James E. Haber
Brandeis University
Analysis of Gene Targeting and Nonhomologous End-Joining
Nonhomologous end-joining (NHEJ) is the primary response to repair double-strand breaks in most organisms, including humans. However, little is known of the mechanisms of this important process and no comprehensive survey has been made of the gene products that participate in NHEJ repair. This research is built around a screen for mutants that affect these gene products and a phenotypic characterization of those mutants.
New mutants will be isolated to more fully describe the number of components necessary for nonhomologous end-joining and related processes. The phenotypes of these mutants will be analyzed for their ability to repair DNA double strand breaks and for two other cellular processes affected by DNA double strand breaks: the formation and maintenance of telomeres and the G2/M checkpoint response. The mechanisms and genetic requirements for both homologous and hit-and-run transformation events will be analyzed. Specific attention will be paid to two sets of proteins that play complicated roles in nonhomologous DNA repair events: the Ku homologues Hdf1/yKu80 and the trio of Mre1/Rad50/Xrs2, proteins that are highly conserved from yeast to humans.
David A. Boothman
Case Western Reserve University, Cleveland OH
Bruce J. Aronow - Co-Principal Investigator
University of Cincinnati
Nuclear ApoJ: A Low Dose Radiation Inducible Regulator of Cell Death
The complex interactions between apoptosis, genomic instability and carcinogenesis induced by low dose ionizing radiation (IR) are poorly understood. Since the ability of any tissue to withstand IR depends on both immediate and delayed stress responses, by both repair and selective programmed cell death, we identified gene products induced by low doses of IR. We identified apoJ (designated xip8/XIP8) as a gene product strongly induced by low dose IR and found that a particular form of the apoJ protein accumulated, in a p53-dependent manner, in the nucleus of irradiated cells. More importantly, this nuclear protein binds Ku70, a critical DNA repair protein subunit; we captured the carboxy-terminal portion of apoJ in 19 out of 25 clones via a yeast two-hybrid screen that used Ku70 as "bait".
Our hypothesis is that apoJ serves as a critical monitor of genomic damage. Nuclear apoJ interacts with the Ku70/Ku80 DNA end-binding protein, which is needed for DNA-PK dependent, nonhomologous DNA double strand break (DSB) repair. ApoJ sequesters Ku70/Ku80, prevents repair, and eliminates genetically unstable cells via apoptosis. Elimination of apoJ will prevent apoptosis, but consequentially increase mutagenesis and tumor formation.
The Specific Aims are as follows:
David J. Brenner
Columbia University
Genetic, Cytogenetic, and Oncogenic Effects of Low doses of Low-Energy (<50 keV) Xrays
Current standards for occupational and residential exposure to ionizing radiation depend crucially on extrapolation of epidemiological data to low dose and low dose rates. While much emphasis is rightly placed on extrapolation to low doses, the uncertainty in the extrapolation to low dose rates involves comparable levels of uncertainty. The two methods currently possible are: a) use of laboratory animal and cellular data, or b) comparing risk estimates generated from the A-bomb survivors (acute exposure) with those from the tuberculosis cohorts that were subjected to repeated fluoroscopies over extended periods of time. However, while the A-bomb survivors were exposed to high energy gamma rays (more than half the dose from photons above 200keV), most of the fluoroscopy dose came from photons with energy less than 30 keV. Thus, it is critical to understand the low-dose relative biological effectiveness of the low-energy x-rays used in the fluoroscopies, in order to make realistic inferences about the effects of prolonged low dose exposure on human populations.
From a public health perspective, screening mammograms are typically given with low doses of 20-25 kVp x rays. Given the increasing emphasis on mammographic screening for breast cancer, it is of societal importance to provide realistic risk estimates for breast cancer induction from mammographic x-rays, in keeping with a recent American Cancer Society recommendation that "the stated 'risks' from mammography should be further quantified."
This project addresses estimation of breast cancer risk from mammography, and in a more general sense, it is concerned with the effects of protracted or highly fractionated exposures to ionizing radiations over long periods of time. Mammograms expose patients to radiation with photon energies much lower than that to which the A-bomb survivors were exposed. Photon energies of concern with mammography are in the range of ~10-26 keV, whereas A-bomb survivors experienced exposures to photons above 150 keV. Since RBE likely depends on photon energy, i.e., increases with decreasing photon energy, mammography risks based on A-bomb survivor data may be too low. Of relevance, results obtained from studies of TB fluoroscopy cohorts where woman received multiple low energy X-rays (most photons <30 keV) over several years have either indicated that, relative to A-bomb survivors, breast cancer risk showed little or no effect of dose rate, or, in another study, a possible protraction-related decrease in risk. Thus, the issue about the risks from low, protracted exposures to ionizing radiations in the human remain unsettled. The objective of this project is to obtain chromosome aberration and transformation data from high energy 137Cs and a range of low energy synchrotron radiation beams. These data could allow a more precise determination of RBE as a function of photon energy that will be used in a reevaluation of risks. Further, the project could affect the so called dose-rate effective factor or DREF, which is the factor by which risk estimates appropriate for a high dose rate should be reduced for application to prolonged exposures.
Charles R. Geard
Columbia University
Site Specific Microbeam Irradiation: Defining a Bystander Effect
Research objectives are directed toward understanding the mechanistic bases for the recognition and processing of radiation induced lesions, with a particular emphasis on their impact in human health. Nuclear DNA changes significantly impact human health with clear relationships to cell death, mutation and oncogenic change. There is uncertainty however as to the potential contributions of stresses induced in non-nuclear targets to long term deleterious effects. It is clear that molecular interplay between membrane, cytoplasm and nucleus contributes to damage recognition, signaling and response pathways. An understanding of the role of cellular or non-cellular components in radiation responses can efficiently be obtained by use of a microbeam. A directed beam of moderate LET charged particles can initiate significant levels of molecular disruption in defined microvolumes.
This collaborative application will use defined fluences (down to 1, equivalent to ~0.002Gy) of keV/ m protons to irradiate cells or the cellular microenvironment or both. Cells proximal to extracellular irradiation (bystanders) may be stressed by radiolysis generated reactive oxygen species. We will ask the question: Are cells irradiated at low dose/fluences hypersensitive? Can non-cellular irradiation initiate a biological response? And, can non-cellular irradiation potentiate or abrogate the cellular trans-nuclear radiation response? We will examine chromosomal clastogenic responses, cell cycle delay and p53 expression and localization in sub-project 1, cell survival, mutagenesis and mutation spectra in sub-project 2 and survival and oncogenic transformation in sub-project 3. Precise site specific microbeam irradiation and relevant biological endpoints will help to define responsiveness at low doses with particular emphasis on the contribution of non targeted bystander cells.
This group will set out to distinguish between the hypotheses that the bystander effect is due to effects secondary to traversal of cell nucleus being traversed by an alpha particle resulting in the expression of effects in other non-traversed cells versus effects due to the creation of ROIs in the medium surrounding the cells leading to effects in cells affected directly by the ROIs. They also introduce the intriguing idea that some component of the bystander effect may be due to hypothesis proposed by Joiner and others that cells may exhibit "induced" radioresistance and may be paradoxically sensitive to radiation in the low dose region, which is after all the region at which the so-called "bystander" effect occurs. The experiments will be done using microbeam irradiation with precisely determined numbers of proptons and cell nuclei or cytoplasms. A number of endpoints will be examined looking at cell survival, cell cycle delays, p53 responses and mutagenicity. The endpoints should allow distinction between the nuclear and non-nuclear responses of cells which has particular relevance to the study of the bystander effect.
John B. Little
Harvard University
Effects of Low-Dose Alpha Irradiation in Human Cells: The Role of Induced Genes and The Bystander Effect
The overall objectives of this research program are to better define the effects of low-dose, low dose-rate irradiation in human cells, with a particular emphasis on non-targeted effects; that is, effects occurring in cells that themselves receive no direct radiation exposure. These included the progeny of the irradiated cell, or cells not traversed by an ionizing particle. In the present project, we will focus on the induction of gene expression by very low doses of alpha radiation in bystander cells, cells in the irradiated population not actually traversed by an alpha particle. Initially, we will study two types of genes, those involved in cell cycle regulation and those involved in DNA damage recognition and repair. The role of factors such as gap junction-mediated intercellular communication and oxidative metabolism in the bystander effect will be examined. We will utilize normal human diploid fibroblasts in culture as an experimental model though, depending upon the initial results, we may extend these studies to other cell lines including radiosensitive disorders and knock-out mouse strains for genes involved in the response to radiation.
Priscilla K. Cooper
Lawrence Berkeley National Laboratory
DNA Damage Responses to Low Dose Ionizing Radiation
The long-term consequences for genetic risk from exposure to intermittent or continuous low dose ionizing radiation are unclear, but clearly of concern to the public. Since measurement of DNA damage is experimentally difficult at doses typical of these exposures, extrapolation of results from higher doses is required. The possibility of a non-linear relationship between dose and effect at very low doses, with either less or more risk than anticipated from linearity, must therefore be considered. A further complication is the probability of genetic heterogeneity within populations with respect to susceptibility to genetic damage from low dose exposures. In order to fully understand the effects of low dose radiation on cells and organisms, it is necessary to identify and characterize the DNA repair mechanisms that repair this damage. Even if many individuals efficiently repair most or all of the DNA damage induced by low doses of radiation or chemicals, there are almost certainly individuals in the population who have reduced repair responses due to subtle alterations in particular DNA damage response genes. In order to understand the effects of low dose radiation on the general population, and particularly to identify susceptible individuals, it is thus necessary to know which different DNA repair pathways function to repair this damage, to understand the way in which this damage is repaired and whether the repair is error-free or error-prone, and to identify and characterize the genes involved.
DNA double-strand breaks (DSBs) constitute the most important primary damage produced by ionizing radiation and are presumed to account for its high lethality, clastogenicity, and predisposition for malignant transformation. They are also formed as initiating steps in normal recombination events, including meiotic exchange and rearrangements to generate immunoglobulin diversity. The correct repair of these lesions, whether endogenously produced or induced by ionizing radiation, is essential for maintenance of genomic integrity. Their failed or inaccurate repair can result in chromosomal translocations, inversions, or loss of genetic information through deletions or chromosomal fragmentation. It is thus crucial to understand the mechanisms of double-strand break repair and the factors that contribute to incorrect repair. In mammalian cells DNA double-strand break repair is the least well understood of all the major DNA repair pathways. Repair of DSBs is distinguished by its requirement for some form of recombination. Illegitimate recombination or end-joining appears to be the predominant mechanism used by mammalian cells both for double-strand break rejoining and for integration of foreign DNA. However, homologous recombination mechanisms do exist that operate in targeted integration events, and mammalian homologs of yeast recombinational repair genes have recently been identified. Although their participation in DSB repair has yet to be directly established, there is increasing evidence for a contribution of homologous recombination to cellular radioresistance during late S/G2 when sister chromatids are present. Investigation of both non-homologous end-joining and homologous recombination pathways for repair of DSBs is included in this project. The very recent discovery that a human genetic disease (Nijmegen breakage syndrome) characterized by cancer-proneness and genomic instability is the result of loss of one protein component of a complex involved in double-strand break repair in yeast and mammalian cells is a dramatic illustration of the importance of these studies for understanding risks to human populations from exposure to low dose ionizing radiation.
Despite their obvious biological importance, DSBs are numerically vastly overshadowed by a large spectrum of other lesions produced in DNA by ionizing radiation. These include single-strand breaks, a large variety of base and sugar damages, DNA interstrand crosslinks, and DNA-protein crosslinks. The hydroxyl radical is the primary reactive species responsible for generation of DNA damage by ionizing radiation under biological conditions, and it and other reactive oxygen species (ROS) are also produced during normal cellular metabolism, as well as in metabolic activation of many carcinogens. Indeed, the DNA damage produced by ionizing radiation and by cellular oxidation is qualitatively similar, and free oxygen radicals have been implicated in degenerative and inflammatory disorders, aging, Alzheimer's disease, and cancer. Although characterization of the biological effects of base damage induced by ionizing radiation or other oxidative damaging agents in mammalian cells has been hampered by lack of available mutants until now, studies with model systems suggest that several of these are potentially lethal or mutagenic if left unrepaired. The process of base excision repair, in which particular damaged bases are removed by specific glycosylases followed by strand scission at the resulting apurinic/apyrimidinic (AP) site, is thought to be the major pathway responsible for repair of most ionizing radiation-induced base lesions and single-strand breaks. Base excision repair (BER) of oxidative lesions has recently been shown by us to occur preferentially in transcriptionally active DNA in human cells, and this transcription-coupled repair process has been found to be specifically defective in a human disease (Cockayne syndrome) characterized by retardation and extreme defects in growth and development. Since both oxidatively damaged bases and AP sites (the ultimate non-instructive lesion) are generated spontaneously in the cell at a significant rate as well as by ionizing radiation, efficient repair mechanisms for this class of damage would seem to be a prerequisite for genomic stability. In agreement with this prediction, our very recent work has found that defects in four different genes that give rise to Cockayne syndrome result in extremely high mutagenesis at an 8-oxoG due to failed repair of this miscoding oxidative lesion. Significantly, several lines of evidence suggest that the relevant repair process is damage-inducible. Thus, elucidation of the mechanism of transcription-coupled repair of oxidative damage and identification of all the genes required for it is also of major importance for understanding human genetic risk from low dose exposures to ionizing radiation.
In summary, this research program presents an integrated approach designed to identify and characterize genes and gene products involved in cellular responses to DNA damage induced by ionizing radiation and to elucidate their role in determining susceptibility. Emphasis is on understanding (a) multiple pathways for DNA double-strand break rejoining that are likely to have different probabilities of error and thus different consequences for maintenance of genomic integrity, and (b) a novel pathway for repairing oxidative lesions in active parts of the genome, defects in which affect mutability. The approaches presented include genetic, molecular and biochemical analyses of these repair pathways in mammalian cells, including analysis of their inducibility by damage. The required genes to be studied are all expected to belong to the class of genes in which mutations or polymorphisms that subtly affect function of the encoded protein might significantly affect susceptibility to genetic alteration by low dose exposures.
Mary Helen Barcellos-Hoff
Lawrence Berkeley National Laboratory
Mechanisms of Tissue Response to Low Dose Ionizing Radiation Exposures: Bioinformatic Tools for Multiparametric Image Analysis
A complex network of physical and chemical signals converge in a coordinated and highly orchestrated fashion to govern form and function in the human body. We hypothesize that tissue response to radiation, and hence risk, is a composite of genetic damage, cell loss and induced gene products. Our studies using highly sensitive and precise cell biology techniques to microscopically map complex patterns of radiation-induced gene expression demonstrate that tissue response to ionizing radiation is rapid, global, tissue specific and sensitive to doses as little as 0.1 Gy. The significance of these biological markers to radiation susceptibility and use in defining dose-response relationships have not been generally appraised. This proposal will examine the dose, tissue and temporal dependence of radiation induced proteins, identify the underlying mechanisms by which tissue responses are implemented and evaluate their relevance in terms of impact on human cancer risk. Murine mammary glands from transgenic and various genetic backgrounds and 3-dimensional models of human mammary epithelial cells will be used to identify common cross-species features of radiation response. We will use digital fluorescence microscopy to map and quantify radiation effects in these models. In order to increase the sensitivity, throughput and accuracy of image analysis, and the subsequent quantitative and statistical analysis, we will develop a bio-informatics framework. Image acquisition, annotation, analysis will be integrated with an image database that registers information about multiple targets, positional references and morphological features. This multidisciplinary and innovative approach will enable construction of phenotype databases necessary to identify critical biological responses to low dose radiation exposure that can be used in computational models of radiation risk.
Larry Thompson
Lawrence Livermore National Laboratory
Assessing Biological Function of DNA-Damage Response Genes
The broad objective of this project is to understand the relative contributions of the individual DNA repair and DNA-damage response pathways to the recovery of mammalian cells from exposure to ionizing radiation (IR). These studies will emphasize cellular responses at low doses in the range of 0-1 Gy for which there is little or no cell killing. In spite of the advances made in discovering many DNA repair genes over the last decade, our current knowledge of these pathways is still quite fragmentary. The goal of this project to provide a better understanding of the molecular mechanisms in areas that are poorly defined by identifying the biological contribution of individual DNA repair genes to cellular recovery from IR exposure. Double-strand breaks (DSBs) are commonly accepted as being the biologically most critical lesion produced by IR, resulting in mutation, chromosomal rearrangements, and cell killing. DSBs are normally repaired with high efficiency unless the cellular repair systems are damaged through mutation or become saturated by excessive levels of damage. However, even at very low doses the repair processes are expected to be imperfect, resulting in genetic damage.
Recently the involvement of a nonhomologous end-joining pathway that eliminates DSBs has been found to be of major importance in mammalian cells although the molecular details are not yet understood. The presence of a second DSB removal pathway, homologous recombinational repair (HRR) pathway, which is analogous to that operating in the yeast Saccharomyces cerevisiae, is just beginning to be documented (reviewed by Thompson and Schild, Biochimie, in press). The relative importance of HRR in repairing DSBs, as well as other forms of IR damage such as base damage, is presently unknown, although the phenotypes of the few available mutants suggest the involvement of this pathway for diverse types of DNA damage. Our first objective is to determine the quantitative importance of HRR in the cellular response to both spontaneous and IR-induced damage. Our initial studies have indicated that HRR plays a much more significant role than has been assumed. In addition to producing strand breaks, IR produces a great variety of base damages (such as thymine glycol and 8-oxy-7, 8-dihydro-guanine) that are acted on by the base-excision repair pathway. The relative biological consequences of base damage resulting from IR exposure, as compared with strand breaks, is poorly understood in mammalian cells. As a second objective, we will assess the contribution of base-excision repair in preventing cell killing and mutagenesis. In addition to DNA repair processes per se, cells exhibit coordinated molecular responses to DNA damage. These responses modulate cell-cycle progression through checkpoint functions that monitor the integrity of the DNA molecules throughout the cycle. We also seek to understand better the nature of these checkpoints and how they are coupled with the DNA repair machinery.
There are three integrated experimental goals. First, we will construct knockout mutations in hamster CHO cells for genes belonging to three major components of DNA repair and damage-response: homologous recombinational repair (HRR), base excision repair, and cell-cycle checkpoint functions. Second, we will characterize these mutants in response to DNA damage in terms of cell survival, chromosomal aberrations, cell-cycle sensitivity, damage and repair (e.g. single-cell DNA comet assay), and, for HRR, Rad51-nuclear focus formation and recombinational repair. Third, we will apply the mutants to evaluate potential dysfunction associated with common human repair-gene alleles that may contribute to cancer risk. CHO cells are uniquely suited to this effort because of their genetic stability and versatility. This approach capitalizes on the abundance of candidate DNA repair gene sequences now in genomic databases. Knockout mutants will be characterized under standardized conditions that allow precise quantification of the contribution of each gene and pathway to low-dose IR exposure. This aspect of the work will overcome current limitations of comparing data from mutants in different cell lines and laboratories. Initially, genes in HRR, particularly RAD51-family genes, will be emphasized because of the limited understanding of this pathway. International distribution of the mutants to other laboratories will greatly augment the mutants' value in contributing toward a detailed understanding of molecular mechanisms.
Andrew Wyrobek
Lawrence Livermore National Laboratory
Molecular Mechanisms and Cellular Consequences of Low-Dose Exposures to DNA-Damaging Agents
Although it is well established that high-dose exposures to physical and chemical DNA damaging agents can lead to diverse tissue pathologies and diseases including cancers, there is little molecular understanding of the early cellular responses, especially for low-dose exposures. Our hypothesis is that genes exhibiting altered expression within the first few hours after low-dose exposure are pivotal in determining cellular damage response and that cell-specific variations in their expression are associated with differential susceptibilities for cytogenetic damage and risks for tissue pathologies and diseases. The proposed research will utilize expression microarrays to identify genes whose expression is modulated after low doses of ionizing radiation (IR), determine the nature of the dose response, and identify cell- and tissue-specific variations in expression response. cDNA microarrays will be developed to survey, in parallel, large numbers of genes involved in stress/damage response and DNA repair, as well as novel genes involved in low dose response. The biological functions of identified genes will be investigated by analyses of regulatory and coding sequence homologies, cytogenetics, and by identifying affected cells in tissue sections. This project will (a) provide important mechanistic and molecular knowledge of the cellular response to low dose IR to help reduce the uncertainty of assessing risk at low-dose levels, (b) identify candidate susceptibility genes important for low-dose IR exposures, and (c) serve as a foundation for a genomics-scale approach to identifying and characterizing cellular responses to chemical and physical DNA damaging agents.
Harvey Mohrenweiser
Lawrence Livermore National Laboratory
DNA Repair Gene Variants: Understanding Mechanisms of Cellular Reponse and Estimating Individual Health Risk from Low-Dose Radiation Exposure
Estimation of health risk from exposures to toxic agents is hampered by unexplained variation among individuals in biomarkers and disease incidence. Some of the variation reflects unknown exposures and shortcomings of biomarkers, but accumulating data suggest that the health consequences of exposure reflect the interaction of dose and the genetic constitution (susceptibility) of the individual. DNA repair genes are critical in protecting the cell from the consequences of radiation and repair gene variants affect cancer susceptibility. This Program will focus on (Aim 1) identification of genetic variation existing at polymorphic frequency in genes of the Base Excision Repair and the Double Strand Break/Recombination Repair pathways. DNA repair capacity assays will be (Aim 2) developed and employed as an intervening phenotype to identify individuals with impaired DNA repair function in these pathways. Molecular epidemiology collaborations will (Aim 3) evaluate the role of variant genes in individual susceptibility to disease from low-level radiation exposure. This Program initiates a coordinated effort to utilize variant genes as tools to learn about the impact of DNA repair on population health. The genetic variation that we identify in human populations will lead to mechanistic studies, by our collaborators, of the variants expected to exhibit impaired function and a multi-laboratory program leading to estimation of individual and population health risk from low-level radiation exposure. The approach has potential to ultimately contribute to regulatory and pharmacological means to ameliorate risk from low-dose radiation exposure and to translate sequence information of the Human Genome Initiative into improved health.
Bobby R. Scott
Lovelace Biomedical & Environmental Research Institute
Advanced Computational Approaches for Characterizing Stochastic Cellular Responses to Low-Dose, Low-Dose-Rate Exposures
The research focuses on the development and use of novel methods to integrate key microdosimetric, molecular, and cellular information into risk assessment for lung cancer induction by radiation and chemicals. The main goal is to develop computational approaches for use in determining the health risks that model new information from cellular and molecular studies together with available data from epidemiological and animal studies. The project is also intended to provide key results of the research in forms easily used by scientists, regulators, legislators, and other stakeholders.
A mechanistic model for high LET radiation induced neoplastic transformation has previously been developed by the PI (NEOTRANS). This research will expand the scope of NEOTRANS for use with low LET radiation, chemicals, mutation induction and to allow a broader array of biological parameters to be used in the model including apoptosis, microdosimetry, and cell cycle progression.
Albert J. Fornace
National Institutes of Health
Development of a Functional Genomics Approach Employing Radiation-Induced Changes in Gene Expression to Monitor Cells After Low Dose & Low Dose-Rate Exposures
This project will explore the utility of gene induction as a means to monitor for exposure to ionizing radiation (IR1). The applicant's laboratory has extensive experience in mammalian genotoxic stress responses and has contributed substantially to the understanding of radiation responses mediated both by the tumor suppressor p53 and other signaling pathways. This laboratory has recently been the first to demonstrate the utility of cDNA microarray hybridization analysis as an approach to quantitatively survey for changes in gene expression after IR. With this functional genomics approach, the responses of a substantial portion of a cell's expressed genes can be monitored. Changes in gene expression after low doses of IR will be characterized in human cells by both single-probe quantitative hybridization and the cDNA microarray approach. These approaches will also be used in low-dose rate studies. Studies will be expanded to responses in irradiated mice using the same approaches. As a first step to in vivo studies in humans, the IR responses of peripheral blood lymphocytes (PBL) irradiated ex vivo will be characterized. A longer term goal will be to characterize the responses from irradiated individuals with focus on accessible cells such as PBL. Neural-net type analysis will be used to sort complex gene expression profiles. The functional-genomics approach should have utility for DOE priorities in radiobiology and toxicology.
Kerry Bloom
University of North Carolina
The Impact of Human Genes and Their Homologues on the Dynamics of Broken Chromosomes in Yeast
The objective of this project is to characterize the functions of eukaryotic genes involved in repair of chromosomal DNA double-strand breaks (DSB) using yeast as a model organism. Experiments will integrate the advanced genetics available in the budding yeast Saccharomyces cerevisiae with recently developed techniques for monitoring changes in the movements of chromosomes and associated structures during normal cell cycling in living cells. It is now established that most yeast genes involved in the two major pathways of DSB repair, homologous recombination and nonhomologous end-joining, have structural homologues in human cells. This conservation between yeast and human repair proteins strongly suggest that many of the results obtained in yeast can be extrapolated to mechanisms of repair in human cells.
Initial studies will examine the cytological fate of both ends of a single chromosomal DSB induced by HO endonuclease using video-imaging of fluorescently tagged chromosomal DNA in living cells. The fate(s) of the ends will be followed in haploid and diploid cells in both G1 and G2 phase to assess the impact of increased or decreased opportunities for DSB break repair by homologous recombination. Real time analysis of the metabolism of a broken chromosome produced by HO endonuclease cleavage will be performed in mutant strains that are defective in both major pathways of DSB break repair. The dynamics of chromosome movement in cells defective in recombination or nonhomologous end joining will be analyzed. Chromosome movement will also be studied in strains containing a deletion of the checkpoint gene RAD9 that controls damage induced transcriptional activation of RAD51 and RAD54. The proposed experiments will enable definition of DNA end kinetics in wild-type cells and identification of cytological alterations in cells containing specific DNA repair deficiencies. This studies will also provide a basis for understanding the mechanisms responsible for additional phenotypes of DSB repair mutants, e.g., the excessive chromosome loss observed in RAD51 and RAD54 mutants following DNA damage.
David G. Hoel
Medical University of South Carolina
Radiation Leukemogenesis: Applying Basic Science to Epidemiological Estimates of Low Dose Risks and Dose-Rate Effects
The goal of this research is to investigate and develop mathematical models that link data from the basic sciences to epidemiological data to estimate risks of chronic myeloid leukemia from low dose, low dose rate exposures to radiation.
The project has three general aims: 1) identify a mathematical model for the low-LET risks of chronic myeloid leukemia (CML); 2) validate the model; and 3) make predictions for other types of radiations and others types of translocation-mediated leukemias.
Michael N. Cornforth
University of Texas
Current Cytogenetic Issues Pertaining to Low Dose and Dose Rate
By providing a quantitative, sensitive and relevant measure of genotoxic damage, the study of chromosomal aberrations serves to guide theoretical models of radiation action. One of the more important and unexpected discoveries in modern radiation cytogenetics occurred when "chromosome painting" showed that a large fraction of exchange aberrations were "complex", involving several chromosomes. Because painting typically reveals only a partial picture of complex exchanges, investigators are forced to make certain assumptions regarding complex aberration formation, in order to model the process. Differing assumptions have led to grossly divergent predictions about the true frequency and degree of aberration complexity. At the heart of this controversy is the major impact such predictions have on fundamental concepts of radiation action, including those upon which protection standards now rely.
The project contains both theoretical and pragmatic goals aimed toward lowering risk uncertainties to human populations following exposure to ionizing radiations. A key aspect of the project is the use of multicolor combinatorial painting (m-FISH) to eliminate discrepancies surrounding the interpretation of complex aberration data. This will allow the testing of two contemporary cytogenetic models. The models chosen are particularly important to the issue of low dose effects, because their predictions have been used to buttress diametrically opposed views of how chromosome aberrations are produced by ionizing radiation. Also described are experiments to examine the feasibility of using complex aberrations as a biomarker of past exposure to alpha-emitting radionuclides and other densely ionizing radiations. Accurate prediction of human risk from low level radiation exposure must rely on extrapolations of models firmly based on radiobiological mechanisms. Dose responses for chromosomal aberration formation have been very helpful here, not only because we know they are prominently involved in carcinogenic and mutagenic processes, but because fairly accurate measurements in low dose regions can be made, along with pertinent mechanistic studies involving such parameters as LET and dose-rate effects, in order to guide and justify the kinds of extrapolations made from the high-dose human or animal data. When whole chromosome-specific FISH painting became available, analysis of radiation induced chromosomal aberrations soon revealed that a surprisingly high proportion of exchanges involved more than just two chromosomes, i.e., were complex. After 3-4 Gy x-rays, it has been estimated that as high as 25% may be complex, and in some cases it was deduced that as many as 4 to 6 chromosomes may be involved. So, the important issue immediately arises as to how these aberrations are being formed, when by classical ideas they shouldn't be seen at such high frequencies at these doses. Do we have to scrap all our ideas about linear-quadratic responses reducing to linear components at low doses and low dose-rates, etc.?