A SIRT1-independent mechanism mediates protection against steroid-induced senescence by resveralogues in equine tenocytes.

Tendinopathy is a common age-related disease which causes significant morbidity for both human athletes and performance horses. In the latter, the superficial digital flexor tendon is an excellent model for human tendinopathies because it is a functional homologue of the human Achilles tendon and a primary site of injuries with strong similarities to the human disease. Corticosteroids have been previously used clinically to treat tendinopathic inflammation, but they upregulate the p53-p21 axis with concomitant reductions in cell proliferation and collagen synthesis in human tenocytes. This phenotype is consistent with the induction of cellular senescence in vitro and in vivo and probably represents an important clinical barrier to their effective use. Because of the many differences in senescence mechanisms between species, this study aimed to evaluate these mechanisms after corticosteroid treatment in equine tenocytes. Exposure to clinically reflective levels of dexamethasone for 48 hours drove equine tenocytes into steroid induced senescence (SIS). This was characterised by permanent growth arrest and upregulation of p53, the cyclin dependent kinase inhibitors p21waf and p16ink4a as well as the matrix degrading enzymes MMP1, MMP2 and MMP13. SIS also induced a distinctive equine senescence associated secretory phenotype (eSASP) characterised by enhanced secretion of IL-8 and MCP-1. Preincubation with resveratrol or the potent SIRT1 activator SRT1720 prevented SIS in equine tenocytes, while treatment with the non-SIRT1 activating resveratrol analogue V29 was equally protective against SIS, consistent with a novel, as yet uncharacterised SIRT1-indendent mechanism which has relevance for the development of future preventative and therapeutic strategies.

Therapeutic Opportunities Presented by Modulation of Cellular Senescence.

Cellular senescence is a permanent state of growth arrest coupled with profound changes in phenotype that can be triggered by multiple extrinsic or intrinsic stimuli. Senescence is a process-level example of the evolution of ageing mechanisms through antagonistic pleiotropy and plays a primary role in tumour suppression, although evidence is mounting for its involvement in other fundamental physiological processes. Evidence from human premature ageing diseases and from transgenic mice in which it is possible to specifically delete senescent cells is consistent with a model in which the accumulation of senescent cells through the life course is responsible for later life chronic disease and impairment. The removal of senescent cells or their reversion to a phenotypically benign state is thus an important emerging goal of translational medicine.Modern bioinformatic approaches based on text mining have compiled co-mentions of cell senescence and age-related diseases allowing an impartial ranking of the impairments most closely associated with this process. Following this schema, the evidence for the involvement of senescence in several highly ranked pathologies is reviewed, alongside potential methods for the ablation of senescent cells or their reversion to their primary phenotype with polyphenolics or inhibitors of p38 MAP kinase. Lastly, the potential for senescence to act as a barrier to the development of bioartificial organs designed to treat some of these conditions is discussed.

Simple Detection Methods for Senescent Cells: Opportunities and Challenges.

Cellular senescence, the irreversible growth arrest of cells from conditional renewal populations combined with a radical shift in their phenotype, is a hallmark of ageing in some mammalian species. In the light of this, interest in the detection of senescent cells in different tissues and different species is increasing. However much of the prior work in this area is heavily slanted towards studies conducted in humans and rodents; and in these species most studies concern primary fibroblasts or cancer cell lines rendered senescent through exposure to a variety of stressors. Complex techniques are now available for the detailed analysis of senescence in these systems. But, rather than focussing on these methods this review instead examines techniques for the simple and reproducible detection of senescent cells. Intended primary for the non-specialist who wishes to quickly detect senescent cells in tissues or species which may lack a significant evidence base on the phenomenon it emphasises the power of the original techniques used to demonstrate the senescence of cells, their interrelationship with other markers and their potential to inform on the senescent state in new species and archival specimens.

Resveralogues: From Novel Ageing Mechanisms to New Therapies?

For much of the 20th century the ageing process was thought to be the result of the interplay of many different biological processes, each with relatively small effects on organismal lifespan. However, this model is no longer tenable. Rather it seems a few biological mechanisms, including nutrient sensing, telomere attrition and cellular senescence, mediate large effects on health and longevity. Biogerontology may have suffered from initial delusions of complexity. However, we argue that it is premature to assume either that the list of biological processes influencing lifespan is now comprehensive or that these mechanisms act independently of each other. A case in point is provided by recent work linking together changes in RNA splicing with advancing age and the ability of polyphenolics based on resveratrol to reverse replicative senescence. In this opinion piece, we propose a novel model in which the factors regulating splice restriction and those controlling cell senescence intersect across chronological and divisional time, giving rise to senescent and growing cells with more diverse properties than previously thought. We also consider therapeutic opportunities and potential problems in the light of this revised conceptual understanding of human cell senescence and ageing.

FOXO1 and ETV6 genes may represent novel regulators of splicing factor expression in cellular senescence.

Cellular plasticity is a key facet of cellular homeostasis requiring correct temporal and spatial patterns of alternative splicing. Splicing factors, which orchestrate this process, demonstrate age-related dysregulation of expression; they are emerging as potential influences on aging and longevity. The upstream drivers of these alterations are still unclear but may involve aberrant cellular signaling. We compared the phosphorylation status of proteins in multiple signaling pathways in early and late passage human primary fibroblasts. We then assessed the impact of chemical inhibition or targeted knockdown of direct downstream targets of the ERK and AKT pathways on splicing factor expression, cellular senescence, and proliferation kinetics in senescent primary human fibroblasts. Components of the ERK and AKT signaling pathways demonstrated altered activation during cellular aging. Inhibition of AKT and ERK pathways led to up-regulation of splicing factor expression, reduction in senescent cell load, and partial reversal of multiple cellular senescence phenotypes in a dose-dependent manner. Furthermore, targeted knockdown of the genes encoding the downstream targets FOXO1 or ETV6 was sufficient to mimic these observations. Our results suggest that age-associated dysregulation of splicing factor expression and cellular senescence may derive in part from altered activity of ERK and AKT signaling and may act in part through the ETV6 and FOXO1 transcription factors. Targeting the activity of downstream effectors of ERK and AKT may therefore represent promising targets for future therapeutic intervention.-Latorre, E., Ostler, E. L., Faragher, R. G. A., Harries, L. W. FOXO1 and ETV6 genes may represent novel regulators of splicing factor expression in cellular senescence.

Telomere elongation through hTERT immortalization leads to chromosome repositioning in control cells and genomic instability in Hutchinson-Gilford progeria syndrome fibroblasts, expressing a novel SUN1 isoform.

Immortalizing primary cells with human telomerase reverse transcriptase (hTERT) has been common practice to enable primary cells to be of extended use in the laboratory because they avoid replicative senescence. Studying exogenously expressed hTERT in cells also affords scientists models of early carcinogenesis and telomere behavior. Control and the premature ageing disease-Hutchinson-Gilford progeria syndrome (HGPS) primary dermal fibroblasts, with and without the classical G608G mutation have been immortalized with exogenous hTERT. However, hTERT immortalization surprisingly elicits genome reorganization not only in disease cells but also in the normal control cells, such that whole chromosome territories normally located at the nuclear periphery in proliferating fibroblasts become mislocalized in the nuclear interior. This includes chromosome 18 in the control fibroblasts and both chromosomes 18 and X in HGPS cells, which physically express an isoform of the LINC complex protein SUN1 that has previously only been theoretical. Additionally, this HGPS cell line has also become genomically unstable and has a tetraploid karyotype, which could be due to the novel SUN1 isoform. Long-term treatment with the hTERT inhibitor BIBR1532 enabled the reduction of telomere length in the immortalized cells and resulted that these mislocalized internal chromosomes to be located at the nuclear periphery, as assessed in actively proliferating cells. Taken together, these findings reveal that elongated telomeres lead to dramatic chromosome mislocalization, which can be restored with a drug treatment that results in telomere reshortening and that a novel SUN1 isoform combined with elongated telomeres leads to genomic instability. Thus, care should be taken when interpreting data from genomic studies in hTERT-immortalized cell lines.

Obesity and type-2 diabetes as inducers of premature cellular senescence and ageing.

Cellular senescence is now considered as a major mechanism in the development and progression of various diseases and this may include metabolic diseases such as obesity and type-2 diabetes. The presence of obesity and diabetes is a major risk factor in the development of additional health conditions, such as cardiovascular disease, kidney disease and cancer. Since senescent cells can drive disease development, obesity and diabetes can potentially create an environment that accelerates cell senescence within other tissues of the body. This can consequently manifest as age-related biological impairments and secondary diseases. Cell senescence in cell types linked with obesity and diabetes, namely adipocytes and pancreatic beta cells will be explored, followed by a discussion on the role of obesity and diabetes in accelerating ageing through induction of premature cell senescence mediated by high glucose levels and oxidised low-density lipoproteins. Particular emphasis will be placed on accelerated cell senescence in endothelial progenitor cells, endothelial cells and vascular smooth muscle cells with relation to cardiovascular disease and proximal tubular cells with relation to kidney disease. A summary of the potential strategies for therapeutically targeting senescent cells for improving health is also presented.

Environmental Screening of Electrode Materials for a Rechargeable Aluminum Battery with an AlCl3/EMIMCl Electrolyte.

Recently, rechargeable aluminum batteries have received much attention due to their low cost, easy operation, and high safety. As the research into rechargeable aluminum batteries with a room-temperature ionic liquid electrolyte is relatively new, research efforts have focused on finding suitable electrode materials. An understanding of the environmental aspects of electrode materials is essential to make informed and conscious decisions in aluminum battery development. The purpose of this study was to evaluate and compare the relative environmental performance of electrode material candidates for rechargeable aluminum batteries with an AlCl3/EMIMCl (1-ethyl-3-methylimidazolium chloride) room-temperature ionic liquid electrolyte. To this end, we used a lifecycle environmental screening framework to evaluate 12 candidate electrode materials. We found that all of the studied materials are associated with one or more drawbacks and therefore do not represent a "silver bullet" for the aluminum battery. Even so, some materials appeared more promising than others did. We also found that aluminum battery technology is likely to face some of the same environmental challenges as Li-ion technology but also offers an opportunity to avoid others. The insights provided here can aid aluminum battery development in an environmentally sustainable direction.

[Global health 2035: a world converging within a generation]. / Salud global 2035: un mundo convergiendo en el lapso de una generación.

Prompted by the 20th anniversary of the 1993 World Development Report, a Lancet Commission revisited the case for investment in health and developed a new investment framework to achieve dramatic health gains by 2035. The Commission's report has four key messages, each accompanied by opportunities for action by national governments of low-income and middle-income countries and by the international community. First, there is an enormous economic payoff from investing in health. The impressive returns make a strong case for both increased domestic financing of health and for allocating a higher proportion of official development assistance to development of health. Second, modeling by the Commission found that a "grand convergence" in health is achievable by 2035-that is, a reduction in infectious, maternal, and child mortality down to universally low levels. Convergence would require aggressive scale up of existing and new health tools, and it could mostly be financed from the expected economic growth of low- and middle-income countries. The international community can best support convergence by funding the development and delivery of new health technologies and by curbing antibiotic resistance. Third, fiscal policies -such as taxation of tobacco and alcohol- are a powerful and underused lever that governments can use to curb non-communicable diseases and injuries while also raising revenue for health. International action on NCDs and injuries should focus on providing technical assistance on fiscal policies, regional cooperation on tobacco, and funding policy and implementation research on scaling-up of interventions to tackle these conditions. Fourth, progressive universalism, a pathway to universal health coverage (UHC) that includes the poor from the outset, is an efficient way to achieve health and financial risk protection. For national governments, progressive universalism would yield high health gains per dollar spent and poor people would gain the most in terms of health and financial protection. The international community can best support countries to implement progressive UHC by financing policy and implementation research, such as on the mechanics of designing and implementing evolution of the benefits package as the resource envelope for public finance grows.

A facile, stereoselective, one-pot synthesis of resveratrol derivatives.

BACKGROUND: Compounds based on trans-1,2-diphenylethene are the subject of intense interest both for their optical properties and as potential leads for drug discovery, as a consequence of their anticancer, anti-inflammatory and antioxidant properties. Perhaps the best known of these is trans-3,5,4'-trihydroxystilbene (resveratrol), that has been identified as a promising lead in the search for anti-ageing therapeutics. RESULTS: We report here a new, convenient, one-pot stereo-selective synthesis of resveratrol and other trans-stilbene derivatives. A wide range of known and novel "Resveralogues" were synthesised by using this simple protocol, including examples with electron donating and electron withdrawing substituents, in uniformly high yield. The structures of all compounds were confirmed by standard methods including (1)H and (13)C NMR, IR and High Resolution Mass spectroscopy. CONCLUSIONS: We have established a simple and convenient protocol for resveralogue synthesis. It is readily scalable, and sufficiently robust and simple for ready use in automated synthesis or for library development of resveralogues. This supersedes previously reported synthetic methods that required inert conditions, extensive purification and/or costly reagents. Graphical abstractOne-pot preparation of diverse Resveralogues - high yields of product with minimal purification.

Mass drug administration for the control and elimination of Plasmodium vivax malaria: an ecological study from Jiangsu province, China.

BACKGROUND: Recent progress in malaria control has caused renewed interest in mass drug administration (MDA) as a potential elimination strategy but the evidence base is limited. China has extensive experience with MDA, but it is not well documented. METHODS: An ecological study was conducted to describe the use of MDA for the control and elimination of Plasmodium vivax in Jiangsu Province and explore the association between MDA and malaria incidence. Two periods were focused on: 1973 to 1983 when malaria burden was high and MDA administered to highly endemic counties province-wide, and 2000 to 2009, when malaria burden was low and a focal approach was used in two counties. All available data about the strategies implemented, MDA coverage, co-interventions, incidence, and adverse events were collected and described. Joinpoint analysis was used to describe trends in incidence and the relationship between MDA coverage and incidence was explored in negative binomial regression models. RESULTS: From 1973 to 1983, MDA with pyrimethamine and primaquine was used on a large scale, with up to 30 million people in target counties covered in a peak year (50% of the total population). Joinpoint analyses identified declines in annual incidence, -56.7% (95% CI -75.5 to -23.7%) from 1973-1976 and -12.4% (95% CI -24.7 to 2.0%) from 1976-1983. Population average negative binomial models identified a relationship between higher total population MDA coverage and lower monthly incidence from 1973-1976, IRR 0.98 (95% CI 0.97 to 1.00), while co-interventions, rainfall and GDP were not associated. From 2000-2009, incidence in two counties declined (annual change -43.7 to -14.0%) during a time when focal MDA using chloroquine and primaquine was targeted to villages and/or individuals residing near passively detected index cases (median 0.04% of total population). Although safety data were not collected systematically, there were rare reports of serious but non-fatal events. CONCLUSIONS: In Jiangsu Province, China, large-scale MDA was implemented and associated with declines in high P. vivax malaria transmission; a more recent focal approach may have contributed to interruption of transmission. MDA should be considered a potential key strategy for malaria control and elimination.

Recapitulation of Werner syndrome sensitivity to camptothecin by limited knockdown of the WRN helicase/exonuclease.

WRN is a RecQ helicase with an associated exonuclease activity important in DNA metabolism, including DNA replication, repair and recombination. In humans, deficiencies in WRN function cause the segmental progeroid Werner syndrome (WS), in which patients show premature onset of many hallmarks of normal human ageing. At the cellular level, WRN loss results in rapid replicative senescence, chromosomal instability and sensitivity to various DNA damaging agents including the topoisomerase inhibitor, camptothecin (CPT). Here, we investigate the potential of using either transient or stable WRN knockdown as a means of sensitising cells to CPT. We show that targeting WRN mRNA for degradation by either RNAi or hammerhead ribozyme catalysis renders human fibroblasts as sensitive to CPT as fibroblasts derived from WS patients, and furthermore, we find altered cell cycle transit and nucleolar destabilisation in these cells following CPT treatment. Such WS-like phenotypes are observed despite very limited decreases in total WRN protein, suggesting that levels of WRN protein are rate-limiting for the cellular response to camptothecin. These findings have major implications for development of anti-WRN agents that may be useful in sensitising tumour cells to clinically relevant topoisomerase inhibitors.

Characterization of cellular senescence mechanisms in human corneal endothelial cells.

The human cornea is a tri-laminar structure composed of several cell types with substantial mitotic potential. Age-related changes in the cornea are associated with declining visual acuity and the onset of overt age-related corneal diseases. Corneal transplantation is commonly used to restore vision in patients with damaged or diseased corneas. However, the supply of donor tissue is limited, and thus there is considerable interest in the development of tissue-engineered alternatives. A major obstacle to these approaches is the short replicative lifespan of primary human corneal endothelial cells (HCEC). Accordingly, a comprehensive investigation of the signalling pathways and mechanisms underpinning proliferative lifespan and senescence in HCEC was undertaken. The effects of exogenous human telomerase reverse transcriptase expression, p53 knockdown, disruption of the pRb pathway by over-expression of CDK4 and reduced oxygen concentration on the lifespan of primary HCEC were evaluated. We provide proof-of-principle that forced expression of telomerase, when combined with either p53 knockdown or CDK4 over-expression, is sufficient to produce immortalized HCEC lines. The resultant cell lines express an HCEC-specific transcriptional fingerprint, and retain expression of the corneal endothelial temperature-sensitive potassium channel, suggesting that significant dedifferentiation does not occur as a result of these modes of immortalization. Exploiting these insights into proliferative lifespan barriers in HCEC will underpin the development of novel strategies for cell-based therapies in the human cornea.

Resveratrol, but not dihydroresveratrol, induces premature senescence in primary human fibroblasts.

Resveratrol, trans-3,5,4'-trihydroxystilbene, is a polyphenolic compound which has been reported to mimic the gene expression patterns seen in whole animals undergoing dietary restriction. The mechanism of action of resveratrol remains poorly understood, but modulation of both cellular proliferation and apoptosis has been proposed as important routes by which the molecule may exert its effects. This study reports the effects of both resveratrol and dihydroresveratrol (a primary in vivo metabolite) on the proliferative capacity of human primary fibroblasts. No generalised reduction in the growth fraction was observed when fibroblasts derived from three different tissues were treated with resveratrol at concentrations of 10 µm or less. However, concentrations above 25 µm produced a dose-dependent reduction in proliferation. This loss of the growth fraction was paralleled by an increase in the senescent fraction as determined by staining for senescence associated beta galactosidase and dose recovery studies conducted over a 7-day period. Entry into senescence in response to treatment with resveratrol could be blocked by a 30-min preincubation with the p38 MAP kinase inhibitor SB203580. No effects on proliferation were observed when cells were treated with dihydroresveratrol at concentrations of up to 100 µm.

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

Little 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 in vitro 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 (p-value<0.5%, minimum effect size of at least 2-fold differential regulation, explore data at http://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 (IL1beta, IL8, ICAM1, TNFAP3, ESM1 and CCL2), tissue remodelling (VEGF, VEGFbeta, ADM and MMP14) and vascular calcification (MGP, BMP2, SPP1, OPG and DCN). The microarray data for IL1beta, 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.

A novel population of repair cells identified in the stroma of the human cornea.

The transmembrane protein CD133 is expressed on somatic stem cells of various adult human tissues. To investigate whether human corneal stroma also contains CD133-expressing cells and to analyze their functional features, stromal cells were isolated by collagenase digestion, immunophenotyped, and transferred to different culture systems to determine their stem cell properties as well as their differentiation potentials. For comparison, the embryonic keratocyte cell line EK1.Br, the dermal stromal cell line NHDF, and stromal cells of diseased corneas were studied. On average, 5.3% of the normal stromal cells expressed the stem cell marker CD133 and 3.6% co-expressed CD34. Expression of CD133 but not CD34 was also demonstrated for EK1.Br cells, whereas NHDF cells were negative for both markers. Further analysis of CD133(+) normal corneal cells revealed that a significant proportion displayed a monocytic phenotype with co-expression of CD45 and CD14. In diseased corneas, up to 26.8% of the stromal cells showed expression of CD133, and virtually all CD133(+) cells co-expressed CD14 but not CD45. Moreover, using a standard clonogenic assay, normal stromal cells had the capacity to form colonies of the macrophage lineage. These colonies could be further differentiated into lumican-expressing keratocytes. Our data suggest that the human corneal stroma harbors CD133(+) monocytic progenitor cells, which possess the potential to differentiate into the fibrocytic lineage. Thus, CD133(+) /CD45(+) /CD14(+) cells might represent stromal repair cells that differentiate into keratocytes via a CD133(+)/CD45()/CD14(+) intermediate stage. The findings from our study may shed new light on regenerative processes of the human corneal stroma.

alpha-Lipoic acid and glutathione protect against the prooxidant activity of SOD/catalase mimetic manganese salen derivatives.

Manganese(III) N,N'-ethylenebis(salicylideneiminato) chloride (Mn-salen chloride) and manganese(III) N,N'-ethylenebis(3-methoxysalicylideneiminato) chloride (Mn-(3,3'-MeO)salen chloride) are in vitro superoxide dismutase and catalase mimetics. They protect against free radical-related disease in animals, but Mn-salen can also be a potent prooxidant, damaging free DNA. Mn-salen protects human fibroblast DNA against hydrogen peroxide damage, however, damage to free DNA was confirmed by the comet assay. The DNA-damaging activity was dramatically reduced by co-administration with glutathione with the combination being less damaging to free DNA than either molecule alone. alpha-Lipoic acid, an antioxidant disulfide commonly used as a dietary supplement, also prevented Mn-salen prooxidant activity. Mn-(3,3'-MeO)salen protected fibroblasts against hydrogen peroxide as efficiently as Mn-salen and showed little damaging activity against free DNA. Protection was invested by both complexes in the presence and in the absence of EDTA, a potential competing chelator. Stabilities of the complexes with respect to decomposition and inactivation were studied by spectroscopic and electrochemical techniques. The complexes' binding to, and cleavage of, DNA was measured using a quartz crystal resonant sensor. Mn-salen was shown to bind strongly to DNA, prior to cleaving it; Mn-(3,3'-MeO)salen bound weakly and left DNA intact. Co-administration of either glutathione or alpha-lipoic acid appears to inhibit binding by Mn-salen thus preventing DNA-cleavage.

Replicative senescence in sheep fibroblasts is a p53 dependent process.

Studies on telomere and telomerase biology are fundamental to the understanding of human ageing, and age-related diseases such as cancer. However, human studies are hampered by the lack of fully reflective animal model systems. Here we describe basic studies of telomere length and telomerase activity in sheep tissues and cells. Terminal restriction fragment lengths from sheep tissues ranged from 9 to 23 kb, with telomerase activity present in testis but suppressed in somatic tissues. Sheep fibroblasts had a finite lifespan in culture, after which the cells entered senescence. During in vitro growth the mean terminal restriction fragment lengths decreased in size at a rate of 210 and 350 bp per population doubling (PD). Senescent skin fibroblasts had increased levels of p53 and p21WAF1 compared to young cells. Incubation of senescent cells with siRNA duplexes specific for p53 suppressed p53 expression and allowed the cells to re-enter the cell cycle. Five PDs beyond senescence the siRNA-treated cells reached a second proliferative barrier. This study shows that telomere biology in sheep is similar to that in humans, with senescence in sheep GM03550 fibroblasts being a telomere-driven, p53-(p21WAF1)-dependent process. Therefore sheep may represent an alternative model system for studying telomere biology, replicative senescence, and by implication human ageing.

Investigation of the signaling pathways involved in the proliferative life span barriers in werner syndrome fibroblasts.

Werner syndrome (WS) fibroblasts enter replicative senescence after a reduced in vitro life span. Although this has been postulated as causal in the accelerated aging seen in this disease, controversy remains as to whether WS is showing the acceleration of a normal cellular aging mechanism or, instead, the occurrence of a novel WS-specific process. To address this, we analyzed the signaling pathways involved in senescence in WS fibroblasts. Cultured WS fibroblasts underwent senescence after approximately 20 population doublings, with the majority of the cells having a 2N DNA content. This was associated with high levels of the CdkIs p16 and p21. Senescent WS cells reentered the cell cycle after microinjection of a p53-neutralizing antibody. Similarly, presenescent WS fibroblasts expressing the E6 and/or E7 oncoproteins bypassed M1 and ultimately reached a second proliferative life span barrier, which strongly resembled the second life span barriers found in normal cells for growth dynamics, cellular morphology, and expression of p16 and p21. The strong similarity between the signaling pathways triggering cell cycle arrest in WS and normal fibroblasts provides support for the defect in WS causing the acceleration of a normal aging mechanism and validates the use of WS as a model for some aspects of human aging.

Distinct expression patterns of polycomb oncoproteins and their binding partners during the germinal center reaction.

Polycomb group (PcG) genes encode two chromatin-binding protein complexes, the PRC1 and the PRC2 PcG complexes, which are essential for the maintenance of cell identity and play a role in oncogenesis. PcG complexes were recently identified as novel regulators of hematopoiesis, and appear to be expressed in a non-overlapping pattern in resting and mature follicular B cells. Using highly specific antisera in combination with immunohistochemistry and triple immunofluorescence, we investigated the expression pattern of nine human PcG genes in germinal center (GC) B cells and highly purified germinal center B cell subpopulations. PcG proteins were detected in characteristic binding patterns that were not necessarily related to mutually exclusive expression of the two PcG complexes. We conclude that the two PcG complexes are expressed throughout GC development, and that the fine composition of each complex is determined by the differentiation status of the cell. In addition, a subset of dividing cells with a centrocyte CD marker profile was identified that co-expresses core components of the PRC1 and PRC2 complex. We propose that these cells reflect a transitional stage between resting and dividing follicular B lymphocytes, and that they possibly represent the healthy precursors of nodal large B cell lymphomas.