Publisher Correction: Transcriptomic alterations during ageing reflect the shift from cancer to degenerative diseases in the elderly.

The original version of this Article contained an error in the spelling of the author Jule Müller, which was incorrectly given as Julia Müller. Additionally, in Fig. 4a, the blue-red colour scale for fold change in ageing/disease regulation included a blue stripe in place of a red stripe at the right-hand end of the scale. These errors have been corrected in both the PDF and HTML versions of the Article.

Raman and infrared spectroscopy reveal that proliferating and quiescent human fibroblast cells age by biochemically similar but not identical processes.

Dermal fibroblast cells can adopt different cell states such as proliferation, quiescence, apoptosis or senescence, in order to ensure tissue homeostasis. Proliferating (dividing) cells pass through the phases of the cell cycle, while quiescent and senescent cells exist in a non-proliferating cell cycle-arrested state. However, the reversible quiescence state is in contrast to the irreversible senescence state. Long-term quiescent cells transit into senescence indicating that cells age also when not passing through the cell cycle. Here, by label-free in vitro vibrational spectroscopy, we studied the biomolecular composition of quiescent dermal fibroblast cells and compared them with those of proliferating and senescent cells. Spectra were examined by multivariate statistical analysis using a PLS-LDA classification model, revealing differences in the biomolecular composition between the cell states mainly associated with protein alterations (variations in the side chain residues of amino acids and protein secondary structure), but also within nucleic acids and lipids. We observed spectral changes in quiescent compared to proliferating cells, which increased with quiescence cultivation time. Raman and infrared spectroscopy, which yield complementary biochemical information, clearly distinguished contact-inhibited from serum-starved quiescent cells. Furthermore, the spectra displayed spectral differences between quiescent cells and proliferating cells, which had recovered from quiescence. This became more distinct with increasing quiescence cultivation time. When comparing proliferating, (in particular long-term) quiescent and senescent cells, we found that Raman as well as infrared spectroscopy can separate these three cellular states from each other due to differences in their biomolecular composition. Our spectroscopic analysis shows that proliferating and quiescent fibroblast cells age by similar but biochemically not identical processes. Despite their aging induced changes, over long time periods quiescent cells can return into the cell cycle. Finally however, the cell cycle arrest becomes irreversible indicating senescence.

CENP-C/H/I/K/M/T/W/N/L and hMis12 but not CENP-S/X participate in complex formation in the nucleoplasm of living human interphase cells outside centromeres.

Kinetochore proteins assemble onto centromeric chromatin and regulate DNA segregation during cell division. The inner kinetochore proteins bind centromeres while most outer kinetochore proteins assemble at centromeres during mitosis, connecting the complex to microtubules. Here, we measured the co-migration between protein pairs of the constitutive centromere associated network (CCAN) and hMis12 complexes by fluorescence cross-correlation spectroscopy (FCCS) in the nucleoplasm outside centromeres in living human interphase cells. FCCS is a method that can tell if in living cells two differently fluorescently labelled molecules migrate independently, or co-migrate and thus are part of one and the same soluble complex. We also determined the apparent dissociation constants (Kd) of the hetero-dimers CENP-T/W and CENP-S/X. We measured co-migration between CENP-K and CENP-T as well as between CENP-M and CENP-T but not between CENP-T/W and CENP-S/X. Furthermore, CENP-C co-migrated with CENP-H, and CENP-K with CENP-N as well as with CENP-L. Thus, in the nucleoplasm outside centromeres, a large fraction of the CENP-H/I/K/M proteins interact with CENP-C, CENP-N/L and CENP-T/W but not with CENP-S/X. Our FCCS analysis of the Mis12 complex showed that hMis12, Nsl1, Dsn1 and Nnf1 also form a complex outside centromeres of which at least hMis12 associated with the CENP-C/H/I/K/M/T/W/N/L complex.

Transcriptomic alterations during ageing reflect the shift from cancer to degenerative diseases in the elderly.

Disease epidemiology during ageing shows a transition from cancer to degenerative chronic disorders as dominant contributors to mortality in the old. Nevertheless, it has remained unclear to what extent molecular signatures of ageing reflect this phenomenon. Here we report on the identification of a conserved transcriptomic signature of ageing based on gene expression data from four vertebrate species across four tissues. We find that ageing-associated transcriptomic changes follow trajectories similar to the transcriptional alterations observed in degenerative ageing diseases but are in opposite direction to the transcriptomic alterations observed in cancer. We confirm the existence of a similar antagonism on the genomic level, where a majority of shared risk alleles which increase the risk of cancer decrease the risk of chronic degenerative disorders and vice versa. These results reveal a fundamental trade-off between cancer and degenerative ageing diseases that sheds light on the pronounced shift in their epidemiology during ageing.

Raman and infrared spectroscopy differentiate senescent from proliferating cells in a human dermal fibroblast 3D skin model.

Senescent cells contribute to tissue aging and dysfunction. Therefore, detecting senescent cells in skin is of interest for skin tumor diagnostics and therapy. Here, we studied the transition into senescence of human dermal fibroblasts (HDFs) in a three-dimensional (3D) human fibroblast-derived matrix (FDM). Senescent and proliferating cells were imaged by Raman spectroscopy (RS) and Fourier transform infrared (FTIR) spectroscopy. The obtained averaged spectra were analyzed using PLS-LDA. For these 3D cultured cells, RS and FTIR could clearly distinguish senescent from proliferating cells. For both techniques, we detected senescence-associated alterations in almost all cellular macromolecules. Furthermore, we identified different biochemical properties of 3D compared to two-dimensional (2D) cultured cells, indicating that cells in their natural, skin-like 3D environment act differently than in (2D) cell cultivations in vitro. Compared to 2D cultured cells, cells grown in 3D models displayed a sharper contrast between the proliferating and senescent state, also affecting the abundance of biomolecules including nucleic acids. The training accuracies of both vibrational spectroscopic techniques were >96%, demonstrating the suitability of these label-free measurements for detecting these cellular states in 3D skin models.

Raman and Infrared Spectroscopy Distinguishing Replicative Senescent from Proliferating Primary Human Fibroblast Cells by Detecting Spectral Differences Mainly Due to Biomolecular Alterations.

Cellular senescence is a terminal cell cycle arrested state, assumed to be involved in tumor suppression. We studied four human fibroblast cell strains (BJ, MRC-5, IMR-90, and WI-38) from proliferation into senescence. Cells were investigated by label-free vibrational Raman and infrared spectroscopy, following their transition into replicative senescence. During the transition into senescence, we observed rather similar biomolecular abundances in all four cell strains and between proliferating and senescent cells; however, in the four aging cell strains, we found common molecular differences dominated by protein and lipid modifications. Hence, aging induces a change in the appearance of biomolecules (including degradation and storage of waste) rather than in their amount present in the cells. For all fibroblast strains combined, the partial least squares-linear discriminant analysis (PLS-LDA) model resulted in 75% and 81% accuracy for the Raman and infrared (IR) data, respectively. Within this validation, senescent cells were recognized with 93% sensitivity and 90% specificity for the Raman and 84% sensitivity and 97% specificity for the IR data. Thus, Raman and infrared spectroscopy can identify replicative senescence on the single cell level, suggesting that vibrational spectroscopy may be suitable for identifying and distinguishing different cellular states in vivo, e.g., in skin.

Conserved genes and pathways in primary human fibroblast strains undergoing replicative and radiation induced senescence.

BACKGROUND: Cellular senescence is induced either internally, for example by replication exhaustion and cell division, or externally, for example by irradiation. In both cases, cellular damages accumulate which, if not successfully repaired, can result in senescence induction. Recently, we determined the transcriptional changes combined with the transition into replicative senescence in primary human fibroblast strains. Here, by γ-irradiation we induced premature cellular senescence in the fibroblast cell strains (HFF and MRC-5) and determined the corresponding transcriptional changes by high-throughput RNA sequencing. RESULTS: Comparing the transcriptomes, we found a high degree of similarity in differential gene expression in replicative as well as in irradiation induced senescence for both cell strains suggesting, in each cell strain, a common cellular response to error accumulation. On the functional pathway level, "Cell cycle" was the only pathway commonly down-regulated in replicative and irradiation-induced senescence in both fibroblast strains, confirming the tight link between DNA repair and cell cycle regulation. However, "DNA repair" and "replication" pathways were down-regulated more strongly in fibroblasts undergoing replicative exhaustion. We also retrieved genes and pathways in each of the cell strains specific for irradiation induced senescence. CONCLUSION: We found the pathways associated with "DNA repair" and "replication" less stringently regulated in irradiation induced compared to replicative senescence. The strong regulation of these pathways in replicative senescence highlights the importance of replication errors for its induction.

Characterization of SKAP/kinastrin isoforms: the N-terminus defines tissue specificity and Pontin binding.

Small Kinetochore-Associated Protein (SKAP)/Kinastrin is a multifunctional protein with proposed roles in mitosis, apoptosis and cell migration. Exact mechanisms underlying its activities in these cellular processes are not completely understood. SKAP is predicted to have different isoforms, however, previous studies did not differentiate between them. Since distinct molecular architectures of protein isoforms often influence their localization and functions, this study aimed to examine the expression profile and functional differences between SKAP isoforms in human and mouse. Analyses of various human tissues and cells of different origin by RT-PCR, and by Western blotting and immunocytochemistry applying newly generated anti-SKAP monoclonal antibodies revealed that human SKAP exists in two protein isoforms: ubiquitously expressed SKAP16 and testis/sperm-specific SKAP1. In mouse, SKAP1 expression is detectable in testis at 4 weeks postnatally, when the first wave of spermatogenesis in mice is complete and the elongated spermatids are present in the testes. Furthermore, we identified Pontin as a new SKAP1 interaction partner. SKAP1 and Pontin co-localized in the flagellar region of human sperm suggesting a functional relevance for SKAP1-Pontin interaction in sperm motility. Since most previous studies on SKAP were performed with the testis-specific isoform SKAP1, our findings provide a new basis for future studies on the role of SKAP in both human somatic cells and male germ cells, including studies on male fertility.

Conserved genes and pathways in primary human fibroblast strains undergoing replicative and radiation induced senescence

BACKGROUND: Cellular senescence is induced either internally, for example by replication exhaustion and cell division, or externally, for example by irradiation. In both cases, cellular damages accumulate which, if not successfully repaired, can result in senescence induction. Recently, we determined the transcriptional changes combined with the transition into replicative senescence in primary human fibroblast strains. Here, by γ-irradiation we induced premature cellular senescence in the fibroblast cell strains (HFF and MRC-5) and determined the corresponding transcriptional changes by high-throughput RNA sequencing. RESULTS: Comparing the transcriptomes, we found a high degree of similarity in differential gene expression in replicative as well as in irradiation induced senescence for both cell strains suggesting, in each cell strain, a common cellular response to error accumulation. On the functional pathway level, "Cell cycle" was the only pathway commonly down-regulated in replicative and irradiation-induced senescence in both fibroblast strains, confirming the tight link between DNA repair and cell cycle regulation. However, "DNA repair" and "replication" pathways were down-regulated more strongly in fibroblasts undergoing replicative exhaustion. We also retrieved genes and pathways in each of the cell strains specific for irradiation induced senescence. CONCLUSION: We found the pathways associated with "DNA repair" and "replication" less stringently regulated in irradiation induced compared to replicative senescence. The strong regulation of these pathways in replicative senescence highlights the importance of replication errors for its induction.

Hormetic effect of rotenone in primary human fibroblasts.

BACKGROUND: Rotenone inhibits the electron transfer from complex I to ubiquinone, in this way interfering with the electron transport chain in mitochondria. This chain of events induces increased levels of intracellular reactive oxygen species, which in turn can contribute to acceleration of telomere shortening and induction of DNA damage, ultimately resulting in aging. In this study, we investigated the effect of rotenone treatment in human fibroblast strains. RESULTS: For the first time we here describe that rotenone treatment induced a hormetic effect in human fibroblast strains. We identified a number of genes which were commonly differentially regulated due to low dose rotenone treatment in fibroblasts independent of their cell origin. However, these genes were not among the most strongly differentially regulated genes in the fibroblast strains on treatment with rotenone. Thus, if there is a common hormesis regulation, it is superimposed by cell strain specific individual responses. We found the rotenone induced differential regulation of pathways common between the two fibroblast strains, being weaker than the pathways individually regulated in the single fibroblast cell strains. Furthermore, within the common pathways different genes were responsible for this different regulation. Thus, rotenone induced hormesis was related to a weak pathway signal, superimposed by a stronger individual cellular response, a situation as found for the differentially expressed genes. CONCLUSION: We found that the concept of hormesis also applies to in vitro aging of primary human fibroblasts. However, in depth analysis of the genes as well as the pathways differentially regulated due to rotenone treatment revealed cellular hormesis being related to weak signals which are superimposed by stronger individual cell-internal responses. This would explain that in general hormesis is a small effect. Our data indicate that the observed hormetic phenotype does not result from a specific strong well-defined gene or pathway regulation but from weak common cellular processes induced by low levels of reactive oxygen species. This conclusion also holds when comparing our results with those obtained for C. elegans in which the same low dose rotenone level induced a life span extending, thus hormetic effect.

Similarities in Gene Expression Profiles during In Vitro Aging of Primary Human Embryonic Lung and Foreskin Fibroblasts.

Replicative senescence is of fundamental importance for the process of cellular aging, since it is a property of most of our somatic cells. Here, we elucidated this process by comparing gene expression changes, measured by RNA-seq, in fibroblasts originating from two different tissues, embryonic lung (MRC-5) and foreskin (HFF), at five different time points during their transition into senescence. Although the expression patterns of both fibroblast cell lines can be clearly distinguished, the similar differential expression of an ensemble of genes was found to correlate well with their transition into senescence, with only a minority of genes being cell line specific. Clustering-based approaches further revealed common signatures between the cell lines. Investigation of the mRNA expression levels at various time points during the lifespan of either of the fibroblasts resulted in a number of monotonically up- and downregulated genes which clearly showed a novel strong link to aging and senescence related processes which might be functional. In terms of expression profiles of differentially expressed genes with age, common genes identified here have the potential to rule the transition into senescence of embryonic lung and foreskin fibroblasts irrespective of their different cellular origin.

The CENP-T C-terminus is exclusively proximal to H3.1 and not to H3.2 or H3.3.

The kinetochore proteins assemble onto centromeric chromatin and regulate DNA segregation during cell division. The inner kinetochore proteins bind centromeres while most outer kinetochore proteins assemble at centromeres during mitosis, connecting the complex to microtubules. The centromere-kinetochore complex contains specific nucleosomes and nucleosomal particles. CENP-A replaces canonical H3 in centromeric nucleosomes, defining centromeric chromatin. Next to CENP-A, the CCAN multi-protein complex settles which contains CENP-T/W/S/X. These four proteins are described to form a nucleosomal particle at centromeres. We had found the CENP-T C-terminus and the CENP-S termini next to histone H3.1 but not to CENP-A, suggesting that the Constitutive Centromere-Associated Network (CCAN) bridges a CENP-A- and a H3-containing nucleosome. Here, we show by in vivo FRET that this proximity between CENP-T and H3 is specific for H3.1 but neither for the H3.1 mutants H3.1(C96A) and H3.1(C110A) nor for H3.2 or H3.3. We also found CENP-M next to H3.1 but not to these H3.1 mutants. Consistently, we detected CENP-M next to CENP-S. These data elucidate the local molecular neighborhood of CCAN proteins next to a H3.1-containing centromeric nucleosome. They also indicate an exclusive position of H3.1 clearly distinct from H3.2, thus documenting a local, and potentially also functional, difference between H3.1 and H3.2.

Long-term quiescent fibroblast cells transit into senescence.

Cellular senescence is described to be a consequence of telomere erosion during the replicative life span of primary human cells. Quiescence should therefore not contribute to cellular aging but rather extend lifespan. Here we tested this hypothesis and demonstrate that cultured long-term quiescent human fibroblasts transit into senescence due to similar cellular mechanisms with similar dynamics and with a similar maximum life span as proliferating controls, even under physiological oxygen conditions. Both, long-term quiescent and senescent fibroblasts almost completely fail to undergo apoptosis. The transition of long-term quiescent fibroblasts into senescence is also independent of HES1 which protects short-term quiescent cells from becoming senescent. Most significantly, DNA damage accumulates during senescence as well as during long-term quiescence at physiological oxygen levels. We suggest that telomere-independent, potentially maintenance driven gradual induction of cellular senescence during quiescence is a counterbalance to tumor development.

A CENP-S/X complex assembles at the centromere in S and G2 phases of the human cell cycle.

The functional identity of centromeres arises from a set of specific nucleoprotein particle subunits of the centromeric chromatin fibre. These include CENP-A and histone H3 nucleosomes and a novel nucleosome-like complex of CENPs -T, -W, -S and -X. Fluorescence cross-correlation spectroscopy and Förster resonance energy transfer (FRET) revealed that human CENP-S and -X exist principally in complex in soluble form and retain proximity when assembled at centromeres. Conditional labelling experiments show that they both assemble de novo during S phase and G2, increasing approximately three- to fourfold in abundance at centromeres. Fluorescence recovery after photobleaching (FRAP) measurements documented steady-state exchange between soluble and assembled pools, with CENP-X exchanging approximately 10 times faster than CENP-S (t1/2 ∼ 10 min versus 120 min). CENP-S binding to sites of DNA damage was quite distinct, with a FRAP half-time of approximately 160 s. Fluorescent two-hybrid analysis identified CENP-T as a uniquely strong CENP-S binding protein and this association was confirmed by FRET, revealing a centromere-bound complex containing CENP-S, CENP-X and CENP-T in proximity to histone H3 but not CENP-A. We propose that deposition of the CENP-T/W/S/X particle reveals a kinetochore-specific chromatin assembly pathway that functions to switch centromeric chromatin to a mitosis-competent state after DNA replication. Centromeres shuttle between CENP-A-rich, replication-competent and H3-CENP-T/W/S/X-rich mitosis-competent compositions in the cell cycle.

Biomolecular dynamics and binding studies in the living cell.

Isolation and preparation of proteins of higher organisms often is a tedious task. In the case of success, the properties of these proteins and their interactions with other proteins can be studied in vitro. If however, these proteins are modified in the cell in order to gain or change function, this is non-trivial to correctly realise in vitro. When, furthermore, the cellular function requires the interplay of more than one or two proteins, in vitro experiments for the analysis of this situation soon become complex. Instead, we thus try to obtain information on the molecular properties of proteins in the living cell. Then, the cell takes care of correct protein folding and modification. A series of molecular techniques are, and new ones become, available which allow for measuring molecular protein properties in the living cell, offering information on concentration (FCS), dynamics (FCS, RICS, FRAP), location (PALM, STED), interactions (F3H, FCCS) and protein proximities (FRET, BRET, FLIM, BiFC). Here, these techniques are presented with their advantages and drawbacks, with examples from our current kinetochore research. The review is supposed to give orientation to researchers planning to enter the field, and inform which techniques help us to gain molecular information on a multi-protein complex. We show that the field of cellular imaging is in a phase of transition: in the future, an increasing amount of physico-chemical data can be determined in the living cell.

Rule-based modeling and simulations of the inner kinetochore structure.

BACKGROUND: Combinatorial complexity is a central problem when modeling biochemical reaction networks, since the association of a few components can give rise to a large variation of protein complexes. Available classical modeling approaches are often insufficient for the analysis of very large and complex networks in detail. Recently, we developed a new rule-based modeling approach that facilitates the analysis of spatial and combinatorially complex problems. Here, we explore for the first time how this approach can be applied to a specific biological system, the human kinetochore, which is a multi-protein complex involving over 100 proteins. RESULTS: Applying our freely available SRSim software to a large data set on kinetochore proteins in human cells, we construct a spatial rule-based simulation model of the human inner kinetochore. The model generates an estimation of the probability distribution of the inner kinetochore 3D architecture and we show how to analyze this distribution using information theory. In our model, the formation of a bridge between CenpA and an H3 containing nucleosome only occurs efficiently for higher protein concentration realized during S-phase but may be not in G1. Above a certain nucleosome distance the protein bridge barely formed pointing towards the importance of chromatin structure for kinetochore complex formation. We define a metric for the distance between structures that allow us to identify structural clusters. Using this modeling technique, we explore different hypothetical chromatin layouts. CONCLUSIONS: Applying a rule-based network analysis to the spatial kinetochore complex geometry allowed us to integrate experimental data on kinetochore proteins, suggesting a 3D model of the human inner kinetochore architecture that is governed by a combinatorial algebraic reaction network. This reaction network can serve as bridge between multiple scales of modeling. Our approach can be applied to other systems beyond kinetochores.

Step-wise assembly, maturation and dynamic behavior of the human CENP-P/O/R/Q/U kinetochore sub-complex.

Kinetochores are multi-protein megadalton assemblies that are required for attachment of microtubules to centromeres and, in turn, the segregation of chromosomes in mitosis. Kinetochore assembly is a cell cycle regulated multi-step process. The initial step occurs during interphase and involves loading of the 15-subunit constitutive centromere associated complex (CCAN), which contains a 5-subunit (CENP-P/O/R/Q/U) sub-complex. Here we show using a fluorescent three-hybrid (F3H) assay and fluorescence resonance energy transfer (FRET) in living mammalian cells that CENP-P/O/R/Q/U subunits exist in a tightly packed arrangement that involves multifold protein-protein interactions. This sub-complex is, however, not pre-assembled in the cytoplasm, but rather assembled on kinetochores through the step-wise recruitment of CENP-O/P heterodimers and the CENP-P, -O, -R, -Q and -U single protein units. SNAP-tag experiments and immuno-staining indicate that these loading events occur during S-phase in a manner similar to the nucleosome binding components of the CCAN, CENP-T/W/N. Furthermore, CENP-P/O/R/Q/U binding to the CCAN is largely mediated through interactions with the CENP-N binding protein CENP-L as well as CENP-K. Once assembled, CENP-P/O/R/Q/U exchanges slowly with the free nucleoplasmic pool indicating a low off-rate for individual CENP-P/O/R/Q/U subunits. Surprisingly, we then find that during late S-phase, following the kinetochore-binding step, both CENP-Q and -U but not -R undergo oligomerization. We propose that CENP-P/O/R/Q/U self-assembles on kinetochores with varying stoichiometry and undergoes a pre-mitotic maturation step that could be important for kinetochores switching into the correct conformation necessary for microtubule-attachment.

Quantitative model of cell cycle arrest and cellular senescence in primary human fibroblasts.

Primary human fibroblasts in tissue culture undergo a limited number of cell divisions before entering a non-replicative "senescent" state. At early population doublings (PD), fibroblasts are proliferation-competent displaying exponential growth. During further cell passaging, an increasing number of cells become cell cycle arrested and finally senescent. This transition from proliferating to senescent cells is driven by a number of endogenous and exogenous stress factors. Here, we have developed a new quantitative model for the stepwise transition from proliferating human fibroblasts (P) via reversibly cell cycle arrested (C) to irreversibly arrested senescent cells (S). In this model, the transition from P to C and to S is driven by a stress function γ and a cellular stress response function F which describes the time-delayed cellular response to experimentally induced irradiation stress. The application of this model based on senescence marker quantification at the single-cell level allowed to discriminate between the cellular states P, C, and S and delivers the transition rates between the P, C and S states for different human fibroblast cell types. Model-derived quantification unexpectedly revealed significant differences in the stress response of different fibroblast cell lines. Evaluating marker specificity, we found that SA-ß-Gal is a good quantitative marker for cellular senescence in WI-38 and BJ cells, however much less so in MRC-5 cells. Furthermore we found that WI-38 cells are more sensitive to stress than BJ and MRC-5 cells. Thus, the explicit separation of stress induction from the cellular stress response, and the differentiation between three cellular states P, C and S allows for the first time to quantitatively assess the response of primary human fibroblasts towards endogenous and exogenous stress during cellular ageing.

Cell-cycle-dependent structural transitions in the human CENP-A nucleosome in vivo.

In eukaryotes, DNA is packaged into chromatin by canonical histone proteins. The specialized histone H3 variant CENP-A provides an epigenetic and structural basis for chromosome segregation by replacing H3 at centromeres. Unlike exclusively octameric canonical H3 nucleosomes, CENP-A nucleosomes have been shown to exist as octamers, hexamers, and tetramers. An intriguing possibility reconciling these observations is that CENP-A nucleosomes cycle between octamers and tetramers in vivo. We tested this hypothesis by tracking CENP-A nucleosomal components, structure, chromatin folding, and covalent modifications across the human cell cycle. We report that CENP-A nucleosomes alter from tetramers to octamers before replication and revert to tetramers after replication. These structural transitions are accompanied by reversible chaperone binding, chromatin fiber folding changes, and previously undescribed modifications within the histone fold domains of CENP-A and H4. Our results reveal a cyclical nature to CENP-A nucleosome structure and have implications for the maintenance of epigenetic memory after centromere replication.

Accumulation of annexin A5 at the nuclear envelope is a biomarker of cellular aging.

Cellular senescence is a permanent cell cycle arrest induced by short telomeres or oncogenic stress in vitro and in vivo. Because no single of the established biomarkers can reliably identify senescent cells, the application of new ones may aid the diagnosis of aged cells. Here we show that annexin A5 accumulates at the nuclear envelope during replicative and drug-induced cellular senescence in primary human fibroblasts. This new cellular aging phenotype that we have termed SA-ANX5 (senescence-associated accumulation at the nuclear envelope of annexin A5) is as efficient and quantitative as the well-established senescence-associated ß-galactosidase activity assay and p21 immunoreactivity. SA-ANX5 is also observed in aged human skin where is exclusively detected in DNA damage foci-positive/Ki-67-negative cells. We also observed that depletion of annexin A5 by siRNA in human fibroblasts accelerates premature senescence through the p38MAP kinase pathway. These observations establish SA-ANX5 as a new biomarker for cellular aging and implicate a functional role for annexin A5 in cellular senescence.