Cellular senescence, a stress induced growth arrest of somatic cells, was first documented in cell cultures over forty years ago, however its physiological significance has only recently been demonstrated. Using novel biomarkers of cellular senescence we examined whether senescent cells accumulate in tissues from baboons of ages encompassing the entire lifespan of this species. We show that dermal fibroblasts, displaying markers of senescence such as telomere damage, active checkpoint kinase ATM, high levels of heterochromatin proteins and elevated levels of p16, accumulate in skin biopsies from baboons with advancing age. The number of dermal fibroblasts containing damaged telomeres reaches a value of over 15% of total fibroblasts, whereas 80% of cells contain high levels of the heterochromatin protein HIRA. In skeletal muscle, a postmitotic tissue, only a small percentage of myonuclei containing damaged telomeres were detected regardless of animal age. The presence of senescent cells in mitotic tissues might therefore be a contributing factor to aging and age related pathology and provides further evidence that cellular senescence is a physiological event.

Cellular senescence, first observed and defined using in vitro cell culture studies, is an irreversible cell cycle arrest which can be triggered by a variety of factors. Emerging evidence suggests that cellular senescence acts as an in vivo tumor suppression mechanism by limiting aberrant proliferation. It has also been postulated that cellular senescence can occur independently of cancer and contribute to the physiological processes of normal organismal aging. Recent data have demonstrated the in vivo accumulation of senescent cells with advancing age. Some characteristics of senescent cells, such as the ability to modify their extracellular environment, could play a role in aging and age-related pathology. In this review, we examine current evidence that links cellular senescence and organismal aging.

SummaryChromatin is highly dynamic and subject to extensive remodeling under many physiologic conditions. Changes in chromatin that occur during the aging process are poorly documented and understood in higher organisms, such as mammals. We developed an immunofluorescence assay to quantitatively detect, at the single cell level, changes in the nuclear content of chromatin-associated proteins. We found increased levels of the heterochromatin-associated proteins histone macro H2A (mH2A) and heterochromatin protein 1 beta (HP1b) in human fibroblasts during replicative senescence in culture, and for the first time, an age-associated increase in these heterochromatin marks in several tissues of mice and primates. Mouse lung was characterized by monophasic mH2A expression histograms at both ages, and an increase in mean staining intensity at old age. In the mouse liver, we observed increased age-associated localization of mH2A to regions of pericentromeric heterochromatin. In the skeletal muscle, we found two populations of cells with either low or high mH2A levels. This pattern of expression was similar in mouse and baboon, and showed a clear increase in the proportion of nuclei with high mH2A levels in older animals. The frequencies of cells displaying evidence of increased heterochromatinization are too high to be readily accounted for by replicative or oncogeneinduced cellular senescence, and are prominently found in terminally differentiated, postmitotic tissues that are not conventionally thought to be susceptible to senescence. Our findings distinguish specific chromatin states in individual cells of mammalian tissues, and provide a foundation to investigate further the progressive epigenetic changes that occur during aging.

The replicative life span of human fibroblasts is heterogeneous, with a fraction of cells senescing at every population doubling. To find out whether this heterogeneity is due to premature senescence, i.e. driven by a nontelomeric mechanism, fibroblasts with a senescent phenotype were isolated from growing cultures and clones by flow cytometry. These senescent cells had shorter telomeres than their cycling counterparts at all population doubling levels and both in mass cultures and in individual subclones, indicating heterogeneity in the rate of telomere shortening. Ectopic expression of telomerase stabilized telomere length in the majority of cells and rescued them from early senescence, suggesting a causal role of telomere shortening. Under standard cell culture conditions, there was a minor fraction of cells that showed a senescent phenotype and short telomeres despite active telomerase. This fraction increased under chronic mild oxidative stress, which is known to accelerate telomere shortening. It is possible that even high telomerase activity cannot fully compensate for telomere shortening in all cells. The data show that heterogeneity of the human fibroblast replicative life span can be caused by significant stochastic cell-to-cell variation in telomere shortening.Heterogeneity is a hallmark of aging. Even genetically identical organisms in a controlled homogeneous environment age at different rates and have vastly different life spans. This is also true for aging of human somatic cells in vitro (1). Replicative life spans differ widely between subclones (2), and there is an ever increasing number of senescent cells in dividing cultures (3). Senescence of human fibroblasts is telomere-driven, i.e. induced by the uncapping of telomeres (4). However, it is often assumed that only the life span of the longest surviving clone(s) is governed by telomere shortening, whereas termination of growth in the early senescing clones might be premature, i.e. caused by stress via telomere-independent mechanisms (5). In fact, a senescent phenotype can be generated in response to a variety of stresses that are telomere-independent and inhibit cell growth, for instance, in response to generalized DNA damage (6), oncogene activation (7), histone acetylation (8), or by induction of the p16 pathway through so far not well characterized mechanisms (9).An alternative to this idea of premature senescence is the proposal that stochastic telomere uncapping can cause heterogeneity in replicative life span (10). The probability of telomere uncapping is dependent on a number of factors, including the presence of active telomerase, the integrity of the singlestranded telomeric G-rich overhang and of the higher order structure of telomeres, the functionality of a number of telomere-binding proteins, and telomere length (for review see Ref. 4). Finally, there is evidence for a substantial interaction between stress and specifically mild oxidative stress and telomeres in cell replicative senescence. Stress accelerates telomere shortening ...

Cellular senescence is a stable proliferation arrest associated with an altered secretory pathway, the senescence-associated secretory phenotype. However, cellular senescence is initiated by diverse molecular triggers, such as activated oncogenes and shortened telomeres, and is associated with varied and complex physiological endpoints, such as tumor suppression and tissue aging. The extent to which distinct triggers activate divergent modes of senescence that might be associated with different physiological endpoints is largely unknown. To begin to address this, we performed gene expression profiling to compare the senescence programs associated with two different modes of senescence, oncogene-induced senescence (OIS) and replicative senescence (RS [in part caused by shortened telomeres]). While both OIS and RS are associated with many common changes in gene expression compared to control proliferating cells, they also exhibit substantial differences. These results are discussed in light of potential physiological consequences, tumor suppression and aging.

Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin.

Etoposide, a topoisomerase II poison is used in the treatment of a number of solid tumors. Contradictory data exist on the role of the telomere/telomerase complex in etoposide induced apoptosis. Therefore we examined the effects of etoposide treatment in the neuroblastoma cell line SHSY5Y, with very short telomeres and the acute lymphoblastic T cell line 1301, which displays extremely long telomeres. Both short-term and continuous exposure to the drug were examined. Etoposide induced widespread DNA damage followed by DNA damage foci formation and ultimately growth arrest and apoptosis in a concentration-dependent manner. However, length of telomeres and of single stranded telomeric G rich overhangs did not change significantly under the treatments in any cell line. There was no significant induction of single-strand breaks in the G-rich strand of telomeres. Telomerase activity was transiently upregulated under low concentrations of etoposide, while high concentrations resulted in decreased telomerase activity only after onset of apoptosis. Telomerase overexpression protected against etoposide induced apoptosis in fibroblasts. The data suggest that telomeres are not major signal transducers towards growth arrest or apoptosis after etoposide treatment. However, upregulation of telomerase might be part of an attempted adaptative response, which protects cells by a mechanism that might be independent of telomere length maintenance.www.landesbioscience.com Cell Cycle

Here we describe a carefully optimized method for the preparation of high quality RNA by flow sorting of formaldehyde fixed senescent cells immunostained for any intracellular antigen. Replicative cellular senescence is a phenomenon of irreversible growth arrest triggered by the accumulation of a discrete number of cell divisions. The underlying cause of senescence due to replicative exhaustion is telomere shortening. We document here a spontaneous and apparently stochastic process that continuously generates senescent cells in cultures fully immortalized with telomerase. In the course of studying this phenomenon we developed a preparative fluorescence activated flow sorting method based on immunofluorescent staining of intracellular antigens that can also deliver RNA suitable for quantitative analysis of global gene expression. The protocols were developed using normal human diploid fibroblasts (HDF) and up to 5×107 cells could be conveniently processed in a single experiment. The methodology is based on formaldehyde crosslinking of cells, followed by permeabilization, antibody staining, flow sorting, reversal of the crosslinks, and recovery of the RNA. We explored key parameters such as crosslink reversal that affect the fragmentation of RNA. The recovered RNA is of high quality for downstream molecular applications based on short range sequence analysis, such qPCR, hybridization microarrays, and next generation sequencing. The RNA was analyzed by Affymetrix Gene Chip expression profiling and compared to RNA prepared by the direct lysis of cells. The correlation between the data sets was very high, indicating that the procedure does not introduce systematic changes in the mRNA transcriptome. The methods presented in this communication should be of interest to many investigators working in diverse model systems.