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Publications (69)
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 i...
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 attra...
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 methodolo...
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 e...
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 in...
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 cellu...
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 c...
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 y...
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 st...
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 s...
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 implicat...
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 i...
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 im...
We investigated the effect of caffeine on low dose γ-radiation-induced chromosomal damage in human T-cell leukaemia cells (Jurkat T-cells) and two normal human fibroblast cell lines (AG1522 and GM2149). Low doses of gamma-radiation were found to increase the levels of chromatid gaps and breaks in a dose-dependent manner in both normal and cancer ce...
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...
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 chromat...
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...
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)-p...
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,...
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 c...
It has been argued that the cell-cell and cell-matrix interaction networks in normal tissues are disrupted by radiation and that this largely controls many of the most important cellular radiation responses. This has led to the broader assertion that individual cells in normal tissue or a 3D normal-tissue-like culture will respond to radiation very...
We have observed that some of the DNA damage or damage product caused by irradiation of interphase cells persisted throughout the cell cycle, and resulted in the expression of gamma-H2AX foci on the mitotic chromosomes. These mitotic expressions of damage after gamma-irradiation of G1 or G2 phase cells were compared in wild-type CHO and their DNA r...
Since deletion of the PU.1 gene on chromosome 2 is a crucial acute myeloid leukemia (AML) initiating step in the mouse model, we quantified PU.1 deleted cells in the bone marrow of gamma-, X- and 56Fe-ion-irradiated mice at various times postirradiation. Although 56Fe ions were initially some two to three times more effective than X or gamma rays i...
Chromosome aberrations in mitotic bone marrow cells of CBA/Ca and C57BL/6 mice were measured 1 day after exposure to 1 Gy of 1 GeV/nucleon 56Fe ions or 3 Gy of gamma rays. The proportion that have lost a region of chromosome 2 containing the PU.1 gene could be explained by a model based on these measurements. The distribution of aberrations among c...
We previously described an enhanced sensitivity for cell killing and G(1)-phase cell cycle arrest after acute gamma irradiation in primary fibroblast strains derived from 14 hereditary-type retinoblastoma family members (both affected RB1(+/-) probands and unaffected RB1(+/+) parents) as well as distinctive gene expression profiles in unirradiated...
The induction and disappearance of DNA double strand breaks (DSBs) after irradiation of G1 and mitotic cells were compared with the gamma-H2AX foci assay and a gel electrophoresis assay. This is to determine whether cell cycle related changes in chromatin structure might influence the gamma-H2AX assay which depends on extensive phosphorylation and...
We reported previously that the homologous recombinational repair (HRR)-deficient Chinese hamster mutant cell line irs3 (deficient in the Rad51 paralog Rad51C) showed only a 50% spontaneous frequency of sister chromatid exchange (SCE) as compared to parental wild-type V79 cells. Furthermore, when irradiated with very low doses of alpha particles, S...
To determine whether cigarette smoke condensate (CSC) without metabolic activation induces direct DNA double strand breaks (DSB) in the G1 phase of various radiosensitive mutants of CHO cells and whether these breaks display collateral hypersensitivity to CSC with respect to cell killing.
We treated the G1-phase cultures of wild-type and DNA repair...
Cells from unaffected parents of retinoblastoma (RB) patients were previously shown to be hypersensitive to radiation induced G(1) arrest and cell killing [1]. The hypersensitivity was similar to that reported for cells from ATM heterozygotes. The latter was consistent with a mild DNA DSB rejoining defect which we demonstrated using a gamma-H2AX fo...
Many, if not the majority of spontaneous or induced mutations in somatic mammalian cells associated with cancer are large chromosome level changes. For exposure to carcinogenic agents, certain specific chromosomal aberrations are likely to lie early along the pathway leading from initial molecular damage to cancer. The kinds of aberrations that occ...
The hereditary form of retinoblastoma (Rb) is associated with a germ line mutation in one RB allele and is characterized by the occurrence of multiple, bilateral Rb tumors and a predisposition to the development of second cancers. In an earlier study, we observed an unexpected hypersensitivity to ionizing radiation in skin fibroblasts derived from...
Homologous recombinational repair (HRR) restores chromatid breaks arising during DNA replication and prevents chromosomal
rearrangements that can occur from the misrepair of such breaks. In vertebrates, five Rad51 paralogs are identified that contribute
in a nonessential but critical manner to HRR proficiency. We constructed and characterized a kno...
DNA fragmentation is a hallmark of apoptosis (programmed cell death). However, the biological function of apoptotic DNA fragmentation remains unclear. Here, we show that DNA fragmentation factor plays an important role for maintaining genomic stability. Inhibition or loss of the DNA fragmentation factor (DFF)/caspase-activated DNase (CAD), whose nu...
Basic to virtually all relevant biological effects of ionizing radiation is the underlying damage produced in DNA and the subsequent cellular processing of such damage. The damage can be qualitatively different for different kinds of radiations, and the genetics of the biological systems exposed can greatly affect damage processing and ultimate out...
Previous reports have suggested a connection between reduced levels of the catalytic subunit of DNA-dependent protein kinases (DNA-PKcs), a component of the nonhomologous DNA double-strand breaks end-joining system, and a reduction in ATM. We studied this possible connection in other DNA-PKcs-deficient cell types, and following knockdown of DNA-PKc...
We have developed a method to suppress the PCR amplification of repetitive sequences in whole chromosome painting probes by adding Cot-1 DNA to the amplification mixture. The repetitive sequences in the Cot-1 DNA bind to their homologous sequences in the probe library, prevent the binding of primers, and interfere with extension of the probe sequen...
The radiosensitizing effect of a phosphoinositide-3 kinase (PI3K) inhibitor, wortmannin, has been studied rather extensively, but there have been few studies on the radiosensitizing effect of another PI3K inhibitor, LY294002. In this report, we present the radiosensitizing effect of LY294002 using normal human cells. Clonogenic cell survival indica...
RNA interference, discovered only five years ago, is an important system for the control of gene expression. It is also quickly becoming a valuable experimental tool as it allows investigators to knock down the level of expression of specific genes. In this paper, we review some applications of this technology for studies in DNA damage processing,...
Radiation-induced genomic instability has been proposed as a very early, if not an initiating, step in radiation carcinogenesis. Numerous studies have established the occurrence of radiation-induced chromosomal instability in various cells of both human and rodent origin. In many of these studies, however, the cells were not "normal" initially, and...
Chinese hamster ovary (CHO-K1) cell line and two of its DNA double strand break (DSB) repair deficient mutant cell lines, xrs-5 (Ku80 mutant) and irs-20 (DNA-PKcs mutant), were treated with various concentrations of sodium arsenite for 2.5h, and the colony forming abilities were studied. The wild type cells showed the highest cell survival, while x...
DNA double strand breaks (DSBs), induced by gamma-irradiation in Chinese hamster ovary cells, were used to examine whether antimony compounds affect the repair of DNA damage. The cells were first incubated with antimony trichloride or antimony potassium tartrate (both Sb(III)) for 2 h, and then irradiated with gamma-rays at a dose of 40 Gy. The DNA...
Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subu...
Around 30 years ago, a very prominent molecular biologist confidently proclaimed that nothing of fundamental importance has ever been learned by irradiating cells! The poor man obviously did not know about discoveries such as DNA repair, mutagenesis, connections between mutagenesis and carcinogenesis, genomic instability, transposable genetic eleme...
The purpose of this study was to measure and compare the effect of d-alpha-tocopheryl succinate (alpha-TS) in modifying radiation-induced chromosomal damage in human normal cells and cancer cells in culture.
Three human normal fibroblast cell lines (GM2149, AG1522 and HF19) and three human cancer cell lines, cervical cancer (HeLa) and ovarian carci...
Bedford, J. S. and Dewey, W. C. Historical and Current Highlights in Radiation Biology: Has Anything Important Been Learned by Irradiating Cells? Radiat. Res. 158, 251–291 (2002). Around 30 years ago, a very prominent molecular biologist confidently proclaimed that nothing of fundamental importance has ever been learned by irradiating cells! The po...
In order to clarify the mechanism underlying the combined effects of radiation and metals, we have studied the cytotoxicity and inhibition of radiation-induced DNA double-strand-break (DNA-DSB) repair by various metals and metalloids. CHO cells were treated with NiCl2, NaAsO2, ZnCl2, CdCl2, CuCl2, and potassium antimonyl tartrate for 2 h, and irrad...
Somatic cell mutants can be informative in the analysis of a wide variety of cellular processes. The use of map-based positional cloning strategies in somatic cell hybrids to analyze genes responsible for recessive mutant phenotypes is often tedious, however, and remains a major obstacle in somatic cell genetics. To fulfill the need for more effici...
It has been known for many years that caffeine reduces or eliminates the G2-phase cell cycle delay normally seen in human HeLa cells or Chinese hamster ovary (CHO) cells after exposure to X or gamma rays. In light of our recent demonstration of a consistent difference between human normal and tumor cells in a G2-phase checkpoint response in the pre...
Jha, M. N., Bamburg, J. R., Bernstein, B. W. and Bedford, J. S. Caffeine Eliminates Gamma-Ray-Induced G2-Phase Delay in Human Tumor Cells but Not in Normal Cells. Radiat. Res. 157, 26–31 (2002). It has been known for many years that caffeine reduces or eliminates the G2-phase cell cycle delay normally seen in human HeLa cells or Chinese hamster ova...
We have studied the efficiency of DNA double strand break (DSB) rejoining in primary cells from mouse strains that show large differences in in vivo radiosensitivity and tumor susceptibility. Cells from radiosen- sitive, cancer-prone BALB/c mice showed inefficient end joining of g ray-induced DSBs as compared with cells from all of the other common...
Sublethal and potentially lethal damage are abstract terms that originated and were defined without reference to molecular and subcellular entities in which such radiation damage was registered. The establishment of a cause-and-effect relationship between chromosome fragment loss and cell killing by ionizing radiations, along with a substantial bod...
A relatively simple method has been developed to improve the resolution for measuring breaks produced in interphase chromosomes by X rays or other agents following the induction of premature chromosome condensation (PCC). Mitotic HeLa cells, which induce PCC when fused with interphase cells, were obtained from cultures grown for several generations...
We show that a recombinational repair mechanism for DNA lesions can be expected to produce exactly the types of exceptions to the usually observed semiconservative segregation of newly synthetized DNA that have been reported in the literature. This removes the obstacles their occurrence appearance to present to the interpretation that the eukaryote...
•A general model for the production of aberrations in eukaryote chromosomes is proposed. The basic assumptions of this model are:•(1) The chromosome is mononeme, containing but a single DNA double helix before replication; (2) Aberrations result only from lesions in DNA; (3) The primary lesions may be either polynucleotide strand breaks or altered...
We have allowed synchronized V79B Chinese hamster tissue culture cells to incorporate 5-bromodeoxyuridine (BUdR) during one DNA synthetic (S) period of the cell cycle and then determined chromosomal aberration yields induced by illumination of the cells with visible light during the succeeding pre- and post-DNA-synthetic (G1 and G2) phases of the c...
Chromosomal inversions resulting from symmetrical chromosome-type exchanges have been shown to occur at similar high frequencies to their asymmetrical counterpart, i.e. interstitial chromosomal deletions (1). Despite their perceived importance, the ability to detect intrachromosomal inversions and especially small inversions has not kept pace with...
Telomeres are tandem arrays of repetitive G-rich sequence that cap the ends of linear chromosomes. As such, they represent an important line of defense against end-to-end fusions and other untoward acts of recombination. In that sense, their ability to function properly may be viewed as diagnostic of genomic stability -or lack thereof. Accurate rep...
