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Results for “replicative lifespan”

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PubMed1999

The effects of nutritional manipulation and laboratory selection on lifespan in Drosophila melanogaster.

There are parallels between the effects of laboratory selection and nutritional manipulation on the expression of lifespan and other fitness-related characters in Drosophila melanogaster. However, little is known about the effects of laboratory selection and nutritional manipulation when applied simultaneously. Given that D. melanogaster is one of the major model organisms for testing theories of aging, simultaneous application of laboratory selection and nutritional manipulation is of considerable interest. To that end we developed six groups of five fold replicated populations selected for either early or late fertility. Each of these groups was maintained on either high- or low-nutrition diets. Comparisons among the groups showed that nutrition is neutral in selecting for lifespan. Moreover, the dietary-restriction response can be broken by simultaneous selection and nutritional manipulation. Finally, characters that respond in a parallel manner under selection or nutritional manipulation may not when the two are applied simultaneously.

Animal Nutritional Physiological Phenomena↗

PubMed2004

Telomerase activity is sufficient to bypass replicative senescence in human limbal and conjunctival but not corneal keratinocytes.

The human ocular surface is covered by the conjunctival, corneal and limbal stratified epithelia. While conjunctival stem cells are distributed in bulbar and forniceal conjunctiva, corneal stem cells are segregated in the basal layer of the limbus, which is the transitional zone between the cornea and the bulbar conjunctiva. Keratinocyte stem and transient amplifying (TA) cells when isolated in culture give rise to holoclones and paraclones, respectively. Keratinocyte replicative senescence ensues when all holoclones have generated paraclones which express high levels of p16(INK4a). In the present study, we show that enforced telomerase activity induces the bypass of replicative senescence in limbal and conjunctival keratinocytes, without the inactivation of the p16(INK4a)/Rb pathway or the abrogation of p53 expression. hTERT-transduced limbal and conjunctival keratinocytes are capable to respond to both growth inhibitory and differentiation stimuli, since they undergo growth arrest in response to phorbol esters, and activate p53 upon DNA damage. Following a sustained PKC stimulation, occasional clones of p16(INK4a)-negative cells emerge and resume ability to proliferate. Telomerase activity, however, is unable to induce the bypass of senescence in corneal TA keratinocytes cultured under the same conditions. These data support the notion that telomere-dependent replicative senescence is a general property of all human somatic cells, including keratinocytes, and suggest that telomerase activity is sufficient to extend the lifespan only of keratinocytes endowed with high proliferative potentials (which include stem cells), but not of TA keratinocytes.

Cell Proliferation↗

PubMed1983

Effects of intermittent feeding upon growth, activity, and lifespan in rats allowed voluntary exercise.

From weaning until death, male Wistar rats were housed in activity-wheel cages with one group maintained on an ad libitum (AL) diet and another provided the diet every-other-day (EOD). EOD-fed rats had a mean lifespan of 124 weeks compared to 103 weeks for AL-fed rats. While post-weaning body weight and growth rates were reduced among the EOD-fed animals compared to AL-fed animals, there was no significant difference in growth duration. Positive correlations were observed between lifespan and estimates of growth rate and duration in the AL group but not in the EOD group; thus, little evidence was produced to support the hypothesis that growth rate is inversely related to longevity. While the EOD feeding regimen resulted in higher activity levels later in life, wheel activity levels were actually lower in this group in early life compared to the AL group. The observation of reduced wheel activity among young rats fed EOD was replicated in a second experiment. Thus, little support was obtained for the hypothesis that increased activity mediates the beneficial effects of dietary restriction on longevity, unless this mechanism is active late in the lifespan.

Animals↗

PubMed2006

Telomerase activity coevolves with body mass not lifespan.

In multicellular organisms, telomerase is required to maintain telomere length in the germline but is dispensable in the soma. Mice, for example, express telomerase in somatic and germline tissues, while humans express telomerase almost exclusively in the germline. As a result, when telomeres of human somatic cells reach a critical length the cells enter irreversible growth arrest called replicative senescence. Replicative senescence is believed to be an anticancer mechanism that limits cell proliferation. The difference between mice and humans led to the hypothesis that repression of telomerase in somatic cells has evolved as a tumor-suppressor adaptation in large, long-lived organisms. We tested whether regulation of telomerase activity coevolves with lifespan and body mass using comparative analysis of 15 rodent species with highly diverse lifespans and body masses. Here we show that telomerase activity does not coevolve with lifespan but instead coevolves with body mass: larger rodents repress telomerase activity in somatic cells. These results suggest that large body mass presents a greater risk of cancer than long lifespan, and large animals evolve repression of telomerase activity to mitigate that risk.

Animals↗

PubMed1981

Evidence for a relationship between longevity of mammalian species and life spans of normal fibroblasts in vitro and erythrocytes in vivo.

The replicative life spans of mammalian fibroblasts in vitro were studied in a number of cell cultures representing eight species. Emphasis was placed on determining the population doubling level at which phase III (a period of decrease in the rate of proliferation) and chromosomal alterations occur. All the cell cultures studied went through a growth crisis, a period of apparent growth cessation lasting for at least 2 weeks. In most cultures, the crisis represented the end of their replicative capacities, but in some cultures cell proliferation was resumed after the crisis. A predominantly diploid chromosome constitution (more than 75%) was demonstrated prior to the growth crisis. In cultures in which cell proliferation was resumed after the crisis, a nondiploid constitution prevailed in all cases except the rat (with 90% or more diploid cells all the time). The growth crisis occurred at population doubling levels that were characteristic for the species and was shown to be related to the species' maximal life span by a strict power law, being proportional to the square root of the maximal life span. Based on data in the literature, the same relationship was also valid for the lifespans of circulating mammalian erythrocytes in vivo. These results may indicate the prevalence of a common functional basis regulating the life span of fibroblasts and erythrocytes and thus operating in replicative as well as postmitotic cells in vitro and in vivo.

Animals↗

PubMed2007

Hallmarks of telomeres in ageing research.

Telomeres are repetitive DNA sequences at the ends of linear chromosomes. Telomerase, a cellular reverse transcriptase, helps maintain telomere length in human stem cells, reproductive cells and cancer cells by adding TTAGGG repeats onto the telomeres. However, most normal human cells do not express telomerase and thus each time a cell divides some telomeric sequences are lost. When telomeres in a subset of cells become short (unprotected), cells enter an irreversible growth arrest state called replicative senescence. Cells in senescence produce a different constellation of proteins compared to normal quiescent cells. This may lead to a change in the homeostatic environment in a tissue-specific manner. In most instances cells become senescent before they can become cancerous; thus, the initial growth arrest induced by short telomeres may be thought of as a potent anti-cancer protection mechanism. When cells can be adequately cultured until they reach telomere-based replicative senescence, introduction of the telomerase catalytic protein component (hTERT) into telomerase-silent cells is sufficient to restore telomerase activity and extend cellular lifespan. Cells with introduced telomerase are not cancer cells, since they have not accumulated the other changes needed to become cancerous. This indicates that telomerase-induced telomere length manipulations may have utility for tissue engineering and for dissecting the molecular mechanisms underlying genetic diseases, including cancer.

Aging↗

PubMed1999

Occult lifelong persistence of infectious hepadnavirus and residual liver inflammation in woodchucks convalescent from acute viral hepatitis.

Traces of hepatitis B virus (HBV) genome can persist for years following recovery from hepatitis B. To determine overall duration, molecular characteristics, and pathological implications of this serologically undetectable form of hepadnaviral carriage, we have analyzed the expression of transcriptionally active virus genomes, their infectivity, and examined liver alterations during the natural lifespan of woodchucks convalescent from acute infection with HBV- related woodchuck hepatitis virus (WHV). In this study, we document lifelong persistence of scanty amounts of replicating virus both in the liver and lymphatic system after spontaneous resolution of an episode of experimental hepadnaviral hepatitis. Antibodies to virus nucleocapsid (core) were found to be the most reliable immunovirological marker coexisting with occult infection. In the majority of convalescent woodchucks, serial liver biopsies showed protracted minimal to mild necroinflammation with periods of normal morphology; however, hepatocellular carcinoma (HCC) ultimately developed in 2 of 9 animals studied. Inocula derived from lymphoid cells of convalescent animals induced classical acute hepatitis in virus-naive woodchucks that progressed to chronic hepatitis and HCC in 1 of the animals, demonstrating infectivity and pathogenic competence of the carried virus. Our results reveal that low levels of infectious WHV and residual hepatic inflammation usually continue for life after resolution of hepatitis and that this recovery does not avert HCC development. They also demonstrate that, in addition to the liver, the lymphatic system is the site of the occult lifelong maintenance of replicating hepadnavirus.

Acute Disease↗

PubMed2002

In vitro differentiation capacity of telomerase immortalized human RPE cells.

PURPOSE: To investigate conditions promoting the differentiation of cultured human retinal pigment epithelial (RPE) cells and assess the differentiation potential of telomerase-immortalized RPE cells. METHODS: Serially passaged RPE 340 (parental) cells have limited replicative ability and senesce after 50 to 60 population doublings (PD)s. RPE 340 cells transfected with the catalytic component of human telomerase (hTERT) have an extended lifespan. RPE 340 and hTERT-transfected RPE (hTERT-RPE) cells were maintained at confluence without serum for up to 12 weeks. Morphologic, immunocytochemical, flow cytometric, and spectrophotometric analyses were performed to examine the extent of RPE differentiation. RESULTS: Parental RPE 340 and hTERT-RPE cells underwent growth arrest and differentiated in the absence of serum. In early-passage parental (PD11) and hTERT-RPE (PD115 and PD300) cells, serum deprivation for 4 weeks or more induced terminal differentiation as characterized by mature, growth-arrested, confluent sheets of polygonal and melanized cells that demonstrated diminished 5'-bromo-2'-deoxyuridine (BrdU) uptake and positive reactivity with antibodies to cellular retinaldehyde-binding protein (CRALBP), cytokeratin, and vimentin. Reintroduction of serum at 4 or 8 weeks allowed the cells to reenter the cell cycle and demelanize. Midpassage (PD 25) parental cells, however, were irreversibly arrested at G(1) after 8 weeks of serum deprivation. CONCLUSIONS: Cultured parental and hTERT-RPE cells express RPE-associated proteins and become stably melanized when density is arrested in the absence of serum. Moreover, hTERT-immortalized RPE cells retain the capacity to undergo terminal differentiation in vitro, even after long-term culture.

Carrier Proteins↗

PubMed1984

Delayed and reduced cell replication and diminishing levels of DNA polymerase-alpha in regenerating liver of aging mice.

Kinetics of cell replication was compared in regenerating livers of Mus musculus at ages ranging between 6 and 32 months. Incorporation of [3H]-thymidine into DNA and autoradiographic analysis showed that the maximal extent of DNA replication following partial hepatectomy became delayed with age. Furthermore, the total fraction of parenchymal and nonparenchymal cells in S phase at different intervals during regeneration diminished as mice aged. The specific activity of DNA polymerase-alpha, the putative replicative enzyme, declined progressively during aging. The specific activity of DNA polymerase-beta, the purported repair enzyme, declined to an appreciably lesser extent during the lifespan of the mouse. No evidence was found for the appearance of a specific inhibitor of polymerase-alpha in senescent mouse liver. Also, the bulk of the activities of both hepatic DNA polymerase-alpha and -beta remained localized in the cell nucleus throughout the lifetime of the animal and were mainly associated with chromatin.

Aging↗

PubMed1998

SV40-mediated transformation and immortalization of human cells.

SV40 infection of human cells results in both transformation and lytic infection. We have used origin-defective viral mutants which are unable to replicate in permissive cells to help analysis of transformation. Expression of large T antigen (T ag) and small t antigen results in the altered growth phenotypes characteristic of transformation in other species. Human diploid fibroblasts (HF) have a limited lifespan and undergo senescence; T ag results in extension of lifespan but only in rare cases are the cells capable of continuous growth and are immortal. We have developed matched sets of non-immortal and immortal transformed HF for assessment of the steps required for immortalization. Results are summarized to characterize both T-dependent and T-independent functions. A novel growth suppressor gene SEN6 has been identified, the inactivation of which is required for immortalization; it may also serve as a marker to distinguish cells in which SV40 is replicating from those in which it is responsible for tumorigenesis.

3T3 Cells↗

PubMed2005

Chaperones and longevity.

That evolution of longevity may depend on alterations in the expression of relatively few regulatory genes has been inferred from the rapid increase in lifespan during evolution of the hominid species (Cutler RG (1979) Mech Ageing Dev 9: 337-354). Also the inherent immortality of the embryonic stem cells implies that replicative senescence (Hayflick L (1997) Biochem Mosc 62: 1180-1190) as possibly aging of species are epigenetic phenomena. Evidence is presented to suggest that the epigenetic changes of the longevity determinants to a significant extend concerns the molecular chaperones. Specific involvement of RNA chaperones in cell immortalization and defective RecQ-DNA chaperones in syndromes of premature aging suggest that DNA/RNA - chaperones probably rank high among the determinants of cellular and species longevity.

Animals↗

PubMed1996

Establishment of permanent human endothelial cells achieved by transfection with SV40 large T antigen that retain typical phenotypical and functional characteristics.

The plasmid pMK16 containing-SV40 replicated origin defective gene was efficiently introduced into early-passage human umbilical vein endothelial cells (HUVEC) using positively charged liposomes. The resulting cell line acquired an almost infinite lifespan, was morphologically unchanged, expressed SV40-antigen, and coexpressed von Willebrand factor (vWF), tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), angiotensin conversion enzyme (ACE), and endothelin converting enzyme (ECE). In addition, these are the first immortalized human endothelial cells, to our knowledge, that biosynthesized and secreted interleukins (IL-1 beta and IL-6) in both a constitutive and regulated fashion and endothelin-1 (ET-1), the most potent vasoactive peptide, which has been suggested to be implicated in the pathogenesis of hypertension. Interestingly enough, both of the immortalized cells and the early-passage HUVEC from which the immortalized cells were obtained biosynthesized and secreted the same levels of ET-1 suggesting full maintenance of its biosynthetic pathway including the presence of active ECE, which cleaves big endothelin-1 (big-ET-1) to ET-1 and regulation factors. Moreover, the immortalized cells retained the ability to express the functional specific amino acid Na(+)-independent system Y+ transporter, which mediates L-arginine transport into endothelial cells from which endothelium-derived relaxing factor (EDRF, nitric oxide) is formed via the action of nitric oxide-synthase. Obtaining these immortalized human endothelial cells without alteration of the differentiated characteristics constitutes a useful model: (a) to study ET-1 secretion, gene regulation, and human ECE, which may be an important therapeutic target in disease conditions in which ET-1 is to be implicated; (b) to study L-arginine transport, which is a key step in the formation of EDRF; (c) to study IL-1 beta and IL-6 secretions, and gene regulations; (d) to substitute large quantities of HUVEC; and, finally, (e) to reproduce, starting with different primary endothelial cells both from human and animal origin.

Antigens, Polyomavirus Transforming↗

PubMedDate not supplied

[The mechanism of replicative and post-replicative DNA methylation as a generator of mutations in a cell].

The functional role of enzymatic DNA methylation in eukaryotes is still obscure even 45 years after its discovery. In the present paper the analysis of various aspects of DNA methylation has been made from the point of view of a common functional model considering the system as a generator of 5mC-->T mutations in the cell. The mechanism and consequences of the reactions have been described in detail, from the 5mC residue deamination during replicative DNA methylation and repair of G.T-mispairs to production of hemimethylated sites in DNA and their postreplicative methylation. It was shown that the loss of the most part of the 5mC residues from DNA during the lifespan proceeds with aging both of organism's tissues and of cell cultures. Evidence was obtained that this is the result of gradual accumulation of 5mC-->T + C transitions in the genome during each cell division. Such substitutions take place 10 times more often in methylated sites *CG than in any other sites of DNA. They disproportionally contribute to general mutagenesis and may be a cause of many hereditary human diseases. The conclusion has been made that DNA methylation can be considered as a genetically programmed mechanism for accumulating mutations with aging.

5-Methylcytosine↗

PubMed2002

Raf-1-induced growth arrest in human mammary epithelial cells is p16-independent and is overcome in immortal cells during conversion.

Using an estrogen-inducible retroviral system, we demonstrate that oncogenic Raf-1 induces growth arrest and morphological changes in finite lifespan human mammary epithelial cells (HMEC). This arrest does not rely on expression of the cyclin-dependent kinase inhibitor (CKI) p16(INK4a), nor on changes in expression of the CKIs p21(Cip1), p14(ARF), p27(Kip1) or p57(Kip2). The Raf-induced arrest is independent of viral oncogene mediated inactivation of p53 and pRB, or c-myc overexpression. Flow cytometric analysis demonstrates that cells arrest in both G1 and G2. The Raf-induced arrest is mitigated or eliminated in some immortally transformed HMEC. Immortal HMEC that have both overcome replicative senescence and undergone the recently described conversion process maintain growth in the presence of transduced oncogenic Raf-1; they also gain EGF-independent growth and a low frequency of anchorage-independent growth. However, HMEC that have overcome replicative senescence but have not undergone conversion and HMEC immortalized by transduction with the catalytic subunit of telomerase, hTERT, remain severely growth arrested. These results indicate that the molecular mechanisms responsible for the Raf-1-induced growth arrest may vary among different finite lifespan cell types, and that in HMEC, this mechanism is altered during the conversion process, rather than as a direct consequence of overcoming senescence or expressing hTERT.

Breast↗

PubMed2003

Biology of the basic multicellular unit and the pathophysiology of osteoporosis.

Bone structural integrity is maintained by removal of old bone by osteoclasts and synthesis of new bone in its place by osteoblasts. This process, called bone remodeling, is accomplished by assembly of osteoclasts and osteoblasts into discrete temporary anatomic structures called basic multicellular units (BMUs). The lifespan of osteoclasts and osteoblasts is short compared to the lifespan of the BMU; therefore, they must be continually replenished for BMU progression to occur. Osteoclasts develop from hematopoeitic progenitors; and osteoblasts are derived from mesenchymal stem cells, also known as marrow stromal fibroblasts. Circulating hormones together with locally produced cytokines and growth factors modulate the replication and differentiation of osteoclast and osteoblast progenitors. The most important locally produced pro-osteoclastogenic cytokine is receptor activator of NF-kappaB (RANK) ligand. It is expressed by stromal/osteoblastic cells, and binds to its receptor (RANK) on osteoclast progenitors to stimulate differentiation. The development of mature osteoblasts is promoted by growth factors released from the bone matrix during resorption, as well as by growth factors produced by osteoblast progenitors themselves. Many of these same locally produced factors and cytokines also govern the lifespan of osteoclasts and osteoblasts by their effects on apoptosis. The orderly genesis and apoptosis of both osteoclasts and osteoblasts is essential for normal bone homeostasis during bone remodeling. Bone loss caused by sex steroid deficiency or glucocorticoid excess is caused by alteration of bone cell production, and by prolonging osteoclast lifespan and shortening osteoblast lifespan. Therapies that prevent or reverse osteoporosis act at least in part by preventing osteoblast apoptosis and/or stimulating osteoclast apoptosis.

Animals↗

PubMed1996

Lifespan and lesions in genetically heterogeneous (four-way cross) mice: a new model for aging research.

Genetically heterogeneous animal models provide many advantages for research on aging but have been used infrequently. We present here lifespan and lesion data from a study of mice bred as a cross between (AKR/J x DBA/2J)F1 females and (C57BL/6J x SJL/J)F1 males. In such a four-way cross population, each mouse is genetically unique, but replicate populations of essentially similar genetic structure can be generated quickly, at low cost, and of arbitrary size from commercially available, genetically stable hybrid parents. We employed a protocol in which mice judged to be severely ill were euthanatized to obtain tissue in optimal condition for necropsy, and we were able to infer a likely cause of illness in 42 of 44 animals. Malignant lymphoma, including at least four histopathologically distinct subtypes, was the most common cause and was also a frequent incidental finding in mice dying of other causes. Neoplastic disease, benign or malignant, was the sole or a contributing cause of illness in 90% of the mice for which a cause could plausibly be assigned. A wide range of lethal and nonlethal degenerative lesions was also noted. The coefficient of variation for lifespan in these genetically heterogeneous mice was 26%, similar to that seen in analyses of recombinant inbred mouse lines. Baseline lifespan and pathology data on four-way cross mice is a useful prelude to the exploitation of this rodent model in tests of genetic and mechanistic hypotheses about aging.

Adenocarcinoma↗

PubMed2006

Glucose-induced replicative senescence in mesenchymal stem cells.

Mesenchymal stem cells (MSCs) show great promise for use in a variety of cell-based therapies. Because isolated primary mesenchymal stem cells are low in numbers, in vitro expansion is necessary. However, the expansion potential is limited and in vitro aging leads to loss of multipotency and replicative senescence. Stress induced by culture conditions is likely to be a major cause of replicative senescence and reduced multipotency of MSC and optimization of culture conditions might be able to reduce this. Caloric restriction (CR) is the only established method to delay aging and extend lifespan. In vitro caloric restriction experiments are rare, but have demonstrated beneficial effects. Therefore, we investigated the effect of culture medium glucose concentration on the proliferative and differentiation potential of mesenchymal stem cells. Reduction in glucose concentrations led to decreased apoptosis and an increased rate of MSC proliferation and increased the number and size of fibroblastic colonies in the colony-forming unit assay.

Animals↗

PubMed1993

Escape from in vitro aging in SV40 large T antigen-transformed human diploid cells: a key event responsible for immortalization occurs during crisis.

A human diploid fibroblast strain MRC-5 was transfected with a replication origin-defective early region of SV40 containing the gene of large T antigen, and 48 clones of T antigen-transformed MRC-5 were isolated. Although T antigen prolonged the lifespan of MRC-5, all the transformed clones were still mortal. From two of the transformed MRC-5 clones, eight independent immortalized clones were obtained in triple experiments of immortalization. The transformed phenotypes of immortalized clones were widely varied and did not always retain those of the parental pre-immortalized clones. The immortalization occurred at the frequency of about 1-3 x 10(-7)/cell. From cell number and population doubling time of the immortalized clones, the immortalization was estimated to occur in or just before the crisis of parental mortal cells. The decreases of modal chromosome numbers and the loss of chromosomes 5 and 10 were found to be common in three independent immortalized clones examined. Thus, the escape from the cellular aging process seemed to be caused by certain genetic events including the loss of chromosomes at the end of lifespan in T antigen-transformed human diploid cells.

Aneuploidy↗