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PubMed2000

Telomerase and the cellular lifespan: implications of the aging process.

The aging process has multiple causes. However, there is now substantial evidence consistent with the hypothesis that (i) all normal mammalian somatic cells have a finite capacity to replicate and (ii) that gradual cell turnover throughout the lifespan of a mammal eventually exhausts this finite capacity. This results in a gradual accumulation of senescent (irreversibly post-mitotic) cells with increasing age. These cells display a radically different phenotype to their growing counterparts, which has the potential to compromise tissue function. Perhaps the best evidence for this is seen in Werner's syndrome, a rare genetic disease, in which patients display most of the features of accelerated aging, together with a profoundly compromised replicative lifespan in certain tissue lineages. Several classes of human cells are now known to count divisions by monitoring the progressive attrition of chromosomal ends (telomeres), leading to the activation of a p53-p21waf-dependent G1 checkpoint. Ectopic expression of telomerase has been shown to prevent senescence in several cell types and offers the potential for interventions in the aging process based on tissue engineering, gene therapy or homeografts. However, this telomere-driven senescence mechanism seems to be absent from rodents, which use telomere-independent means (perhaps based upon p14arf) to count divisions. Similar senescence pathways are now being reported in humans, and this, coupled with the demonstration of tissue-specific telomeric loss rates, has the potential to render strategies based on the use of telomerase dependent on the characteristics of the target tissue. Werner's syndrome may provide strong clues regarding the potential limitations and prospects of such future treatments.

Aging↗
PubMed2002

Termination of lifespan of SV40-transformed human fibroblasts in crisis is due to apoptosis.

Normal human fibroblasts in culture have a limited lifespan, ending in replicative senescence. Introduction of SV40 sequences encoding large T antigen and small t antigen into pre-senescent cells results in an extension of lifespan for an additional 20-30 population doublings. Rare clones of SV40-transformed cells are capable of indefinite growth and are described as immortal; however, the great majority of SV40-transformed cells terminate this extended lifespan in cell death, termed "crisis." We have examined the properties of cells in crisis to obtain further insights into mechanism of cell death and immortalization. Populations at the terminal cell passage show a balance between cell replication and cell death over a period of several weeks, with a progressive increase in cells undergoing cell death. During this period, there is less than a 3-fold increase in attached cell number, with two stages being identifiable on the basis of the focal pattern of cell survival. We also demonstrate that cells in crisis are undergoing apoptosis based on TUNEL assay, subG1 DNA content, annexin V reactivity, and activation of caspases 3 and 8. We suggest a model whereby SV40-transformed cells acquire increased sensitivity to apoptosis based on changes in properties which activate caspase 8 in addition to changes previously described involving shortening of telomeric sequences. While only telomere stabilization could be clearly shown to be essential for survival of cells through crisis, the extended period of cell replication and altered gene expression observed in SV40-transformed cells during crisis are compatible with other genetic alterations in immortal cells.

Antigens, Polyomavirus Transforming↗
PubMed1991

Autosomal dominant polycystic kidney disease--in vitro culture of cyst-lining epithelial cells.

The major form of autosomal dominant polycystic kidney disease (ADPKD) in humans is linked to the PKD1 gene on chromosome 16p. The identity of the gene and the underlying pathogenetic mechanisms are not yet defined. Cyst-lining epithelial cells derived from a polycystic kidney were successfully grown in culture and designated MZ-PKD-1 cells. By linkage analysis, the related pedigree of the nephrectomized patient could be linked to the PKD1 gene on chromosome 16p. Thus, these cells exhibit the genotype of a mutated PKD1 gene and represent an in vitro culture model for ADPKD involving chromosome 16p. The antigenic phenotype was characterized immunohistologically by epithelial differentiation antigens and markers of individual nephron segments. An essentially identical antigenic pattern of proximal tubular cells was observed both in vitro and in fresh frozen tissue. Electron microscopy showed the formation of a microvillous-like coating. During growth phases in vitro successive changes in the cell shape were observed. MZ-PKD-1 cells exhibited a limited lifespan ending in replicative senescence. Northern blot analysis of kidney-growth-related genes, c-myc, TGF-alpha, TGF-beta 1, and EGF receptor revealed abundant expression of all of these genes in MZ-PKD-1 cells.

Antibodies, Monoclonal↗
PubMed2003

Positive and negative regulation of deltaN-p63 promoter activity by p53 and deltaN-p63-alpha contributes to differential regulation of p53 target genes.

Mammary epithelial regeneration implies the existence of cellular progenitors with retained replicative capacity, prolonged lifespan and developmental potency. Evidence exists that deltaN-p63 isoforms preserve these features by modulating p53 activity in basal epithelia. deltaN-p63 mRNA levels decline at the onset of differentiation suggesting that its transcriptional regulation may contribute to the initiation of differentiation. To study transcriptional regulation of deltaN-p63, a 10.3 kbp fragment containing the deltaN-p63 promoter was isolated. We report here that deltaN-p63 is a positive and negative transcriptional target of p53 and deltaN-p63-alpha, respectively. Disruption of p53 activity or expression abolishes the expression of deltaN-p63-alpha. This regulation is mediated by a p53-binding element sufficient to confer these activities to a heterologous promoter. Chromatin immune-precipitation indicates that, in asynchronously growing cells, p53 occupies this element. In response to DNA damage, deltaN-p63-alpha is recruited to this element as transcription of deltaN-p63 declines. Disruption of deltaN-p63-alpha expression had differential effects on the transcriptional regulation of several p53-target genes. These findings indicate that p53 contributes to the preservation of basal epithelia by driving the expression of deltaN-p63 isoforms. These studies also suggest that in response to genotoxic stress, deltaN-p63-alpha mediates the silencing of its own promoter thereby altering the pattern of p53-target gene expression.

Amino Acid Sequence↗
PubMed2002

Wolbachia density and virulence attenuation after transfer into a novel host.

The factors that control replication rate of the intracellular bacterium Wolbachia pipientis in its insect hosts are unknown and difficult to explore, given the complex interaction of symbiont and host genotypes. Using a strain of Wolbachia that is known to over-replicate and shorten the lifespan of its Drosophila melanogaster host, we have tracked the evolution of replication control in both somatic and reproductive tissues in a novel host/Wolbachia association. After transinfection (the transfer of a Wolbachia strain into a different species) of the over-replicating Wolbachia popcorn strain from D. melanogaster to Drosophila simulans, we demonstrated that initial high densities in the ovaries were in excess of what was required for perfect maternal transmission, and were likely causing reductions in reproductive fitness. Both densities and fitness costs associated with ovary infection rapidly declined in the generations after transinfection. The early death effect in D. simulans attenuated only slightly and was comparable to that induced in D. melanogaster. This study reveals a strong host involvement in Wolbachia replication rates, the independence of density control responses in different tissues, and the strength of natural selection acting on reproductive fitness.

Adaptation, Physiological↗
PubMed1976

The relationship between in vitro cellular aging and in vivo human age.

Differences between early and late passage cell cultures on the organelle and macromolecular levels have been attributed to cellular "aging". However, concern has been expressed over whether changes in diploid cell populations after serial passage in vitro accurately reflect human cellular aging in vivo. Studies were therefore undertaken to determine if significant differences would be observed in the in vitro lifespans of skin fibroblast cultures from old and young normal, non-hospitalized volunteers and to examine if parameters that change with in vitro "aging" are altered as a function of age in vivo. Statistically signigificant (P less than 0.05) decreases were found in the rate of fibroblast migration, onset of cell culture senescence, in vitro lifespan, cell population replication rate, and cell number at confluency of fibroblast cultures derived from the old donor group when compared to parallel cultures from young donors. No significant differences were observed in modal cell volumes and cellular macromolecular contents. The differences observed in cell cultures from old and young donors were quantitatively and qualitatively distinct from those cellular alterations observed in early and late passage WI-38 cells (in vitro "aging"). Therefore, although early and late passage cultures of human diploid cells may provide an important cell system for examining loss of replicative potential, fibroblast cultures derived from old and young human donors may be a more appropriate model system for studying human cellular aging.

Aging↗
PubMed2005

hTERT extends proliferative lifespan and prevents oxidative stress-induced apoptosis in human lens epithelial cells.

PURPOSE: Telomerase is a specialized polymerase that catalyzes synthesis of telomeres in most eukaryotes. When introduced into somatic cells, it extends the proliferative lifespan and prevents replicative senescence. Whether it has similar functions in lens epithelial cells, especially in human lens epithelial cells (HLECs) remains to be determined. In this study, the human telomerase reverse transcriptase (hTERT) catalytic subunit was introduced into HLECs. A stable cell line expressing hTERT was established and the functions of hTERT were studied. METHODS: The telomeric repeat amplification protocol (TRAP) assay was used to analyze the telomerase activity. Western blot analysis was used to examine hTERT expression. Southern blot analysis was used to detect telomere length. HLECs isolated from intact lenses were cultured in DMEM and transfected with hTERT cDNA. The expression of the exogenous hTERT was examined with RT-PCR, Western blot analysis, and TRAP assay. The functions of hTERT were examined with various techniques. RESULTS: Among the human, bovine, and rabbit lenses examined, only the central epithelium from the 6-month rabbit lens displayed telomerase activity. In both transparent and cataractous human lenses, hTERT activity and expression were not detected. However, the template RNA was present in both types of human lenses. The telomeres in transparent lenses were approximately 1 kb longer than those in cataractous lenses. The primary cultures and later passages of HLECs also displayed no detectable telomerase activity. Introduction of hTERTcDNA into HLECs followed by G418 selection yielded a stable line of HLECs expressing hTERT. In this line, hTERT has supported normal growth after 48 population doublings (PDs) to date and also enhanced antiapoptotic activity against oxidative stress. CONCLUSIONS: Telomere lengths may be associated with cataractogenesis. hTERT introduced into HLECs prevents replicative senescence through telomere synthesis. Furthermore, hTERT displays functions beyond telomere synthesis in normal HLECs.

Adult↗
PubMed2002

Cellular longevity: role of apoptosis and replicative senescence.

Cellular longevity refers to the lifespan of an individual cell. Normal cells have a finite lifespan and typically die by undergoing apoptosis, or enter into a state of irreversible growth arrest, termed replicative senescence, at the end of that lifespan. The lifespan of a cell is a balance between pro-survival/anti-apoptotic and pro-apoptotic death-promoting factors. The role of heat shock proteins, Bcl-2 family members, antioxidant molecules, and telomere length and telomerase activity in the regulation of apoptosis and replicative senescence, will be discussed.

Animals↗
PubMed1999

[Replicative senescence as a model of aging: the role of oxidative stress and telomere shortening--an overview].

Replicative senescence is characterized by the irreversible loss of division potential of cultivated human and animal cells. Correlations between the replicative potential in vitro and the age of the donor or the maximal lifespan of the species suggest replicative senescence to be an appropriate model for aging. Telomeres of human somatic cells shorten with each cell division but are stabilized at constant length in tumors and immortal cells by the enzyme telomerase. The assumption of a causal role of telomere shortening for the limited lifespan of cells in vitro was borne out recently. We could demonstrate oxidative stress as a main reason for telomere shortening. Telomeres are sensors for oxidative damage in the genome. Telomeres shorten during in vivo aging as well; however, there are significant differences between individuals. Telomere erosion might play a major role for the aging of the immune system. Our data suggest that telomere shortening in vivo could reflect the cumulative amount of oxidative damage to the organism. It might be useful as a biomarker of aging.

Animals↗
PubMed2006

Recruitment and proliferation of CD4+ T cells in synovium following adoptive transfer of adjuvant-induced arthritis.

Adjuvant-induced arthritis can be transferred to naive Dark Agouti (DA) strain (DA.CD45.1) rats by thoracic duct (TD) lymphocytes. Disease can be re-induced in convalescent rats by further transfer of arthritogenic cells, suggesting that resolution of the adoptive disease is not due to active regulation. To examine whether resolution is due to exhaustion of effector cells, we transferred the disease to DA.CD45.1 recipients, using CD4+ T cells from DA.CD45.2 donors. At the height of the adoptively transferred disease, donor cells comprised only 5-10% of recirculating CD4+ T cells but they accounted for approximately 40% of the CD4+ T cells in synovium-rich tissues of the hind paws. Approximately 65% of the donor cells in the synovium expressed a marker of proliferation (Ki-67 antigen). Division of CD4+ T cells continued in shielded paws after suppression of the recirculating pool of lymphocytes by selective irradiation. Intravenously injected CD4+ TD T lymphoblasts from arthritic donors were recruited to normal paws and, in greater numbers, to paws of animals with existing arthritis. Survival of the [125I]iodo-deoxyuridine-labeled lymphoblasts was greater in animals with existing arthritis. We conclude that effector CD4+ T cells in target tissues can proliferate in response to autoantigens and exhibit enhanced survival. However, without a continuous supply, adoptively transferred effector cells do not produce autonomous local disease, due to limits to their lifespan and ability to replicate indefinitely.

Adjuvants, Immunologic↗
PubMed2006

Regulation of intracellular signalling by the terminal membrane proteins of members of the Gammaherpesvirinae.

The human gamma(1)-herpesvirus Epstein-Barr virus (EBV) and the gamma(2)-herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV), rhesus rhadinovirus (RRV), herpesvirus saimiri (HVS) and herpesvirus ateles (HVA) all contain genes located adjacent to the terminal-repeat region of their genomes, encoding membrane proteins involved in signal transduction. Designated 'terminal membrane proteins' (TMPs) because of their localization in the viral genome, they interact with a variety of cellular signalling molecules, such as non-receptor protein tyrosine kinases, tumour-necrosis factor receptor-associated factors, Ras and Janus kinase (JAK), thereby initiating further downstream signalling cascades, such as the MAPK, PI3K/Akt, NF-kappaB and JAK/STAT pathways. In the case of TMPs expressed during latent persistence of EBV and HVS (LMP1, LMP2A, Stp and Tip), their modulation of intracellular signalling pathways has been linked to the provision of survival signals to latently infected cells and, hence, a contribution to occasional cellular transformation. In contrast, activation of similar pathways by TMPs of KSHV (K1 and K15) and RRV (R1), expressed during lytic replication, may extend the lifespan of virus-producing cells, alter their migration and/or modulate antiviral immune responses. Whether R1 and K1 contribute to the oncogenic properties of KSHV and RRV has not been established satisfactorily, despite their transforming qualities in experimental settings.

Animals↗
PubMed1993

E6 of human papillomavirus type 16 can overcome the M1 stage of immortalization in human mammary epithelial cells but not in human fibroblasts.

Immortalization is the consequence of the inactivation or bypass of two mortality stage mechanisms, M1 and M2, which are controlled by several genes including Rb and p53 in human fibroblasts. Abrogation of the M1 controls can be obtained through the activity of DNA tumor virus genes such as E6 and E7 of human papillomavirus 16 (HPV16). Fibroblasts expressing both E6 (which binds p53) and E7 (which binds Rb) bypass M1 and continue replicating (exhibit an extended lifespan) until an independent mechanism, M2, is activated. The inactivation of the M2 mechanism finally results in cell immortalization. The present study establishes a difference in the tissue-specific mechanisms for the control of the M1 stage of cellular senescence. The expression of HPV16 E6 was sufficient to bypass the M1 stage of cellular senescence and confer an extended lifespan in human mammary epithelial cells but not in fibroblasts. This implies that the M1 mechanism in human mammary epithelial cells does not involve the constitutive activation of Rb function as it does in fibroblasts. In addition, the results confirmed that the expression of HPV16 E6 (or both E6 and E7) did not directly immortalize the human mammary epithelial cells, since the inactivation of a second event, M2, was required to achieve immortalization. These observations are considered in the context of the telomere shortening model of cellular senescence.

Blotting, Northern↗
PubMed1999

Differences in locomotor activity across the lifespan of Drosophila melanogaster.

The identification of differential patterns of change across the lifespan in quantitative traits is of abiding interest in the biological and gerontological research communities. These differential phenotypic patterns in complex systems illuminate developmental processes and form the foundation for the identification of putative biomarkers of aging. The goal of the present study was to explore changes in locomotor activity through the lifespan of Drosophila melanogaster. A replicated serial cross-sectional sampling design was used to test activity in five genetically independent inbred strains at 7, 14, 21, 28, 35, and 42 days of age. Differences were observed in activity level across ages and strains, suggesting that patterns of activity throughout the lifespan of D. melanogaster are influenced by genetic factors. Observed sex differences in change in activity level indicate that the aging processes may proceed differently in males and females.

Animals↗
PubMed2001

Characterization of human myocardial fibroblasts immortalized by HPV16 E6--E7 genes.

Human myocardial fibroblasts (HMF) have proved to be useful as a species specific cell culture system in various studies on myocarditis and cardiac remodelling. However, their use is limited, since they are hard to obtain and lifespan is short due to replicative senescence. To overcome these disadvantages, we transfected primary HMF with the E6 and E7 genes of the oncogenic human papillomavirus (HPV) 16. Successful transfection was demonstrated in 3 of 12 experiments by detection of E6-E7 gene transcription with nucleic acid sequence based amplification (NASBA). No significant change of phenotype was noted in the emerging cell lines (HMF(1226D), HMF(1321D), HMF(1226K)), but their in vitro lifespan was increased by 20 to 30 population doublings until cells entered crisis. A single subclone of HMF(1226K) had a transformed phenotype and continued to proliferate indefinitely. This subclone (HMF(1226K/I)) was considered to be immortalized and telomerase activity was detected. Despite the increased risk of mutations due to abrogation of p53 function, HMF(1226K/I) and the HMF lines with an increased lifespan retained the properties of primary HMF cells, as they expressed fibroblast markers (prolyl-4-hydroxylase, vimentin), cytokines (interleukin 1 alpha, 6, 8), and angiotensin II receptors and still were permissive for coxsackievirus B3 infection.

Cell Line↗
PubMed2002

The effect of telomerase expression on the escape from M2 crisis in virus-transformed human retinal pigment epithelial cells.

Transformation with viral oncogene extends the lifespan of normal cells beyond replicative senescence called M1, but most of them eventually succumb to second crisis called M2 when telomeres become critically short. To acquire an infinite growth capacity, these cells have to overcome M2 crisis, which is known to follow telomerase activation. We have investigated if telomerase expression is required for virus-transformed pre-M2 cells to avert M2 crisis. Human retinal pigment epithelial (RPE) cells were transformed with simian virus 40 large T antigen and a VR3 clone in pre-M2 stage was obtained. Then, VR3 cells were transfected with a telomerase-containing vector and two cell lines that expressed telomerase temporarily or continuously were cloned and designated as ST1 and ST2, respectively. Normal RPE cells went into senescence after 36 population doublings. Although the lifespan was extended in the VR3 clone about 20 times more, it eventually underwent second crisis. The telomere length of VR3 decreased compared to that of normal RPE cells and the decrease continued during subculture. However, the ST1 and ST2 clones that expressed both T antigen and telomerase could avert this crisis. The initial telomere length of ST1 and ST2 was longer than that of normal cells. The ST1 underwent growth arrest again as telomerase expression faded out and elongated telomere was shortened, but the ST2 that maintained telomerase activity and telomere length proliferated continuously. In conclusion, telomerase activation is definitely required to overcome M2 crisis and acquire an infinite lifespan in human somatic epithelial cells and this mechanism is independent from M1 crisis escape in cell immortalization.

Antigens, Viral, Tumor↗
PubMed2004

L-carnosine reduces telomere damage and shortening rate in cultured normal fibroblasts.

Telomere is the repetitive DNA sequence at the end of chromosomes, which shortens progressively with cell division and limits the replicative potential of normal human somatic cells. L-carnosine, a naturally occurring dipeptide, has been reported to delay the replicative senescence, and extend the lifespan of cultured human diploid fibroblasts. In this work, we studied the effect of carnosine on the telomeric DNA of cultured human fetal lung fibroblast cells. Cells continuously grown in 20 mM carnosine exhibited a slower telomere shortening rate and extended lifespan in population doublings. When kept in a long-term nonproliferating state, they accumulated much less damages in the telomeric DNA when cultured in the presence of carnosine. We suggest that the reduction in telomere shortening rate and damages in telomeric DNA made an important contribution to the life-extension effect of carnosine.

Aging↗
PubMed2005

Roy Walford and the immunologic theory of aging.

Roy Walford died on April 27, 2004, at the age of 79. His contributions to gerontological research in such diverse areas as caloric restriction, genetics of lifespan, immunosenescence, DNA repair and replicative senescence were truly remarkable in their depth and innovation. Significantly, most of the areas that he pioneered during his illustrious research career remain the "hot" areas of current gerontological research. In this sense, he has achieved the most important type of immortality. His death was a major personal and professional loss to numerous scientists within the gerontological community. In launching this new journal on Immunity and Ageing, it is highly fitting, therefore, to remember him on the anniversary of his death by briefly reviewing the contributions of Roy Walford to this important facet of gerontology. Indeed, it was Roy who actually first coined the commonly used term "immunosenescence".

Journal Article↗