THE HERPESVIRUS NUCLEAR EGRESS COMPLEX COMPONENT, UL31, IS RECRUITED TO SITES OF DNA DAMAGE THROUGH POLY(ADP-RIBOSE) BINDING

The herpes simplex virus (HSV) UL31 gene encodes a conserved member of the herpesvirus nuclear egress complex that not only functions in the egress of DNA-containing capsids from the nucleus, but is also required for optimal viral genome expression, replication and packaging into capsids. Here, we report that the UL31 protein from HSV-2 and the orthologous protein, ORF69, from Kaposi's sarcoma-associated herpesvirus (KSHV) are recruited to sites of DNA damage. Recruitment of UL31 to sites of DNA damage occurred in HSV-2 infected cells, but did not require other viral proteins. The N-terminus of UL31 contains sequences resembling a poly(ADP-ribose) (PAR) binding motif. As protein poly-ADP ribosylation (PARylation) is a hallmark of the DNA damage response we examined the relationship between PARylation and UL31 recruitment to DNA damage. While the PAR polymerase (PARP)1/2 inhibitor, olaparib, prevented UL31 recruitment to damaged DNA, KU55933 inhibition of signaling through the ataxia telangiectasia mutated (ATM) DNA damage response pathway had no effect. These findings were further supported by experiments demonstrating direct and specific interaction between HSV-2 UL31 and PAR using purified components. Co-transfection with the viral kinase Us3, known to phosphorylate UL31, inhibited UL31 recruitment to DNA damage but also prevented the recruitment of other proteins recruited to DNA damage sites. The viral E3 ubiquitin ligase ICP0 was observed to co-localize with UL31 in transfected cells in a manner that is independent of the PAR-binding ability of UL31. However, inhibition of PARP1/2/3 did not reduce the ability of HSV-2 to replicate and we observed reduced PAR levels in the nuclei of infected cells. This study reveals a previously unrecognized function for UL31 orthologs and may suggest that the recognition of PAR by UL31 is coupled to the nuclear egress of herpesvirus capsids, influences viral DNA replication and packaging, or possibly modulates the DNA damage response mounted by virally infected cells.

Activation of STING-Dependent Innate Immune Signaling By S-Phase-Specific DNA Damage in Breast Cancer

BACKGROUND: Previously we identified a DNA damage response-deficient (DDRD) molecular subtype within breast cancer. A 44-gene assay identifying this subtype was validated as predicting benefit from DNA-damaging chemotherapy. This subtype was defined by interferon signaling. In this study, we address the mechanism of this immune response and its possible clinical significance.

METHODS: We used immunohistochemistry (IHC) to characterize immune infiltration in 184 breast cancer samples, of which 65 were within the DDRD subtype. Isogenic cell lines, which represent DDRD-positive and -negative, were used to study the effects of chemokine release on peripheral blood mononuclear cell (PBMC) migration and the mechanism of immune signaling activation. Finally, we studied the association between the DDRD subtype and expression of the immune-checkpoint protein PD-L1 as detected by IHC. All statistical tests were two-sided.

RESULTS: We found that DDRD breast tumors were associated with CD4+ and CD8+ lymphocytic infiltration (Fisher's exact test P < .001) and that DDRD cells expressed the chemokines CXCL10 and CCL5 3.5- to 11.9-fold more than DNA damage response-proficient cells (P < .01). Conditioned medium from DDRD cells statistically significantly attracted PBMCs when compared with medium from DNA damage response-proficient cells (P < .05), and this was dependent on CXCL10 and CCL5. DDRD cells demonstrated increased cytosolic DNA and constitutive activation of the viral response cGAS/STING/TBK1/IRF3 pathway. Importantly, this pathway was activated in a cell cycle-specific manner. Finally, we demonstrated that S-phase DNA damage activated expression of PD-L1 in a STING-dependent manner.

CONCLUSIONS: We propose a novel mechanism of immune infiltration in DDRD tumors, independent of neoantigen production. Activation of this pathway and associated PD-L1 expression may explain the paradoxical lack of T-cell-mediated cytotoxicity observed in DDRD tumors. We provide a rationale for exploration of DDRD in the stratification of patients for immune checkpoint-based therapies.

Study of DNA damage with a new system for irradiation of samples in a nuclear reactor

FAPESP:97/5550

DNA damage induced by low energy electrons (LEEs)

Abstract : The major objective of our study is to investigate DNA damage induced by soft X-rays (1.5 keV) and low-energy electrons (˂ 30 eV) using a novel irradiation system created by Prof. Sanche’s group. Thin films of double-stranded DNA are deposited on either glass and tantalum substrates and irradiated under standard temperature and pressure surrounded by a N[subscript 2] environment. Base release (cytosine, thymine, adenine and guanine) and base modifications (8-oxo-7,8-dihydro -2’-deoxyguanosine, 5-hydroxymethyl-2’-deoxyuridine, 5-formyl-2’-deoxyuridine, 5,6-dihydrothymidine and 5,6-dihydro-2’-deoxy uridine) are analyzed and quantified by LC-MS/MS. Our results reveal larger damage yields in the sample deposited on tantalum than those on glass. This can be explained by an enhancement of damage due to low-energy electrons, which are emitted from the metal substrate. From a comparison of the yield of products, base release is the major type of damage especially for purine bases, which are 3-fold greater than base modifications. A proposed pathway leading to base release involves the formation of a transient negative ion (TNI) followed by dissociative electron attachment (DEA) at the N-g lycosidic bond. On the other hand, base modification products consist of two major types of chemical modifications, which include thymine methyl oxidation products that likely arises from DEA from the methyl group of thymine, and 5,6-dihydropyrimidine that can involve the initial addition of electrons, H atoms, or hydride ions to the 5,6-pyrimidine double bond.

Exploiting DNA damage repair defects for effective targeting of acute myeloid leukaemia by PARP inhibitors

Abstract for 24th Biennial Congress of the European Association for Cancer Research, 9–12 July 2016, Manchester, UK. Poster Session: Cancer Genomics, Epigenetics and Genome Instability II: Monday 11 July 2016

Parental exposure to the herbicide diuron results in oxidative DNA damage to germinal cells of the Pacific oyster Crassostrea gigas

Chemical pollution by pesticides has been identified as a possible contributing factor to the massive mortality outbreaks observed in Crassostrea gigas for several years. A previous study demonstrated the vertical transmission of DNA damage by subjecting oyster genitors to the herbicide diuron at environmental concentrations during gametogenesis. This trans-generational effect occurs through damage to genitor-exposed gametes, as measured by the comet-assay. The presence of DNA damage in gametes could be linked to the formation of DNA damage in other germ cells. In order to explore this question, the levels and cell distribution of the oxidized base lesion 8-oxodGuo were studied in the gonads of exposed genitors. High-performance liquid chromatography coupled with UV and electrochemical detection analysis showed an increase in 8-oxodGuo levels in both male and female gonads after exposure to diuron. Immunohistochemistry analysis showed the presence of 8-oxodGuo at all stages of male germ cells, from early to mature stages. Conversely, the oxidized base was only present in early germ cell stages in female gonads. These results indicate that male and female genitors underwent oxidative stress following exposure to diuron, resulting in DNA oxidation in both early germ cells and gametes, such as spermatozoa, which could explain the transmission of diuron-induced DNA damage to offspring. Furthermore, immunostaining of early germ cells seems indicates that damages caused by exposure to diuron on germ line not only affect the current sexual cycle but also could affect future gametogenesis.

Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.

The Herpesvirus Nuclear Egress Complex Component, UL31, is Recruited to Sites of DNA Damage Through Poly(ADP-RIBOSE) Binding

Thesis (Master, Biomedical & Molecular Sciences) -- Queen's University, 2016-08-23 15:03:30.807

Polymorphisms and DNA methylation of gene TP53 associated with extra-axial brain tumors

The p53 tumor suppressor gene is the most frequently mutated gene in human cancer; this gene is mutated in up to 50% of human tumors. It has a critical role in the cell cycle, apoptosis and cell senescence, and it participates in many crucial physiological and pathological processes. Polymorphisms of p53 have been suggested to be associated with genetically determined susceptibility in various types of cancer. Another process involved with the development and progression of tumors is DNA hypermethylation. Aberrant methylation of the promoter is an alternative epigenetic change in genetic mechanisms, leading to tumor suppressor gene inactivation. In the present study, we examined the TP53 Arg72Pro and Pro47Ser polymorphisms using PCR-RFLP and the pattern of methylation of the p53 gene by methylation-specific PCR in 90 extra-axial brain tumor samples. Patients who had the allele Pro of the TP53 Arg72Pro polymorphism had an increased risk of tumor development ( odds ratio, OR = 3.23; confidence interval at 95%, 95% CI = 1.71-6.08; P = 0.003), as did the allele Ser of TP53 Pro47Ser polymorphism (OR = 1.28; 95% CI = 0.03-2.10; P = 0.01). Comparison of overall survival of patients did not show significant differences. In the analysis of DNA methylation, we observed that 37.5% of meningiomas, 30% of schwannomas and 52.6% of metastases were hypermethylated, suggesting that methylation is important for tumor progression. We suggest that TP53 Pro47Ser and Arg72Pro polymorphisms and DNA hypermethylation are involved in susceptibility for developing extra-axial brain tumors.

Chrysotile effects on human lung cell carcinoma in culture: 3-D reconstruction and DNA quantification by image analysis

Background: Chrysotile is considered less harmful to human health than other types of asbestos fibers. Its clearance from the lung is faster and, in comparison to amphibole forms of asbestos, chrysotile asbestos fail to accumulate in the lung tissue due to a mechanism involving fibers fragmentation in short pieces. Short exposure to chrysotile has not been associated with any histopathological alteration of lung tissue. Methods: The present work focuses on the association of small chrysotile fibers with interphasic and mitotic human lung cancer cells in culture, using for analyses confocal laser scanning microscopy and 3D reconstructions. The main goal was to perform the analysis of abnormalities in mitosis of fibers-containing cells as well as to quantify nuclear DNA content of treated cells during their recovery in fiber-free culture medium. Results: HK2 cells treated with chrysotile for 48 h and recovered in additional periods of 24, 48 and 72 h in normal medium showed increased frequency of multinucleated and apoptotic cells. DNA ploidy of the cells submitted to the same chrysotile treatment schedules showed enhanced aneuploidy values. The results were consistent with the high frequency of multipolar spindles observed and with the presence of fibers in the intercellular bridge during cytokinesis. Conclusion: The present data show that 48 h chrysotile exposure can cause centrosome amplification, apoptosis and aneuploid cell formation even when long periods of recovery were provided. Internalized fibers seem to interact with the chromatin during mitosis, and they could also interfere in cytokinesis, leading to cytokinesis failure which forms aneuploid or multinucleated cells with centrosome amplification.

GEITLERINEMA SPECIES (OSCILLATORIALES, CYANOBACTERIA) REVEALED BY CELLULAR MORPHOLOGY, ULTRASTRUCTURE, AND DNA SEQUENCING

Geitlerinema amphibium (C. Agardh ex Gomont) Anagn. and G. unigranulatum (Rama N. Singh) Komarek et M. T. P. Azevedo are morphologically close species with characteristics frequently overlapping. Ten strains of Geitlerinema (six of G. amphibium and four of G. unigranulatum) were analyzed by DNA sequencing and transmission electronic and optical microscopy. Among the investigated strains, the two species were not separated with respect to cellular dimensions, and cellular width was the most varying characteristic. The number and localization of granules, as well as other ultrastructural characteristics, did not provide a means to discriminate between the two species. The two species were not separated either by geography or environment. These results were further corroborated by the analysis of the cpcB-cpcA intergenic spacer (PC-IGS) sequences. Given the fact that morphology is very uniform, plus the coexistence of these populations in the same habitat, it would be nearly impossible to distinguish between them in nature. On the other hand, two of the analyzed strains were distinct from all others based on the PC-IGS sequences, in spite of their morphological similarity. PC-IGS sequences indicate that these two strains could be a different species of Geitlerinema. Using morphology, cell ultrastructure, and PC-IGS sequences, it is not possible to distinguish G. amphibium and G. unigranulatum. Therefore, they should be treated as one species, G. unigranulatum as a synonym of G. amphibium.

An evaluation, using the comet assay and the micronucleus test, of the antigenotoxic effects of chlorophyll b in mice

We investigated the effects of the dietary pigment chlorophyll b (CLb) on cisplatin (cDDP)-induced oxidative stress and DNA damage, using the comet assay in mouse peripheral blood cells and the micronucleus (MN) test in bone marrow and peripheral blood cells. We also tested for thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH) in liver and kidney tissues, as well as catalase (CAT) activity and GSH in total blood. CLb (0.2 and 0.5 mg/kg b.w.) was administrated by gavage every day for 13 days. On the 14th day of the experiment, 6 mg/kg cDDP or saline was delivered intraperitoneally. Treatment with cDDP led to a significant decrease in DNA migration and an increase in MN frequency in both cell types, bone marrow and peripheral blood cells. In the kidneys of mice treated with cDDP, TBARS levels were increased, whereas GSH levels were depleted in kidney and liver. In mice that were pretreated with CLb and then treated with cDDP, TBARS levels maintained normal concentrations and GSH did not differ from cDDP group. The improvement of oxidative stress biomarkers after CLb pre-treatment was associated with a decrease in DNA damage, mainly for the highest dose evaluated. Furthermore, CLb also slightly reduced the frequency of chromosomal breakage and micronucleus formation in mouse bone marrow and peripheral blood cells. These results show that pre-treatment with CLb attenuates cDDP-induced oxidative stress, chromosome instability, and lipid peroxidation. (C) 2011 Elsevier B.V. All rights reserved.

Cytogenetic biomonitoring of inhabitants of a large uranium mineralization area: the municipalities of Monte Alegre, Prainha, and Alenquer, in the State of Para, Brazil

Uranium is a natural radioactive metallic element; its effect on the organism is cumulative, and chronic exposure to this element can induce carcinogenesis. Three cities of the Amazon region-Monte Alegre, Prainha, and Alenquer-in North Brazil, are located in one of the largest uranium mineralization areas of the world. Radon is a radioactive gas, part of uranium decay series and readily diffuses through rock. In Monte Alegre, most of the houses are built of rocks removed from the Earth`s crust in the forest, where the uranium reserves lie. The objective of the present work is to determine the presence or absence of genotoxicity and risk of carcinogenesis induced by natural exposure to uranium and radon in the populations of these three cities. The frequency of micronuclei (MN) and chromosomal aberrations (CA) showed no statistically significant differences between the control population and the three study populations (P > 0.05). MN was also analyzed using the fluorescence in situ hybridization (FISH) technique, with a centromere-specific probe. No clastogenic and/or aneugenic effects were found in the populations. Using FISH analysis, other carcinogenesis biomarkers were analyzed, but neither the presence of the IGH/BCL2 translocation nor an amplification of the MYC gene and 22q21 region was detected. Clastogenicity and DNA damage were also not found in the populations analyzed using the alkaline comet assay. The mitotic index showed no cytotoxicity in the analyzed individuals` lymphocytes. Once we do not have data concerning radiation doses from other sources, such as cosmic rays, potassium, thorium, or anthropogenic sources, it is hard to determine if uranium emissions in this geographic region where our study population lives are too low to cause significant DNA damage. Regardless, genetic analyses suggest that the radiation in our study area is not high enough to induce DNA alterations or to interfere with mitotic apparatus formation. It is also possible that damages caused by radiation doses undergo cellular repair.

Evaluation of the genotoxic and antigenotoxic effects after acute and subacute treatments with acai pulp (Euterpe oleracea Mart.) on mice using the erythrocytes micronucleus test and the comet assay

Acai, the fruit of a palm native to the Amazonian basin, is widely distributed in northern South America, where it has considerable economic importance. Whereas individual polyphenolics compounds in Acai have been extensively evaluated, studies of the intact fruit and its biological properties are lacking. Therefore, the present study was undertaken to investigate the in vivo genotoxicity of Acai and its possible antigenotoxicity on doxorubicin (DXR)-induced DNA damage. The Acai pulp doses selected were 3.33, 10.0 and 16.67 g/kg b.w. administered by gavage alone or prior to DXR (16 mg/kg b.w.) administered by intraperitoneal injection. Swiss albino mice were distributed in eight groups for acute treatment with acai pulp (24 h) and eight groups for subacute treatment (daily for 14 consecutive days) before euthanasia. The negative control groups were treated in a similar way. The results of chemical analysis suggested the presence of carotenoids, anthocyanins, phenolic. and flavonoids in Acai pulp. The endpoints analyzed were micronucleus induction in bone marrow and peripheral blood cells polychromatic erythrocytes, and DNA damage in peripheral blood, liver and kidney cells assessed using the alkaline (pH > 13) comet assay. There were no statistically significant differences (p > 0.05) between the negative control and the groups treated with the three doses of Acai pulp alone in all endpoints analyzed, demonstrating the absence of genotoxic effects. The protective effects of Acai pulp were observed in both acute and subacute treatments, when administered prior to DXR. In general, subacute treatment provided greater efficiency in protecting against DXR-induced DNA damage in liver and kidney cells. These protective effects can be explained as the result of the phytochemicals present in Acai pulp. These results will be applied to the developmental of food with functional characteristics, as well as to explore the characteristics of Acai as a health promoter. (C) 2009 Elsevier B.V. All rights reserved.

Chromosomal fragile site FRA16D and DNA instability in cancer

It has been proposed that common aphidicolin-inducible fragile sites, in general, predispose to specific chromosomal breakage associated with deletion, amplification, and/or translocation in certain forms of cancer. Although this appears to be the case for the fragile site FRA3B and may be the case for FRA7G, it is not Set clear whether this association is a general property of this class of fragile site. The major aim of the present study was to determine whether the FRA16D chromosomal fragile site locus has a role to play in predisposing DNA sequences within and adjacent to the fragile site to DNA instability (such as deletion or translocation), which could lead to or be associated with neoplasia. We report the localization of FRA16D within a contig of cloned DNA and demonstrate that this fragile site coincides with a region of homozygous deletion in a gastric adenocarcinoma cell line and is bracketed by translocation breakpoints in multiple myeloma, as reported previously (Chesi, M., et al., Blood, 91: 4457-4463, 1998), Therefore, given similar findings at the FRA3B and FRA7G fragile sites, it is likely that common aphidicolin-inducible fragile sites exhibit the general property of localized DNA instability in cancer cells.