Analysis of telomere maintenance in artemis defective human cell lines

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.Telomeres are physical ends of chromosomes consisting of (TTAGGG)n DNA sequence and a specialized set of proteins that protect chromosomal ends from degradation and from eliciting DNA damage response. These specialized set of proteins, known as shelterin, directly bind to telomeric DNA. In addition, some DNA double-strand break (DSB) repair proteins such as, DNA-PKcs and KU70/80, play active roles in telomere maintenance. Mouse knock-out experiments have revealed that deletion of either DNA-PKcs or Ku70/80 resulted in elevated levels of telomeric fusion, indicative of dysfunctional telomeres. Artemis protein is involved in DNA DSB repair through non-homologous end joining (NHEJ) and it is phosphorylated by DNAPKcs. Human cells defective in Artemis have been identified and shown to be radiosensitive and patients with an Artemis defective gene suffer from radiosensitive severe-combined immune deficiency syndrome (RS-SCID). Mouse cells defective in Artemis have elevated levels of telomeric fusion. We have demonstrated in this thesis that Artemis defective human cell lines show a mild telomeric dysfunction phenotype detectable at the cytological level. The nature of telomere dysfunction phenotype appears to be similar to that observed in DNAPKcs defective cells as exemplified by the presence of IR induced chromatid telomeric fusions. We have also shown that (a) DNA damage occurring within the telomeric DNA is difficult to repair or irreparable in older cells and that (b) Artemis defective older cells show higher proportion of DNA damage at telomeres than their normal counterparts. Finally, we have demonstrated that inhibition of DNA-PKcs causes (a) an increase in telomeric fusions in Artemis defective cell lines relative to both normal cell lines after inhibition and Artemis cell lines before inhibition and (b)elevated levels of DNA damage at telomeres following exposure of cells to radiation relative to both irradiated normal cells exposed to a DNA-PKcs inhibitor and irradiated Artemis defective cells but not exposed to the DNA-PKcs inhibitor. These results suggest that the effects of Artemis and DNA-PKcs on telomeres are cumulative. We have also performed (a) experiments to examine telomere function in Artemis defective cell lines after knocking down DNA-PKcs levels by RNAi and b) preliminary experiments to knock-down Artemis in DNA-PKcs defective cells. Taken together, our results suggest that the Artemis defect causes mild telomere dysfunction phenotype in human cells

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology

Disruptive chemicals, senescence and immortality

Cell death is a process of dying within biological cells that are ceasing to function. This process is essential in regulating organism development, tissue homeostasis, and to eliminate cells in the body that are irreparably damaged. In general, dysfunction in normal cellular death is tightly linked to cancer progression. Specifically, the up-regulation of prosurvival factors, including oncogenic factors and antiapoptotic signaling pathways, and the down-regulation of proapoptotic factors, including tumor suppressive factors, confers resistance to cell death in tumor cells, which supports the emergence of a fully immortalized cellular phenotype. This review considers the potential relevance of ubiquitous environmental chemical exposures that have been shown to disrupt key pathways and mechanisms associated with this sort of dysfunction. Specifically, bisphenol A, chlorothalonil, dibutyl phthalate, dichlorvos, lindane, linuron, methoxychlor and oxyfluorfen are discussed as prototypical chemical disruptors; as their effects relate to resistance to cell death, as constituents within environmental mixtures and as potential contributors to environmental carcinogenesis.The publisher and the author(s) have made this article open access. This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by Oxford University Press. The published article can be found at: http://carcin.oxfordjournals.org

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety ‘Mode of Action’ framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/ mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety ‘Mode of Action’ framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology