Monitoring Genomic Structural Rearrangements Resulting from Gene Editing.

The cytogenomics-based methodology of directional genomic hybridization (dGH) enables the detection and quantification of a more comprehensive spectrum of genomic structural variants than any other approach currently available, and importantly, does so on a single-cell basis. Thus, dGH is well-suited for testing and/or validating new advancements in CRISPR-Cas9 gene editing systems. In addition to aberrations detected by traditional cytogenetic approaches, the strand specificity of dGH facilitates detection of otherwise cryptic intra-chromosomal rearrangements, specifically small inversions. As such, dGH represents a powerful, high-resolution approach for the quantitative monitoring of potentially detrimental genomic structural rearrangements resulting from exposure to agents that induce DNA double-strand breaks (DSBs), including restriction endonucleases and ionizing radiations. For intentional genome editing strategies, it is critical that any undesired effects of DSBs induced either by the editing system itself or by mis-repair with other endogenous DSBs are recognized and minimized. In this paper, we discuss the application of dGH for assessing gene editing-associated structural variants and the potential heterogeneity of such rearrangements among cells within an edited population, highlighting its relevance to personalized medicine strategies.

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. These may occur spontaneously through endogenous cellular processes or as a result of exposure to mutagenic environmental agents. It is in this context that we discuss the rather unique destabilizing effects of ionizing radiation (IR) in terms of its ability to cause large-scale structural rearrangements to the genome. We present arguments supporting the conclusion that these and other important effects of IR originate largely from microscopically visible chromosome aberrations.

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). Directional genomic hybridization (dGH) is a relatively new cytogenomics-based methodology that combines the strand-specific strategy of Chromosome Orientation-FISH (CO-FISH) with bioinformatics-driven design of single-stranded DNA probe sets that are unique and of like orientation. Such a strategy produces directional probe sets that hybridize to one-and only one-chromatid of prepared (single-stranded) metaphase chromosomes, thereby facilitating high-resolution visualization of intra-chromosomal rearrangements, specifically inversions, and greatly improving our ability to detect such otherwise cryptic structural variants within the genome. In addition to its usefulness in the study of various disease states, including cancer, relevant applications of dGH include monitoring cytogenetic damage caused by exposure to clastogenic agents (e.g., ionizing radiation). dGH can be applied as a discovery tool to globally assess the integrity of the genome, but it can also be used in a more targeted fashion to interrogate fine structural changes at the kilobase level. Consequently, dGH is capable of providing significant mechanistic insight and information not easily obtainable by other approaches.

Persistence of Gamma-H2AX Foci in Bronchial Cells Correlates with Susceptibility to Radiation Associated Lung Cancer in Mice.

The risk of developing radiation-induced lung cancer differs between different strains of mice, but the underlying cause of the strain differences is unknown. Strains of mice also differ in how quickly they repair radiation-induced DNA double-strand breaks (DSBs). We assayed mouse strains from the CcS/Dem recombinant congenic strain set for their efficacy in repairing DNA DSBs during protracted irradiation. We measured unrepaired γ-H2AX radiation-induced foci (RIF), which persisted after chronic 24-h gamma irradiation, as a surrogate marker for repair efficiency in bronchial epithelial cells for 17 of the CcS/Dem strains and the BALB/c founder strain. We observed a very strong correlation (R2 = 79.18%, P < 0.001) between the level of unrepaired RIF and radiogenic lung cancer incidence measured in the same strains. Interestingly, spontaneous levels of foci in nonirradiated mice also showed good correlation with lung cancer incidence when incidence data from male and female mice were combined. These results suggest that genetic differences in DNA repair capacity largely account for differing susceptibilities to radiation-induced lung cancer among CcS/Dem mouse strains, and that high levels of spontaneous DNA damage are also a relatively good marker of cancer predisposition. In a smaller pilot study, we found that the repair capacity measured in peripheral blood leucocytes also correlated well with radiogenic lung cancer susceptibility, raising the possibility that the assay could be used to detect radiogenic lung cancer susceptibility in humans.

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. We sought to characterize the breakpoint junctions of chromosomal translocations and inversions in the clonal derivatives of human cells exposed to ionizing radiation. Here, we describe the first successful use of DNA paired-end analysis to locate and sequence across the breakpoint junctions of a radiation-induced reciprocal translocation. The analyses employed, with varying degrees of success, several well-known bioinformatics algorithms, a task made difficult by the involvement of repetitive DNA sequences. As for underlying mechanisms, the results of Sanger sequencing suggested that the translocation in question was likely formed via microhomology-mediated non-homologous end joining (mmNHEJ). To our knowledge, this represents the first use of MPS to characterize the breakpoint junctions of a radiation-induced chromosomal translocation in human cells. Curiously, these same approaches were unsuccessful when applied to the analysis of inversions previously identified by directional genomic hybridization (dGH). We conclude that molecular cytogenetics continues to provide critical guidance for structural variant discovery, validation and in "tuning" analysis filters to enable robust breakpoint identification at the base pair level.

Coordination of the Ser2056 and Thr2609 Clusters of DNA-PKcs in Regulating Gamma Rays and Extremely Low Fluencies of Alpha-Particle Irradiation to G0/G1 Phase Cells.

The catalytic subunit of DNA dependent protein kinase (DNA-PKcs) and its kinase activity are critical for mediation of non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSB) in mammalian cells after gamma-ray irradiation. Additionally, DNA-PKcs phosphorylations at the T2609 cluster and the S2056 cluster also affect DSB repair and cellular sensitivity to gamma radiation. Previously we reported that phosphorylations within these two regions affect not only NHEJ but also homologous recombination repair (HRR) dependent DSB repair. In this study, we further examine phenotypic effects on cells bearing various combinations of mutations within either or both regions. Effects studied included cell killing as well as chromosomal aberration induction after 0.5-8 Gy gamma-ray irradiation delivered to synchronized cells during the G0/G1 phase of the cell cycle. Blocking phosphorylation within the T2609 cluster was most critical regarding sensitization and depended on the number of available phosphorylation sites. It was also especially interesting that only one substitution of alanine in each of the two clusters separately abolished the restoration of wild-type sensitivity by DNA-PKcs. Similar patterns were seen for induction of chromosomal aberrations, reflecting their connection to cell killing. To study possible change in coordination between HRR and NHEJ directed repair in these DNA-PKcs mutant cell lines, we compared the induction of sister chromatid exchanges (SCEs) by very low fluencies of alpha particles with mutant cells defective in the HRR pathway that is required for induction of SCEs. Levels of true SCEs induced by very low fluence of alpha-particle irradiation normally seen in wild-type cells were only slightly decreased in the S2056 cluster mutants, but were completely abolished in the T2609 cluster mutants and were indistinguishable from levels seen in HRR deficient cells. Again, a single substitution in the S2056 together with a single substitution in the T2609 cluster abolished SCE formation and thus also effectively interferes with HRR.

Differential radiosensitivity phenotypes of DNA-PKcs mutations affecting NHEJ and HRR systems following irradiation with gamma-rays or very low fluences of alpha particles.

We have examined cell-cycle dependence of chromosomal aberration induction and cell killing after high or low dose-rate γ irradiation in cells bearing DNA-PKcs mutations in the S2056 cluster, the T2609 cluster, or the kinase domain. We also compared sister chromatid exchanges (SCE) production by very low fluences of α-particles in DNA-PKcs mutant cells, and in homologous recombination repair (HRR) mutant cells including Rad51C, Rad51D, and Fancg/xrcc9. Generally, chromosomal aberrations and cell killing by γ-rays were similarly affected by mutations in DNA-PKcs, and these mutant cells were more sensitive in G1 than in S/G2 phase. In G1-irradiated DNA-PKcs mutant cells, both chromosome- and chromatid-type breaks and exchanges were in excess than wild-type cells. For cells irradiated in late S/G2 phase, mutant cells showed very high yields of chromatid breaks compared to wild-type cells. Few exchanges were seen in DNA-PKcs-null, Ku80-null, or DNA-PKcs kinase dead mutants, but exchanges in excess were detected in the S2506 or T2609 cluster mutants. SCE induction by very low doses of α-particles is resulted from bystander effects in cells not traversed by α-particles. SCE seen in wild-type cells was completely abolished in Rad51C- or Rad51D-deficient cells, but near normal in Fancg/xrcc9 cells. In marked contrast, very high levels of SCEs were observed in DNA-PKcs-null, DNA-PKcs kinase-dead and Ku80-null mutants. SCE induction was also abolished in T2609 cluster mutant cells, but was only slightly reduced in the S2056 cluster mutant cells. Since both non-homologous end-joining (NHEJ) and HRR systems utilize initial DNA lesions as a substrate, these results suggest the possibility of a competitive interference phenomenon operating between NHEJ and at least the Rad51C/D components of HRR; the level of interaction between damaged DNA and a particular DNA-PK component may determine the level of interaction of such DNA with a relevant HRR component.

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. We developed a cytogenetic assay for potential use in retrospective biodosimetry that is based on the detection of chromosomal inversions, another symmetrical aberration whose transmissibility (stability) is also ostensibly high. Many of the well-known difficulties associated with inversion detection were circumvented through the use of directional genomic hybridization, a method of molecular cytogenetics that is less labor intensive and better able to detect small chromosomal inversions than other currently available approaches. Here, we report the dose-dependent induction of inversions following exposure to radiations with vastly different ionization densities [i.e., linear energy transfer (LET)]. Our results show a dramatic dose-dependent difference in the yields of inversions induced by low-LET gamma rays, as compared to more damaging high-LET charged particles similar to those encountered in deep space.

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. Even with significant advances in whole genome approaches, changes in the absolute orientation of DNA remain difficult to detect routinely. Consequently, our understanding of inversions is still surprisingly limited, as is our appreciation for their frequency and involvement in human disease. Here, we introduce the directional genomic hybridization methodology of chromatid painting-a whole new way of looking at structural features of the genome-that can be employed with high resolution on a cell-by-cell basis, and demonstrate its basic capabilities for genome-wide discovery and targeted detection of inversions. Bioinformatics enabled development of sequence- and strand-specific directional probe sets, which when coupled with single-stranded hybridization, greatly improved the resolution and ease of inversion detection. We highlight examples of the far-ranging applicability of this cytogenomics-based approach, which include confirmation of the alignment of the human genome database and evidence that individuals themselves share similar sequence directionality, as well as use in comparative and evolutionary studies for any species whose genome has been sequenced. In addition to applications related to basic mechanistic studies, the information obtainable with strand-specific hybridization strategies may ultimately enable novel gene discovery, thereby benefitting the diagnosis and treatment of a variety of human disease states and disorders including cancer, autism, and idiopathic infertility.

Molecular characterisation of murine acute myeloid leukaemia induced by 56Fe ion and 137Cs gamma ray irradiation.

Exposure to sparsely ionising gamma- or X-ray irradiation is known to increase the risk of leukaemia in humans. However, heavy ion radiotherapy and extended space exploration will expose humans to densely ionising high linear energy transfer (LET) radiation for which there is currently no understanding of leukaemia risk. Murine models have implicated chromosomal deletion that includes the hematopoietic transcription factor gene, PU.1 (Sfpi1), and point mutation of the second PU.1 allele as the primary cause of low-LET radiation-induced murine acute myeloid leukaemia (rAML). Using array comparative genomic hybridisation, fluorescence in situ hybridisation and high resolution melt analysis, we have confirmed that biallelic PU.1 mutations are common in low-LET rAML, occurring in 88% of samples. Biallelic PU.1 mutations were also detected in the majority of high-LET rAML samples. Microsatellite instability was identified in 42% of all rAML samples, and 89% of samples carried increased microsatellite mutant frequencies at the single-cell level, indicative of ongoing instability. Instability was also observed cytogenetically as a 2-fold increase in chromatid-type aberrations. These data highlight the similarities in molecular characteristics of high-LET and low-LET rAML and confirm the presence of ongoing chromosomal and microsatellite instability in murine rAML.

Genetic susceptibility: radiation effects relevant to space travel.

Genetic variation in the capacity to repair radiation damage is an important factor influencing both cellular and tissue radiosensitivity variation among individuals as well as dose rate effects associated with such damage. This paper consists of two parts. The first part reviews some of the available data relating to genetic components governing such variability among individuals in susceptibility to radiation damage relevant for radiation protection and discusses the possibility and extent to which these may also apply for space radiations. The second part focuses on the importance of dose rate effects and genetic-based variations that influence them. Very few dose rate effect studies have been carried out for the kinds of radiations encountered in space. The authors present here new data on the production of chromosomal aberrations in noncycling low passage human ATM+/+ or ATM+/- cells following irradiations with protons (50 MeV or 1 GeV), 1 GeV(-1) n iron ions and gamma rays, where doses were delivered at a high dose rate of 700 mGy(-1) min, or a lower dose rate of 5 mGy min(-1). Dose responses were essentially linear over the dose ranges tested and not significantly different for the two cell strains. Values of the dose rate effectiveness factor (DREF) were expressed as the ratio of the slopes of the dose-response curves for the high versus the lower (5 mGy min(-1)) dose rate exposures. The authors refer to this as the DREF5. For the gamma ray standard, DREF5 values of approximately two were observed. Similar dose rate effects were seen for both energies of protons (DREF5 ≈ 2.2 in both cases). For 1 GeV(-1) n iron ions [linear energy transfer (LET) ≈ 150 keV µ(-1)], the DREF5 was not 1 as might have been expected on the basis of LET alone but was approximately 1.3. From these results and conditions, the authors estimate that the relative biological effectiveness for 1 GeV(-1) n iron ions for high and low dose rates, respectively, were about 10 and 15 rather than around 20 for low dose rates, as has been assumed by most recommendations from radiation protection organizations for charged particles of this LET. The authors suggest that similar studies using appropriate animal models of carcinogenesis would be valuable.

Caspase 3-mediated stimulation of tumor cell repopulation during cancer radiotherapy.

In cancer treatment, apoptosis is a well-recognized cell death mechanism through which cytotoxic agents kill tumor cells. Here we report that dying tumor cells use the apoptotic process to generate potent growth-stimulating signals to stimulate the repopulation of tumors undergoing radiotherapy. Furthermore, activated caspase 3, a key executioner in apoptosis, is involved in the growth stimulation. One downstream effector that caspase 3 regulates is prostaglandin E(2) (PGE(2)), which can potently stimulate growth of surviving tumor cells. Deficiency of caspase 3 either in tumor cells or in tumor stroma caused substantial tumor sensitivity to radiotherapy in xenograft or mouse tumors. In human subjects with cancer, higher amounts of activated caspase 3 in tumor tissues are correlated with markedly increased rate of recurrence and death. We propose the existence of a cell death-induced tumor repopulation pathway in which caspase 3 has a major role.

Quantitative, noninvasive imaging of radiation-induced DNA double-strand breaks in vivo.

DNA double-strand breaks (DSB) are a major form of DNA damage and a key mechanism through which radiotherapy and some chemotherapeutic agents kill cancer cells. Despite its importance, measuring DNA DSBs is still a tedious task that is normally carried out by gel electrophoresis or immunofluorescence staining. Here, we report a novel approach to image and quantify DSBs in live mammalian cells through bifragment luciferase reconstitution. N- and C-terminal fragments of firefly luciferase genes were fused with H2AX and MDC1 genes, respectively. Our strategy was based on the established fact that at the sites of DSBs, H2AX protein is phosphoryated and physically associates with the MDC1 protein, thus bringing together N- and C-luciferase fragments and reconstituting luciferase activity. Our strategy allowed serial, noninvasive quantification of DSBs in cells irradiated with X-rays and (56)Fe ions. Furthermore, it allowed for the evaluation of DSBs noninvasively in vivo in irradiated tumors over 2 weeks. Surprisingly, we detected a second wave of DSB induction in irradiated tumor cells days after radiation exposure in addition to the initial rapid induction of DSBs. We conclude that our new split-luciferase-based method for imaging γ-H2AX-MDC1 interaction is a powerful new tool to study DSB repair kinetics in vivo with considerable advantage for experiments requiring observations over an extended period of time.

Differential role of DNA-PKcs phosphorylations and kinase activity in radiosensitivity and chromosomal instability.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is the key functional element in the DNA-PK complex that drives nonhomologous end joining (NHEJ), the predominant DNA double-strand break (DSB) repair mechanism operating to rejoin such breaks in mammalian cells after exposure to ionizing radiation. It has been reported that DNA-PKcs phosphorylation and kinase activity are critical determinants of radiosensitivity, based on responses reported after irradiation of asynchronously dividing populations of various mutant cell lines. In the present study, the relative radiosensitivity to cell killing as well as chromosomal instability of 13 DNA-PKcs site-directed mutant cell lines (defective at phosphorylation sites or kinase activity) were examined after exposure of synchronized G(1) cells to (137)Cs γ rays. DNA-PKcs mutant cells defective in phosphorylation at multiple sites within the T2609 cluster or within the PI3K domain displayed extreme radiosensitivity. Cells defective at the S2056 cluster or T2609 single site alone were only mildly radiosensitive, but cells defective at even one site in both the S2056 and T2609 clusters were maximally radiosensitive. Thus a synergism between the capacity for phosphorylation at the S2056 and T2609 clusters was found to be critical for induction of radiosensitivity.

Some unsolved problems and unresolved issues in radiation cytogenetics: a review and new data on roles of homologous recombination and non-homologous end joining.

New data and historical evidence from our own and other laboratories are summarized and discussed bearing on several issues relating to mechanisms and processes involved in the formation of chromosomal aberrations following exposure to ionizing radiations. Specifically addressed are: (1) the lesions and processes affecting the appearance of chromatid-type and/or chromosome-type aberrations after radiation, (2) DNA double strand break rejoining processes and the restitution of breaks vs. the formation of exchanges, (3) the role of homologous recombinational repair in protecting cells from induction of chromatid-type aberrations after irradiation of late S/G2 cells, (4) the role of interphase chromatin structure and nuclear organization in aberration induction, (5) cellular responses for aberration induction in relation to their tissue context, and (6) approaches to the detection of aberrations previously known as "cryptic".

Apoptotic cells activate the "phoenix rising" pathway to promote wound healing and tissue regeneration.

The ability to regenerate damaged tissues is a common characteristic of multicellular organisms. We report a role for apoptotic cell death in promoting wound healing and tissue regeneration in mice. Apoptotic cells released growth signals that stimulated the proliferation of progenitor or stem cells. Key players in this process were caspases 3 and 7, proteases activated during the execution phase of apoptosis that contribute to cell death. Mice lacking either of these caspases were deficient in skin wound healing and in liver regeneration. Prostaglandin E(2), a promoter of stem or progenitor cell proliferation and tissue regeneration, acted downstream of the caspases. We propose to call the pathway by which executioner caspases in apoptotic cells promote wound healing and tissue regeneration in multicellular organisms the "phoenix rising" pathway.

G2-phase chromosomal radiosensitivity of primary fibroblasts from hereditary retinoblastoma family members and some apparently normal controls.

We previously described an enhanced sensitivity for cell killing and gamma-H2AX focus induction after both high-dose-rate and continuous low-dose-rate gamma irradiation in 14 primary fibroblast strains derived from hereditary-type retinoblastoma family members (both affected RB1(+/-) probands and unaffected RB1(+/+) parents). Here we present G(2)-phase chromosomal radiosensitivity assay data for primary fibroblasts derived from these RB family members and five Coriell cell bank controls (four apparently normal individuals and one bilateral RB patient). The RB family members and two normal Coriell strains had significantly higher ( approximately 1.5-fold, P < 0.05) chromatid-type aberration frequencies in the first postirradiation mitosis after doses of 50 cGy and 1 Gy of (137)Cs gamma radiation compared to the remaining Coriell strains. The induction of chromatid-type aberrations by high-dose-rate G(2)-phase gamma irradiation is significantly correlated to the proliferative ability of these cells exposed to continuous low-dose-rate gamma irradiation (reported in Wilson et al., Radiat. Res. 169, 483-494, 2008). Our results suggest that these moderately radiosensitive individuals may harbor hypomorphic genetic variants in genomic maintenance and/or DNA repair genes or may carry epigenetic changes involving genes that more broadly modulate such systems, including G(2)-phase-specific DNA damage responses.

Variations in radiosensitivity among individuals: a potential impact on risk assessment?

To have an impact on risk assessment for purposes of radiation protection recommendations, significantly broad variations in carcinogenic radiosensitivity would have to exist in significant proportions in the human population. Even if we knew all the genes where mutations would have major effects, individual genome sequencing does not seem useful, since we do not know all these genes, nor can we be certain of the phenotypic effect of polymorphisms discovered. Further, sequencing would not reveal epigenetic changes in gene expression. Another approach to develop phenotypic biomarkers for cells or tissues for which variations in radiation response may reflect the variations in carcinogenic sensitivity. To be useful, experimental evidence for such a correlation would be crucial, and it is also evident that correlations may be tissue or tumor specific. Some cellular markers are discussed that have shown promise in this regard. They include chromosome aberration induction and DNA repair assays that are sufficiently sensitive to measure after modest or low doses or dose rates. To this end we summarize here some of these assays and review the results of a number of experiments from our laboratory that show clear differences in DNA repair capacity reflected by gamma-H2AX foci formation in cells from a high proportion (perhaps 1/3) of apparently normal individuals. A low dose-rate assay was used to amplify such differences. Another promising assay combines G(2) chromosomal radiosensitivity with the above gamma-H2AX foci on mitotic chromosomes. There are other potentially useful assays as well.

Incidence of acute myeloid leukemia and hepatocellular carcinoma in mice irradiated with 1 GeV/nucleon (56)Fe ions.

Abstract Estimates of cancer risks posed to space-flight crews by exposure to high atomic number, high-energy (HZE) ions are subject to considerable uncertainty because epidemiological data do not exist for human populations exposed to similar radiation qualities. We assessed the leukemogenic efficacy of one such HZE species, 1 GeV (56)Fe ions, a component of space radiation, in a mouse model for radiation-induced acute myeloid leukemia. CBA/CaJ mice were irradiated with 1 GeV/nucleon (56)Fe ions or (137)Cs gamma rays and followed until they were moribund or to 800 days of age. We found that 1 GeV/nucleon (56)Fe ions do not appear to be substantially more effective than gamma rays for the induction of acute myeloid leukemia (AML). However, (56)Fe-ion-irradiated mice had a much higher incidence of hepatocellular carcinoma (HCC) than gamma-irradiated mice, with an estimated RBE of approximately 50. These data suggest a difference in the effects of HZE iron ions on the induction of leukemia compared to solid tumors, suggesting potentially different mechanisms of tumorigenesis.