Role of Mammalian Rad9 in Genomic Stability and Ionizing Radiation Response

Eukaryotic cells have evolved DNA damage response mechanisms utilizing proficient DNA repair and cell cycle checkpoints in order to maintain genomic stability. The Schizosaccharomyces pombe Rad9 gene was initially identified as encoding a cell cycle checkpoint protein. When the mammalian homologue of S. pombe Rad9 was inactivated, however, chromosomal instability was observed even in the absence of DNA damaging agents. Both an increase in chromosome end-to-end associations and telomere loss were observed in cells with inactivated mammalian Rad9. This telomere instability correlated with enhanced S- and G2-phase specific cell killing, delayed kinetics of γ-H2AX foci appearance and disappearance, and reduced chromosomal repair after ionizing radiation (IR) exposure, suggesting that Rad9 plays a role in cell cycle phase specific DNA damage repair. Inactivation of mammalian Rad9 also resulted in decreased homologous recombinational (HR) repair, which occurs predominantly in the S- and G2-phase of the cell cycle. These newly defined functions of mammalian Rad9 are discussed in relation to telomere stability and HR repair as a mechanism for promoting cell survival after IR exposure.     

Integrin Signalling and the Cellular Response to Ionizing Radiation

Cell survival and cycling in mammalian cells are both greatly affected by ionizing radiation and are both strictly controlled by integrated integrin-mediated adhesion to extracellular matrix (ECM) proteins and by binding of growth factors to their cognate receptors. Recent emerging findings show a diverse panel of integrin-dependent signals that are channelled into the regulation and modification of the cellular response to ionizing radiation. Cell adhesion-mediated radioresistance and alteration of DNA damage-induced cell cycle arrest in cells attached to the ECM can be linked to focal adhesion protein signalling. This review summarizes the latest radiobiological and radiooncological findings about integrins and their signal transduction pathways.     

The Mechanical Characteristics of Human Endothelial Cells in Response to Single Ionizing Radiation Doses By Using Micropipette Aspiration Technique

The mechanical properties of living cells are known to be promising biomarkers when investigating the health and functions of the human body. Ionizing irradiation results in vascular injury due to endothelial damage. Thus, the current study objective was to evaluate the influence of continuous radiation doses on the mechanical properties of human umbilical vein endothelial cells (HUVECs), and to identify Young’s modulus (E) and viscoelastic behavior. Single-dose (0, 2, 4, 6, and 8 Gy) radiation was applied to HUVECs using a Cobalt-60 treatment machine in the current vitro irradiation study. Thereafter, a micropipette-aspiration technique was used to measure the elastic modulus of the HUVECs in control and radiation-induced samples. Confocal imaging was then performed for following of the cytoskeletal reorganization of the HUVECs in response to the different radiation doses. Significant enhanced adhesion of the elastic modulus of the HUVECs was observed. The dose value was seen to increase from 0 Gy to 8 Gy. A linear relationship was observed between the 0 Gy and 8 Gy doses following an examination of the dose-response curve for elastic modulus after irradiation. The correlation coefficient was found to be 0.955 and the sensitivity of the dose-elastic modulus to be 7.69 Pa..Gy-1 following analysis of the linear portion of the response curve. Also, a significant increment in stiffness accompanied with the considerable drop in creep compliance curve was detected in radiation-induced groups. Biomechanics-based analysis can provide a platform from which to assess the response of the endothelium to radiation when studying vascular system behavior during the cancer therapy process.     

Dynamics of Tumor Hypoxia in Response to Patupilone and Ionizing Radiation

Tumor hypoxia is one of the most important parameters that determines treatment sensitivity and is mainly due to insufficient tumor angiogenesis. However, the local oxygen concentration in a tumor can also be shifted in response to different treatment modalities such as cytotoxic agents or ionizing radiation. Thus, combined treatment modalities including microtubule stabilizing agents could create an additional challenge for an effective treatment response due to treatment-induced shifts in tumor oxygenation. Tumor hypoxia was probed over a prolonged observation period in response to treatment with different cytotoxic agents, using a non-invasive bioluminescent ODD-Luc reporter system, in which part of the oxygen-dependent degradation (ODD) domain of HIF-1α is fused to luciferase. As demonstrated in vitro, this system not only detects hypoxia at an ambient oxygen concentration of 1% O₂, but also discriminates low oxygen concentrations in the range from 0.2 to 1% O₂. Treatment of A549 lung adenocarcinoma-derived tumor xenografts with the microtubule stabilizing agent patupilone resulted in a prolonged increase in tumor hypoxia, which could be used as marker for its antitumoral treatment response, while irradiation did not induce detectable changes in tumor hypoxia. Furthermore, despite patupilone-induced hypoxia, the potency of ionizing radiation (IR) was not reduced as part of a concomitant or adjuvant combined treatment modality.     

Proteomic Analysis of Trypanosoma cruzi Response to Ionizing Radiation Stress

Trypanosoma cruzi, the causative agent of Chagas disease, is extremely resistant to ionizing radiation, enduring up to 1.5 kGy of gamma rays. Ionizing radiation can damage the DNA molecule both directly, resulting in double-strand breaks, and indirectly, as a consequence of reactive oxygen species production. After a dose of 500 Gy of gamma rays, the parasite genome is fragmented, but the chromosomal bands are restored within 48 hours. Under such conditions, cell growth arrests for up to 120 hours and the parasites resume normal growth after this period. To better understand the parasite response to ionizing radiation, we analyzed the proteome of irradiated (4, 24, and 96 hours after irradiation) and non-irradiated T. cruzi using two-dimensional differential gel electrophoresis followed by mass spectrometry for protein identification. A total of 543 spots were found to be differentially expressed, from which 215 were identified. These identified protein spots represent different isoforms of only 53 proteins. We observed a tendency for overexpression of proteins with molecular weights below predicted, indicating that these may be processed, yielding shorter polypeptides. The presence of shorter protein isoforms after irradiation suggests the occurrence of post-translational modifications and/or processing in response to gamma radiation stress. Our results also indicate that active translation is essential for the recovery of parasites from ionizing radiation damage. This study therefore reveals the peculiar response of T. cruzi to ionizing radiation, raising questions about how this organism can change its protein expression to survive such a harmful stress.     

TIP-1 Translocation onto the Cell Plasma Membrane Is a Molecular Biomarker of Tumor Response to Ionizing Radiation

Background

Tumor response to treatment has been generally assessed with anatomic and functional imaging. Recent development of in vivo molecular and cellular imaging showed promise in time-efficient assessment of the therapeutic efficacy of a prescribed regimen. Currently, the in vivo molecular imaging is limited with shortage of biomarkers and probes with sound biological relevance. We have previously shown in tumor-bearing mice that a hexapeptide (HVGGSSV) demonstrated potentials as a molecular imaging probe to distinguish the tumors responding to ionizing radiation (IR) and/or tyrosine kinase inhibitor treatment from those of non-responding tumors.

Methodology/Principal Findings

In this study we have studied biological basis of the HVGGSSV peptide binding within the irradiated tumors by use of tumor-bearing mice and cultured cancer cells. The results indicated that Tax interacting protein 1 (TIP-1, also known as Tax1BP3) is a molecular target that enables the selective binding of the HVGGSSV peptide within irradiated xenograft tumors. Optical imaging and immunohistochemical staining indicated that a TIP-1 specific antibody demonstrated similar biodistribution as the peptide in tumor-bearing mice. The TIP-1 antibody blocked the peptide from binding within irradiated tumors. Studies on both of human and mouse lung cancer cells showed that the intracellular TIP-1 relocated to the plasma membrane surface within the first few hours after exposure to IR and before the onset of treatment associated apoptosis and cell death. TIP-1 relocation onto the cell surface is associated with the reduced proliferation and the enhanced susceptibility to the subsequent IR treatment.

Conclusions/Significance

This study by use of tumor-bearing mice and cultured cancer cells suggested that imaging of the radiation-inducible TIP-1 translocation onto the cancer cell surface may predict the tumor responsiveness to radiation in a time-efficient manner and thus tailor radiotherapy of cancer.     

Phenotypic Plasticity of Invasive Edge Glioma Stem-like Cells in Response to Ionizing Radiation

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FHL2参与调控MCF-7细胞周期

目的：研究FHL2对细胞周期阻滞的影响。方法：应用pSR-GFP/Neo载体构建FHL2-siRNA干扰载体,转染MCF-7细胞,G418筛选稳定细胞系,通过流式细胞仪检测离子辐射后细胞周期的变化。结果：利用设计的FHL2-siRNA干扰载体能够干扰细胞中FHL2的表达;当离子辐射后,FHL2敲除细胞G2/M期阻滞的程度比野生型细胞显著降低。结论：FHL2参与调控MCF-7细胞离子辐射后细胞周期G2/M期阻滞。     

Ionizing Radiation Impairs T Cell Activation by Affecting Metabolic Reprogramming

Ionizing radiation has a variety of acute and long-lasting adverse effects on the immune system. Whereas measureable effects of radiation on immune cell cytotoxicity and population change have been well studied in human and animal models, little is known about the functional alterations of the surviving immune cells after ionizing radiation. The objective of this study was to delineate the effects of radiation on T cell function by studying the alterations of T cell receptor activation and metabolic changes in activated T cells isolated from previously irradiated animals. Using a global metabolomics profiling approach, for the first time we demonstrate that ionizing radiation impairs metabolic reprogramming of T cell activation, which leads to substantial decreases in the efficiency of key metabolic processes required for activation, such as glucose uptake, glycolysis, and energy metabolism. In-depth understanding of how radiation impacts T cell function highlighting modulation of metabolism during activation is not only a novel approach to investigate the pivotal processes in the shift of T cell homeostasis after radiation, it also may lead to new targets for therapeutic manipulation in the combination of radiotherapy and immune therapy. Given that appreciable effects were observed with as low as 10 cGy, our results also have implications for low dose environmental exposures.     

Human Stefin B Role in Cell's Response to Misfolded Proteins and Autophagy

Alternative functions, apart from cathepsins inhibition, are being discovered for stefin B. Here, we investigate its role in vesicular trafficking and autophagy. Astrocytes isolated from stefin B knock-out (KO) mice exhibited an increased level of protein aggregates scattered throughout the cytoplasm. Addition of stefin B monomers or small oligomers to the cell medium reverted this phenotype, as imaged by confocal microscopy. To monitor the identity of proteins embedded within aggregates in wild type (wt) and KO cells, the insoluble cell lysate fractions were isolated and analyzed by mass spectrometry. Chaperones, tubulins, dyneins, and proteosomal components were detected in the insoluble fraction of wt cells but not in KO aggregates. In contrast, the insoluble fraction of KO cells exhibited increased levels of apolipoprotein E, fibronectin, clusterin, major prion protein, and serpins H1 and I2 and some proteins of lysosomal origin, such as cathepsin D and CD63, relative to wt astrocytes. Analysis of autophagy activity demonstrated that this pathway was less functional in KO astrocytes. In addition, synthetic dosage lethality (SDL) gene interactions analysis in Saccharomyces cerevisiae expressing human stefin B suggests a role in transport of vesicles and vacuoles These activities would contribute, directly or indirectly to completion of autophagy in wt astrocytes and would account for the accumulation of protein aggregates in KO cells, since autophagy is a key pathway for the clearance of intracellular protein aggregates.     

内质网应激与自噬及其交互作用影响内皮细胞凋亡

内质网应激是普遍存在于真核细胞中的应激-防御机制。在内环境稳态遭到破坏的情况下,未折叠蛋白质反应的3条信号通路,分别通过增强蛋白质折叠能力、减少蛋白质生成和促进内质网相关蛋白质降解等途径缓解细胞内压力。同时,也通过多种分子信号机制调控细胞凋亡。自噬是一种生理性的降解机制。通过形成自噬泡并与溶酶体结合摄取并水解胞内受损细胞器和蛋白质等,清除代谢废物,维持细胞正常功能。自噬缺陷或过度激活均可导致细胞凋亡或非程序性死亡。自噬的程度和细胞内压力水平有关。内质网应激通过未折叠蛋白质反应和Ca²⁺浓度变化及其相关分子信号调控自噬。自噬又可反馈性调节内质网应激反应,二者相互作用,在内皮细胞凋亡过程中发挥重要作用。未来内质网应激和自噬可作为药物靶点为内皮相关性疾病提供诊疗策略。     

Vinculin在细胞响应力学刺激中的作用

Vinculin是一种细胞骨架蛋白兼粘着斑组成蛋白,主要分布于细胞 细胞连接处及细胞 细胞外基质(extracellular matrix, ECM)粘着斑部位．Vinculin通过与多种粘着斑蛋白、细胞骨架蛋白及细胞骨架F-肌动蛋白相结合并相互作用,参与细胞的力 化学信号转导,在细胞粘附、伸展、运动、增殖、存活等过程中起重要作用．本文结合本课题组研究工作,在介绍vinculin分子结构的基础上,对其在细胞力 化学信号转导中的作用做一综述．     

Genome-wide Reinforcement of Cohesin Binding at Pre-existing Cohesin Sites in Response to Ionizing Radiation in Human Cells

The cohesin complex plays a central role in genome maintenance by regulation of chromosome segregation in mitosis and DNA damage response (DDR) in other phases of the cell cycle. The ATM/ATR phosphorylates SMC1 and SMC3, two core components of the cohesin complex to regulate checkpoint signaling and DNA repair. In this report, we show that the genome-wide binding of SMC1 and SMC3 after ionizing radiation (IR) is enhanced by reinforcing pre-existing cohesin binding sites in human cancer cells. We demonstrate that ATM and SMC3 phosphorylation at Ser¹⁰⁸³ regulate this process. We also demonstrate that acetylation of SMC3 at Lys¹⁰⁵ and Lys¹⁰⁶ is induced by IR and this induction depends on the acetyltransferase ESCO1 as well as the ATM/ATR kinases. Consistently, both ESCO1 and SMC3 acetylation are required for intra-S phase checkpoint and cellular survival after IR. Although both IR-induced acetylation and phosphorylation of SMC3 are under the control of ATM/ATR, the two forms of modification are independent of each other and both are required to promote reinforcement of SMC3 binding to cohesin sites. Thus, SMC3 modifications is a mechanism for genome-wide reinforcement of cohesin binding in response to DNA damage response in human cells and enhanced cohesion is a downstream event of DDR.     

Some Short-Term Cell Kinetic Effects of Ionizing Radiation On Mouse Bladder Urothelium

Abstract. Early morphological changes and the pattern of reactive proliferation of the hairless mouse urinary bladder urothelium after irradiation are reported. Groups of female hairless mice were anaesthetized with sodium pentobarbital and irradiated over the bladder region with 0, 10, 20 and 30 Gy. Control groups were sham-treated. Short-term cell kinetic changes were monitored using incorporation of tritiated thymidine and flow cytometry. Only minor radiation-induced alterations in the cell kinetic pattern were recorded, and no significant histomorphological changes were seen. However, a marked increase in the thymidine incorporation was seen in the control animals on the first day after anaesthesia. Radiation proctitis induced early deaths in the 30 Gy irradiated animals. the present results are in accordance with commonly accepted radiobiological theories, but not in agreement with results previously published by others     

Some Long-Term Cell Kinetic Effects of Ionizing Radiation On Mouse Bladder Urothelium

Abstract. The cell kinetics of the mouse bladder urothelium were followed with tritiated thymidine pulse labelling and flow cytometry for one year after irradiation with electrons. No perturbations were registered after 10 Gy. Three to four weeks after 20 Gy an elevation of the labelling index with a subsequent return to normal was seen. Flow cytometry revealed some increase in the proportion of octaploid nuclei at the same time. From about six months after irradiation the normal polyploidization decreased. the urothelium turned into a mainly diploid cell population. the proportion of diploid S phase cells also increased. the data give some support to the model hypothesis of reactive proliferation in a 'flexible' tissue, according to Wheldon et al. (1982).     

Pathways that Regulate Autophagy and their Role in Mediating Tumor Response to Treatment

Addenda to:

Rapamycin-Sensitive Pathway Regulates Mitochondrial Membrane Potential, Autophagy and Survival in Irradiated MCF-7 Cells

Paglin S, Lee N-Y, Nakar C, Fitzgerald M, Plotkin J, Deuel B, Hackett N, McMahill M, Sphicas E, Lampen N and Yahalom J.

Cancer Res 2005; 65:11061-70.

In addition to their role in cellular homeostasis, pathways that regulate autophagy affect both tumorigenesis and tumor response to treatment. Therefore, understanding regulation of autophagy in treated cancer cells is relevant to discovery of molecular targets for development of anti-cancer drugs. Our recent report points to radiation-induced inactivation of mTOR pathway as an underlying mechanism of radiation-induced autophagy in the human breast cancer cell line MCF-7. Most importantly, radiation-induced inactivation of this pathway was detrimental to cell survival and was associated with reversal of mitochondrial ATPase activity and mitochondrial hyperpolarization, decreased level of eukaryotic initiation factor 4G (eIF4G) and increased phosphorylation of p53. Future analysis of the interrelationship among these events and the role each of them plays in cell survival following radiation will increase our ability to employ the mTOR pathway in anti-cancer therapy.