Extension Of Replicative Life Span Research Articles

Effect of Antioxidants on the Fibroblast Replicative Lifespan In Vitro.

Replicative senescence is an unalterable growth arrest of primary cells in the culture system. It has been reported that aging in vivo is related to the limited replicative capacity that normal somatic cells show in vitro. If oxidative damage contributes to the lifespan limitation, antioxidants are expected to extend the replicative lifespan of fibroblasts. This article critically reviews the results of experiments devoted to this problem performed within the last decades under conditions of in vitro culture. The results of studied are heterogeneous, some papers showing no effects of antioxidants; most finding limited enhancement of reproductive capacity of fibroblasts, some reporting a significant extension of replicative lifespan (RLS). Both natural and synthetic antioxidants were found to extend the RLS of fibroblasts, either by a direct antioxidant effect or, indirectly, by activation of signaling pathways and activation of proteasomes or hormetic effects. Most significant prolongation of RLS was reported so far for nicotinamide, N-hydroxylamines, carnosine and Methylene Blue. These results may be of importance for the design of skin-protecting cosmetics.

New drugs for pharmacological extension of replicative life span in normal and progeroid cells

A high-throughput anti-aging drug screen was developed that simultaneously measures senescence-associated β-galactosidase activity and proliferation. Applied to replicatively pre-aged fibroblasts, this screen yielded violuric acid (VA) and 1-naphthoquinone-2-monoxime (N2N1) as its top two hits. These lead compounds extended the replicative life spans of normal and progeroid human cells in a dose-dependent manner and also extended the chronological life spans of mice and C. elegans. They are further shown here to function as redox catalysts in oxidations of NAD(P)H. They thus slow age-related declines in NAD(P)+/NAD(P)H ratios. VA participates in non-enzymatic electron transfers from NAD(P)H to oxidized glutathione or peroxides. N2N1 transfers electrons from NAD(P)H to cytochrome c or CoQ10 via NAD(P)H dehydrogenase (quinone) 1 (NQO1). Our results indicate that pharmacologic manipulation of NQO1 activity via redox catalysts may reveal mechanisms of senescence and aging.

Methionine restriction activates the retrograde response and confers both stress tolerance and lifespan extension to yeast, mouse and human cells.

A methionine-restricted diet robustly improves healthspan in key model organisms. For example, methionine restriction reduces age-related pathologies and extends lifespan up to 45% in rodents. However, the mechanisms underlying these benefits remain largely unknown. We tested whether the yeast chronological aging assay could model the benefits of methionine restriction, and found that this intervention extends lifespan when enforced by either dietary or genetic approaches, and furthermore, that the observed lifespan extension is due primarily to reduced acid accumulation. In addition, methionine restriction-induced lifespan extension requires the activity of the retrograde response, which regulates nuclear gene expression in response to changes in mitochondrial function. Consistent with an involvement of stress-responsive retrograde signaling, we also found that methionine-restricted yeast are more stress tolerant than control cells. Prompted by these findings in yeast, we tested the effects of genetic methionine restriction on the stress tolerance and replicative lifespans of cultured mouse and human fibroblasts. We found that such methionine-restricted mammalian cells are resistant to numerous cytotoxic stresses, and are substantially longer-lived than control cells. In addition, similar to yeast, the extended lifespan of methionine-restricted mammalian cells is associated with NFκB-mediated retrograde signaling. Overall, our data suggest that improved stress tolerance and extension of replicative lifespan may contribute to the improved healthspan observed in methionine-restricted rodents, and also support the possibility that manipulation of the pathways engaged by methionine restriction may improve healthspan in humans.

Immortality, but not oncogenic transformation, of primary human cells leads to epigenetic reprogramming of DNA methylation and gene expression

Tumourigenic transformation of normal cells into cancer typically involves several steps resulting in acquisition of unlimited growth potential, evasion of apoptosis and non-responsiveness to growth inhibitory signals. Both genetic and epigenetic changes can contribute to cancer development and progression. Given the vast genetic heterogeneity of human cancers and difficulty to monitor cancer-initiating events in vivo, the precise relationship between acquisition of genetic mutations and the temporal progression of epigenetic alterations in transformed cells is largely unclear. Here, we use an in vitro model system to investigate the contribution of cellular immortality and oncogenic transformation of primary human cells to epigenetic reprogramming of DNA methylation and gene expression. Our data demonstrate that extension of replicative life span of the cells is sufficient to induce accumulation of DNA methylation at gene promoters and large-scale changes in gene expression in a time-dependent manner. In contrast, continuous expression of cooperating oncogenes in immortalized cells, although essential for anchorage-independent growth and evasion of apoptosis, does not affect de novo DNA methylation at promoters and induces subtle expression changes. Taken together, these observations imply that cellular immortality promotes epigenetic adaptation to highly proliferative state, whereas transforming oncogenes confer additional properties to transformed human cells.

Baculoviral expression of telomerase in primary human fibroblasts to rejuvenate cells for tissue engineering

Tissue engineering involves the use of synthetic or natural materials as a scaffold to support the growth of replacement tissue or organs. The use of autologous cells to populate the scaffold avoids problems associated with rejection; however, a major limitation of this approach is the finite lifespan of primary cells in culture. This finite lifespan is due to the shortening of telomeres, short repetitive sequences of DNA located at the ends of eukaryotic chromosomes. Ectopic expression of telomerase reverse transcriptase (hTERT) is able to reconstitute telomerase activity and maintain the length of telomeres. This study investigated an alternative gene delivery vector, baculovirus, for the expression of hTERT in primary human cells. A recombinant baculovirus was used to efficiently deliver the hTERT gene to primary fibroblasts and the telomerase enzyme was found to be active. Although no increase in telomere length was detected, expression of hTERT in primary fibroblasts resulted in a significant extension of replicative lifespan. To our knowledge this is a novel attempt to use a recombinant baculovirus for the extension of cellular lifespan by exogenous expression of telomerase.

Absence of Mitochondrial Translation Control Proteins Extends Life Span by Activating Sirtuin-Dependent Silencing

Altered mitochondrial functionality can extend organism life span, but the underlying mechanisms are obscure. Here we report that inactivating SOV1, a member of the yeast mitochondrial translation control (MTC) module, causes a robust Sir2-dependent extension of replicative life span in the absence of respiration and without affecting oxidative damage. We found that SOV1 interacts genetically with the cAMP-PKA pathway and the chromatin remodeling apparatus. Consistently, Sov1p-deficient cells displayed reduced cAMP-PKA signaling and an elevated, Sir2p-dependent, genomic silencing. Both increased silencing and life span extension in sov1Δ cells require the PKA/Msn2/4p target Pnc1p, which scavenges nicotinamide, a Sir2p inhibitor. Inactivating other members of the MTC module also resulted in Sir2p-dependent life span extension. The data demonstrate that the nuclear silencing apparatus senses and responds to the absence of MTC proteins and that this response converges with a pathway for life span extension elicited by reducing TOR signaling.

Grainyhead-like 2 Enhances the Human Telomerase Reverse Transcriptase Gene Expression by Inhibiting DNA Methylation at the 5′-CpG Island in Normal Human Keratinocytes

We recently identified Grainyhead-like 2 (GRHL2) as a novel transcription factor that binds to and regulates the activity of the human telomerase reverse transcriptase (hTERT) gene promoter. In this study, we investigated the biological functions of GRHL2 and the molecular mechanism underlying hTERT gene regulation by GRHL2. Retroviral transduction of GRHL2 in normal human keratinocytes (NHK) led to a significant extension of replicative life span, whereas GRHL2 knockdown notably repressed telomerase activity and cell proliferation. Using promoter magnetic precipitation coupled with Western blotting, we confirmed the binding of GRHL2 to the hTERT promoter and mapped the minimal binding region at -53 to -13 of the promoter. Furthermore, mutation analysis revealed the three nucleotides from -21 to -19 to be critical for GRHL2 binding. Because hTERT expression is regulated in part by DNA methylation, we determined the effects of GRHL2 on the methylation status of the hTERT promoter. Senescent NHK exhibited hypermethylation of the CpG island, which occurred with the loss of hTERT expression. On the contrary, the promoter remained hypomethylated in GRHL2-transduced NHK, irrespective of cell proliferation status. Also, knockdown of endogenous GRHL2 led to hypermethylation of the promoter. These results indicate that GRHL2 regulates the hTERT expression through an epigenetic mechanism and controls the cellular life span.

Rapamycin increases rDNA stability by enhancing association of Sir2 with rDNA in Saccharomyces cerevisiae

The target of rapamycin (TOR) kinase is an evolutionarily conserved key regulator of eukaryotic cell growth and proliferation. Recently, it has been reported that inhibition of TOR signaling pathway can delay aging and extend lifespan in several eukaryotic organisms, but how lifespan extension is mediated by inhibition of TOR signaling is poorly understood. Here we report that rapamycin treatment and nitrogen starvation, both of which cause inactivation of TOR complex 1 (TORC1), lead to enhanced association of Sir2 with ribosomal DNA (rDNA) in Saccharomyces cerevisiae. TORC1 inhibition increases transcriptional silencing of RNA polymerase II-transcribed gene integrated at the rDNA locus and reduces homologous recombination between rDNA repeats that causes formation of toxic extrachromosomal rDNA circles. In addition, TORC1 inhibition induces deacetylation of histones at rDNA. We also found that Pnc1 and Net1 are required for enhancement of association of Sir2 with rDNA under TORC1 inhibition. Taken together, our findings suggest that inhibition of TORC1 signaling stabilizes the rDNA locus by enhancing association of Sir2 with rDNA, thereby leading to extension of replicative lifespan in S. cerevisiae.

Targeting the age‐related occurrence, removal, and accumulation of molecular damage by hormesis

Strategies for testing and developing effective means of intervention, prevention, and modulation of aging incorporate means to minimize the occurrence and accumulation of molecular damage, to reduce molecular heterogeneity, and to evaluate the relevance of the type and extent of damage with respect to its role in aging and age-related diseases. One such approach is that of mild stress-induced hormesis, which stimulates maintenance and repair systems and strengthens the homeodynamic space of cells and organisms. Hormesis through mild heat shock, natural and synthetic hormetins, and other stressors brings about several antiaging effects in human fibroblasts, keratinocytes, and telomerase-immortalized bone marrow stem cells. Depending on the cell type, these antiaging hormetic effects include extension of replicative life span, enhanced proteasomal activities, increased chaperone levels, and improved wound healing, angiogenesis, and differentiation. The main molecular pathways for achieving such hormetic effects are through targeting the processes for the repair and removal of molecular damage, which can slow aging.

Gene regulatory changes in yeast during life extension by nutrient limitation

Genetic analyses aimed at identification of the pathways and downstream effectors of calorie restriction (CR) in the yeast Saccharomyces cerevisiae suggest the importance of central metabolism for the extension of replicative life span by CR. However, the limited gene expression studies to date are not informative, because they have been conducted using cells grown in batch culture which markedly departs from the conditions under which yeasts are grown during life span determinations. In this study, we have examined the gene expression changes that occur during either glucose limitation or elimination of nonessential-amino acids, both of which enhance yeast longevity, culturing cells in a chemostat at equilibrium, which closely mimicks conditions they encounter during life span determinations. Expression of 59 genes was examined quantitatively by real-time, reverse transcriptase polymerase chain reaction (qRT-PCR), and the physiological state of the cultures was monitored. Extensive gene expression changes were detected, some of which were common to both CR regimes. The most striking of these was the induction of tricarboxylic acid (TCA) cycle and retrograde response target genes, which appears to be at least partially due to the up-regulation of the HAP4 gene. These gene regulatory events portend an increase in the generation of biosynthetic intermediates necessary for the production of daughter cells, which is the measure of yeast replicative life span.

SIRT1 markedly extends replicative lifespan if the NAD + salvage pathway is enhanced

Sir2 mediates lifespan extension in lower eukaryotes but whether its mammalian homolog, sirtuin 1, silent mating type information regulation 2 homolog (SIRT1), is a longevity protein is controversial. We stably introduced the SIRT1 gene into human vascular smooth muscle cells (SMCs) and observed minimal extension of replicative lifespan. However, SIRT1 activity was found to be exquisitely dependent on nicotinamide phosphoribosyltransferase (Nampt) activity. Moreover, overexpression of Nampt converted SIRT1-overexpressing SMCs to senescence-resistant cells together with heightened SIRT1 activity, suppressed p21, and strikingly lengthened replicative lifespan. Thus, SIRT1 can markedly postpone SMC senescence, but this requires overcoming an otherwise vulnerable nicotinamide adenine dinucleotide salvage reaction in aging SMCs.

Mitochondrial reactive oxygen species trigger hypoxia-inducible factor-dependent extension of the replicative life span during hypoxia.

Physiological hypoxia extends the replicative life span of human cells in culture. Here, we report that hypoxic extension of replicative life span is associated with an increase in mitochondrial reactive oxygen species (ROS) in primary human lung fibroblasts. The generation of mitochondrial ROS is necessary for hypoxic activation of the transcription factor hypoxia-inducible factor (HIF). The hypoxic extension of replicative life span is ablated by a dominant negative HIF. HIF is sufficient to induce telomerase reverse transcriptase mRNA and telomerase activity and to extend replicative life span. Furthermore, the down-regulation of the von Hippel-Lindau tumor suppressor protein by RNA interference increases HIF activity and extends replicative life span under normoxia. These findings provide genetic evidence that hypoxia utilizes mitochondrial ROS as signaling molecules to activate HIF-dependent extension of replicative life span.

The histone acetyltransferase GCN5 modulates the retrograde response and genome stability determining yeast longevity

Transcriptional silencing decreases at both subtelomeric and silent mating-type loci and increases at the ribosomal DNA locus during the replicative life span of the yeast Saccharomyces cerevisiae . Evidence exists that epigenetic changes in the regulatory state of chromatin may be a causal factor in determining yeast longevity and that histone deacetylases play a role. The significance of histone acetylation has been examined here in more detail. Deletion of the histone acetyltransferase gene GCN5 suppressed the extension of replicative life span afforded by the induction of the retrograde response, which signals mitochondrial dysfunction and leads to changes in nuclear gene expression. It was difficult to ascribe this effect to changes in transcriptional silencing in any of the three known types of heterochromatin. However, a promoter related effect was uncovered by the participation of GCN5 in the induction of the retrograde response. Gcn5p and the retrograde signal transducer Rtg2p are components of the histone acetyltransferase coactivator complex SLIK. Rtg2p blocks the production of extrachromosomal ribosomal DNA circles when it is not engaged in transmission of the retrograde signal. Deletion of GCN5 , which disrupts the integrity of SLIK, suppressed circle accumulation. The results indicate that Gcn5p and SLIK impact the interplay between the retrograde response signal and Rtg2p with consequences for the induction of the response and circle production. Rtg2p and Gcn5p in the SLIK complex link metabolism to stress responses, chromatin-dependent gene regulation, and genome stability in yeast aging.

Extension of replicative lifespan in WI-38 human fibroblasts by dexamethasone treatment is accompanied by suppression of p21 Waf1/Cip1/Sdi1 levels

Numerous studies have shown that supplementation of the growth medium of human fibroblasts with dexamethasone at physiologic concentrations extends replicative lifespan up to 30%. While this extension of lifespan has been used to probe various aspects of the senescent phenotype, no mechanism for the increased lifespan of human fibroblasts grown in the presence of dexamethasone has ever been identified. In the present study we present evidence that the extended lifespan of human lung fibroblasts (WI-38 cells) that occurs when these cells are maintained in culture medium supplemented with dexamethasone is accompanied by a suppression of p21 Waf1/Cip1/Sdi1 levels, which normally increase as these cells enter senescence, while p16 INK4a levels are unaffected. These results suggest that the delay of senescence in cultures grown in the presence of dexamethasone is due to a suppression of the senescence related increase in p21 Waf1/Cip1/Sdi1. These results are consistent with models of replicative senescence in which p53 and p21 Waf1/Cip1/Sdi1 play a role in the establishment of the senescent arrest.

In vitro aging of macaque adherent cells: similar pattern of cellular aging between human and macaque

To explore new models for human cellular aging as well as to evaluate aging of the macaques, profiles of cellular aging in macaques were studied. Adherent cells were obtained from five Japanese macaques ( Macaca fuscata), 14 long-tailed macaques ( Macaca fascicularis), two bonnet monkeys ( Macaca radiata) and a rhesus monkey ( Macaca mulatta). A total of 35 cultures were performed and cell morphology, doubling time, telomere length and telomerase activity were studied. They were classified into three groups; group I: cell strains with a definite replicative life-span (−41 PDLs) (presence of M1), group II: cell strains with a limited extension of replicative life-span (79–106 PDLs) with p53 mutation(s) (presence of M2), and group III: a cell strain with an indefinite replicative life-span (>150 PDLs) with characteristics of transformation. Except for the last group, telomerase activity was not observed. Macaque cells demonstrated three chronological patterns comprising both human and rodent patterns, however, presence of the two limits of proliferation in vitro grants macaque cells to be more appropriate than rodents in both studying human aging and oncogenesis.

Somatic mutations in the p53 gene account for the extension of replicative life span of macaque cells

To identify the underlying molecular bases that enable macaque cells to extend their replicative life span (RLS), somatic mutations in p53 were studied in two Japanese macaque ( Macaca fuscata) and one long-tailed macaque ( Macaca fascicularis) cell strains with extended RLS. Nucleotide sequences of the p53 whole coding region of each species were determined in early passaged cells and somatic mutations were studied in cells with extended RLS. Different type of genomic alteration which may disrupt normal p53 function was observed in each strain: (1) introduction of a premature stop codon in one chromosome and loss of heterozygosity for the other; (2) introduction of a missense mutation into each chromosome independently; (3) generation of a novel splice donor site to delete four amino acid residues in the presence of silencing of the normal p53 gene. Critical roles of p53 in cellular aging among macaques in terms of extention of RLS were demonstrated.

Differentiation between Senescence (M1) and Crisis (M2) in Human Fibroblast Cultures

Normal human fibroblasts undergo only a limited number of divisions in culture and eventually enter a nonreplicative state designated senescence or mortality stage 1 (M1). Expression of certain viral oncogenes, such as the SV40 large T antigen (SV40 T-Ag), can elicit a significant extension of replicative life span, but these cultures eventually also cease dividing. This proliferative decline has been designated crisis or mortality stage 2 (M2). BrdU incorporation assays are commonly used to distinguish between senescence (<5% labeling index) and crisis (>30% labeling index). It has not been possible, however, to ascertain whether the high labeling index, indicative of ongoing DNA replication, was caused by the presence of T-Ag. We used gene targeting to knock out both copies of the p21CIP1/WAF1 gene in presenescent human fibroblasts. p21 −/− cells displayed an extended life span but eventually entered a nonproliferative state. In their terminally nonproliferative state both p21 +/+ and p21 −/− cultures were positive for the senescence-associated β-galactosidase (SA-β-gal) activity; in contrast, the labeling index of p21 +/+ cells was low (<5%) whereas the labeling index of p21 −/− cells was high (>30%). The observation that p21 −/− and SV40 T-Ag-expressing cells behave identically with respect to life span extension as well as the high labeling index in the terminally nonproliferative state indicates that crisis is not a phenomenon induced solely by viral oncogenes, but a physiological state resulting from the bypass of normal senescence mechanisms. The widely used biomarker for senescence, SA-β-gal, cannot distinguish between senescence and crisis. We propose that all SA-β-gal-positive cultures should be further examined for their BrdU labeling index.

Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span.

Normal somatic cells have a finite life span [1] and lose telomeric DNA, present at the ends of chromosomes, each time they divide as a function of age in vivo or in culture [2-4]. In contrast, many cancer cells and cell lines established from tumours maintain their telomere length by activation of an RNA-protein complex called telomerase, an enzyme originally discovered in Tetrahymena [5], that synthesizes telomeric repeats [6-8]. These findings have led to the formation of the 'telomere hypothesis', which proposes that critical shortening of telomeric DNA due to the end-replication problem [9] is the signal for the initiation of cellular senescence [10,11]. In yeast, the EST2 gene product, the catalytic subunit of telomerase, is essential for telomere maintenance in vivo [12-14]. The recent cloning of the cDNA encoding the catalytic subunit of human telomerase (hTERT) [15,16] makes it possible to test the telomere hypothesis. In this study, we expressed hTERT in normal human diploid fibroblasts, which lack telomerase activity, to determine whether telomerase activity could be reconstituted leading to extension of replicative life span. Our results show that retroviral-mediated expression of hTERT resulted in functional telomerase activity in normal aging human cells. Moreover, reconstitution of telomerase activity in vivo led to an increase in the length of telomeric DNA and to extension of cellular life span. These findings provide direct evidence in support of the telomere hypothesis, indicating that telomere length is one factor that can determine the replicative life span of human cells.