Cyclin D1 Overexpression Permits the Reproducible Detection of Senescent Human Vascular Smooth Muscle Cells

Burton, Dominick G. A.; Sheerin, Angela; Ostler, Elizabeth L.; Smith, K R; Giles, Peter; Lowe, J.; Rhys‐Williams, William; Kipling, David; Faragher, R. G. A.

doi:10.1196/annals.1404.026

Cited by 42 publications

(34 citation statements)

References 15 publications

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“…Although large cell size correlates with senescence in many cell types, it is known that not all senescent cells within a given culture are large (Mitsui et al 1981). Thus the SA-β-Gal assay almost certainly underestimates the number of senescent cells present in a given sample (Burton et al, 2007). This needs to be borne in mind when the assay is used in morphometric estimates of 'senescent' cell number.…”

Section: Discussionmentioning

confidence: 99%

Microarray analysis of senescent vascular smooth muscle cells: A link to atherosclerosis and vascular calcification

Burton¹,

Giles²,

Sheerin³

et al. 2009

Experimental Gerontology

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International audienceLittle is known about the senescent phenotype of human vascular smooth muscle cells (VSMCs) and the potential involvement of senescent VSMCs in age-related vascular disease, such as atherosclerosis. As such, VSMCs were grown and characterised to generate senescent VSMCs needed for microarray analysis (Affymetrix). Comparative analysis of the transcriptome profiles of early (14 CPD) and late (39-42 CPD) passage VSMCs found a total of 327 probesets called as differentially expressed: 149 are up-regulated in senescence and 178 repressed (-value < 0.5%, minimum effect size of at least 2-fold differential regulation, explore data at www.madras.cf.ac.uk/vsmc). Data mining shows a differential regulation of genes at senescence associated with the development of atherosclerosis and vascular calcification. These included genes with roles in inflammation (IL1β, IL8, ICAM1, TNFAP3, ESM1 and CCL2), tissue remodelling (VEGF, VEGFβ, ADM and MMP14) and vascular calcification (MGP, BMP2, SPP1, OPG and DCN). The microarray data for IL1β, IL8 and MGP were validated by either, ELISA, Western blot analysis or RT-PCR. These data thus provide the first evidence for a role of VSMC senescence in the development of vascular calcification and provides further support for the involvement of senescent VSMCs in the progression of atherosclerosis

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Section: Discussionmentioning

confidence: 99%

Microarray analysis of senescent vascular smooth muscle cells: A link to atherosclerosis and vascular calcification

Burton¹,

Giles²,

Sheerin³

et al. 2009

Experimental Gerontology

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“…Senescent cells have undergone an irreversible cell cycle arrest, and display a radically altered phenotype: genetically, morphologically and behaviourally distinct from its growth-competent counterparts. These changes are thought to have a detrimental impact on neighbouring cells, the surrounding extracellular matrix and other structural components, leading to aged tissues, disease, and an increased risk of cancer (Burton et al 2005(Burton et al , 2007Campisi 1997a, b).…”

Section: Introductionmentioning

confidence: 99%

Cellular senescence, ageing and disease

Burton

2008

AGE

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118

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Cellular senescence is the irreversible growth arrest of individual mitotic cells, which as a consequence display a radically altered phenotype that is thought to impair tissue function and predispose tissues to disease development and/or progression as they gradually accumulate. However, in the past, research into mechanisms of ageing has commonly been researched and treated separately from disease development. This may partly be due to the lack of understanding concerning mechanisms of ageing and the difficulty in implementing what was known into models of disease development. Only in the last 10 years, with increasing knowledge of the senescent phenotype and the ability to detect senescent cells in human tissues, have biologists been able to investigate the relationship between cellular senescence and disease. This review therefore brings together and discusses recent findings which suggest that cellular senescence does contribute to ageing and the development/ progression of disease.

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“…Paradoxically, senescent cells have extremely high levels of cyclin D1. 14,[32][33][34][35][36][37][38][39][40] The levels of cyclins D1 and E far exceeded their levels in proliferating cells. 14,41 Furthermore, hyperelevated cyclins were the earliest markers of geroconversion.…”

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confidence: 99%

MEK drives cyclin D1 hyperelevation during geroconversion

2013

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When the cell cycle becomes arrested, MTOR (mechanistic Target of Rapamycin) converts reversible arrest into senescence (geroconversion). Hyperexpression of cyclin D1 is a universal marker of senescence along with hypertrophy, beta-Gal staining and loss of replicative/regenerative potential (RP), namely, the ability to restart proliferation when the cell cycle is released. Inhibition of MTOR decelerates geroconversion, although only partially decreases cyclin D1. Here we show that in p21-and p16-induced senescence, inhibitors of mitogen-activated/extracellular signal-regulated kinase (MEK) (U0126, PD184352 and siRNA) completely prevented cyclin D1 accumulation, making it undetectable. We also used MEL10 cells in which MEK inhibitors do not inhibit MTOR. In such cells, U0126 by itself induced senescence that was remarkably cyclin D1 negative. In contrast, inhibition of cyclin-dependent kinase (CDK) 4/6 by PD0332991 caused cyclin D1-positive senescence in MEL10 cells. Both types of senescence were suppressed by rapamycin, converting it into reversible arrest. We confirmed that the inhibitor of CDK4/6 caused cyclin D1 positive senescence in normal RPE cells, whereas U0126 prevented cyclin D1 expression. Elimination of cyclin D1 by siRNA did not prevent other markers of senescence that are consistent with the lack of its effect on MTOR. Our data confirmed that a mere inhibition of the cell cycle was sufficient to cause senescence, providing MTOR was active, and inhibition of MEK partially inhibited MTOR in a cell-type-dependent manner. Second, hallmarks of senescence may be dissociated, and hyperelevated cyclin D1, a marker of hyperactivation of senescent cells, did not necessarily determine other markers of senescence. Third, inhibition of MEK was sufficient to eliminate cyclin D1, regardless of MTOR. In proliferating cells, MTOR (mechanistic Target of Rapamycin) stimulates cellular mass growth, whereas cyclins D and E initiate S-phase transition.1-4 Withdrawal of growth factors causes reversible cell-cycle arrest (quiescence) with low levels of cyclins and MTOR activity, reversible by re-addition of growth factors. In contrast, cell-cycle arrest in the presence of growth stimuli, which activate MTOR, leads to cellular senescence. 5,6 In other words, when the cell cycle is blocked, while growth-promoting pathways such as MTOR remain active, cells continue to grow in size and undergo senescence. A process of conversion from normal reversible arrest into senescence was named as gerogenic conversion (geroconversion).7 Cellular senescence is characterized by large flat morphology (hypertrophy), beta-Gal staining, very high levels of cyclins D1 and E, senescence-associated secretory phenotype and loss of replicative/regenerative potential (RP).7-12 Loss of RP means that cells cannot proliferate even when cell-cycle block is removed and cells re-enter the cell cycle.13,14 Rapamycin decreases hypertrophy and loss of RP, in a dose-dependent manner proportional to S6 dephosphorylation (a marker of MTORC1 activity). 15Other ...

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Cyclin D1 Overexpression Permits the Reproducible Detection of Senescent Human Vascular Smooth Muscle Cells

Cited by 42 publications

References 15 publications

Microarray analysis of senescent vascular smooth muscle cells: A link to atherosclerosis and vascular calcification

Microarray analysis of senescent vascular smooth muscle cells: A link to atherosclerosis and vascular calcification

Cellular senescence, ageing and disease

MEK drives cyclin D1 hyperelevation during geroconversion

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