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At least 127 records · Page 7Linked to original sources
PubMed2006

Activation of peroxisome proliferator-activated receptor gamma suppresses telomerase activity in vascular smooth muscle cells.

Activation of the peroxisome proliferator-activated receptor (PPAR) gamma, the molecular target for insulin sensitizing thiazolidinediones used in patients with type 2 diabetes, inhibits vascular smooth muscle cell (VSMC) proliferation and prevents atherosclerosis and neointima formation. Emerging evidence indicates that telomerase controls key cellular functions including replicative lifespan, differentiation, and cell proliferation. In the present study, we demonstrate that ligand-induced and constitutive PPARgamma activation inhibits telomerase activity in VSMCs. Telomerase reverse transcriptase (TERT) confers the catalytic activity of telomerase, and PPARgamma ligands inhibit TERT expression through a receptor-dependent suppression of the TERT promoter. 5'-deletion analysis, site-directed mutagenesis, and transactivation studies using overexpression of Ets-1 revealed that suppression of TERT transcription by PPARgamma is mediated through negative cross-talk with Ets-1-dependent transactivation of the TERT promoter. Chromatin immunoprecipitation assays further demonstrated that PPARgamma ligands inhibit Ets-1 binding to the TERT promoter, which is mediated at least in part through an inhibition of Ets-1 expression by PPARgamma ligands. In VSMCs overexpressing TERT, the efficacy of PPARgamma ligands to inhibit cell proliferation is lost, indicating that TERT constitutes an important molecular target for the antiproliferative effects of PPARgamma ligands. Finally, we demonstrate that telomerase activation during the proliferative response after vascular injury is effectively inhibited by PPARgamma ligands. These findings provide a previously unrecognized mechanism for the antiproliferative effects of PPARgamma ligands and support the concept that PPARgamma ligands may constitute a novel therapeutic approach for the treatment of proliferative cardiovascular diseases.

Animals↗
PubMed2004

Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion.

Human marrow stromal cells (MSCs) can be isolated from bone marrow and differentiate into multiple tissues in vitro and in vivo. These properties make them promising tools in cell and gene therapy. The lack of a specific MSC marker and the low frequency of MSCs in bone marrow necessitate their isolation by in vitro expansion prior to clinical use. This may severely reduce MSC proliferative capacity to the point that the residual proliferative potential is insufficient to maintain long-term tissue regeneration upon reinfusion. In this study we determined the effect of in vitro expansion on the replicative capacity of MSCs by correlating their rate of telomere loss during in vitro expansion with their behavior in vivo. We report that even protocols that involve minimal expansion induce a rapid aging of MSCs, with losses equivalent to about half their total replicative lifespan.

Bone Marrow Cells↗
PubMed2005

Telomere and telomerase dynamics in human cells.

Accumulating evidence now implicates telomeres and telomerase as critical regulators genomic stability and replicative lifespan in mammalian cells. Disruption of telomere maintenance and/or telomerase expression contributes to the etiology of some degenerative diseases and may participate in the process of aging. Although telomere dysfunction and aberrant telomerase expression clearly play important roles in cancer development, the contribution of telomere biology to cancer is complex and involves both positive and negative influences on tumor development. Indeed, recent work from several laboratories suggests additional roles for telomeres and telomerase in both normal and malignant physiology. Understanding the complexity of telomere biology will provide further insights into chromosome biology in both normal and malignant cells.

Cellular Senescence↗
PubMed1998

Telomerase in cancer: clinical applications.

The biology of telomeres and telomerase has been the subject of intensive investigative effort since it became evident that they play a significant role in two important biological processes, the loss of cellular replicative capacity inherent to organismal ageing and the unrestricted cell proliferation characteristic of carcinogenesis. Telomere shortening in normal cells is a result of DNA replication events, and reduction beyond a critical length is a signal for cellular senescence. One of the cellular mechanisms used to overcome proliferative restriction is the activation of the enzyme telomerase, which replaces the loss of telomeric DNA that occurs at each cell division. Studies have demonstrated that tumours have shorter telomeres than normal tissue and that telomerase is activated in up to 90% of all human cancers while it is present only in a limited range of normal adult tissues. The role of telomerase in the extension of the cellular replicative lifespan has recently been shown by ectopic expression of the enzyme, being consistent with the oncogenesis model whereby the acquisition of an 'immortal' phenotype is a requirement for advanced tumour progression. In this article we review the present knowledge of telomeres and telomerase in cancer and discuss the potential use of this enzyme as a diagnostic and prognostic tumour marker and as a target for cancer therapy.

Adult↗
PubMed2004

Enhanced long-term survival, but no increase in replicative capacity, following retroviral transduction of human cord blood CD34+ cells with human telomerase reverse transcriptase.

Overexpression of telomerase reverse transcriptase (hTERT) can immortalize some primary human mesenchymal cells. We investigated whether retrovirally-mediated expression of hTERT in CD34+ umbilical cord blood (UCB) cells can extend the replicative lifespan of human hematopoietic progenitor cells. Overexpression of hTERT did not immortalize these cells but did lead to enhanced survival of mature hematopoietic cells.

Antigens, CD34↗
PubMedDate not supplied

Normal human mesothelial cells and fibroblasts transfected with the EJras oncogene become EGF-independent, but are not malignantly transformed.

The nature of the lesion in growth control exerted by the cancer-derived c-H-ras mutation, EJ-ras, and its transforming potential in diploid cells are both poorly understood. We introduced EJ-ras into normal, diploid human mesothelial cells and fibroblasts and obtained transfectants expressing p21EJ-ras. All clones examined were independent of EGF for rapid growth, and all secreted an EGF-like mitogen into the medium at levels sufficient to satisfy the EGF requirement of normal cells. The EJ-ras transfectants were not altered with respect to any other growth requirement, and they were not transformed. Eleven clones tested all retained a finite replicative lifespan which, in most cases, was the same as that of the parent cell strain. Three transfectants tested were not tumorigenic in nude mice. Thus p21EJ-ras can circumvent an important mitogenic signal pathway in human cells. Nevertheless, neither the secretion of an autocrine growth factor nor any other effect of p21EJ-ras serves to malignantly transform normal human cells, in contrast to the susceptibility of some established rodent cell lines to transformation by these mechanisms.

Cell Division↗
PubMed1994

The cellular and molecular biology of skin aging.

BACKGROUND: The dramatic alterations in the appearance of the integument with increasing age are due in part to a progressive destruction of the delicate architecture of the connective tissue components of the dermis. Both collagenous and elastic components display a degeneration consistent with the overexpression of proteolytic activity. Recent advances in the field of molecular gerontology, using in vitro models of cellular aging, are yielding clues as to the fundamental causes of dermal aging. OBSERVATIONS: Dermal fibroblasts possess a finite replicative capacity of 50 to 100 doublings, then cease replicating in response to growth factors. Cells cultivated to the end of their replicative lifespan in vitro display alterations consistent with their playing a role in aging in vivo. In particular, senescent dermal fibroblasts overexpress metalloproteinase activities that may explain the age-related atrophy of extracellular matrix architecture. CONCLUSIONS: The recent discovery of a structural change in the telomeric region of the genome with cellular aging and new insights into DNA damage checkpoint mechanisms offer new opportunities to uncover both the molecular mechanisms regulating cellular aging and possibly to devise new strategies to manipulate these molecular events for therapeutic effect.

Cellular Senescence↗
PubMed1997

Copy number, epigenetic state and expression of the rRNA genes in young and senescent rat embryo fibroblasts.

The recent cloning of the gene that causes the premature aging in Werner syndrome patients has evoked speculations that deficits in expression of the ribosomal RNA genes could be related to cellular aging in general. Here we compare the state of the rRNA genes and the rRNA metabolism in young and senescent (aged) rat embryo fibroblasts (REF). Southern blot analysis revealed that the copy number and the methylation state of the genes did not change significantly with increasing cumulative population doublings (CPD) of the culture. Hence, young (low numbers of CPD) and senescent REF (high numbers of CPD), respectively, have the same repertoire of rDNA units that can be transcribed. The rRNA synthesis in these cells was analyzed by incorporation of labeled uridine at conditions allowing the measurement of absolute rather than relative rRNA synthesis rates. We revealed that the cell density dependence of the rRNA synthesis diminishes in senescent cells. Exponentially growing young REF exhibited an rRNA synthesis of 16 amol uridine incorporation per minute and cell. The rRNA synthesis decreased 10-fold in quiescent cells at saturating cell densities. Exponentially growing REF near the end of their replicative lifespan exhibited a 2-fold lower rRNA synthesis rate compared to young cells. However, in senescent REF the rRNA synthesis rate decreased only 2-fold with increasing cell densities resulting in a 3-fold higher rRNA synthesis rate compared to young cells at saturating cell densities. These data could be confirmed by calculating the rRNA synthesis rates from the rRNA content, the rRNA half-life, and the proliferation rate of the cells. Hence, senescent REF exhibited a higher rRNA synthesis rate when compared to young cells at similar growth rates resulting in the generally observed higher rRNA content (and cell size) of senescent cells. We conclude that cellular senescence of REF is not accompanied by rRNA expression deficiencies.

Animals↗
PubMed1995

A biomarker that identifies senescent human cells in culture and in aging skin in vivo.

Normal somatic cells invariably enter a state of irreversibly arrested growth and altered function after a finite number of divisions. This process, termed replicative senescence, is thought to be a tumor-suppressive mechanism and an underlying cause of aging. There is ample evidence that escape from senescence, or immortality, is important for malignant transformation. By contrast, the role of replicative senescence in organismic aging is controversial. Studies on cells cultured from donors of different ages, genetic backgrounds, or species suggest that senescence occurs in vivo and that organismic lifespan and cell replicative lifespan are under common genetic control. However, senescent cells cannot be distinguished from quiescent or terminally differentiated cells in tissues. Thus, evidence that senescent cells exist and accumulate with age in vivo is lacking. We show that several human cells express a beta-galactosidase, histochemically detectable at pH 6, upon senescence in culture. This marker was expressed by senescent, but not presenescent, fibroblasts and keratinocytes but was absent from quiescent fibroblasts and terminally differentiated keratinocytes. It was also absent from immortal cells but was induced by genetic manipulations that reversed immortality. In skin samples from human donors of different age, there was an age-dependent increase in this marker in dermal fibroblasts and epidermal keratinocytes. This marker provides in situ evidence that senescent cells may exist and accumulate with age in vivo.

Adult↗
PubMed2000

The impact of media composition and petite mutation on the longevity of a polyploid brewing yeast strain.

Ageing in Saccharomyces cerevisiae is a finite phenomenon, determined by replicative, rather than chronological lifespan. Yeast physiological condition is known to influence industrial fermentation performance, however, until recently cellular senescence has not been considered as a brewing yeast stress factor. A polyploid lager yeast (BB11) and a brewery isolate, exhibiting petite mutation were analysed for longevity. It was observed that mitochondrial deficiency induced a reduction in lifespan. In addition, replicative capacity was perceived to be dependent on environmental conditions.

Culture Media↗
PubMed2001

Chronological lifespan of stationary phase yeast cells; a model for investigating the factors that might influence the ageing of postmitotic tissues in higher organisms.

Budding yeast can be considered to have two distinct lifespans: (a) a replicative (budding, non-chronological) lifespan, measured as the number of daughters produced by each actively dividing mother cell; and (ii) a chronological lifespan, measured as the ability of stationary cultures to maintain viability over time. In non-dividing cells, essential components that become damaged cannot be diluted out through cell division but must, of necessity, be turned over and renewed. By elevating stress resistances, many of the activities needed for such renewal should be elevated with commensurate reduction in the steady-state levels of damaged cell components. Therefore, chronological lifespan in particular might be expected to relate to stress resistance. For yeast to attain a full chronological lifespan requires the expression of the general stress response. It is more important, though, that the cells should be efficiently adapted to respiratory maintenance, since it is cultures grown to stationary phase on respiratory media that usually display the longest chronological lifespans. For this reason, respiration-adapted cells potentially provide a better model of chronological ageing than cultures pre-grown on glucose.

Animals↗
PubMed1998

Gompertzian mortality originates in the winding-down of the mitotic clock.

UNLABELLED: Gompertz' age-related exponential increase in mortality rate and the obdurately flat mortality trajectory of Drosophila are paradoxical notions for metazoan aging theory. A multiclonal model of Gompertzian organisms provides a resolution by assuming that (a) conception initiates a stochastic process producing a train of replications of fixed length (the Hayflick limit); (b) unique stem cells arise early on to generate multiple vital clones; (c) life continues until one such clone critically depletes its replicative potential. Lifespan is thus governed by the time it takes to reach the terminal branches of the mitotic tree. Although these times are not independent, asymptotic independence can be justified. This clears the way for asymptotic extreme-value theory to guarantee: (1) a non-increasing failure rate, under unlimited replicability; (2) an exponentially increasing failure rate, under limited replicability. However, to obtain an exact fit to the human force-of-mortality function also requires the inclusion of the phenomenon of mitotic deceleration (implemented with a lognormal model of replication). CONCLUSION: the sine qua non of Gompertzian mortality is cellular aging, expressed through these two mitotic phenomena. Conversely, those metazoa with unlimited cellular replicability, by staving off clonal failure would succumb only to catastrophic, age-independent events, yielding a constant mortality rate, the signature of a mitotic clock that does not run down.

Animals↗
PubMed2004

Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts.

Limitation of lifespan in replicative senescence is related to oxidative stress, which is probably both the cause and consequence of impaired mitochondrial respiratory function. The respiration of senescent human diploid fibroblasts was analysed by high-resolution respirometry. To rule out cell-cycle effects, proliferating and growth-arrested young fibroblasts were used as controls. Uncoupled respiration, as normalized to citrate synthase activity, remained unchanged, reflecting a constant capacity of the respiratory chain. Oligomycin-inhibited respiration, however, was significantly increased in mitochondria of senescent cells, indicating a lower coupling of electron transport with phosphorylation. In contrast, growth-arrested young fibroblasts exhibited a higher coupling state compared with proliferating controls. In intact cells, partial uncoupling may lead to either decreased oxidative ATP production or a compensatory increase in routine respiration. To distinguish between these alternatives, we subtracted oligomycin-inhibited respiration from routine respiration, which allowed us to determine the part of respiratory activity coupled with ATP production. Despite substantial differences in the respiratory control ratio, ranging from 4 to 11 in the different experimental groups, a fixed proportion of respiratory capacity was maintained for coupled oxidative phosphorylation in all the experimental groups. This finding indicates that the senescent cells fully compensate for increased proton leakage by enhanced electron-transport activity in the routine state. These results provide a new insight into age-associated defects in mitochondrial function and compensatory mechanisms in intact cells.

Aging↗
PubMed2024

Nonlethal deleterious mutation-induced stress accelerates bacterial aging.

Random mutagenesis, including when it leads to loss of gene function, is a key mechanism enabling microorganisms' long-term adaptation to new environments. However, loss-of-function mutations are often deleterious, triggering, in turn, cellular stress and complex homeostatic stress responses, called "allostasis," to promote cell survival. Here, we characterize the differential impacts of 65 nonlethal, deleterious single-gene deletions on Escherichia coli growth in three different growth environments. Further assessments of select mutants, namely, those bearing single adenosine triphosphate (ATP) synthase subunit deletions, reveal that mutants display reorganized transcriptome profiles that reflect both the environment and the specific gene deletion. We also find that ATP synthase α-subunit deleted (ΔatpA) cells exhibit elevated metabolic rates while having slower growth compared to wild-type (wt) E. coli cells. At the single-cell level, compared to wt cells, individual ΔatpA cells display near normal proliferation profiles but enter a postreplicative state earlier and exhibit a distinct senescence phenotype. These results highlight the complex interplay between genomic diversity, adaptation, and stress response and uncover an "aging cost" to individual bacterial cells for maintaining population-level resilience to environmental and genetic stress; they also suggest potential bacteriostatic antibiotic targets and -as select human genetic diseases display highly similar phenotypes, - a bacterial origin of some human diseases.

Escherichia coli↗
PubMed1983

Transformation and immortalization of human keratinocytes by SV40.

We have studied the appearance of transformed properties following infection of human epidermal keratinocytes by the oncogenic virus SV40. Shortly after infection, only a small fraction of the cells are positive for SV40 T antigen by immunofluorescence; this fraction progressively increases upon serial subcultivation concomitant with an increase in plating/colony-forming efficiency and growth rate. The capacity of the cells to differentiate progressively decreases, as indicated by cytochemical staining and cornified cell-envelope formation induced by suspension in methyl cellulose. The infected cells enter a period of growth crisis characterized by cytopathology and cell death as the level of T antigen synthesis reaches about 90 percent positive cells at about the tenth serial passage. Viable cells emerging from the crisis period are found to exhibit anchorage-independent growth, as indicated by the formation of viable colonies in semisolid media, but there is considerable variability in colony formation among clones isolated from anchorage-independent populations. The emergent population also manifests phenotypic instability in terms of the appearance of variants, which, in contrast to uninfected cells, expresses a well-defined actin cytoskeleton. The infected cells eventually become "immortalized," as evidenced by an indefinite lifespan, i.e., replication capability maintained well beyond the ordinary time of senescence for uninfected cells. We present these findings in the context of a stage-specific model of epithelial transformation in vitro.

Actins↗
PubMed2005

Generation of an immortalized human endothelial cell line as a model of neovascular proliferating endothelial cells to assess chemosensitivity to anticancer drugs.

Assessment of chemosensitivity of neovessel endo-thelium associated to tumor mass is hindered by the limited availability of experimental models of actively proliferating endothelial cells. In fact, primary endothelial cells possess a limited lifespan and replicative senescence represents a major limit to their long-term culture. Moreover, non-dividing senescent cells undergo a gradual loss of phenotypic markers and become unable to respond to mitogenic stimuli. We report the generation of an immortalized human endothelial cell line by transfection of human umbilical vein endothelial cells (HUVEC) with both SV40 large/small T antigens and the catalytic subunit of human telomerase. This cell line (HUV-ST) possesses stabilized telomere length and increased proliferation rate with respect to parental cells or to cells transfected with SV40 T antigens only (HUV-S). Nevertheless, even at PD > 100 it is not tumorigenic and displays all major endothelial phenotypic markers, such as von Willebrand factor, CD31, vascular endothelial growth factor (VEGF) receptors (VEGFR1/Flt-1, VEGR2/KDR) and CD105/endoglin. HUV-ST cells are capable of organizing into tubule-like networks with branching morphology in response to appropriate stimuli and migrate upon exposure to VEGF. Interestingly, HUV-ST cells over-express the tumor endothelial marker-1/endosialin which is regarded as the most differentially expressed molecule in tumor-derived endothelium versus normal-derived endothelium. Analysis of chemosensitivity to the wide spectrum methylating agent temozolomide (TMZ), an anticancer drug more effective against actively dividing cells than against resting or slowing proliferating cells, indicated that HUV-ST cells are more susceptible to the drug with respect to HUVEC or HUV-S cells. Abrogation of poly(ADP-ribose) polymerase activity significantly enhances growth inhibition induced by TMZ. In conclusion, the immortalized human endothelial line HUV-ST represents a suitable model for studying the efficacy of anti-neovascular therapy, mimicking proliferating neovascular endothelial cells associated to the tumor mass.

Animals↗
PubMed2006

Preadaptation to efficient respiratory maintenance is essential both for maximal longevity and the retention of replicative potential in chronologically ageing yeast.

Only recently have the studies of yeast ageing started to focus on the S288c-derived strains used extensively in genomics and on the longest lifespans. Chronological longevity (stationary (G(0)) survival) of such strains is greater when cells are pre-grown on a respiratory carbon source, as compared to when they are pre-grown on glucose (the latter a respiration-repressing sugar). Prior adaptation to efficient respiratory maintenance also ensures that such chronologically aged yeast cells still display a full replicative lifespan should they reenter the cell cycle. In contrast, cells that are pre-grown on glucose exhibit marked and progressive losses of replicative potential as they age chronologically in stationary phase. Increasing the respiratory activity in glucose-grown cultures by HAP4 gene overexpression increased survival and reversed the loss of replicative potential during a subsequent stationary phase. Adaptation to efficient respiratory maintenance is therefore important, not just for maximal longevity, but also for the maintenance of a full replicative lifespan by chronologically ageing cultures of yeast. In such respiration-adapted cultures, losses of the Sch9 protein kinase or Yca1 caspase both shortened lifespan. In contrast loss of Yap1, the major transcriptional regulator of the oxidative stress response, generated a small increase in chronological lifespan in certain strain backgrounds. It would appear, therefore, that any induction of oxidative stress response genes in chronologically ageing yeast is not operating to generate an increase in longevity, even though such protective effects might be expected from the increased proxidant status of these cells over time.

Adaptation, Physiological↗