0.05Gy时有显著增加，脾脏NK细胞活性随着照射剂量的增加而减弱。 2重离子照射后，小鼠淋巴细胞DNA和染色体的损伤随辐射剂量和照射后时间的延长而加剧。脾脏NK细胞活性在照射后各个时间点减弱，血清中IFN-γ水平和SOD酶活性随着重离子照射剂量的增加而降低。预防性给予NAC，12C6+离子辐射对淋巴细胞DNA和染色体所致损伤，胸腺细胞周期和凋亡，脾脏NK细胞活性，血清中IFN-γ的水平和SOD酶的活性的损伤与盐水组比较均有显著改善。 3 X射线照射对肺腺癌H1299细胞周期和凋亡率未产生明显影响，重离子照射后随着照射剂量的增加细胞周期被阻滞在G2/M期，细胞凋亡率也呈剂量依赖性；X射线和12C6+离子照射A549细胞后，细胞周期均被阻滞在G2/M期，凋亡率剂量依赖性增加。A549细胞P53蛋白的表达水平随着重离子照射剂量的增加而增加。结论： 1重离子辐射造成细胞DNA和染色体损伤随着照射剂量的增加和照射后时间的延长而增加，比X射线辐射损伤复杂和难以修复，产生这种现象的机理为辐射导致活性氧分子簇的产生，细胞因子和与细胞氧化反应有关的酶活性的变化，同时这种损伤对胸腺细胞周期、凋亡和胸腺、脾脏指数以及机体免疫系统都有影响；低剂量重离子辐射（0.05Gy）对小鼠机体的免疫力有刺激作用，机体免疫能力随着照射剂量增加和照射后时间的推移而减弱，不同的免疫器官对辐射的敏感性也不同； 2 200mg/kg 的NAC对辐射所致小鼠免疫系统损伤有很好的保护作用； 3 肺腺癌细胞H1299比同系A549具有较强的辐射敏感性，A549细胞凋亡的增加与P53蛋白表达水平升高有关

Evaluation of distortions in laser welded shipbuilding parts using local-global finite element approach

In recognition of the differences of scale between the welding pool and the heat affected zone along the welding line on one hand, and the overall size of the components being welded on the other, a local-global finite element approach was developed for the evaluation of distortions in laser welded shipbuilding parts. The approach involves the tandem use of a 'local' and a 'global' step. The local step involves a three-dimensional finite element model for the simulation of the laser welding process using the Sysweld finite element code, which takes into account thermal, metallurgical, and mechanical aspects. The simulation of the laser welding process was performed using a non-linear heat transfer analysis, based on a keyhole formation model, and a coupled transient thermomechanical analysis, which takes into account metallurgical transformations using the temperature dependent material properties and the continuous cooling transformation diagram. The size and shape of the keyhole used in the local finite element analysis was evaluated using a keyhole formation model and the Physica finite volume code. The global step involves the transfer of residual plastic strains and the stiffness of the weld obtained from the local model to the global analysis, which then provides the predicted distortions for the whole part. This newly developed methodology was applied to the evaluation of global distortions due to laser welding of stiffeners on a shipbuilding part. The approach has been proved reliable in comparison with experiments and of practical industrial use in terms of computing time and storage.

Inactivation of V79 cells by low-energy protons, deuterons and helium-3 ions

Previous work by ourselves and by others has demonstrated that protons with a linear energy transfer (LET) about 30 V mu m(-1) are more effective at killing cells than doubly charged particles of the same LET. In this work we show that by using deuterons, which have about twice the range of protons with the same LET, it is possible to extend measurements of the RBE of singly charged particles to higher LET (up to 50 keV mu m(-1)). We report the design and use of a new arrangement for irradiating V79 mammalian cells. Cell survival. measurements have been made using protons in the energy range 1.0-3.7 MeV, deuterons in the energy range 0.9-3.4 MeV and He-3(2+) ions in the energy range 3.4-6.9 MeV;. This corresponds to volume-averaged LET (within the cell nucleus) between 10 and 28 keV mu m(-1) for protons, 18-50 keV mu m(-1) for deuterons, and 59-106 keV mu m(-1) for helium ions. Our results show no difference in the effectiveness of protons and deuterons matched for LET. However, for LET above about 30 keV mu m(-1) singly charged particles are more effective at inactivating cells than doubly-charged particles of the same LET and that this difference can be understood in terms of the radial dose distribution around the primary ion track.

A review of dsb induction data for varying quality radiations

Purpose: This short review summarizes the data obtained with various techniques for measuring the yields of double strand breaks (dsb) produced by particle radiations of differing linear energy transfer (LET) in order to obtain relative biological effectiveness (RBE) values.

A study of endonuclease III-sensitive sites in irradiated DNA: detection of alpha-particle-induced oxidative damage

An important difference between chemical agents that induce oxidative damage in DNA and ionizing radiation is that radiation-induced damage is clustered locally on the DNA, Both modelling and experimental studies have predicted the importance of clustering of lesions induced by ionizing radiation and its dependence on radiation quality. With increasing linear energy transfer, it is predicted that complex lesions will be formed within 1-20 bp regions of the DNA, As well as strand breaks, these sites may contain multiple damaged bases, We have compared the yields of single strand breaks (ssb) and double strand breaks (dsb) along with those produced by treatment of irradiated DNA with the enzyme endonuclease III, which recognizes a number of oxidized pyrimidines in DNA and converts them to strand breaks. Plasmid DNA was irradiated under two different scavenging conditions to test the involvement of OH radicals with either Co-60 gamma-rays or alpha-particles from a Pu-238 source. Under low scavenging conditions (10 mM Tris) gamma-irradiation induced 7.1x10(-7) ssb Gy/bp, which increased 3.7-fold to 2.6 x 10(-6) ssb Gy/bp with endo III treatment. In contrast the yields of dsb increased by 4.2-fold from 1.5 x 10(-8) to 6.3 x 10(-8) dsb Gy/bp, This equates to an additional 2.5% of the endo III-sensitive sites being converted to dsb on enzyme treatment. For alpha-particles this increased to 9%. Given that endo III sensitive sites may only constitute similar to 40% of the base lesions induced in DNA, this suggests that up to 6% of the ssb measured in X- and 22% in alpha-particle-irradiated DNA could have damaged bases associated with them contributing to lesion complexity.

Relative Biological Effectiveness Variation Along Monoenergetic and Modulated Bragg Peaks of a 62-MeV Therapeutic Proton Beam: A Preclinical Assessment

iological optimization of proton therapy critically depends on detailed evaluation of relative biological effectiveness (RBE) variations along the Bragg curve. The clinically accepted RBE value of 1.1 is an oversimplification, which disregards the steep rise of linear energy transfer (LET) at the distal end of the spread-out Bragg peak. We observed significant cell killing RBE variations dependent on beam modulation, intrinsic radiosensitivity, and LET in agreement with the LEM predicted values, indicating dose-averaged LET as a suitable parameter for biological effectiveness. Data have also been used to validate a RBE parameterized model.

(223)Ra and other bone-targeting radiopharmaceuticals-the translation of radiation biology into clinical practice

Osseous metastases are a source of significant morbidity for patients with a variety of cancers. Radiotherapy is well established as an effective means of palliating symptoms associated with such metastases. The role of external beam radiotherapy is limited where sites of metastases are numerous and widespread. Low linear energy transfer (LET) radionuclides have been utilized to allow targeted delivery of radiotherapy to disparate sites of disease, with evidence of palliative benefit. More recently, the bone targeting, high LET radionuclide (223)Ra has been shown to not only have a palliative effect but also a survival prolonging effect in metastatic, castration-resistant prostate cancer with bone metastases. This article reviews the different radionuclide-based approaches for targeting bone metastases, with an emphasis on (223)Ra, and key elements of the underlying radiobiology of these that will impact their clinical effectiveness. Consideration is given to the remaining unknowns of both the basic radiobiological and applied clinical effects of (223)Ra as targets for future research.

Alpha particles induce pan-nuclear phosphorylation of H2AX in primary human lymphocytes mediated through ATM

The use of high linear energy transfer radiations in the form of carbon ions in heavy ion beam lines or alpha particles in new radionuclide treatments has increased substantially over the past decade and will continue to do so due to the favourable dose distributions they can offer versus conventional therapies. Previously it has been shown that exposure to heavy ions induces pan-nuclear phosphorylation of several DNA repair proteins such as H2AX and ATM in vitro. Here we describe similar effects of alpha particles on ex vivo irradiated primary human peripheral blood lymphocytes. Following alpha particle irradiation pan-nuclear phosphorylation of H2AX and ATM, but not DNA-PK and 53BP1, was observed throughout the nucleus. Inhibition of ATM, but not DNA-PK, resulted in the loss of pan-nuclear phosphorylation of H2AX in alpha particle irradiated lymphocytes. Pan-nuclear gamma-H2AX signal was rapidly lost over 24h at a much greater rate than foci loss. Surprisingly, pan-nuclear gamma-H2AX intensity was not dependent on the number of alpha particle induced double strand breaks, rather the number of alpha particles which had traversed the cell nucleus. This distinct fluence dependent damage signature of particle radiation is important in both the fields of radioprotection and clinical oncology in determining radionuclide biological dosimetry and may be indicative of patient response to new radionuclide cancer therapies.

Variations in the Processing of DNA Double-Strand Breaks Along 60-MeV Therapeutic Proton Beams

PURPOSE: To investigate the variations in induction and repair of DNA damage along the proton path, after a previous report on the increasing biological effectiveness along clinically modulated 60-MeV proton beams.

METHODS AND MATERIALS: Human skin fibroblast (AG01522) cells were irradiated along a monoenergetic and a modulated spread-out Bragg peak (SOBP) proton beam used for treating ocular melanoma at the Douglas Cyclotron, Clatterbridge Centre for Oncology, Wirral, Liverpool, United Kingdom. The DNA damage response was studied using the 53BP1 foci formation assay. The linear energy transfer (LET) dependence was studied by irradiating the cells at depths corresponding to entrance, proximal, middle, and distal positions of SOBP and the entrance and peak position for the pristine beam.

RESULTS: A significant amount of persistent foci was observed at the distal end of the SOBP, suggesting complex residual DNA double-strand break damage induction corresponding to the highest LET values achievable by modulated proton beams. Unlike the directly irradiated, medium-sharing bystander cells did not show any significant increase in residual foci.

CONCLUSIONS: The DNA damage response along the proton beam path was similar to the response of X rays, confirming the low-LET quality of the proton exposure. However, at the distal end of SOBP our data indicate an increased complexity of DNA lesions and slower repair kinetics. A lack of significant induction of 53BP1 foci in the bystander cells suggests a minor role of cell signaling for DNA damage under these conditions.

Investigating the Implications of a Variable RBE on Proton Dose Fractionation Across a Clinical Pencil Beam Scanned Spread-Out Bragg Peak

Purpose: To investigate the clinical implications of a variable relative biological effectiveness (RBE) on proton dose fractionation. Using acute exposures, the current clinical adoption of a generic, constant cell killing RBE has been shown to underestimate the effect of the sharp increase in linear energy transfer (LET) in the distal regions of the spread-out Bragg peak (SOBP). However, experimental data for the impact of dose fractionation in such scenarios are still limited.

Methods and Materials: Human fibroblasts (AG01522) at 4 key depth positions on a clinical SOBP of maximum energy 219.65 MeV were subjected to various fractionation regimens with an interfraction period of 24 hours at Proton Therapy Center in Prague, Czech Republic. Cell killing RBE variations were measured using standard clonogenic assays and were further validated using Monte Carlo simulations and parameterized using a linear quadratic formalism.

Results: Significant variations in the cell killing RBE for fractionated exposures along the proton dose profile were observed. RBE increased sharply toward the distal position, corresponding to a reduction in cell sparing effectiveness of fractionated proton exposures at higher LET. The effect was more pronounced at smaller doses per fraction. Experimental survival fractions were adequately predicted using a linear quadratic formalism assuming full repair between fractions. Data were also used to validate a parameterized variable RBE model based on linear α parameter response with LET that showed considerable deviations from clinically predicted isoeffective fractionation regimens.

Conclusions: The RBE-weighted absorbed dose calculated using the clinically adopted generic RBE of 1.1 significantly underestimates the biological effective dose from variable RBE, particularly in fractionation regimens with low doses per fraction. Coupled with an increase in effective range in fractionated exposures, our study provides an RBE dataset that can be used by the modeling community for the optimization of fractionated proton therapy.

Contribution to fundamental aspects of biophysics, radiobiology, and modeling of cellular response to low radiation doses

Tese de doutoramento, Engenharia Biomédica e Biofísica, Universidade de Lisboa, Faculdade de Ciências, 2014