American journal of physiology. Lung cellular and molecular physiology, Jan 18, 2015
Cellular senescence has been associated with the structural and functional decline observed durin... more Cellular senescence has been associated with the structural and functional decline observed during physiological lung ageing and in chronic obstructive pulmonary disease (COPD). Airway epithelial cells are the first line of defense in the lungs and are important to COPD pathogenesis. However, the mechanisms underlying airway epithelial cell senescence, and particularly the role of telomere dysfunction in this process, are poorly understood. We aimed to investigate telomere dysfunction in airway epithelial cells from patients with COPD, in the ageing murine lung and following cigarette smoke exposure. We evaluated co-localization of γH2A.X and telomeres and telomere length in small airway epithelial cells from patients with COPD, during murine lung ageing and following cigarette smoke exposure in vivo and in vitro. We found that telomere-associated DNA damage foci increase in small airway epithelial cells from patients with COPD, without significant telomere shortening detected. With...
Telomeres are specialized nucleoprotein structures, which protect chromosome ends and have been i... more Telomeres are specialized nucleoprotein structures, which protect chromosome ends and have been implicated in the ageing process. Telomere shortening has been shown to contribute to a persistent DNA damage response (DDR) during replicative senescence, the irreversible loss of division potential of somatic cells. Similarly, persistent DDR foci can be found in stress-induced senescence, although their nature is not understood. Here we show, using immuno-fluorescent in situ hybridization and ChIP, that up to half of the DNA damage foci in stress-induced senescence are located at telomeres irrespective of telomerase activity. Moreover, live-cell imaging experiments reveal that all persistent foci are associated with telomeres. Finally, we report an age-dependent increase in frequencies of telomere-associated foci in gut and liver of mice, occurring irrespectively of telomere length. We conclude that telomeres are important targets for stress in vitro and in vivo and this has important c...
Cellular senescence is the irreversible loss of proliferative potential and is accompanied by a n... more Cellular senescence is the irreversible loss of proliferative potential and is accompanied by a number of phenotypic changes. First described by Hayflick and Moorhead in 1961, it has since become a popular model to study cellular aging. The replicative lifespan of human fibroblasts is heterogeneous even in clonal populations, with the fraction of senescent cells increasing with each population doubling (PD). Thus, the study of individual cells in mass culture is necessary in order to properly understand senescence and its associated phenotype. Cell sorting is a process that allows the physical separation of cells based on different characteristics which can be measured by flow cytometry. Here, we describe various methods by which senescent cells can be sorted from mixed cultures and discuss how different methods impact on the posterior analysis of sorted populations.
Telomeres are specialized nucleoprotein structures, which protect chromosome ends and have been i... more Telomeres are specialized nucleoprotein structures, which protect chromosome ends and have been implicated in the ageing process. Telomere shortening has been shown to contribute to a persistent DNA damage response (DDR) during replicative senescence, the irreversible loss of division potential of somatic cells. Similarly, persistent DDR foci can be found in stress-induced senescence, although their nature is not understood. Here we show, using immuno-fluorescent in situ hybridization and ChIP, that up to half of the DNA damage foci in stress-induced senescence are located at telomeres irrespective of telomerase activity. Moreover, live-cell imaging experiments reveal that all persistent foci are associated with telomeres. Finally, we report an age-dependent increase in frequencies of telomere-associated foci in gut and liver of mice, occurring irrespectively of telomere length. We conclude that telomeres are important targets for stress in vitro and in vivo and this has important consequences for the ageing process.
Cellular senescence is a stable proliferation arrest, a potent tumor suppressor mechanism, and a ... more Cellular senescence is a stable proliferation arrest, a potent tumor suppressor mechanism, and a likely contributor to tissue aging. Cellular senescence involves extensive cellular remodeling, including of chromatin structure. Autophagy and lysosomes are important for recycling of cellular constituents and cell remodeling. Here we show that an autophagy/lysosomal pathway processes chromatin in senescent cells. In senescent cells, lamin A/C-negative, but strongly γ-H2AX-positive and H3K27me3-positive, cytoplasmic chromatin fragments (CCFs) budded off nuclei, and this was associated with lamin B1 down-regulation and the loss of nuclear envelope integrity. In the cytoplasm, CCFs were targeted by the autophagy machinery. Senescent cells exhibited markers of lysosomal-mediated proteolytic processing of histones and were progressively depleted of total histone content in a lysosome-dependent manner. In vivo, depletion of histones correlated with nevus maturation, an established histopathologic parameter associated with proliferation arrest and clinical benignancy. We conclude that senescent cells process their chromatin via an autophagy/lysosomal pathway and that this might contribute to stability of senescence and tumor suppression.
American journal of physiology. Lung cellular and molecular physiology, Jan 18, 2015
Cellular senescence has been associated with the structural and functional decline observed durin... more Cellular senescence has been associated with the structural and functional decline observed during physiological lung ageing and in chronic obstructive pulmonary disease (COPD). Airway epithelial cells are the first line of defense in the lungs and are important to COPD pathogenesis. However, the mechanisms underlying airway epithelial cell senescence, and particularly the role of telomere dysfunction in this process, are poorly understood. We aimed to investigate telomere dysfunction in airway epithelial cells from patients with COPD, in the ageing murine lung and following cigarette smoke exposure. We evaluated co-localization of γH2A.X and telomeres and telomere length in small airway epithelial cells from patients with COPD, during murine lung ageing and following cigarette smoke exposure in vivo and in vitro. We found that telomere-associated DNA damage foci increase in small airway epithelial cells from patients with COPD, without significant telomere shortening detected. With...
Telomeres are specialized nucleoprotein structures, which protect chromosome ends and have been i... more Telomeres are specialized nucleoprotein structures, which protect chromosome ends and have been implicated in the ageing process. Telomere shortening has been shown to contribute to a persistent DNA damage response (DDR) during replicative senescence, the irreversible loss of division potential of somatic cells. Similarly, persistent DDR foci can be found in stress-induced senescence, although their nature is not understood. Here we show, using immuno-fluorescent in situ hybridization and ChIP, that up to half of the DNA damage foci in stress-induced senescence are located at telomeres irrespective of telomerase activity. Moreover, live-cell imaging experiments reveal that all persistent foci are associated with telomeres. Finally, we report an age-dependent increase in frequencies of telomere-associated foci in gut and liver of mice, occurring irrespectively of telomere length. We conclude that telomeres are important targets for stress in vitro and in vivo and this has important c...
Cellular senescence is the irreversible loss of proliferative potential and is accompanied by a n... more Cellular senescence is the irreversible loss of proliferative potential and is accompanied by a number of phenotypic changes. First described by Hayflick and Moorhead in 1961, it has since become a popular model to study cellular aging. The replicative lifespan of human fibroblasts is heterogeneous even in clonal populations, with the fraction of senescent cells increasing with each population doubling (PD). Thus, the study of individual cells in mass culture is necessary in order to properly understand senescence and its associated phenotype. Cell sorting is a process that allows the physical separation of cells based on different characteristics which can be measured by flow cytometry. Here, we describe various methods by which senescent cells can be sorted from mixed cultures and discuss how different methods impact on the posterior analysis of sorted populations.
Telomeres are specialized nucleoprotein structures, which protect chromosome ends and have been i... more Telomeres are specialized nucleoprotein structures, which protect chromosome ends and have been implicated in the ageing process. Telomere shortening has been shown to contribute to a persistent DNA damage response (DDR) during replicative senescence, the irreversible loss of division potential of somatic cells. Similarly, persistent DDR foci can be found in stress-induced senescence, although their nature is not understood. Here we show, using immuno-fluorescent in situ hybridization and ChIP, that up to half of the DNA damage foci in stress-induced senescence are located at telomeres irrespective of telomerase activity. Moreover, live-cell imaging experiments reveal that all persistent foci are associated with telomeres. Finally, we report an age-dependent increase in frequencies of telomere-associated foci in gut and liver of mice, occurring irrespectively of telomere length. We conclude that telomeres are important targets for stress in vitro and in vivo and this has important consequences for the ageing process.
Cellular senescence is a stable proliferation arrest, a potent tumor suppressor mechanism, and a ... more Cellular senescence is a stable proliferation arrest, a potent tumor suppressor mechanism, and a likely contributor to tissue aging. Cellular senescence involves extensive cellular remodeling, including of chromatin structure. Autophagy and lysosomes are important for recycling of cellular constituents and cell remodeling. Here we show that an autophagy/lysosomal pathway processes chromatin in senescent cells. In senescent cells, lamin A/C-negative, but strongly γ-H2AX-positive and H3K27me3-positive, cytoplasmic chromatin fragments (CCFs) budded off nuclei, and this was associated with lamin B1 down-regulation and the loss of nuclear envelope integrity. In the cytoplasm, CCFs were targeted by the autophagy machinery. Senescent cells exhibited markers of lysosomal-mediated proteolytic processing of histones and were progressively depleted of total histone content in a lysosome-dependent manner. In vivo, depletion of histones correlated with nevus maturation, an established histopathologic parameter associated with proliferation arrest and clinical benignancy. We conclude that senescent cells process their chromatin via an autophagy/lysosomal pathway and that this might contribute to stability of senescence and tumor suppression.
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Papers by Graeme Hewitt