Mechanisms of increased risk of tumorigenesis in Atm and Brca1 double heterozygosity

<p>Abstract</p> <p>Background</p> <p>Both epidemiological and experimental studies suggest that heterozygosity for a single gene is linked with tumorigenesis and heterozygosity for two genes increases the risk of tumor incidence. Our previous work has demonstrated that <it>Atm/Brca1 </it>double heterozygosity leads to higher cell transformation rate than single heterozygosity. However, the underlying mechanisms have not been fully understood yet. In the present study, a series of pathways were investigated to clarify the possible mechanisms of increased risk of tumorigenesis in <it>Atm </it>and <it>Brca1 </it>heterozygosity.</p> <p>Methods</p> <p>Wild type cells, <it>Atm </it>or <it>Brca1 </it>single heterozygous cells, and <it>Atm</it>/<it>Brca1 </it>double heterozygous cells were used to investigate DNA damage and repair, cell cycle, micronuclei, and cell transformation after photon irradiation.</p> <p>Results</p> <p>Remarkable high transformation frequency was confirmed in <it>Atm</it>/<it>Brca1 </it>double heterozygous cells compared to wild type cells. It was observed that delayed DNA damage recognition, disturbed cell cycle checkpoint, incomplete DNA repair, and increased genomic instability were involved in the biological networks. Haploinsufficiency of either ATM or BRCA1 negatively impacts these pathways.</p> <p>Conclusions</p> <p>The quantity of critical proteins such as ATM and BRCA1 plays an important role in determination of the fate of cells exposed to ionizing radiation and double heterozygosity increases the risk of tumorigenesis. These findings also benefit understanding of the individual susceptibility to tumor initiation.</p

Genetic Control of the Trigger for the G2/M Checkpoint

The work undertaken in this project addressed two seminal areas of low dose radiation biology that are poorly understood and controversial. These areas are the challenge to the linear-no-threshold (LNT) paradigm at low doses of radiation and, the fundamental elements of radiation bystander effect biology Genetic contributions to low dose checkpoint engagement: The LNT paradigm is an extrapolation of known, measured cancer induction endpoints. Importantly, data for lower doses is often not available. Debatably, radiation protection standards have been introduced which are prudently contingent on the adherence of cancer risk to the established trend seen at higher doses. Intriguing findings from other labs have hinted at separate DNA damage response programs that engage at low or high levels of radiation. Individual radiation sensitivity commensurate with hemizygosity for a radiation sensitivity gene has been estimated at 1-2% in the U.S.. Careful interrogation of the DNA damage response at low doses of radiation became important and served as the basis for this grant. Several genes were tested in combinations to determine if combined haploinsufficiency for multiple radiosensitizing genes could render a cell more sensitive to lower levels of acute radiation exposure. We measured a classical radiation response endpoint, cell cycle arrest prior to mitosis. Mouse embryo fibroblasts were used and provided a uniform, rapidly dividing and genetically manipulable population of study. Our system did not report checkpoint engagement at acute doses of gamma rays below 100 mGy. The system did report checkpoint engagement reproducibly at 500 mGy establishing a threshold for activation between 100 and 500 mGy. Engagement of the checkpoint was ablated in cells nullizygous for ATM but was otherwise unperturbed in cells combinatorially haploinsufficient for ATM and Rad9, ATM and PTEN or PTEN and Rad9. Taken together, these experiments tell us that, in a sensitive fibroblast culture system, the engagement of the G2/M checkpoint only occurs at doses where most of the cells are bound for mitotic catastrophe. Further, compound haploinsufficiency of various radiosensitizing genes does not impact the threshold of activation. The experiments confirm a threshold of activation for the G2/M checkpoint, hinting at two separate radiation response programs acting below and above this threshold. Small RNA transfer in bystander effect biology: Small regulatory RNA molecules have now risen in prominence and utility. Specific examples are small interfering RNAs (siRNA) which are employed in cell level expression ablation projects and micro-RNAs (miRNA) which are a pool of short transcription products which serve to modulate the expression of other transcripts emerging from the genome in a meta-regulatory fine tuning of gene expression. The existing tenets of bystander effect radiation biology involve the communication of inflammatory mediators or direct intercellular communication of reactive oxygen/nitrogen species in cell-to-cell communicative organelles called gap junctions. By ablating gap junctions, reducing the ROS/inflammatory cytokine expression one can attenuate bystander effect signaling in cell culture systems. We hypothesized that miRNAs are a competent intercellular communication molecule and therefore a possible component of the bystander response. This view is supported by the observation that miRNA are secreted from cells in exosomes found in the circulation. This circulating pool reports disease type and severity in humans. We proposed use of microbeam irradiation technology at our facilities and enhancement of this capability with a new sorting technology which would allow us to sort irradiated and non-irradiated cells with absolute fidelity. Pursuing direct quantitative transfer assessment, we succeeded in designing and constructing a new add-on sorting appliance which harmonized with our existing instruments. The sorter allowed us to gently sort single fluorescently labeled cells. The plans for this appliance were published and are now available for use in other laboratories for single-cell analyses. Our microfluidic cell sorting modality is being integrated into subsequent microbeam irradiation experiments that are planned and ongoing. We generated and irradiated pools of specially engineered Donor-Recipient cell lines in co-culture that would report a small RNA transfer event by modulation of fluorescent protein expression. Both induction and reciprocal silencing designs were tested. We observed elevation of miRNA/siRNA transfer in response to radiation at doses of 5Gy in experiments to date. The reproducibility of these findings has not been good. Future studies will involve refinement of the reporting systems and a decrease in acute dose of radiation used to determine the lowest dose at which miRNA transfer between cells contributes to radiation bystander effect biology

A role for Syk-kinase in the control of the binding cycle of the β2 integrins (CD11/CD18) in human polymorphonuclear neutrophils

A fine control of β2 integrin (CD11/CD18)-mediated firm adhesion of human neutrophils to the endothelial cell monolayer is required to allow ordered emigration. To elucidate the molecular mechanisms that control this process, intracellular protein tyrosine signaling subsequent to β2 integrin-mediated ligand binding was studied by immunoprecipitation and Western blotting techniques. The 72-kDa Syk-kinase, which was tyrosine-phosphorylated upon adhesion, was found to coprecipitate with CD18, the β-subunit of the β2 integrins. Moreover, inhibition of Syk-kinase by piceatannol enhanced adhesion and spreading but diminished N-formyl-Met-Leu-Phe-induced chemotactic migration. The enhancement of adhesiveness was associated with integrin clustering, which results in increased integrin avidity. In contrast, piceatannol had no effect on the surface expression or on the affinity of β2 integrins. Altogether, this suggests that Syk-kinase controls alternation of β2 integrin-mediated ligand binding with integrin detachment

207-nm UV Light—A Promising Tool for Safe Low-Cost Reduction of Surgical Site Infections. II: In-Vivo Safety Studies

Background UVC light generated by conventional germicidal lamps is a well-established anti-microbial modality, effective against both bacteria and viruses. However, it is a human health hazard, being both carcinogenic and cataractogenic. Earlier studies showed that single-wavelength far-UVC light (207 nm) generated by excimer lamps kills bacteria without apparent harm to human skin tissue in vitro. The biophysical explanation is that, due to its extremely short range in biological material, 207 nm UV light cannot penetrate the human stratum corneum (the outer dead-cell skin layer, thickness 5–20 μm) nor even the cytoplasm of individual human cells. By contrast, 207 nm UV light can penetrate bacteria and viruses because these cells are physically much smaller. Aims To test the biophysically-based hypothesis that 207 nm UV light is not cytotoxic to exposed mammalian skin in vivo. Methods Hairless mice were exposed to a bactericidal UV fluence of 157 mJ/cm2 delivered by a filtered Kr-Br excimer lamp producing monoenergetic 207-nm UV light, or delivered by a conventional 254-nm UV germicidal lamp. Sham irradiations constituted the negative control. Eight relevant cellular and molecular damage endpoints including epidermal hyperplasia, pre-mutagenic UV-associated DNA lesions, skin inflammation, and normal cell proliferation and differentiation were evaluated in mice dorsal skin harvested 48 h after UV exposure. Results While conventional germicidal UV (254 nm) exposure produced significant effects for all the studied skin damage endpoints, the same fluence of 207 nm UV light produced results that were not statistically distinguishable from the zero exposure controls. Conclusions As predicted by biophysical considerations and in agreement with earlier in vitro studies, 207-nm light does not appear to be significantly cytotoxic to mouse skin. These results suggest that excimer-based far-UVC light could potentially be used for its anti-microbial properties, but without the associated hazards to skin of conventional germicidal UV lamps

Radiation dose-rate effects on gene expression for human biodosimetry

Background: The effects of dose-rate and its implications on radiation biodosimetry methods are not well studied in the context of large-scale radiological scenarios. There are significant health risks to individuals exposed to an acute dose, but a realistic scenario would include exposure to both high and low dose-rates, from both external and internal radioactivity. It is important therefore, to understand the biological response to prolonged exposure; and further, discover biomarkers that can be used to estimate damage from low-dose rate exposures and propose appropriate clinical treatment. Methods: We irradiated human whole blood ex vivo to three doses, 0.56 Gy, 2.23 Gy and 4.45 Gy, using two dose rates: acute, 1.03 Gy/min and a low dose-rate, 3.1 mGy/min. After 24 h, we isolated RNA from blood cells and these were hybridized to Agilent Whole Human genome microarrays. We validated the microarray results using qRT-PCR. Results: Microarray results showed that there were 454 significantly differentially expressed genes after prolonged exposure to all doses. After acute exposure, 598 genes were differentially expressed in response to all doses. Gene ontology terms enriched in both sets of genes were related to immune processes and B-cell mediated immunity. Genes responding to acute exposure were also enriched in functions related to natural killer cell activation and cell-to-cell signaling. As expected, the p53 pathway was found to be significantly enriched at all doses and by both dose-rates of radiation. A support vectors machine classifier was able to distinguish between dose-rates with 100% accuracy using leave-one-out cross-validation. Conclusions: In this study we found that low dose-rate exposure can result in distinctive gene expression patterns compared with acute exposures. We were able to successfully distinguish low dose-rate exposed samples from acute dose exposed samples at 24 h, using a gene expression-based classifier. These genes are candidates for further testing as markers to classify exposure based on dose-rate

Role of ATM in the telomere response to the G-quadruplex ligand 360A

Telomeres are known to prevent chromosome ends from being recognized as DNA double-strand breaks. Conversely, many DNA damage response proteins, including ATM, are thought to participate to telomere maintenance. However, the precise roles of ATM at telomeres remain unclear due to its multiple functions in cell checkpoints and apoptosis. To gain more insights into the role of ATM in telomere maintenance, we determined the effects of the G-quadruplex ligand 360A in various cell lines lacking functional ATM. We showed, by using Fluorescence in situ hybridization (FISH) and Chromosome Orientation-FISH using telomere PNA probes, that 360A induced specific telomere aberrations occurring during or after replication, mainly consisting in sister telomere fusions and also recombinations that involved preferentially the lagging strand telomeres. We demonstrate that ATM reduced telomere instability independently of apoptosis induction. Our results suggest thus that ATM has a direct role in preventing inappropriate DNA repair at telomeres, which could be related to its possible participation to the formation of protected structures at telomeres

Hemizygosity for Atm and Brca1 influence the balance between cell transformation and apoptosis

In recent years data from both mouse models and human tumors suggest that loss of one allele of genes involved in DNA repair pathways may play a central role in genomic instability and carcinogenesis. Additionally several examples in mouse models confirmed that loss of one allele of two functionally related genes may have an additive effect on tumor development. To understand some of the mechanisms involved, we examined the role of monoallelic loss or Atm and Brca1 on cell transformation and apoptosis induced by radiation. Cell transformation and apoptosis were measured in mouse embryo fibroblasts (MEF) and thymocytes respectively. Combinations of wild type and hemizygous genotypes for ATM and BRCA1 were tested in various comparisons. Haploinsufficiency of either ATM or BRCA1 resulted in an increase in the incidence of radiation-induced transformation of MEF and a corresponding decrease in the proportion of thymocytes dying an apoptotic death, compared with cells from wild-type animals. Combined haploinsufficiency for both genes resulted in an even larger effect on apoptosis. Under stress, the efficiency and capacity for DNA repair mediated by the ATM/BRCA1 cell signalling network depends on the expression levels of both proteins

Alterations to nuclear architecture and genome behavior in senescent cells.

The organization of the genome within interphase nuclei, and how it interacts with nuclear structures is important for the regulation of nuclear functions. Many of the studies researching the importance of genome organization and nuclear structure are performed in young, proliferating, and often transformed cells. These studies do not reveal anything about the nucleus or genome in nonproliferating cells, which may be relevant for the regulation of both proliferation and replicative senescence. Here, we provide an overview of what is known about the genome and nuclear structure in senescent cells. We review the evidence that nuclear structures, such as the nuclear lamina, nucleoli, the nuclear matrix, nuclear bodies (such as promyelocytic leukemia bodies), and nuclear morphology all become altered within growth-arrested or senescent cells. Specific alterations to the genome in senescent cells, as compared to young proliferating cells, are described, including aneuploidy, chromatin modifications, chromosome positioning, relocation of heterochromatin, and changes to telomeres

New Insight into Intrachromosomal Deletions Induced by Chrysotile in the gpt delta Transgenic Mutation Assay

BACKGROUND: Genotoxicity is often a prerequisite to the development of malignancy. Considerable evidence has shown that exposure to asbestos fibers results in the generation of chromosomal aberrations and multilocus mutations using various in vitro approaches. However, there is less evidence to demonstrate the contribution of deletions to the mutagenicity of asbestos fibers in vivo. OBJECTIVES: In the present study, we investigated the mutant fractions and the patterns induced by chrysotile fibers in gpt delta transgenic mouse primary embryo fibroblasts (MEFs) and compared the results obtained with hydrogen peroxide (H(2)O(2)) in an attempt to illustrate the role of oxyradicals in fiber mutagenesis. RESULTS: Chrysotile fibers induced a dose-dependent increase in mutation yield at the redBA/gam loci in transgenic MEF cells. The number of λ mutants losing both redBA and gam loci induced by chrysotiles at a dose of 1 μg/cm(2) increased by > 5-fold relative to nontreated controls (p < 0.005). Mutation spectra analyses showed that the ratio of λ mutants losing the redBA/gam region induced by chrysotiles was similar to those induced by equitoxic doses of H(2)O(2). Moreover, treatment with catalase abrogated the accumulation of γ-H2AX, a biomarker of DNA double-strand breaks, induced by chrysotile fibers. CONCLUSIONS: Our results provide novel information on the frequencies and types of mutations induced by asbestos fibers in the gpt delta transgenic mouse mutagenic assay, which shows great promise for evaluating fiber/particle mutagenicity in vivo

Focal Adhesion Remodeling Is Crucial for Glucose-Stimulated Insulin Secretion and Involves Activation of Focal Adhesion Kinase and Paxillin

Actin cytoskeleton remodeling is known to be involved in glucose-stimulated insulin secretion (GSIS). We have observed glucose-stimulated changes at the β-cell basal membrane similar to focal adhesion remodeling in cell migration. This led us to study the role of two key focal adhesion proteins, focal adhesion kinase (FAK) and paxillin, in GSIS