Long telomeric C-rich 5'-tails in human replicating cells.

Telomeres protect the ends of linear chromosomes from abnormal recombination events and buffer them against terminal DNA loss. Models of telomere replication predict that two daughter molecules have one end that is blunt, the product of leading-strand synthesis, and one end with a short G-rich 3′-overhang. However, experimental data from proliferating cells are not completely consistent with this model. For example, telomeres of human chromosomes have long G-rich 3′-overhangs, and the persistence of blunt ends is uncertain. Here we show that the product of leading-strand synthesis is not always blunt but can contain a long C-rich 5′-tail, the incompletely replicated template of the leading strand. We examined the presence of G-rich and C-rich single-strand DNA in fibroblasts and HeLa cells. Although there were no significant changes in the length distribution of the 3′-overhang, the 5′-overhangs were mostly present in S phase. Similar results were obtained using telomerase-negative fibroblasts. The amount and the length distribution of the 5′ C-rich tails strongly correlate with the proliferative rate of the cell cultures. Our results suggest that, contrary to what has commonly been supposed, completion of leading-strand synthesis is inefficient and could well drive telomere shortening

The Oncogenic Signaling Pathways in BRAF-Mutant Melanoma Cells are Modulated by Naphthalene Diimide-Like G-Quadruplex Ligands

Melanoma is the most aggressive and deadly type of skin cancer. Despite the advent of targeted therapies directed against specific oncogene mutations, melanoma remains a tumor that is very difficult to treat, and ultimately remains incurable. In the past two decades, stabilization of the non-canonical nucleic acid G-quadruplex structures within oncogene promoters has stood out as a promising approach to interfere with oncogenic signaling pathways in cancer cells, paving the way toward the development of G-quadruplex ligands as antitumor drugs. Here, we present the synthesis and screening of a library of differently functionalized core-extended naphthalene diimides for their activity against the BRAFV600E-mutant melanoma cell line. The most promising compound was able to stabilize G-quadruplexes that formed in the promoter regions of two target genes relevant to melanoma, KIT and BCL-2. This activity led to the suppression of protein expression and thus to interference with oncogenic signaling pathways involved in BRAF-mutant melanoma cell survival, apoptosis, and resistance to drugs. This G-quadruplex ligand thus represents a suitable candidate for the development of melanoma treatment options based on a new mechanism of action and could reveal particular significance in the context of resistance to targeted therapies of BRAF-mutant melanoma cells

European Parliament Pilot Project on Exposure to Indoor air Chemicals and Possible Health Risks

This report outlines the results of the 2-year pilot project on indoor air quality and potential health effects executed by the Joint Research Centre and funded by the European Parliament via the Directorate-General Health and Consumer Protection. It had four distinct objectives as follows: 1) to identify and quantify the main air pollutants present in public buildings, including indoor environments where children frequently stay, like schools and kindergartens, 2) to identify the main sources of these pollutants, applying source apportionment analyses, 3) to estimate people¿s exposure to these pollutants while working and/or living in these areas and combined with micro-environmental activity patterns during the day, 4) to evaluate possible health risks due to (chronic) exposure to air pollutants, in particular, for children. The results indicate that indoor air pollution concentrations are consistently higher than the respective outdoor ones for the chemical families this study focused on. Differences attributable to variation in consumer behaviour, climate and type of building materials used, have been identified in the indoor:outdoor ratio of primary pollutants across Europe. These differences account for small variance in the corresponding health risk to the local population across the EU.JRC.I.5-Physical and chemical exposure

The arsenic in mice as experimental model for risk modifiers.

Studies on the relevance of host factors in modulating the physiological responses following chronic exposure to xenobiotics were carried out according to a \u201cToxicogenomic Model on Arsenic in Mice\u201d developed at thte JRC. This model is focused on chronic exposure to arsenate given alone or in combination with other xenobiotics, to assess potential \u201ccocktail effects\u201d and related cumulative risks. DNA-macroarrays technology is applied to evaluate physiological responses at transcriptional level and assessing possible biochemical responses. A cluster of 1200 cancer genes was used for screening purposes, while quantitative PCR on selected genes applied for validation. The exposure varied from in-utero and post-lactation up to adult age (4 months), the chemical forms (arsenate and dimethylarsenate) and doses from 0.1 up to 10 mg As/L in drinking water. Comparison between acute single doses and chronic exposure was also performed. Chronic exposure to arsenate and atrazine in drinking water was selected as an example of multiple chronic exposure. The liver, kidney, lung, bone marrow, adrenals, uterus, and testis were the tissues considered. In the tissues of mice chronically exposed to arsenate, the modulation of gene expression was not only depending on the levels, types and length of exposure, while differently regulated also by the sex, age and diet. The main gene functional families modulated were covering a wide range of biochemical and physiological regulations, like cell cycle modulation, cell adhesion, apoptosis, xenobiotic metabolism, DNA repair, protein turnover, and proto-oncogenes. The patterns of gene expression were strongly influenced by co-exposure to other xenobiotics like atrazine and naphthalene, particularly for genes involved in the metabolism and in neuroendocrine regulation. These effects varied according to the tissue considered, supporting the needs for coherent and specifically designed studies to assess relevant biomarkers of long-term exposure to low levels of xenobiotics and their mixtures

Sex as a major determinant of gene expression in tissues of mice exposed to arsenate.

Inorganic arsenic, frequently found as contaminant of ground water used for drinking purposes in many areas of the world, is a well-known potent human toxicant and carcinogen. Chronic exposure to inorganic arsenic has been associated with cancer of skin, lung, bladder and kidney and, probably, liver. The mechanism of arsenic action in vivo is poorly understood, in particular in relation to dose, type of tissue and gender. To elucidate tissue- and gender dependent biological responses in the genome of mice, we have used cDNA macroarrays for investigation on the expression of 1185 cancer-related genes in mice after exposure to arsenate in drinking water. Continuous exposures of mice to arsenate in drinking water modulate the gene expression in tissues. Interestingly, there were remarkable sex differences: male and female mice show completely different changes in the expression of cancer-related genes. The main gene functional families modulated, were covering a wide range of biochemical and physiological regulations, like cell cycle modulation, cell adhesion, apoptosis, xenobiotic metabolism, DNA repair, protein turnover and proto-oncogens. This result demonstrates important gene-environmental interactions: the molecular mechanisms triggered by arsenic levels frequently experienced following exposure via drinking water, are totally different in males and females. The results obtained using cancer-related genes will be compared with the profiles of over 30.000 genes using the Applied Biosystems expression Array System, to clarify the sex-specific gene pathways

Dietary proteins modulates the gene expression in mice chronically exposed to arsenate.

In the frame of a project on the assessment of risk modifying factors modulating the health effects of environmental chemicals we are developing a toxicogenomic approach using an \u201carsenic in mice\u201d experimental model, considering multistressors exposure, genetics, age, levels and length of exposure, etc. In the present study, we used cDNA Macroarrays to investigate the effects of low protein intake on the expression of 1185 cancer-related genes in the liver of male and female mice transplacentary exposed to different levels of arsenate in drinking water during gestation and developmental age. The results of this study support the relevance of dietary factors in modulating the physiological responses in gene expression following chronic exposure to xenobiotics. In mice chronically exposed to arsenate in drinking water, the modulation of gene expression in different tissues was not only depending on the levels of the xenobiotic under investigation, but mainly regulated by the content of proteins in diet

Clonogenicity and gene expression modulation in the bone marrow of mice chronically exposed to arsenic and atrazine.

The clonogenicity of myeloid progenitors (CFU-GM) and the modulation of gene expression of 1185 cancer-related genes by DNA-macroarrays in bone marrow were used to investigate in male and female mice the combined effects of continuous exposure to arsenate and atrazine in drinking water. In male mice, the exposure to arsenate or to atrazine alone and the combined exposure did not change the clonogenicity of the progenitors. In females the percentage of CFU-GM decreased significantly after atrazine exposure, did not change with arsenic treatment, but dramatically increased after the combined exposure to the two chemicals. Results from microarrays indicate that atrazine alone didn\u2019t stimulate the expression of any of the cancer genes analyzed in both male and female. Arsenic induced gene expression modulation only in female and had no effects on male. Major significant changes on the gene expression in bone marrow cells resulted following the co-exposure to arsenic and atrazine in both male and female. These results indicate that co-exposure of mice to atrazine and arsenate induces significant effects at the level of transcriptional activation of genes in bone marrow cells, as well as stimulating the myeloid progenitors to proliferate, particularly when co-administered in drinking water to female mice

Combined in-utero and juvenile exposure of mice to arsenate and atrazine in drinking water modulates the gene expression and clonogenicity of myeloid progenitors in bone marrow.

Increasing evidence proves that human fetuses are exposed to multiple risk factors and major concerns have been expressed towards exposure to potential endocrine modulating chemicals at early stage of life and during growth. Understanding that exposures occur as mixture of chemicals and that they converge on other inherent and environmental risk-modulating factors, there is a need to develop experimental models to assess the effects of exposure to multiple chemicals during different stage of life. In the present study, the clonogenicity of myeloid progenitors (CFU-GM) and the modulation of gene expression of 1197 cancer-related genes (DNA macroarrays) in bone marrow were used to investigate in male and female young mice the combined effects of continuous exposure to arsenate and atrazine in drinking water. Female adult mice were treated with arsenate in drinking water (1 mg As/L) for 10 days before mating and during the gestation. Offspring were randomly put into separate groups of males and females. One group of arsenic exposed offspring were exposed for 4 months to atrazine (1mg Atr/L) and arsenate (1 mg As/L) in drinking water (As+Atr). One group of each of arsenic unexposed offspring were exposed for 4 months to atrazine (1mg Atr/L) in drinking water (Atr). Additional arsenate (1 mg As/L) was given to one group of arsenic exposed offspring (As). Control mice without any treatment were also analysed (Ctrl). In male mice the exposure to arsenate or to atrazine alone did not result in significant changes on the gene expression in bone marrow cells, whereas, co-exposure to arsenic and atrazine (As+Atr) resulted in a significant up-modulation of gene expression. The percentage of CFU-GM weakly decreased after exposure to individual compounds, while the co-exposure did not change the clonogenicity of the progenitors. In female mice, the co-exposure to both chemicals resulted in a drastic up-modulation of gene expression, while in these cells the single treatments showed a up-modulation of few genes as well. The percentage of CFU-GM decreased significantly after atrazine exposure, did not change with arsenic treatment, but dramatically increased after the combined administration. These results indicate that in-utero and juvenile co-exposure of mice to atrazine and arsenate induce significant effects at the level of transcriptional activation of genes in bone marrow cells, as well as stimulating the myeloid progenitors to proliferate, particularly when co-administered in drinking water to female mice

Toxicogenomic Study of Indoor and Outdoor Air Chemical Mixtures

Although environmental exposures occur to mixtures of chemicals rather than to individual agents, most of the toxic effects of air pollutants are ascribed to single chemicals. There is a growing feeling in both the scientific and regulatory communities, however that, as the need for better air quality increases, there is also a need for more comprehensive toxicological approaches on the potential impact of complex environmental chemical mixtures on human health. In this perspective, it is expected that the toxicogenomics approach would be the appropriate screening method for assessing biological effects of complex chemical mixtures, allowing us to review the whole spectrum of potential biological response rather than focusing on a pre-defined number of endpoints as in classical toxicological analysis. In this study, we focused on a typical indoor air mixture as defined in the EU-wide review study INDEX and on a mixture of polyaromatic hydrocarbons (PAHs) isolated from urban air in the city of Milan with the aim to identify specific sets of biomarkers for each type of exposure (indoor or outdoor). A human cell line derived from a bronco-pulmonary system (A549) was used. Applying a Total Gene Expression assay by Applied Biosystems Microarrays, we profiled large sets of genes modulated by single mixtures exposure and identified common biochemical pathways and specific molecular responses. Indoor air mixtures induced a higher gene modulation than PAHs, confirming major differences in toxic mode of action of the two mixtures. Indoor air induced primarily modulation of genes associated to protein targeting and localization including in particular cytoskeletal organization; PAHs modulated mostly the expression of genes related to cell motility and gene networks regulating cell-cell signaling, as well as cell proliferation and differentiation. These results provide biological information useful for articulating mechanistic hypotheses of exposure to xenobiotic mixtures and physiological responses.JRC.I.5-Physical and chemical exposure