Inhibited effects of veliparib combined doxorubicin for BEL-7404 proliferation of human liver cancer cell line

AbstractObjectiveTo explore inhibition effects of veliparib as PARP inhibitor combined doxorubicin for BEL-7404 proliferation of human liver cancer cell line.MethodsBEL-7404 was taken as the object of study and conventional culture was performed. It wastreated by doxorubicin and (or) veliparib after 24 h. Cell proliferation rate was detected by four methyl thiazolyl tetrazolium (MTT) assay, cell apoptosis was measured with annexin V-FITC/PI double staining method by flow cytometry, DNA damage degree evaluation by single cell gel electrophoresis assay, and cytosolic C levels of the mitochondrial and cytosol by polyacrylamide gel electrophoresis (Western blotting).ResultsCell proliferation rate of doxorubicin combined veliparib group was lower than that of the control group and doxorubicin alone treated group significantly (P<0.01), the apoptosis rate was significantly higher than that of the control group and doxorubicin alone treated group (P<0.05). At the same time, DNA damage level of doxorubicin combined with veliparib group was significantly higher than doxorubicin alone treatment group and the control group (P<0.01), and cytochrome C in the cytosol was significantly higher than that of control group and doxorubicin alone treated group (P<0.01).ConclusionsVeliparib, PARP inhibitor could inhibit PARP activity, block tumor cell DNA repair, and have significant sensitizing effect for hepatocellular carcinoma cell line BEL-7404 treated with doxorubicin. This might provide a new target for clinical treatment of hepatic carcinoma

Association between the c.*229C>T polymorphism of the topoisomerase IIb binding protein 1 (TopBP1) gene and breast cancer

Topoisomerase IIb binding protein 1 (TopBP1) is involved in cell survival, DNA replication, DNA damage repair and cell cycle checkpoint control. The biological function of TopBP1 and its close relation with BRCA1 prompted us to investigate whether alterations in the TopBP1 gene can influence the risk of breast cancer. The aim of this study was to examine the association between five polymorphisms (rs185903567, rs116645643, rs115160714, rs116195487, and rs112843513) located in the 30UTR region of the TopBP1 gene and breast cancer risk as well as allele-specific gene expression. Five hundred thirty-four breast cancer patients and 556 population controls were genotyped for these SNPs. Allele-specific Top- BP1 mRNA and protein expressions were determined by using real time PCR and western blotting methods, respectively. Only one SNP (rs115160714) showed an association with breast cancer. Compared to homozygous common allele carriers, heterozygous and homozygous for the T variant had significantly increased risk of breast cancer (adjusted odds ratio = 3.81, 95 % confidence interval: 1.63–8.34, p = 0.001). Mean TopBP1 mRNA and protein expression were higher in the individuals with the CT or TT genotype. There was a significant association between the rs115160714 and tumor grade and stage. Most carriers of minor allele had a high grade (G3) tumors classified as T2-T4N1M0. Our study raises a possibility that a genetic variation of TopBP1 may be implicated in the etiology of breast cancer

Dynamique des populations de caribous migrateurs (Rangifer tarandus) basée sur des indices de condition corporelle

Monitoring and understanding wildlife population dynamics is key to their management and conservation. High population size or density can negatively affect demographic parameters including reproduction, recruitment and survival. These parameters are intricately linked to individual body size and condition. Migratory caribou ( Rangifer tarandus ) are a keystone species in the tundra. Populations can fluctuate drastically and rapidly and are challenging to monitor and manage. As an alternative to population estimates, indirect ecological indicators, which are density-dependent indices based on individual physical characteristics or performance, have been proposed to monitor ungulate populations. I amalgamated morphological data measured on newborns, yearlings and adult females for four migratory caribou herds; the Rivière-George, Rivière-aux-Feuilles, Beverly and Porcupine. I investigated how body condition relates to population dynamics at the individual level by determining how body size, condition and population size impact female reproductive success in the Rivière-George herd. At the population level, I determined the efficacy of body condition indices to estimate demographic trends for all four herds. Body condition of adult females was positively related to the probability of gestation for the Rivière-George herd. Although population size negatively affected gestation rates, females did not adopt a conservative reproductive strategy as predicted, because the threshold adult female mass required for gestation did not vary with population size. At the population level, physical traits showed negative density dependence in three of the four herds. Physical traits, however, did not consistently correlate with population size nor did they predict numerical changes in population size. Physical traits often showed density dependence stronger, or even exclusively, during periods of demographic growth. Physical traits of juveniles seemed to respond to changes in population size more readily than those of adult females, and the effect of population size at birth persisted in skeletal measures of adults. Density dependence was apparent only in some herds, highlighting the importance of determining the drivers of population dynamics, particularly during periods of decline. Relationships between body condition, demographic rates and population dynamics are complex, so that changes in population size cannot be predicted reliably by monitoring physical traits

Functional studies of accessory factors associated with base excision repair

Exposure to environmental and cellular mutagens is ubiquitous and, as a consequence, DNA is constantly faced with the possibility of becoming damaged. Base excision repair (BER) removes some of this damage to limit the impact of these exposures on cell physiology and ultimately human health. The function of core BER enzymes may be enhanced by other protein accessory factors, namely poly(ADP-ribose) polymerase-1 (PARP-1) and x-ray repair cross complementing gene 1 (XRCC1). The main hypothesis of this research was that genetic approaches using cellular knockout and complementation models can evaluate whether the accessory proteins PARP-1 and XRCC1 are determinants of BER efficiency. While numerous biochemical studies have implicated PARP-1 in BER, the role of this protein in BER is somewhat uncertain. The first aim of this research was to evaluate the role of PARP-1 in BER in vertebrate cells. Chicken cells lacking PARP-1 were treated with an alkylating agent under different scenarios with subsequent endpoint measurements. PARP-1 was necessary as a survival factor during chronic exposure but did not appear relevant in acute exposures until the late stages of BER. In the absence of exposure, the DNA lesions measured were equal between PARP-1 proficient and deficient cells. XRCC1 acts as a scaffold for numerous protein interactions necessary for proficient BER. However, the presence of polymorphic forms of XRCC1 in the human population may influence DNA repair and disease susceptibility. The second aim of this research was to demonstrate the applicability of using transgenic cells in a combined study design for determining the biological significance of XRCC1 polymorphisms. Isogenic, mammalian cells lacking XRCC1 were transfected with various forms of the human XRCC1 gene, exposed to different genotoxicants, and assessed for single strand break repair capacity. Only cells expressing the 280His variant showed a repair defect. Subsequently, evaluation of data from the Carolina Breast Cancer Study demonstrated associations between XRCC1 280His, smoking, and breast cancer. Together these studies demonstrate that accessory factors can influence BER efficiency and illustrate the importance of a multi-disciplinary approach for investigating the link between genes, the environment, and disease risk

Exploitation of people with mental health problems: a critical discourse analysis of the role of mental health service policy in Scotland

This thesis engages with the phenomenon of abuse and exploitation of people with a diagnosis or experience of mental health problems. The thesis focuses especially on exploitation that does not meet legal thresholds of criminality. In this thesis, this type of exploitation is called “noncriminal exploitation”. The research project was designed to answer the research questions: RQ1) How does the relationship between society and people with mental health problems affect or allow noncriminal exploitation of people with mental health problems? RQ2) How are mental health service providers represented in this relationship? RQ3) What are the obligations – moral or otherwise - of mental health service providers in addressing noncriminal exploitation of people with mental health problems? RQ4) Are existing policies and procedures effective in protecting the rights of people with mental health problems from exploitation? To answer these questions, Foucauldian influenced critical discourse analysis is applied as a theoretical orientation and methodological framework to examine the policies that shape mental health services and practice in Scotland; and the potential effects of these texts on the lives of people who use those services, especially as it relates to exploitation of people who have mental health problems. The actual and potential roles of mental health services in preventing and addressing exploitation are examined. An archive of policy and related documents relevant to mental health services in Scotland was selected; 10 representative documents from this archive were analysed for discursive themes, especially related to exploitation. A focused analysis of the current Mental Health Strategy of Scotland was conducted, to examine it both for internal discursive themes and for the presence of broader themes which emerged from the archive. Competing discursive narratives embedded within the archive were discovered: Discourses that can be described as emancipatory and empowering of people with mental health problems (Rights-Autonomy discourses) are countered by discourses that embody paternalistic attitudes, medicalization, and stigma (Harms-Paternalist discourses). This thesis critically examines sample texts for the presence and effects of these discourses; traces the discourses throughout the Scottish Mental Health Strategy and discusses potential effects of these discourses. When assessed by the stated goals of the Strategy and related policies, these discourses impede progress towards development of services that are person-centred and rights-based. This reinforces the marginalization of people with mental health problems and can impede progress towards the explicitly stated goals of the Strategy. The thesis discusses the impact of these discursive realities on the issue of non-criminal exploitation. Roles and obligations of mental health workers encountering exploitation are explored. Policy solutions, as well as actions aimed at mitigating Harms-Paternalist Discourses, are proposed

N-methylpurine DNA glycosylase overexpression increases alkylation sensitivity by rapidly removing non-toxic 7-methylguanine adducts

Previous studies indicate that overexpression of N-methylpurine DNA glycosylase (MPG) dramatically sensitizes cells to alkylating agent-induced cytotoxicity. We recently demonstrated that this sensitivity is preceded by an increased production of AP sites and strand breaks, confirming that overexpression of MPG disrupts normal base excision repair and causes cell death through overproduction of toxic repair intermediates. Here we establish through site-directed mutagenesis that MPG-induced sensitivity to alkylation is dependent on enzyme glycosylase activity. However, in contrast to the sensitivity seen to heterogeneous alkylating agents, MPG overexpression generates no cellular sensitivity to MeOSO(2)(CH(2))(2)-lexitropsin, an alkylator which exclusively induces 3-meA lesions. Indeed, MPG overexpression has been shown to increase the toxicity of alkylating agents that produce 7-meG adducts, and here we demonstrate that MPG-overexpressing cells have dramatically increased removal of 7-meG from their DNA. These data suggest that the mechanism of MPG-induced cytotoxicity involves the conversion of non-toxic 7-meG lesions into highly toxic repair intermediates. This study establishes a mechanism by which a benign DNA modification can be made toxic through the overexpression of an otherwise well-tolerated gene product, and the application of this principle could lead to improved chemotherapeutic strategies that reduce the peripheral toxicity of alkylating agents

The formation and biological significance of N7-guanine adducts

DNA alkylation or adduct formation occurs at nucleophilic sites in DNA, mainly the N7-position of guanine. Ever since identification of the first N7-guanine adduct, several hundred studies on DNA adducts have been reported. Major issues addressed include the relationships between N7-guanine adducts and exposure, mutagenesis, and other biological endpoints. It became quickly apparent that N7-guanine adducts are frequently formed, but may have minimal biological relevance, since they are chemically unstable and do not participate in Watson Crick base pairing. However, N7-guanine adducts have been shown to be excellent biomarkers for internal exposure to direct acting and metabolically activated carcinogens. Questions arise, however, regarding the biological significance for N7-guanine adducts that are readily formed, do not persist, and are not likely to be mutagenic. Thus, we set out to review the current literature to evaluate their formation and the mechanistic evidence for the involvement of N7-guanine adducts in mutagenesis or other biological processes. It was concluded that there is insufficient evidence that N7-guanine adducts can be used beyond confirmation of exposure to the target tissue and demonstration of the molecular dose. There is little to no evidence that N7-guanine adducts or their depurination product, apurinic sites, are the cause of mutations in cells and tissues, since increases in AP sites have not been shown unless toxicity is extant. However, more research is needed to define the extent of chemical depurination versus removal by DNA repair proteins. Interestingly, N7-guanine adducts are clearly present as endogenous background adducts and the endogenous background amounts appear to increase with age. Furthermore, the N7-guanine adducts have been shown to convert to ring opened lesions (FAPy), which are much more persistent and have higher mutagenic potency. Studies in humans are limited in sample size and differences between controls and study groups are small. Future investigations should involve human studies with larger numbers of individuals and analysis should include the corresponding ring opened FAPy derivatives

Analysis of 8-oxo-7,8-dihydro-2′-deoxyguanosine by ultra high pressure liquid chromatography–heat assisted electrospray ionization–tandem mass spectrometry

Increased amounts of reactive oxygen species (ROS), generally termed oxidative stress, are frequently hypothesized to be causally associated with many diseases. Analyses of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) in DNA and urine are widely used biomarkers for oxidative stress. Over the years it became clear that analysis of 8-oxo-dG in DNA is challenging due to artifactual formation during sample work up. The present study demonstrates that 8-oxo-dG can be measured reliably and accurately when appropriate precautions are taken. First, the presence of an antioxidant, metal chelator, or free radical trapping agent during sample preparation improves reproducibility. Second, sample enrichment by HPLC fraction collection was used to optimize sensitivity. Third, heat assisted electrospray ionization (HESI) eliminated potential interferences and improved assay performance and sensitivity. Subsequently, the UPLC–HESI–MS/MS method was applied to show the biphasic dose response of 8-oxo-dG in H2O2-treated HeLa cells. Application of this method to human lymphocyte DNA (n = 156) gave a mean±SD endogenous amount of 1.57±0.88 adducts per 106 dG, a value that is in agreement with the suggested amount previously estimated by European Standard Committee on Oxidative DNA Damage (ESCODD) and others. These results suggest that the present method is well suited for application to molecular toxicology and epidemiology studies investigating the role of oxidative stress