DNA-PKcs and ATM influence generation of ionizing radiation-induced bystander signals.

The phenomenon by which irradiated cells influence non-irradiated neighboring cells, referred to as the bystander effect (BSE), is not well understood in terms of the underlying pathways involved. We sought to enlighten connections between DNA damage repair and the BSE. Utilizing sister chromatid exchange (SCE) frequencies as a marker of the BSE, we performed cell transfer strategies that enabled us to distinguish between generation versus reception of a bystander signal. We find that DNA-dependent Protein Kinase catalytic subunit (DNA-PKcs) and Ataxia Telangectasia Mutated (ATM) are necessary for the generation of such a bystander signal in normal human cells following gamma (gamma)-ray exposure, but are not required for its reception. Importantly, we also show that directly irradiated human cells do not respond to receipt of a bystander signal, helping to explain why the BSE is a low-dose phenomenon. These studies provide the first evidence for a role of the DNA damage response proteins DNA-PKcs and ATM specifically in the generation of a bystander signal and intercellular signaling.

Ultra-violet light induced changes in DNA dynamics may enhance TT-dimer recognition.

Short-wave ultra-violet light promotes the formation of DNA dimers between adjacent thymine bases, and if unrepaired these dimers may induce skin cancer. Living cells have a very robust repair system capable of repairing hundreds of lesions every day. Although many of the details of the dimer repair mechanism are known, it is still a mystery how the dimers are recognized. Because the dimers are hidden from repair proteins diffusing in the cell nucleus, it has been surmised that dimer recognition is indirect. In this paper, a new recognition signal is suggested by a theory of the dimer-induced large amplitude, prolonged oscillations in the motion of the two strands in double-stranded DNA molecules. These large amplitude oscillations of the two DNA strands, localized around the dimer will unveil the dimer allowing the repair proteins to bind to the dimer site. The temperature dependence of the recognition rate is correlated with the inter-strand fluctuations and must decrease with decreasing temperature according to the findings in this paper. Moreover the probability for finding a large opening is localized to the dimer neighbourhood and these large openings may play an important role in dimer-repair protein biochemistry.

Strand-specific postreplicative processing of mammalian telomeres.

Telomeres are specialized nucleoprotein structures that stabilize the ends of linear eukaryotic chromosomes. In mammalian cells, abrogation of telomeric repeat binding factor TRF2 or DNA-dependent protein kinase (DNA-PK) activity causes end-to-end chromosomal fusion, thus establishing an essential role for these proteins in telomere function. Here we show that TRF2-mediated end-capping occurs after telomere replication. The postreplicative requirement for TRF2 and DNA-PKcs, the catalytic subunit of DNA-PK, is confined to only half of the telomeres, namely, those that were produced by leading-strand DNA synthesis. These results demonstrate a crucial difference in postreplicative processing of telomeres that is linked to their mode of replication.

DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes.

Recent findings intriguingly place DNA double-strand break repair proteins at chromosome ends in yeast, where they help maintain normal telomere length and structure. In the present study, an essential telomere function, the ability to cap and thereby protect chromosomes from end-to-end fusions, was assessed in repair-deficient mouse cell lines. By using fluorescence in situ hybridization with a probe to telomeric DNA, spontaneously occurring chromosome aberrations were examined for telomere signal at the points of fusion, a clear indication of impaired end-capping. Telomeric fusions were not observed in any of the repair-proficient controls and occurred only rarely in a p53 null mutant. In striking contrast, chromosomal end fusions that retained telomeric sequence were observed in nontransformed DNA-PK(cs)-deficient cells, where they were a major source of chromosomal instability. Metacentric chromosomes created by telomeric fusion became even more abundant in these cells after spontaneous immortalization. Restoration of repair proficiency through transfection with a functional cDNA copy of the human DNA-PK(cs) gene reduced the number of fusions compared with a negative transfection control. Virally transformed cells derived from Ku70 and Ku80 knockout mice also displayed end-to-end fusions. These studies demonstrate that DNA double-strand break repair genes play a dual role in maintaining chromosomal stability in mammalian cells, the known role in repairing incidental DNA damage, as well as a new protective role in telomeric end-capping.

Alpha particles induce the production of interleukin-8 by human cells.

The pulmonary microenvironment is a primary target for alpha particles like those emitted by inhaled radon and its progeny. While exposure to alpha particles has recently been associated with the generation of extracellular and intracellular reactive oxygen species (ROS; Cancer Res. 57, 3963-3971, 1997), little is known about how exposure to alpha particles may affect the generation of oxidative stress-related mediators in the respiratory tract. Interleukin-8 (IL8) is a cytokine recognized for its potent role as a chemoattractant and activator of polymorphonuclear leukocytes. Oxidative stress can up-regulate expression of the gene that encodes IL8 (IL8) in a variety of cell types. In this study, we set out to investigate a potential linkage between the generation of ROS and production of IL8 in alpha-particle-irradiated normal human lung fibroblasts. ELISA revealed that exposure of the fibroblasts to low doses of alpha particles (3.6-19 cGy) caused significant increases in generation of the IL8 protein as early as 30 min after irradiation. Northern blot analyses revealed that such increases were associated with increased IL8 mRNA levels. Cells exposed to alpha particles in the presence of antioxidants, i.e. superoxide dismutase and dimethyl sulfoxide, resulted in significant decreases in extracellular IL8 protein levels. Similar results were obtained with cells treated with dexamethasone, an inhibitor of transcription. Our results indicate that alpha-particle-induced increases in production of IL8 occur temporally in parallel with elevated production of ROS. Conceivably, such production of IL8 induced by alpha particles may contribute to an inflammatory response in the lower respiratory tract. Additionally, the promitogenic effects of IL8 may be a factor in hyperplastic responses in the airway epithelial cells to inhaled radon and radon progeny and perhaps other stresses associated with ROS.

A new mechanism for DNA alterations induced by alpha particles such as those emitted by radon and radon progeny.

The mechanism(s) by which alpha (alpha) particles like those emitted from inhaled radon and radon progeny cause their carcinogenic effects in the lung remains unclear. Although direct nuclear traversals by alpha-particles may be involved in mediating these outcomes, increasing evidence indicates that a particles can cause alterations in DNA in the absence of direct hits to cell nuclei. Using the occurrence of excessive sister chromatid exchanges (SCE) as an index of DNA damage in human lung fibroblasts, we investigated the hypothesis that alpha-particles may induce DNA damage through the generation of extracellular factors. We have found that a relatively low dose of alpha-particles can result in the generation of extracellular factors, which, upon transfer to unexposed normal human cells, can cause excessive SCE to an extent equivalent to that observed when the cells are directly irradiated with the same irradiation dose. A short-lived, SCE-inducing factor(s) is generated in alpha-irradiated culture medium containing serum in the absence of cells. A more persistent SCE-inducing factor(s), which can survive freeze-thaw and is heat labile is produced by fibroblasts after exposure to the alpha-particles. These results indicate that the initiating target for alpha-particle-induced genetic changes can be larger than a cell's nucleus or even a whole cell. How transmissible factors like those observed here in vitro may extend to the in vivo condition in the context of a-particle-induced carcinogenesis in the respiratory tract remains to be determined.

Alpha particles initiate biological production of superoxide anions and hydrogen peroxide in human cells.

The mechanism(s) by which high-linear energy transfer a particles, like those emitted by inhaled radon and radon daughters, cause lung cancer has not been elucidated. Conceivably, DNA damage that is induced by a particles may be mediated by the metabolic generation of reactive oxygen species (ROS), in addition to direct a particle-DNA interactions and hydroxyl radical-DNA interactions. Using normal human lung fibroblasts, we investigated the hypothesis that densely ionizing alpha particles may induce the intracellular generation of superoxide (O2.-) and hydrogen peroxide (H2O2). Ethidium bromide and 2',7'-dichlorofluorescein, fluorescent products of the membrane-permeable dyes hydroethidine and 2',7'-dichlorofluorescin diacetate, respectively, were used to monitor the intracellular production of O2.- and H2O2, respectively, by flow cytometry. Compared to sham-irradiated cells, fibroblasts that were exposed to alpha particles (0.4-19 cGy) had significant increases in intracellular O2.- production, along with concomitant increases in H2O2 production. Further analyses suggest that the plasma membrane-bound NADPH-oxidase is primarily responsible for this increased intracellular generation of ROS and that the ROS response does not require direct nuclear or cellular "hits" by the a particles. In this latter regard, we additionally report that unirradiated cells also show the ROS response when they are incubated with serum-containing culture medium that has been exposed to a particles or when they are incubated with supernatants from a-irradiated cells. Our overall results support the possibility that a particles, at least in part, may mediate their DNA-damaging effects indirectly via a ROS-related mechanism.

Extracellular factor(s) following exposure to alpha particles can cause sister chromatid exchanges in normal human cells.

The mechanism(s) by which alpha particles like those emitted from inhaled radon and radon progeny cause their mutagenic and carcinogenic effects remains unclear. Although direct nuclear traversals by alpha particles may be involved in mediating these outcomes, increasing evidence indicates that alpha particles can cause alterations in DNA in the absence of direct "hits" to cell nuclei. Using the occurrence of excessive sister chromatid exchanges (SCEs) as an index of DNA damage in human lung fibroblasts, we investigated the hypothesis that alpha particles may induce DNA damage via the generation of extracellular factors. We have found that a relatively low dose of alpha particles indeed results in the generation of extracellular factors, which, upon transfer to unexposed normal human cells, can cause excessive SCEs to an extent equivalent to that observed when the cells are directly irradiated with the same irradiation dose. A short-lived, SCE-inducing factor(s) was generated in alpha-irradiated culture medium containing serum in the absence of cells; it was found that the activity of this factor can be promptly inhibited by superoxide dismutase. A more persistent SCE-inducing factor(s), which can survive freeze-thawing, is heat labile and also can be inhibited by superoxide dismutase, was produced by fibroblasts after exposure to alpha particles. These results indicate that the initiating target for alpha-particle-induced genetic changes can be larger than the nuclear compartment alone and even larger than the cytoplasmic compartment. How transmissible factors like those observed here in vitro may extend to the in vivo condition in the context of alpha-particle-induced carcinogenesis in the respiratory tract and elsewhere remains to be determined.

Rejoining and misrejoining of radiation-induced chromatin breaks. II. Biophysical Model.

A biophysical model for the kinetics of the formation of radiation-induced chromosome aberrations is developed to account for the recent experimental results obtained with a combination of the premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH) techniques. In this model, we consider the broken ends of DNA double-strand breaks (DSBs) to be reactant and make use of the interaction distance hypothesis. The repair/misrepair process between broken ends is suggested to consist of two steps; the first step represents the two break ends approaching each other, and the second step represents the enzymatic processes leading to DNA end-to-end rejoining. Only the second step is reflected in the kinetics observed in experiments using PCC. The model appears to be able to fit existing data for human cells. It is shown that the kinetics of the formation of chromosome aberrations can be explained by a single rate that characterizes both rejoining and misrejoining of DSBs, suggesting that repair and misrepair share the same mechanism. Fast repair (completed in minutes) in a subset of DSBs is suggested as an explanation of the complete exchanges observed with PCC in human lymphocytes immediately after irradiation. The fast repair component seems to be absent in human fibroblasts.

Transmission of radiation-induced acentric chromosomal fragments to micronuclei in normal human fibroblasts.

A simplifying assumption made when calculating the probability of a chromosomal aberration resulting in a micronucleus is that virtually all radiation-induced micronuclei result from acentric fragments. In the present study we used antibodies to chromosomal centromeres (kinetochores) to determine the frequency of centric versus acentric micronuclei in normal human fibroblasts exposed to 6 Gy of 60Co gamma rays while they were in density-inhibited growth. Up to 14% of the micronuclei induced by this exposure contained one or more kinetochores; i.e., they were not composed of acentric chromatin. By deleting kinetochore-positive micronuclei from the analysis, and by reconstructing micronucleus frequencies based on the fraction of cells that had divided following radiation exposure, a direct comparison between micronuclei and acentric chromosome fragments was made. On that basis, the probability of an acentric fragment becoming a visible micronucleus in either daughter cell of a dividing pair was estimated to be about 0.6. The distribution of acentric fragments among mitotic cells conformed to Poisson expectation, while the distribution of micronuclei among daughter cells was significantly overdispersed. The phenomenon of overdispersion is discussed in connection with proposed cellular processes that effect a nonrandom segregation of acentric fragments.

Cell-cycle dependence of X-ray oxygen effect: role of endogenous glutathione.

The oxygen effect was measured in human T-1 cell populations synchronized by mitotic selection and x-irradiated in vitro after they were allowed to progress to six different ages during the division cycle. Survival curves and dose-ratio calculations with 95% confidence intervals were obtained from computer fits of the data to the linear-quadratic model. The oxygen enhancement ratio (OER) values at the 1% survival increased level were 2.6 +/- 0.08 in G1/early S phase and increased to 3.0 +/- 0.15 in late S/G2 phase. The OER values at 10% survival increased linearly from 2.6 +/- 0.2 for G1-phase cells to 3.2 +/- 0.2 for late S/G2-phase cells. The increased OER in S-phase cells was the result of a greater hypoxic radioresistance compared with that measured with G1-phase cells. In parallel experiments with synchronized cell populations, glutathione (GSH) and glutathione disulfide levels were measured by the Tietze assay and also were found to increase over the same period. The molecular mechanisms responsible for the radiation response involve a number of factors, one of which in this cell line may be GSH levels, especially under conditions of hypoxic exposure. Our data are consistent with the hypothesis that G1- to late S-phase, age-dependent fluctuations in GSH content may be correlated with changes in OER during the human T-1 cell cycle. Changes in GSH content relative to its constitutive levels in the cell and alternative reductive factors (i.e., protein thiols), as well as their cellular location, may be important factors in the comparison of these findings to other cell lines.