Newly Funded Project Descriptions

03-07 Low Dose Basic Research

Identification of Mouse Genetic Susceptibility to Radiation Carcinogenesis
(Jointly funded by NASA and DOE)
Allan Balmain
University of California, San Francisco
San Francisco, CA 94143

Dr. Balmain will use the power of mouse genetics together with novel developments in genomics to identify pathways that control genetic susceptibility to radiation-induced DNA damage and tumor development. This research will identify genetic loci that trigger rapid tumor development of mice after radiation. By identifying somatic genetic alterations in these loci, it will be possible to characterize loci that act as tumor suppressor genes and oncogenes. Dr. Balmain will identify candidate radiation susceptibility genes using a novel haplotyping approach. Using BAC microarrays, it is possible to detect changes in gene copy number in the DNA of radiation-induced lymphoma and to identify genes involved in radiation response in both normal tissues and tumors. Such research will help define the genetic basis for increased or decreased radiation risk in humans. (Jointly funded by NASA and DOE)

An Expression Array Strategy to Identify Mouse Strain-Specific DNA Damage Response Pathways in Mammary Tissue
Co-P.I.s:

Allan Balmain,
UCSF Cancer Research Institute,
San Francisco California

Mary Helen Barcellos-Hoff
Lawrence Berkeley National Laboratory
Berkeley California

This study will expand the use of genome-wide expression array technology to identify candidate genes that modify DNA damage and repair pathways. Drs. Balmain and Barcellos-Hoff will use mouse mammary tissue from animals whose genetic background results in either a high or low levels of susceptibility for radiation-induced mammary cancer. These studies will identify genetic modifiers of radiation response and provide information that will be useful in prediction of risk for radiation-induced mammary cancer.

Multidimensional Analysis of Human Epithelial Cell Response to Low Dose Radiation
Mary Helen Barcellos-Hoff
Lawrence Berkeley National Laboratory
Berkeley, CA 94720

A consortium of LBNL investigators will come together for this project to study the changes in gene expression response to low-dose radiation in a physiologically relevant three-dimensional human mammary epithelial cell model. This system will make it possible to study the signal transduction associated with cell-cell and cell-matrix interactions. The consortium will employ high-throughput analysis of the gene expression patterns to identify underlying molecular signature pathways of radiation responses. The proposed research will determine both quantitative and qualitative differences in patterns of gene expression as a function of dose. The gene families and likely signaling pathways operational in rapid responses will be identified. Persistent changes in gene expression and alterations will be compared in cells hit and in those not hit directly by radiation (bystanders). The research will be extended to studies of the soluble proteins that mediate secondary responses to radiation. This will provide cross-platform comparisons among responses using the same cell model and facilitate the ability to define increased interactions between cells and their microenvironment.

Transgenerational Radiation Genetics: A feasibility Study for the Use of the Japanese Medaka to Investigate Adaptive Responses and Genomic Instability
Joel S. Bedford
Department of Environmental and Radiological Health Sciences
Colorado State University, Fort Collins, Colorado

This proposal will determine if it is possible for either radiation-induced adaptive responses or genomic instability to be transmitted from one generation to the next. Studies will be carried out using a small fish the Japanese Medaka (Oryzias letipes), to test the assumption outlined by UNSCEAR that these phenomena are not transmitted from one generation to the next. The data from this study will provide preliminary information needed to determine if more extensive studies in mammals are necessary to determine the importance of trans-generational transmission of these radiation responsiveness traits. If these traits can be transmitted, the radiation risk in offspring may be increased or decreased and should be considered in future risk assessments.

Is Increased Low-Dose Radiosensitivity Associated with Increased Transgenerational Germline Mutation Radiosensitivity?
David Brenner
Columbia University
New York, NY 10032

Dr. Brenner’s group will investigate whether mice heterozygous for genes that confer somatic radio-sensitivity show an increased low-dose radio-sensitivity for germline mutations. His team will also determine whether mice that are heterozygous for the ATM and BRAC1 genes produce transgenerational germline instability in the offspring of irradiated males. They will use genome-wide screening to study the potential mechanisms of radiation-induced transgenerational mutation effects. They will use a new sensitive single-molecule PCR system to assess germline mutation rates in an expanded tandem repeat locus following very low doses. One hypothesis to be tested is that a primary mechanism for epigenetic transgenerational germline effects results from changes in DNA methylation.

Studies of Bystander effects in 3-D Tissue Systems Using a Low-LET Microbeam
David Brenner
Columbia University
New York, NY 10032

Bystander effects represent natural communication pathways and thus should be studied in three-dimensional tissue systems with normal micro-architectures and microenvironments. Dr. Brenner’s group will conduct a pilot study using the microbeam to evaluate bystander responses induced by protons in three-dimensional artificial skin tissues of both humans and rats. The measured cellular endpoints will reflect potentially damaging or protective effects: apoptosis and terminal cell differentiation will be considered protective while increased cell proliferation will be measured as an endpoint that may reflect increased risk. These biological endpoints will be studied both in cells directly irradiated with protons and in “bystander” cells. This approach makes it possible to look for systematic variations in gene regulation following low-LET exposure in cells which are capable of bystander responses.

MFISH Measurements of Chromosomal Aberrations in Individual Exposed in Utero to Gamma-Ray Doses from 5 to 20 cGy
David J. Brenner
Columbia University
New York, NY

The risk for late effects in individuals exposed to low doses of gamma radiation during in utero development has been a concern. This study will measure the chromosome aberration frequency in the blood lymphocytes of individuals that were identified as having been exposed to low doses of gamma rays in utero while their mothers were working at the nuclear facility at Mayak in Russia. The results can be compared to sex-gender and age-matched controls to determine if an excess aberration frequency can be detected in these individuals. Dr. Brenners' group will also evaluate the sensitivity of this special population by comparing chromosome aberration frequency in those exposed in utero to that observed in young adults exposed to the same radiation doses.

Cellular and Molecular Studies of Radio-Adaptive Responses
Judith Campisi
Lawrence Berkeley National Laboratory
Berkeley, CA 94720

Radiation hormesis is postulated to result from positive feedback stimulation of repair and /or cell defense mechanisms. Dr. Campisi will test the hypothesis that in mammalian cells, radiation-induced responses may differ depending on the cell type, growth and/or differentiation status and genotype of the cell. Using both human fibroblast and mammary epithelial cells, as well as the simple multi-cellular nematode, Caenorhabditis elegans, Dr. Campisi will explore the mechanisms involved in radiation-induced hormesis. These studies will provide a broad cellular and genetic framework on which to better understand the genetic regulation of the radio-adaptive response.

Low Dose Radiation Damage and Radioprotection in the Vertebrate Embryo
William Dynan
Medical College of Georgia
Augusta, GA 30912

This project will identify mechanisms that control sensitivity and resistance to the effects of ionizing radiation in cells of the developing vertebrate embryo using zebra fish, Danio rerio, as a new radiobiological model. In these studies, Dr. Dynan will determine if radiation-induced cell death is responsible for the radiation–induced damage in the central nervous system of the embryo. He will investigate the underlying mechanisms of low dose radiation injury to the embryo. The research will test the hypothesis that DNA-double strand break repair is the primary mechanism of protection against radiation-induced cell death. Finally, using transplanted irradiated cells, the studies will determine whether radiation alterations in development occur in vivo only as a result of direct damage to an irradiated cell, or if it can be induced as a bystander effect.

Genomic Instability Induced by a Bystander Signal
Edwin Goodwin
Los Alamos National Laboratory
Los Alamos, NM 87545

Dr. Goodwin will use high throughput microarray technology to determine the temporal evolution of gene expression occurring in cells experiencing bystander effects. Radiation-induced cytokines will be examined as potential signaling molecules. Microarrays will be used to test the hypothesis that radiation–induced genomic instability is the result of a bystander effect that results in the failure of important genes to switch off in animals or cells with certain genetic backgrounds. This may identify new tumor suppressor genes of particular significance to radiation-induced cancer. Dr. Goodwin will determine if there are interactions between macrophages and lung fibroblasts that produce a threshold dose below which there is no bystander effect. Studies will determine if the threshold for induction of bystander effects depends on the radiation dose-rate. These studies will also determine if interactions between different cell types will alter the length of time that a bystander response can be measured.

Individual Genetic Susceptibility
(Jointly funded by NASA and DOE)
Eric Hall
Columbia University
New York, NY 100032

The identification of radiosensitive subgroups in the human population is of considerable societal importance. To determine if there are interactions between different genes in producing radio-sensitivity, Dr. Hall will compare the sensitivity of ATM knockout and double knockout mice to the sensitivity of wild type mice. Mice that are heterozygous for Mrad9, BRCA2, BRCA1 and ATM will be bred to produce cells with a number of double heterozygotes, such as ATM /het /Mrad9/het and will measure oncogenic transformation in cultured embryo fibroblasts derived from these genetically unique mice to determine radio-sensitivity. Identification of single and combinations of genetic factors that predispose cells to radiation-induced deleterious health effects provides a basis for predicting increased sensitivity in human subgroups. (Jointly funded by NASA and DOE)

A Quantitative Assessment of Bystander Mutagenesis in the Mouse Mammary Gland In vivo
Amy Kronenberg
Lawrence Berkeley National Laboratory
Berkeley, CA 94720

Dr. Kronenberg will use a DR-Green Fluorescent Protein (GFP) mouse model, to quantify gene conversion in bystander cells from the mouse mammary gland in vivo following stromal irradiation of the fat pads. Using innovative quantitative microscopy and flow cytometry, these studies will quantify gene conversion in bystander cells. The bystander effects will be produced by irradiation of the fat pad prior to transplantation with non-exposed DR-GFP mammary epithelial cells. The production of EGFP+ mammary epithelial cells represents gene conversion and provides an indicator of the repair of replication errors via homologous recombinational repair. Dr. Kronenberg, will define the baseline frequencies of gene conversion in bystander cells and assess the role of these changes in mammary cancer. These studies will aid in development of models for breast cancer risk in women exposed to low doses of radiation.

Molecular Characterization of the Role of SOD Genes in Mammalian Cellular Response to Low Dose Ionizing Radiation
Chaun-Yuan Li
Duke University Medical Center
Durham, NC 27710

This project will focus on the role of superoxide dismutase (SOD) genes and oxygen metabolism on the production of stress in the response to low doses of ionizing radiation. This research create cell lines where the genes for SOD are down regulated by RNA interference. The role of these down-regulated genes in the response of cells to radiation will be determined. Dr. Li will evaluate the roles of the SOD genes and oxidative stress on both adaptive responses and bystander effects. Changes in the level of normal oxidative stress in the response of cells to low doses of radiation will be characterized in these studies.

Regulation of NF-kB and MnSOD in Low Dose Radiation-Induced Adaptive Responses in Mouse and Human Skin Cells
Jian Jian Li
City of Hope National Medical Center
Duarte, CA 91010

Dr. Li will determine if low dose ionizing radiation induced adaptive responses in skin cells are mediated by activation of the signaling networks This research will evaluate the networks involving transcription factor NF-kB and the mitochondrial antioxidant protein MnSOD. He will use cells transfected with NF-kB luciferase responder genes to define a dose-response for activation of the NF-kB gene. NF-kB activation and the expression of MnSOD will be evaluated and their role in the adaptive response evaluated. The correlation between NF-kB activation and redox imbalances induced by low doses of radiation will be studied in vivo using irradiated NF-kB reporter mice. These studies will determine if NF-kB is activated by low dose radiation in vivo and determine if its activation is involved in the adaptive response.

Real-Time Molecular Study of Bystander Effects Using Imaging and Nano-Particle Optics
Mohan Natarajan
University of Texas Health Science Center

Dr. Natarajan will develop two novel approaches to study bystander effects. The first approach is to grow endothelial cells on matri-gel and subjecting them to flow that simulates blood flow and creates shear stresses that are similar to those that endothelial cells experience in vivo. This creates a model system with both cell/matrix interaction as well as physical stresses that can modify gene expression. This system will be exposed to low doses of radiation. Direct and bystander effects will be studied as a function of dose. The second approach provides the needed tools to study bystander effects in the model system to detect real time changes in single cells. This approach will develop nanoparticles as unique probes and use single molecule microscopy and spectroscopy as tools to detect cellular changes, with the early focus on radiation-induced changes in TNF-à and NF-êB. This makes it possible to follow both changes in irradiated cells and mediators that are responsible for initiating the signaling to the neighboring cells. Studies will be conducted that define the ligan-receptor interactions on single live-cell surfaces. These approaches further define the biochemical mechanisms, targets and time frame associated with TNF-à and NF-êB mediated bystander effects.

Methods for Deriving Gene and Pathway Specific Dose Response Curves from Gene Expression Experiments Using DNA Microarrays
David O. Nelson
Lawerence Livermore National Laboratory

It is necessary to have well-defined analytical methods to handle the very large data bases generated from microarrays that define the changes in gene expression as a function of multiple variables. Dr. Nelson will determine appropriate models for describing the associations and dependencies of the changes in gene expression levels as a function of dose, dose-rate and time after exposure. Studies will define new method on ways to extend the current analytical clustering techniques on data derived from microarrays using simple experimental designs to more complex designs that produce data where the observations are dependent on a number of variables. The study will develop new analytical and statistical tools for estimating gene and pathway-specific dose-response profiles and will help define relationships between different sets of genes. This project will make it possible to conduct and evaluate the very large data-bases created by the complex large-scale experiments on gene expression needed to understand the relationships between radiation exposures and changes in gene expression.

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Mechanisms(s) of Three-dimensional Intercellular Signaling in Mammary Epithelial Cells in Response to Low Dose, Low-LET Radiation: Implications for the Radiation-Induced Bystander Effects
Lee Opresko
Pacific Northwest National Laboratory
Richland, WA 99352

Dr. Opresko will make use of novel high-speed confocal microscopy in situ to visualize the transmission of radiation-induced bystander signaling in a three-dimensional mammary epithelial system. She will develop tools for real-time monitoring of signals in bystander cells following exposure to low doses of low-LET radiation. Cell survival, growth, cell differentiation, radiation-induced activation of signaling pathways and changes in patterns of gene induction will be determined as a function of both dose and the proportion of cell population irradiated. Bystander-induced changes in cell signaling in this three-dimensional system will be compared to bystander effects in simple tissue culture systems.

Bystander and Adaptive Responses in Tissue Models Exposed to Low Radiation Doses
Kevin Prise
Gray Cancer Institute
Middlesex ENG HA62JR
United Kingdom

Dr. Prise will characterize the three-dimensional urothelial cell model to be used for investigation of mechanisms underlying radiation-induced bystander effect at low radiation doses. He will use a focused soft X-ray microbeam to examine whether the passage of a single electron track can trigger bystander responses in the three dimensional tissue models and compare these responses with the response of isolated urothelial cells and fibroblasts. Research will be conducted to determine if responses are altered by increased or decreased levels of oxidative stress. He will determine the dose-response for bystander effects in a urothelial tissue model and test the hypothesis that altering cellular oxidative stress levels impacts the ability of the cells to produce adaptive responses or bystander effects. The potential protective role of direct and bystander responses on premature cell differentiation and apoptosis will be evaluated.

Low Dose Suppression of Neoplastic Transformation in vitro
(Jointly funded by NASA and DOE)
Leslie Redpath
University of California, Irvine
Irvine, CA 92697

Dr. Redpath will quantify the dose rate dependence of low dose suppression of neoplastic transformation in vitro, for gamma-ray exposure. In addition, he will examine the linear energy transfer (LET) dependence of low-dose suppression of cell transformation by determining the cell transformation frequency following either high-energy proton irradiation or high energy, heavy ion irradiation. These studies will make it possible to compare the effectiveness of these higher LET radiation exposures in reducing cell transformation to that of 60 kVp X-rays and 137Cs gamma rays. Such information is important for NASA as they evaluate the radiation risk associated with long-term space travel. (Jointly funded by NASA and DOE)

Quantification of Repair of Low-Dose-Induced DNA Double-Strand Breaks in Diploid Human Cells
David Schild
Lawrence Berkeley National Laboratory
Berkeley, CA 94720

Dr. Schild will focus on methods to detect the production and repair of DNA double strand breaks induced by very low levels of ionizing radiation. This will be done in immortalized diploid human cell lines. This study will use fluorescent ?H2AX nuclear foci formation and a second co-localizing protein marker to evaluate the frequency of induction and the rate of repair of DNA double strand breaks. These protocols will define efficiency of repair of DNA double strand breaks over the dose range of 0.1-10 cGy.

The characterization of genetic responses to low dose radiation using a genome-wide insertional approach.
Katherine A. Vallis

This project will determine the usefulness of the gene trap mutagenesis screen method for detecting genes with altered levels of expression induced by low doses of radiation. Gene trap mutagenesis has been shown to be a powerful functional genomics technique. It allows scientists to simultaneously identify genes that are responsive to low doses of radiation, create mutations in these genes and conduct a functional analysis of the genes in vivo. This method is very useful in discovery of genes and pathways that are modulated by moderate doses of radiation. Dr. Vallis' study will determine if these techniques can be extended for use in the low dose and dose-rate region.