NGS Detects Extensive Genomic Alterations in Survivors of Irradiated Normal Human Fibroblast Cells.

It is thought that cells surviving ionizing radiation exposure repair DNA double-strand breaks (DSBs) and restore their genomes. However, the recent biochemical and genetic characterization of DSB repair pathways reveals that only homologous recombination (HR) can function in an error-free manner and that the non-homologous end joining (NHEJ) pathways canonical NHEJ (c-NHEJ), alternative end joining (alt-EJ), and single-strand annealing (SSA) are error-prone, and potentially leave behind genomic scars and altered genomes. The strong cell cycle restriction of HR to S/G2 phases and the unparalleled efficiency of c-NHEJ throughout the cell cycle, raise the intriguing question as to how far a surviving cell "reaches" after repairing the genome back to its pre-irradiation state.

The Central Role of Cytogenetics in Radiation Biology.

Radiation cytogenetics has a rich history seldom appreciated by those outside the field. Early radiobiology was dominated by physics and biophysical concepts that borrowed heavily from the study of radiation-induced chromosome aberrations. From such studies, quantitative relationships between biological effect and changes in absorbed dose, dose rate and ionization density were codified into key concepts of radiobiological theory that have persisted for nearly a century.

Dose-Dependent Transmissibility of Chromosome Aberrations in Human Lymphocytes at First Mitosis. II. Biological Effectiveness of Heavy Charged Particles Versus Gamma Rays.

Chromosome aberrations (CAs) are large scale structural rearrangements to the genome that have been used as a proxy endpoint of mutagenic and carcinogenic potential. And yet, many types of CAs are incapable of causing either of these effects simply because they are lethal. Using 24-color multi-fluor combinatorial painting (mFISH), we examined CAs in normal human lymphocytes exposed to graded doses of 1 GeV/nucleon accelerated 56Fe ions and 662 keV 137Cs gamma rays.

Dose-dependent Transmissibility of Chromosome Aberrations at First Mitosis after Exposure to Gamma Rays. I. Modeling and Implications Related to Risk Assessment.

The relationship between certain chromosomal aberration (CA) types and cell lethality is well established. On that basis we used multi-fluor in situ hybridization (mFISH) to tally the number of mitotic human lymphocytes exposed to graded doses of gamma rays that carried either lethal or nonlethal CA types. Despite the fact that a number of nonlethal complex exchanges were observed, the cells containing them were seldom deemed viable, due to coincident lethal chromosome damage.

Destabilizing Effects of Ionizing Radiation on Chromosomes: Sizing up the Damage.

For long-term survival and evolution, all organisms have depended on a delicate balance between processes involved in maintaining stability of their genomes and opposing processes that lead toward destabilization. At the level of mammalian somatic cells in renewal tissues, events or conditions that can tip this balance toward instability have attracted special interest in connection with carcinogenesis. Mutations affecting DNA (and its subsequent repair) would, of course, be a major consideration here.

Robbing Peter to Pay Paul: Competition for Radiogenic Breaks During Rejoining Diminishes Curvature in the Dose Response for Simple Chromosome Exchanges.

The large majority of chromosome damage produced by ionizing radiations takes the form of exchange aberrations. For simple exchanges between two chromosomes, multi-fluor fluorescence in situ hybridization (mFISH) studies confirm that the dose response to X rays or gamma rays is quasilinear with dose. This result is in seeming conflict with generalized theories of radiation action that depend on the interaction of lesions as the source of curvature in dose-response relationships.

Accounting for overdispersion of lethal lesions in the linear quadratic model improves performance at both high and low radiation doses.

Purpose: The linear-quadratic (LQ) model represents a simple and robust approximation for many mechanistically-motivated models of radiation effects. We believe its tendency to overestimate cell killing at high doses derives from the usual assumption that radiogenic lesions are distributed according to Poisson statistics.

Materials And Methods: In that context, we investigated the effects of overdispersed lesion distributions, such as might occur from considerations of microdosimetric energy deposition patterns, differences in DNA damage complexities and repair pathways, and/or heterogeneity of cell responses to radiation.

Occam's broom and the dirty DSB: cytogenetic perspectives on cellular response to changes in track structure and ionization density.

Given equal doses, it is well-known that densely ionizing radiations are more potent in causing a number of biological effects compared to sparsely ionizing radiations, such as x- or gamma rays. According to classical models of radiation action, this results from differences in the spatial distribution of lesions along charged particle tracks. In recent years investigators have been barraged with the alternative narrative that this is instead due to 'qualitative' differences in the types of molecular lesions that each type of radiation produces.

Directional Genomic Hybridization (dGH) for Detection of Intrachromosomal Rearrangements.

Fluorescence in situ Hybridization (FISH) techniques, including whole chromosome painting (WCP), spectral karyotyping (SKY), and multicolor FISH (mFISH), are used extensively to characterize and enumerate inter-chromosomal rearrangements (e.g., translocations).

Estimation of Radiation Doses to U.S. Military Test Participants from Nuclear Testing: A Comparison of Historical Film-Badge Measurements, Dose Reconstruction and Retrospective Biodosimetry.

Retrospective radiation dose estimations, whether based on physical or biological measurements, or on theoretical dose reconstruction, are limited in their precision and reliability, particularly for exposures that occurred many decades ago. Here, we studied living U.S.

Chromosome Translocations, Inversions and Telomere Length for Retrospective Biodosimetry on Exposed U.S. Atomic Veterans.

It has now been over 60 years since U.S. nuclear testing was conducted in the Pacific islands and Nevada, exposing military personnel to varying levels of ionizing radiation.

A Cytogenetic Profile of Radiation Damage.

Most of the important biological effects associated with the exposure to ionizing radiations are mirrored at the chromosomal level. In all cases, changes in the levels of cytogenetic effects are associated with changes in absorbed dose, dose rate and radiation quality. Some of the complexities associated with the quantitative description of such changes in response can be circumvented by appealing to concepts embodied in what has been called the "mean inactivation dose".

Radiation quality and intra-chromosomal aberrations: Size matters.

The shift from plant to mammalian cell models in radiation cytogenetics hastened the development of methods suitable for the analysis of chromosome-type aberrations. These included methods to detect interchanges that take place between different chromosomes (dicentrics and translocations), and intrachanges occurring within a given chromosome (rings, interstitial deletions and inversions). In this review we consider the relationship between chromosome-type interchanges and intrachanges in response to changes in ionization density (linear energy transfer; LET).

Molecular Cytogenetics Guides Massively Parallel Sequencing of a Radiation-Induced Chromosome Translocation in Human Cells.

Chromosome rearrangements are large-scale structural variants that are recognized drivers of oncogenic events in cancers of all types. Cytogenetics allows for their rapid, genome-wide detection, but does not provide gene-level resolution. Massively parallel sequencing (MPS) promises DNA sequence-level characterization of the specific breakpoints involved, but is strongly influenced by bioinformatics filters that affect detection efficiency.

Straightening Beta: Overdispersion of Lethal Chromosome Aberrations following Radiotherapeutic Doses Leads to Terminal Linearity in the Alpha-Beta Model.

Recent technological advances allow precise radiation delivery to tumor targets. As opposed to more conventional radiotherapy-where multiple small fractions are given-in some cases, the preferred course of treatment may involve only a few (or even one) large dose(s) per fraction. Under these conditions, the choice of appropriate radiobiological model complicates the tasks of predicting radiotherapy outcomes and designing new treatment regimens.

Telomeres and NextGen CO-FISH: Directional Genomic Hybridization (Telo-dGH™).

The cytogenomics-based methodology of Directional Genomic Hybridization (dGH™) emerged from the concept of strand-specific hybridization, first made possible by Chromosome Orientation FISH (CO-FISH), the utility of which was demonstrated in a variety of early applications, often involving telomeres. Similar to standard whole chromosome painting (FISH), dGH™ is capable of identifying inter-chromosomal rearrangements (translocations between chromosomes), but its distinctive strength stems from its ability to detect intra-chromosomal rearrangements (inversions within chromosomes), and to do so at higher resolution than previously possible. dGH™ brings together the strand specificity and directionality of CO-FISH with sophisticated bioinformatics-based oligonucleotide probe design to unique sequences.

Seeking Beta: Experimental Considerations and Theoretical Implications Regarding the Detection of Curvature in Dose-Response Relationships for Chromosome Aberrations.

The concept of curvature in dose-response relationships figures prominently in radiation biology, encompassing a wide range of interests including radiation protection, radiotherapy and fundamental models of radiation action. In this context, the ability to detect even small amounts of curvature becomes important. Standard (ST) statistical approaches used for this purpose typically involve least-squares regression, followed by a test on sums of squares.

Three-Color Chromosome Painting as Seen through the Eyes of mFISH: Another Look at Radiation-Induced Exchanges and Their Conversion to Whole-Genome Equivalency.

Whole-chromosome painting (WCP) typically involves the fluorescent staining of a small number of chromosomes. Consequently, it is capable of detecting only a fraction of exchanges that occur among the full complement of chromosomes in a genome. Mathematical corrections are commonly applied to WCP data in order to extrapolate the frequency of exchanges occurring in the entire genome [whole-genome equivalency (WGE)].

Directional genomic hybridization: inversions as a potential biodosimeter for retrospective radiation exposure.

Chromosome aberrations in blood lymphocytes provide a useful measure of past exposure to ionizing radiation. Despite the widespread and successful use of the dicentric assay for retrospective biodosimetry, the approach suffers substantial drawbacks, including the fact that dicentrics in circulating blood have a rather short half-life (roughly 1-2 years by most estimates). So-called symmetrical aberrations such as translocations are far more stable in that regard, but their high background frequency, which increases with age, also makes them less than ideal for biodosimetry.

From DNA damage to chromosome aberrations: joining the break.

Despite many years of experimental studies on radiation-induced chromosomal aberrations, and the recent progress in elucidating the molecular mechanisms of the DNA damage response, the link between DNA double-strand break repair and its expression as microscopically visible chromosomal rearrangements remains, in many ways, obscure. Some long standing controversies have partially been resolved to the satisfaction of most investigators, including the linearity of the dose-response for DNA double-strand break induction, the necessity of pairwise interaction of radiogenic damaged sites in the formation of exchange aberrations, and the importance of proximity between lesions in misrejoining. However, the contribution of different molecular DNA repair mechanisms (e.

The LET dependence of unrepaired chromosome damage in human cells: a break too far?

Cytogenetic damage is among the few radiobiological end points that allow a precise distinction to be made between misrepaired damage, represented by exchange-type aberrations such as dicentrics and translocations, and unrepaired damage that leads to "open breaks". This latter category includes both terminal deletions and incomplete exchanges, whose different mechanisms of formation can be recognized by multicolor fluorescence in situ hybridization (mFISH). mFISH was used to examine the yields of chromosome aberrations at the first postirradiation mitosis in human fibroblasts and lymphocytes irradiated with ¹³⁷Cs γ rays, a radiation of low-linear energy transfer (LET), and two sources of high-LET radiation: α particles from ²³⁸Pu and 1 GeV/amu ⁵⁶Fe ions.

Directional genomic hybridization for chromosomal inversion discovery and detection.

Chromosomal rearrangements are a source of structural variation within the genome that figure prominently in human disease, where the importance of translocations and deletions is well recognized. In principle, inversions-reversals in the orientation of DNA sequences within a chromosome-should have similar detrimental potential. However, the study of inversions has been hampered by traditional approaches used for their detection, which are not particularly robust.

The LET Dependence of Unrepaired Chromosome Damage in Human Cells: A Break Too Far?

Cytogenetic damage is among the few radiobiological end points that allow a precise distinction to be made between misrepaired damage, represented by exchange-type aberrations such as dicentrics and translocations, and unrepaired damage that leads to "open breaks". This latter category includes both terminal deletions and incomplete exchanges, whose different mechanisms of formation can be recognized by multicolor fluorescence in situ fluorescence hybridization (mFISH). mFISH was used to examine the yields of chromosome aberrations at the first postirradiation mitosis in human fibroblasts and lymphocytes irradiated with (137)Cs γ rays, a radiation of low-linear energy transfer (LET), and two sources of high-LET radiation: α particles from (238)Pu and 1 GeV/amu (56)Fe ions.

Chromosome damage in human cells by γ rays, α particles and heavy ions: track interactions in basic dose-response relationships.

We irradiated normal human lymphocytes and fibroblasts with (137)Cs γ rays, 3.5 MeV α particles and 1 GeV/amu (56)Fe ions and measured the subsequent formation of chromosome-type aberrations by mFISH at the first mitosis following irradiation. This was done for the purposes of characterizing the shape of dose-response relationships and determining the frequency distribution of various aberration types with respect to the parameters of dose, radiation quality and cell type.