Marianne B. Sowa

Lack of evidence for low-LET radiation induced bystander response in normal human fibroblasts and colon carcinoma cells

Purpose: To investigate radiation-induced bystander responses and to determine the role of gap junction intercellular communication and the radiation environment in propagating this response. Materials and methods: We used medium transfer and targeted irradiation to examine radiation-induced bystander effects in primary human fibroblast (AG01522) and human colon carcinoma (RKO36) cells. We examined the effect of variables such as gap junction intercellular communication, linear energy transfer (LET), and the role of the radiation environment in non-targeted responses. Endpoints included clonogenic survival, micronucleus formation and foci formation at histone 2AX over doses ranging from 10–100 cGy. Results: The results showed no evidence of a low-LET radiation-induced bystander response for the endpoints of clonogenic survival and induction of DNA damage. Nor did we see evidence of a high-LET, Fe ion radiation (1 GeV/n) induced bystander effect. However, direct comparison for 3.2 MeV α-particle exposures showed a statistically significant medium transfer bystander effect for this high-LET radiation. Conclusions: From our results, it is evident that there are many confounding factors influencing bystander responses as reported in the literature. Our observations reflect the inherent variability in biological systems and the difficulties in extrapolating from in vitro models to radiation risks in humans.

NON-TARGETED EFFECTS OF IONIZING RADIATION: IMPLICATIONS FOR RISK ASSESSMENT AND THE RADIATION DOSE RESPONSE PROFILE

Radiation risks at low doses remain a hotly debated topic. Recent experimental advances in our understanding of effects occurring in the progeny of irradiated cells, and/or the non-irradiated neighbors of irradiated cells (i.e., non-targeted effects associated with exposure to ionizing radiation), have influenced this debate. The goal of this document is to summarize the current status of this debate and speculate on the potential impact of non-targeted effects on radiation risk assessment and the radiation dose response profile.

Effects of ionizing radiation on cellular structures, induced instability and carcinogenesis

Ionizing radiation is perhaps the most extensively studied human carcinogen. There have been a number of epidemiological studies on human populations exposed to radiation for medical or occupational reasons, as a result of protracted environmental exposures due to radiation accidents, or after atomic bombings. As a result of these studies exposure to ionizing radiation has been unambiguously linked to cancer causation. While cancer induction is the primary concern and the most important somatic effect of exposure to ionizing radiation, potential health risks do not only involve neoplastic diseases but also somatic mutations that might contribute to birth defects and ocular maladies, and heritable mutations that might impact on disease risks in future generations. Consequantly it is important we understand the long-term health risks associated with exposure to ionizing radiation.

Metabolomic Response of Human Skin Tissue to Low Dose Ionizing Radiation

Understanding how human organs respond to ionizing radiation (IR) at a systems biology level and identifying biomarkers for IR exposure at low doses can help provide a scientific basis for establishing radiation protection standards. Little is known regarding the physiological responses to low dose IR at the metabolite level, which represents the end-point of biochemical processes inside cells. Using a full thickness human skin tissue model and GC-MS-based metabolomic analysis, we examined the metabolic perturbations at three time points (3, 24 and 48 h) after exposure to 3, 10 and 200 cGy of X-rays. PLS-DA score plots revealed dose- and time-dependent clustering between sham and irradiated groups. Importantly, delayed metabolic responses were observed at low dose IR. When compared with the high dose at 200 cGy, a comparable number of significantly changed metabolites were detected 48 h after exposure to low doses (3 and 10 cGy) of irradiation. Biochemical pathway analysis showed perturbations to DNA/RNA damage and repair, lipid and energy metabolisms, even at low doses of IR.

Three-dimensional culture conditions lead to decreased radiation induced cytotoxicity in human mammary epithelial cells.

For both targeted and non-targeted exposures, the cellular responses to ionizing radiation have predominantly been measured in two-dimensional monolayer cultures. Although convenient for biochemical analysis, the true interactions in vivo depend upon complex interactions between cells themselves and the surrounding extracellular matrix. This study directly compares the influence of culture conditions on radiation induced cytotoxicity following exposure to low-LET ionizing radiation. Using a three-dimensional (3D) human mammary epithelial tissue model, we have found a protective effect of 3D cell culture on cell survival after irradiation. The initial state of the cells (i.e., 2D versus 3D culture) at the time of irradiation does not alter survival, nor does the presence of extracellular matrix during and after exposure to dose, but long term culture in 3D which offers significant reduction in cytotoxicity at a given dose (e.g. approximately 4-fold increased survival at 5Gy). The cell cycle delay induced following exposure to 2 and 5Gy was almost identical between 2D and 3D culture conditions and cannot account for the observed differences in radiation responses. However the amount of apoptosis following radiation exposure is significantly decreased in 3D culture relative to the 2D monolayer after the same dose. A likely mechanism of the cytoprotective effect afforded by 3D culture conditions is the down regulation of radiation induced apoptosis in 3D structures.

A variable-energy electron microbeam: a unique modality for targeted low-LET radiation.

Abstract Sowa, M. B., Murphy, M. K., Miller, J. H., McDonald, J. C., Strom, D. J. and Kimmel, G. A. A Variable-Energy Electron Microbeam: A Unique Modality for Targeted Low-LET Radiation. Radiat. Res. 164, 695–700 (2005). We have designed and constructed a low-cost, variable-energy low-LET electron microbeam that uses energetic electrons to mimic radiation damage produced by γ and X rays. The microbeam can access lower regions of the LET spectrum, similar to conventional X-ray or 60Co γ-ray sources. The device has two operating modes, as a conventional microbeam targeting single cells or subpopulations of cells or as a pseudo broad-beam source allowing for direct comparison with conventional sources. By varying the incident electron energy, the target cells can be selectively exposed to different parts of the energetic electron tracks, including the track ends.

Annexin A2 Modulates Radiation-Sensitive Transcriptional Programming and Cell Fate

We previously established annexin A2 as a radioresponsive protein associated with anchorage independent growth in murine epidermal cells. In this study, we demonstrate annexin A2 nuclear translocation in human skin organotypic culture and murine epidermal cells after exposure to X radiation (10–200 cGy), supporting a conserved nuclear function for annexin A2. Whole genome expression profiling in the presence and absence of annexin A2 [shRNA] identified fundamentally altered transcriptional programming that changes the radioresponsive transcriptome. Bioinformatics predicted that silencing AnxA2 may enhance cell death responses to stress in association with reduced activation of pro-survival signals such as nuclear factor kappa B. This prediction was validated by demonstrating a significant increase in sensitivity toward tumor necrosis factor alpha-induced cell death in annexin A2 silenced cells, relative to vector controls, associated with reduced nuclear translocation of RelA (p65) following tumor necrosis factor alpha treatment. These observations implicate an annexin A2 niche in cell fate regulation such that AnxA2 protects cells from radiation-induced apoptosis to maintain cellular homeostasis at low-dose radiation.

Quantitative Phosphoproteomics Identifies Filaggrin and other Targets of Ionizing Radiation in a Human Skin Model

Abstract:  Our objective here was to perform a quantitative phosphoproteomic study on a reconstituted human skin tissue to identify low‐ and high‐dose ionizing radiation‐dependent signalling in a complex three‐dimensional setting. Application of an isobaric labelling strategy using sham and three radiation doses (3, 10, 200 cGy) resulted in the identification of 1052 unique phosphopeptides. Statistical analyses identified 176 phosphopeptides showing significant changes in response to radiation and radiation dose. Proteins responsible for maintaining skin structural integrity including keratins and desmosomal proteins (desmoglein, desmoplakin, plakophilin 1, 2 and 3) had altered phosphorylation levels following exposure to both low and high doses of radiation. Altered phosphorylation of multiple sites in profilaggrin linker domains coincided with altered profilaggrin processing suggesting a role for linker phosphorylation in human profilaggrin regulation. These studies demonstrate that the reconstituted human skin system undergoes a coordinated response to both low and high doses of ionizing radiation involving multiple layers of the stratified epithelium that serve to maintain tissue integrity and mitigate effects of radiation exposure.

Cell type-dependent gene transcription profile in a three-dimensional human skin tissue model exposed to low doses of ionizing radiation: implications for medical exposures.

The concern over possible health risks from exposures to low doses of ionizing radiation has been driven largely by the increase in medical exposures, the routine implementation of X‐ray backscatter devices for airport security screening, and, most recently, the nuclear incident in Japan. Because of a paucity of direct epidemiological data at very low doses, cancer risk must be estimated from high dose exposure scenarios. However, there is increasing evidence that low and high dose exposures result in different signaling events and may have different response mechanisms than higher doses. We have examined the radiation‐induced temporal response after exposure to 10 cGy of an in vitro three dimensional (3D) human skin tissue model using microarray‐based transcriptional profiling. Cell type‐specific analysis showed significant changes in gene expression with the levels of >1,400 genes altered in the dermis and >400 genes regulated in the epidermis. The two cell layers rarely exhibited overlapping responses at the mRNA level. Quantitative reverse transcription polymerase chain reaction (qRT‐PCR) measurements validated the microarray data in both regulation direction and value. Key pathways identified relate to cell cycle regulation, immune responses, hypoxia, reactive oxygen signaling, and DNA damage repair. The proliferation status as well as the expression of PCNA was examined in histological samples. We discuss in particular the role of proliferation, emphasizing how the disregulation of cellular signaling in normal tissue may impact progression toward radiation‐induced secondary diseases. Environ. Mol. Mutagen. 2012.

William F. Morgan (1952-2015).

Dr. William Francis Morgan, an eminent scientist and the Director of Radiation Biology and Biophysics in the Biological Sciences Division of the Pacific Northwest National Laboratory, died on Friday evening, November 13, 2015 at the Kadlec Medical Center near his home in Richland, Washington of a pulmonary embolism. He was 62. Dr. Morgan, Bill to many of his friends, was a leading figure in the study of the biological effects of ionizing radiation and had a distinguished career that spanned more than 35 years. Born and raised in Christchurch, New Zealand, Bill received his bachelor’s degree in botany and his master’s and doctoral degrees in cytogenetics from the University of Canterbury. After completing a postdoctoral fellowship at the University of California, San Francisco (UCSF), Bill joined the faculty at the Laboratory of Radiobiology and Environmental Health at UCSF and later obtained a joint appointment at the Lawrence Berkeley National Laboratory (LBNL) in 1995. In 1999, he left California to become Director of the Radiation Oncology Research Laboratory at the University of Maryland School of Medicine in Baltimore. In 2008, Bill moved to Pacific Northwest National Laboratory and took on the role of Director until his untimely death. He held joint appointments in the Department of Radiation Oncology at both Oregon Health Science University and the University of Washington. Dr. Morgan’s research focused on elucidating the long-term biological effects of ionizing radiation, with a significant emphasis on the study of low dose effects on human health. Dr. Morgan’s research was funded through multiple agencies, most notably from the Department of Energy’s Low Dose Radiation Research Program, as well as from the NIH, NASA and other state and federal government agencies. His work had a profound impact at many scientific levels, where he made significant strides in understanding the mechanisms of DNA double-strand break repair, and how deficiencies in repair pathways culminated in cytogenetic abnormalities, mutations, genomic instability and cancer predisposition. He was a champion in the field of radiation-induced genomic instability and pioneered major breakthroughs in our understanding of how the unstable phenotype was initiated and perpetuated. His worked defined genomic instability and linked radiation exposure to the onset and persistence of oxidative stress. Dr. Morgan had a keen interest in nontargeted effects and was instrumental in linking radiation exposure to metabolic abnormalities and other radiomimetic changes in unexposed cells. This work provided the intellectual backdrop that cemented his long-standing interest in low dose effects, and his relentless