Abstract: Accumulation of molecular damage and increased molecular heterogeneity are hallmarks of cellular aging. Mild stress-induced hormesis can be an effective way for reducing the accumulation of molecular damage, and thus slowing down aging from within. We have shown that repeated mild heat stress (RMHS) has anti-aging effects on growth and various other cellular and biochemical characteristics of normal human skin fibroblasts and keratinocytes undergoing aging in vitro. RMHS given to human cells increased the basal levels of various chaperones, reduced the accumulation of damaged proteins, stimulated proteasomal activities, increased the cellular resistance to other stresses, enhanced the levels of various antioxidant enzymes, enhanced the activity and amounts of sodium-potassium pump, and increased the phosphorylation-mediated activities of various stress kinases. We have now observed novel hormetic effects of mild heat stress on improving the wound healing capacity of skin fibroblasts and on enhancing the angiogenic ability of endothelial cells. We have also tested potential hormetins, such as curcumin and rosmarinic acid in bringing about their beneficial effects in human cells by inducing stress response pathways involving heat shock proteins and hemeoxygenase HO-1. These data further support the view that mild stress-induced hormesis can be applied for the modulation, intervention and prevention of aging and age-related impairments.
Abstract: Dicarbonyls glyoxal (GO) and methylglyoxal (MGO) produced during the autoxidation of reducing sugars are a source of macromolecular damage in cells. Since an accumulation of damaged macromolecules is a universal characteristic of aging, we have tested whether GO and MGO which cause oxidative damage to proteins and other macromolecules can bring about accelerated aging in normal human skin fibroblasts in vitro. A treatment of cells with 1.0 mM GO or 400 microM MGO leads to the appearance of senescent phenotype within 3 days, as judged by the following criteria: morphological phenotype, irreversible growth arrest and G2 arrest, increased senescence-associated beta-galactosidase (SABG) activity, increased H2O2 level, increased Nxi-(carboxymethyl)-lysine (CML) protein level, and altered activities of superoxide dismutase and catalase antioxidant enzymes. This experimental model of accelerated cellular aging in vitro can be useful for studies on testing the effects of various physical, chemical and biological conditions, including natural and synthetic molecules, for the modulation of aging.
Abstract: Studies performed on various experimental model systems indicate that genetic interventions can increase longevity, even if in a highly protected laboratory condition. Generally, such interventions required partial or complete switching off of the gene and inhibiting the activity of its gene products, which normally have other well-defined roles in metabolic processes. Overexpression of some genes, such as stress response and antioxidant genes, in some model systems also extends their longevity. Such genetic interventions may not be easily applicable to humans without knowing their effects on human growth, development, maturation, reproduction and other characteristics. Studies on the association of single nucleotide polymorphisms and multiple polymorphisms (haplotype) in genes with human longevity have identified several genes whose frequencies increase or decrease with age. Whether genetic redesigning can be achieved in the wake of numerous and complex epigenetic factors that effectively determine the life course and the life span of an individual still appears to be a 'mission impossible'.
Abstract: Hormesis in aging is represented by mild stress-induced stimulation of protective mechanisms in cells and organisms resulting in biologically beneficial effects. Single or multiple exposure to low doses of otherwise harmful agents, such as irradiation, food limitation, heat stress, hypergravity, reactive oxygen species and other free radicals have a variety of anti-aging and longevity-extending hormetic effects. Detailed molecular mechanisms that bring about the hormetic effects are being increasingly understood, and comprise a cascade of stress response and other pathways of maintenance and repair. Although the extent of immediate hormetic effects after exposure to a particular stress may only be moderate, the chain of events following initial hormesis leads to biologically amplified effects that are much larger, synergistic and pleiotropic. A consequence of hormetic amplification is an increase in the homeodynamic space of a living system in terms of increased defence capacity and reduced load of damaged macromolecules. Hormetic strengthening of the homeodynamic space provides wider margins for metabolic fluctuation, stress tolerance, adaptation and survival. Hormesis thus counter-balances the progressive shrinkage of the homeodynamic space, which is the ultimate cause of aging, diseases and death. Healthy aging may be achieved by hormesis through mild and periodic, but not severe or chronic, physical and mental challenges, and by the use of nutritional hormesis incorporating mild stress-inducing molecules called hormetins. The established scientific foundations of hormesis are ready to pave the way for new and effective approaches in aging research and intervention.
Abstract: Aging at the molecular level is characterized by the progressive accumulation of molecular damage. The sources of damage act randomly through environmental and metabolically generated free radicals, through spontaneous errors in biochemical reactions, and through nutritional components. However, damage to a macromolecule may depend on its structure, localization and interactions with other macromolecules. Damage to the maintenance and repair pathways comprising homeodynamic machinery leads to age-related failure of homeodynamics, increased molecular heterogeneity, altered cellular functioning, reduced stress tolerance, diseases and ultimate death. Novel approaches for testing and developing effective means of intervention, prevention and modulation of aging involve means to minimize the occurrence and accumulation of molecular damage. Mild stress-induced hormesis by physical, biological and nutritional methods, including hormetins, represents a promising strategy for achieving healthy aging and for preventing age-related diseases.
Abstract: Normal human epidermal keratinocytes (NHEK) show both the Hayflick phenomenon of replicative senescence and differentiation in vitro, depending upon the culture conditions. Using this experimental model system, we have studied age-related changes in the ability of serially passaged NHEK to enter into differentiation in the presence of calcium, as measured by the levels of differentiation markers involucrin, p38 and Hsp27. The results obtained in these studies show that calcium-induced differentiation of NHEK becomes progressively delayed during cellular aging in vitro, which can be modulated by treatments such as mild heat stress, kinetin and curcumin. Whereas all these treatments on their own were able to increase the levels of various differentiation markers to varying extents, their effects were synergistic and rapid in the presence of calcium. Furthermore, all three modulators tested in the present study bring about their effects by inducing stress response pathways in terms of an increase in the levels of stress proteins Hsp90, Hsp70 and heme-oxygenase-1 (HO-1), which is indicative of stress-induced hormesis bringing about the biologically beneficial effects.
Abstract: Aging is characterized by stochastic accumulation of molecular damage, progressive failure of maintenance and repair, and consequent onset of age-related diseases. Applying hormesis in aging research and therapy is based on the principle of stimulation of maintenance and repair pathways by repeated exposure to mild stress. Studies on the beneficial biological effects of repeated mild heat shock on human cells in culture, and other studies on the anti-aging and life-prolonging effects of proxidants, hypergravity, irradiation and ethanol on cells and organisms suggest that hormesis as an antiaging and gerontomodulatory approach has a promising future. Its clinical applications include prevention and treatment of diabetes, cataract, osteoporosis, dementia and some cancers.
Abstract: Mild stress-induced hormesis is an effective strategy to intervene in the aging process. Repeated exposure of human skin fibroblasts to 41 degrees C heat shock for 1 h twice a week is an example of mild stress that has many hormetic effects, including improved resistance to other stressors. We are now developing an experimental model system of sugar-induced premature senescence, which can be useful to test the hormetic and antiaging effects of other stresses. Our present studies show that early-passage human skin fibroblasts treated with 1 mM glyoxal for 72 h undergo premature senescence in terms of enlarged cell size, inhibition of cell division, slowing down of cell growth, a decrease in the number of DNA synthesizing cells, and increased resistance to apoptosis.
Abstract: Progressive accumulation of molecular damage is a hallmark of cellular aging, which is amenable to intervention and prevention by hormesis through mild stress. Our studies have shown that repeated mild heat stress (RMHS) has antiaging effects on growth and various other cellular and biochemical characteristics of normal human skin fibroblasts undergoing aging in vitro. RMHS at 41 degrees C, for 1 h twice a week, increased the basal levels of various chaperones, reduced the accumulation of oxidatively and glycoxidatively damaged proteins, stimulated proteasomal activities for the degradation of abnormal proteins, improved cellular resistance to ethanol, hydrogen peroxide, and UV-B rays, enhanced the levels of various antioxidant enzymes, and increased the phosphorylation-mediated activities of various stress kinases. RMHS-exposed human fibroblasts are also better protected against glucose- and glyoxal-induced growth inhibition and apoptosis. We have also observed various hormetic effects of RMHS on normal human epidermal keratinocytes, which include increased replicative life span, increased proteasomal activity, and enhanced levels of Na/K-ATPase pump. We are also testing the above effects of RMHS in combination with potential hormetic molecules, such as curcumin, on aging, longevity, and differentiation of human cells in culture.
Abstract: Many biological subdisciplines that regularly assess dose-response relationships have identified an evolutionarily conserved process in which a low dose of a stressful stimulus activates an adaptive response that increases the resistance of the cell or organism to a moderate to severe level of stress. Due to a lack of frequent interaction among scientists in these many areas, there has emerged a broad range of terms that describe such dose-response relationships. This situation has become problematic because the different terms describe a family of similar biological responses (e.g., adaptive response, preconditioning, hormesis), adversely affecting interdisciplinary communication, and possibly even obscuring generalizable features and central biological concepts. With support from scientists in a broad range of disciplines, this article offers a set of recommendations we believe can achieve greater conceptual harmony in dose-response terminology, as well as better understanding and communication across the broad spectrum of biological disciplines.
Abstract: Normal human epidermal keratinocytes (NHEK) show both the Hayflick phenomenon and differentiation in vitro. The aim of this study was to induce senescence in keratinocytes using two sugars, glucose and glyoxal. Induction of senescence in early-passage NHEK was characterized by monitoring cell morphology, short-term growth characteristics, cell proliferation, and viability assay. In addition, apoptosis, senescence-associated (SA) beta-gal activity, proteasomal activity and glycation, and glycoxidation of total proteins were determined. Our results show that a 3-day treatment with 100 mM glucose or 0.1 mM glyoxal induces in early-passage NHEK various cellular and biochemical characteristics comparable to those observed in serially subcultured late passage NHEK. Furthermore, sugar-treated prematurely aged NHEK showed impaired differentiation, as measured by the quantification of involucrin. There is preliminary evidence that a preexposure of NHEK to mild heat shock (41 degrees C, 1 h, 6 h in advance) can abrogate some of the sugar-induced negative effects, which is an example of mild stress-induced hormesis. This experimental model can be useful to study the effects of potential antiaging interventions.
Abstract: 5-Methylcytosine (m(5)C) has a plethora of functions and roles in various biological processes including human diseases and aging. A TLC-based fast and simple method for quantitative determination of total genomic levels of m(5)C in DNA is described, which can be applicable to aging research with respect to rapid and high throughput screening and comparison. Using this method, an example of the analysis of global alternations of m(5)C in serially passaged human skin fibroblasts is provided, which shows age-related global hypomethylation during cellular aging in vitro. This method can be useful for screening potential modulators of aging at the level of epigenetic alterations.
Abstract: Human longevity is determined to a certain extent by genetic factors. Several candidate genes have been studied for their association with human longevity, but the data collected so far are inconclusive. One of the reasons is the choice of the candidate genes in addition to the choice of an appropriate study design and methodology. Since aging is characterized by a progressive accumulation of molecular damage and an attenuation of the cellular defense mechanisms, the focus of studies on human longevity association with genes has now shifted to the pathways of cellular maintenance and repair mechanisms. One such pathway includes the battery of stress response genes, especially the heat shock protein HSP70 genes. Three such genes, HSPA1A, HSPA1B and HSPA1L, are present within the MHC-III region on the short arm of chromosome 6. We and others have found alleles, genotypes and haplotypes which have been significantly associated with human longevity and survival. We have also provided some functional evidence for these genetic associations by showing that isolated peripheral blood cells from those genotypes which are negatively associated with human longevity also have less ability to respond to heat shock. Stress response genes, particularly HSP70, are now the major candidates in the gene-longevity association studies.
Abstract: Aging is amenable to intervention and prevention by mild stress-induced hormesis. Previously, we have reported that repeated mild heat stress has antiaging and other beneficial effects on growth and a range of cellular and biochemical characteristics of normal human skin fibroblasts and keratinocytes undergoing aging in vitro. We have also established a model system of sugar-induced premature senescence in human cells, which can be useful for monitoring the protective and hormetic effects of other treatments. We have now initiated studies on testing the hormetic effects of glyoxal and heat shock on the wound-healing capacity of skin fibroblasts and on the angiogenic ability of endothelial cells. The effects of glyoxal on the extent of wound closure in vitro showed a typical biphasic hormetic curve with 20-40% stimulation at lower doses (up to 0.125 mmol) and more than 50% inhibition at concentrations above 0.5 mmol. In the case of angiogenesis by endothelial cells, measured by the standard tube formation assay on Matrigel, a prior exposure to mild heat shock at 41 degrees C for 1 h increased the total tube length and total number of junctions by 30-60% and 10-14%, respectively. In contrast, a severe heat shock at 42.5 degrees C had slightly inhibitory effects on total tube length and the number of junctions. These data add to the ever-growing body of evidence in support of the view that mild stress-induced hormesis can be a useful approach for the modulation, intervention, and prevention of aging and age-related impairments.
Abstract: Healthy aging and longevity depend on successful and dynamic interactions among biological, psychological, and environmental factors. Biological aging occurs mainly during the period of survival beyond the evolutionarily required essential lifespan (ELS). Natural selection processes for survival and successful reproduction have selected for a range of genetically determined ELS-assuring maintenance and repair systems (MRSs). The progressive failure of MRSs, and the consequent accumulation of molecular heterogeneity and damage, underlie the biological basis of aging, age-related diseases, and eventual death. However, the genetic processes of MRSs operate in a complex hierarchy of factors which range from intracellular molecular factors to physiological, psychological, environmental, and other stochastic factors, including chance. This view also facilitates setting up a framework for understanding, researching, and developing effective and realistic strategies for aging intervention, prevention, and therapies. Manipulating genes and the milieu in which genes and gene products operate opens up novel possibilities of aging intervention and prevention. Gene therapy, stem cells, and modulation through functional foods, nutriceuticals, cosmeceuticals and lifestyle alterations, including mild stress-induced hormesis, are examples of such strategies at various levels of development and practice.
Abstract: Adult human skin fibroblasts were exposed repeatedly to 41 degrees C or 42 degrees C heat shock (HS) for 1 h twice a week during serial passaging throughout their replicative life span. On the basis of longevity curves, cell size, and morphology, we observed that repeated mild heat shock (RMHS) at 41, degrees C had strong anti-aging hormetic effects, including 20% extension of cellular longevity. The basal levels of the MAP kinases JNK1, JNK2, and p38 increased during serial passaging, while that of ERK2 decreased. RMHS further exaggerated these effects, which suggests that age-related changes in MAP kinases may be an adaptive response for better cell survival.
Abstract: Curcumin (diferuloylmethane), is a component of the yellow powder prepared from the roots of Curcuma longa (Zingiberaceae), also known as tumeric or turmeric. It is widely cultivated and used as a food ingredient in tropical areas of Asia and Central America. Treatment of mid-passage human epidermal keratinocytes with curcumin resulted in a biphasic hormetic dose-response with respect to proteasome activity. Curcumin treatment (up to 1 microM for 24 h) increased chymotrypsin-like activity by 46% compared to that in untreated keratinocytes. However, higher concentrations of curcumin were inhibitory, and at 10 microM the proteasome activity decreased to 46% of its initial value. Furthermore, the preincubation of human keratinocytes at 43 degrees C for 1 h, followed by 24-h treatment with 3 microM curcumin, led to an increase in heat-shock protein (hsp70 and hsp90) levels by 24% and 19%, respectively, and the effect was sustained at concentrations up to 10 microM. On the other hand, the level of the small hsp27 was unaffected by curcumin concentrations of 0.3-1 microM, while it decreased by 34% at 10 microM.
Abstract: Kinetin (N(6)-furfuryladenine) is a cytokinin growth factor having several anti-aging effects reported for human cells and fruit flies. We have observed that short-term culturing of human keratinocytes in the presence of 40 to 200 microM kinetin results in a significant inhibition of cell growth. Studies were undertaken to analyze the process of differentiation as a reason for growth inhibition. Keratinocytes at different passage levels were treated with fetal calf serum (FCS) and calcium as differentiation-inducing positive controls, with different concentrations of kinetin, and with a combination of kinetin and calcium. The induction and progression of differentiation was monitored by morphological observations and by using several differentiation markers, including keratins (K10 and K14), involucrin, epidermal transglutaminase, and some new keratinocyte-specific antibodies isolated by the phage display method. In young keratinocytes, two days of calcium treatment reduced the K14 level by 78%, and increased the levels of K10 and involucrin by 40% and 29%, respectively. In comparison, 40 microM kinetin had no effect on the K14 level, but increased the K10 level by 28% and that of involucrin by four-fold. The combination of calcium and 40 microM kinetin led to a decrease by 23% in the K14 level, to an increase in the level of K10 by 55%, and to a two-fold rise in the involucrin level. These results suggest that the rate, extent, and quality of differentiation depend on the inducing agent, and that kinetin may be useful in promoting the differentiation of human keratinocytes, especially in the presence of calcium.
Abstract: This article introduces a special issue of Biogerontology, for which a number of international experts who are still active or have been active in research on the effects of dietary restriction (DR) were asked to answer the following question. Do you think that DR can increase longevity in all species, particularly in human beings? Twelve scientists responded with their opinion articles of which roughly half of them taking the position that yes, DR can be applicable to human beings, while the other half arguing for the inapplicability of DR to humans. The conclusions of these learned opinions are summarized here.
Abstract: Strategies that lead to the upregulation of the proteasome are known to elicit beneficial consequences to the organism by countering oxidative stress-associated disorders, such as protein conformational diseases, cancer, and aging. Mild treatment with proteasome inhibitors has been previously demonstrated to stimulate proteasome activity and cellular resistance against oxidative injury. However, the mechanism for this action has not been clearly defined. We examined the role of the nuclear factor-E2-related factor 2 (Nrf2) in fibroblasts, a key transactivator of the antioxidant response pathway, in the regulation of the proteasome by its inhibitor MG-132. Here, we demonstrate that the stimulation of the proteasome by low levels of MG-132 can be abrogated by small interfering RNAs (siRNAs) targeted against Nrf2. Consistently, cells that constitutively express Nrf2 exhibit elevated levels of proteasome activities. We further investigate how its beneficial effects, that is, proteasome stimulation, are manifested in young and replicative-senescent cells. Our data underscore that manipulation of Nrf2 by the administration of pharmacologically low levels of proteasome inhibitors may prove to be an alternatively potent strategy for inducing long-term protective effects against oxidative stress.
Abstract: We have studied the association of three single nucleotide polymorphisms (SNPs) present in the three HSP70 (heat-shock protein) genes on 6p21 with human longevity. The availability of biological samples from various population cohorts in Denmark has given us the opportunity to try novel methods of gene association with human longevity. A significant association of one haplotype with male longevity was observed. Furthermore, a significant difference in the survival of the carriers of the different genotypes in females was observed. We also found an age-dependant decline in the ability of peripheral blood mononuclear cells to respond to heat stress in terms of Hsp70 induction.
Abstract: Augmentation of proteasome machinery is emerging as a significant gerontomodulatory consequence of hormetic stimulation, such as mild heat stress. This study describes the phenomenon we term hormetic proteasomal oscillation, wherein mildly heat-stressed human fibroblasts (41 degrees C, 1 h) display an adaptation response pattern in proteasome activity. Remarkably, such response appears to be diverse in severely heat-stressed or senescent fibroblasts. This proteasomal oscillation, as an innate cellular reaction to heat and aging, however, is independent of 20S proteasome protein levels and nuclear factor-E2-related factor 2 (Nrf2) transactivation.
Abstract: Heat shock (HS)-induced stress response in human cells results in a variety of biological effects and is known to induce the transcription of heat-shock proteins, which help the cells to cope with different kinds of stress. We have studied the effects of HS on the differentiation of human mesenchymal stem cells (hMSCs) into osteoblastic cells. As a model for hMSCs we used a telomerase-immortalized hMSC line designated hMSC-TERT. Cells were exposed to 1 h HS at 41 degrees C, 42.5 degrees C, or 44 degrees C prior to incubation in a medium containing either 10(-8) M 1alpha,25-dihydroxy-vitamin-D(3) (calcitriol) or 10(-8) M calcitriol, 50 microg/mL L-ascorbic acid, and 10 mM beta-glycerophosphate followed by an analysis of induction of osteoblast differentiation and the formation of mineralized matrix, respectively. Our results indicate that the exposure of cells to mild heat stress enhances the extent of differentiation of hMSCs by 12% to 42%. These effects are an expression of the phenomenon of mild stress-induced hormesis.
Abstract: Traditional categorization of theories of aging into programmed and stochastic ones is outdated and obsolete. Biological aging is considered to occur mainly during the period of survival beyond the natural or essential lifespan (ELS) in Darwinian terms. Organisms survive to achieve ELS by virtue of genetically determined longevity assuring maintenance and repair systems (MRS). Aging at the molecular level is characterized by the progressive accumulation of molecular damage caused by environmental and metabolically generated free radicals, by spontaneous errors in biochemical reactions, and by nutritional components. Damages in the MRS and other pathways lead to age-related failure of MRS, molecular heterogeneity, cellular dysfunctioning, reduced stress tolerance, diseases and ultimate death. A unified theory of biological aging in terms of failure of homeodynamics comprising of MRS, and involving genes, milieu and chance, is acquiring a definitive shape and wider acceptance. Such a theory also establishes the basis for testing and developing effective means of intervention, prevention and modulation of aging.
Abstract: Age-dependent changes in heat shock response (HSR) were studied in mononuclear cells (monocytes and lymphocytes) collected from young (mean age = 22.6 +/- 1.7 years) and middle-aged (mean age = 56.3 +/- 4.7 years) subjects after 1 hour of heat shock at 42 degrees C. Genotype-specific HSR was measured by genotyping the subjects for 3 single nucleotide polymorphisms, HSPA1A(A-110C), HSPA1B(A1267G), and HSPA1L(T2437C), 1 each in the 3 HSP70 genes. A significant age-related decrease in the induction of Hsp70 occurred after heat shock in both monocytes and lymphocytes. The noninducible and inducible forms of Hsp70 decreased 1.3-fold (P < 0.001) and 1.4-fold (P < 0.001), respectively, in the monocytes with age. In the young subjects, a positive association was found between HSPA1L(T2437C) polymorphism and HSR. CC carriers had a significantly lower induction than TT carriers in both monocytes (P = 0.015) and lymphocytes (P = 0.044). This polymorphism, which is present in the coding region of HSPA1L gene, can affect the chaperoning function of Hsp70. These data consolidate our other observations that the CC genotype is unfavorable for human longevity and provide a functional explanation in terms of variations in HSR.
Abstract: Accumulation of posttranslationally damaged proteins during aging could explain the decline of cell performance with age. N(epsilon)-carboxymethyllysine (CML) is the major glycation product on damaged proteins, causing dysfunction and cross-linking. The proteasome, a multicatalytic degradation complex, is one of the pathways for eliminating damaged proteins, and thus regulating their accumulation within the cell. However, the proteinase activities of the proteasome decline during aging. This may be due to posttranslational modifications of the subunits forming the proteasome complex. Using phage display technology, we have selected 16 single-chain variable fragments (scFv) recognizing the CML-modified alpha7 subunit of the proteasome. Using one of them, Ab3, we have observed a five-fold increase of CML-alpha7 in old human skin fibroblasts in comparison with young fibroblasts and telomerase-immortalized bone marrow cells (hTERT-BMCs).
Abstract: Chaperones, particularly the heat-shock proteins, are considered as key players in the maintenance of protein homeostasis and are associated with longevity and cellular immortalization. In this study, we investigated the geroprotective activity of the chemical chaperone glycerol. Glycerol showed significant chaperoning activity in refolding heat-denatured luciferase in vivo and in protecting cells from heat stress-induced cytotoxicity. This was accompanied by decrease in p53, an upregulation of a stress chaperone mortalin/mtHsp70, and an increase in proteasome activity in the presence of oxidative stress.
Abstract: Our studies have shown that zeatin, (6-[4-hydroxy-3-methyl-but-2-enylamino]adenine), a cytokinin plant growth factor, has gerontomodulatory, youth preserving and anti-aging effects on serially passaged human adult skin fibroblasts undergoing aging in vitro. There were no immediate negative or toxic effects in terms of cell attachment, cell proliferation, cell survival, cytoskeletal organization, and cellular growth by treatment with zeatin concentrations between 1 and 200 microM. During long-term treatment, cells could be maintained throughout their replicative lifespan in the presence of 40, 80, and 200 microM zeatin, but the optimal concentration of zeatin's anti-aging and youth preserving effects was found to be 80 microM. Life-long serial passaging of human skin fibroblasts in the presence of zeatin resulted in the prevention of cell enlargement, reduction of intracellular debris, prevention of actin polymerization, and enhancement of cellular ability to decompose hydrogen peroxide and to cope with ethanol and oxidative stresses. Most importantly, anti-aging and beneficial effects of zeatin were observed without any induction of additional cell proliferation or an increase in the maximum proliferative capacity, thus ruling out any potentially harmful and carcinogenic effects.
Abstract: Ageing is characterized by a progressive accumulation of molecular damage in nucleic acids, proteins and lipids. The inefficiency and failure of maintenance, repair and turnover pathways is the main cause of age-related accumulation of damage. Research in molecular gerontology is aimed at understanding the genetic and epigenetic regulation of survival and maintenance mechanisms at the levels of transcription, post-transcriptional processing, post-translational modifications, and interactions among various gene products. Concurrently, several approaches are being tried and tested to modulate ageing in a wide variety of organisms. The ultimate aim of such studies is to improve the quality of human life in old age and prolong the health-span. Various gerontomodulatory approaches include gene therapy, hormonal supplementation, nutritional modulation and intervention by free radical scavengers and other molecules. A recent approach is that of applying hormesis in ageing research and therapy, which is based on the principle of stimulation of maintenance and repair pathways by repeated exposure to mild stress. A combination of molecular, physiological and psychological modulatory approaches can realize "healthy ageing" as an achievable goal in the not-so-distant future.
Abstract: Foundational changes in science are rare, but in the field of biogerontology there is a new theory of aging that may shake things up. The conventional wisdom about duration of life is based on an old idea known as the "rate of living" theory, which suggests that aging is caused by the loss of some vital substance. The modern version of this theory is that duration of life is influenced by the relative speed of a species' resting metabolism. However, empirical evidence does not consistently support this hypothesis. In an article published recently by mathematician/biologist Lloyd Demetrius, it is suggested that the most important factor involved in duration of life is not metabolic rate or oxidative stress, but metabolic stability. If Demetrius is correct, his theory will have important implications for intervention research. For example, if the metabolic rate/oxidative stress theory is correct, efforts to intervene in the aging process should be directed at finding ways to reduce metabolic rate, lessen the production of reactive oxygen species (ROS), improve antioxidant defenses, or increase the quantity of antioxidants. If the metabolic stability hypothesis is correct, efforts to intervene in the aging process should be directed at finding ways to increase the stability of the steady state values of ROS, increase the robustness of metabolic networks, or improve the stability of antioxidant enzymes. For now there is reason to believe that Demetrius' theory deserves further consideration - whether it meets the test of a paradigm shift has yet to be determined.
Abstract: Aging is characterized by a stochastic accumulation of molecular damage, progressive failure of maintenance and repair, and consequent onset of age-related diseases. Applying hormesis in aging research and therapy is based on the principle of stimulation of maintenance and repair pathways by repeated exposure to mild stress. In a series of experimental studies we have shown that repetitive mild heat stress has anti-aging hormetic effects on growth and various other cellular and biochemical characteristics of human skin fibroblasts undergoing aging in vitro. These effects include the maintenance of stress protein profiles, reduction in the accumulation of oxidatively and glycoxidatively damaged proteins, stimulation of the proteasomal activities for the degradation of abnormal proteins, improved cellular resistance to ethanol, hydrogenperoxide and ultraviolet-B rays, and enhanced levels of various antioxidant enzymes. Anti-aging hormetic effects of mild heat shock appear to be facilitated by reducing protein damage and protein aggregation by activating internal antioxidant, repair and degradation processes.
Abstract: We have studied the possible association between the -110A > C polymorphism in the promoter region of one of the heat shock protein genes HSP70-1 with human longevity in a cohort of aged Danish twins. This cohort includes individuals aged between 70 and 91 years (mean = 75.6 years), who are categorized according to the presence or absence of various diseases and according to the various, age-related parameters for which a genetic component has already been defined. Four hundred DNA samples from the cohort were genotyped using real-time PCR. Aging phenotypes (diseases, physical and cognitive functioning) were compared with regard to genotype. Of all the aging phenotypes studied, self-rated health and relative self-rated health, which represent an individual's overall sense of physical well-being and which have been shown to be both predictors of survival at older ages and better indicators of future survival than objectively measured health status, were associated with the polymorphism. An association was found between low self-rated health and heterozygosity for -110A > C polymorphism in the promoter region of HSP70-1 in aged Danish twins.
Abstract: In a series of experimental studies we have shown that repetitive mild heat stress has anti-aging hormetic effects on growth and various other cellular and biochemical characteristics of human skin fibroblasts undergoing aging in vitro. We have reported the hormetic effects of repeated challenge at the levels of maintenance of stress protein profile; reduction in the accumulation of oxidatively and glycoxidatively damaged proteins; stimulation of the proteasomal activities for the degradation of abnormal proteins; improved cellular resistance to ethanol, hydrogenperoxide, and ultraviolet-B rays; and enhanced levels of various antioxidant enzymes. We are now undertaking a detailed analysis of the signal transduction pathways to determine alterations in the phosphorylation and dephosphorylation states of extracellular signal-related kinase, c-Jun terminal kinase and p38 MAP-kinases as a measure of cellular responsiveness to mild and severe heat stress. Furthermore, we are also undertaking comparative studies using non-aging immortal cell lines, such as SV40-transformed human fibroblasts, spontaneous osteosarcoma cells, and telomerase-immortalized human bone marrow cells for establishing differences in normal and cancerous cells with respect to their responsiveness to mild and severe stresses.
Abstract: As a result of almost 50 years of efforts in collecting descriptive data, biogerontologists are now able to construct general principles of aging and to explore possibilities of gerontomodulation. Most of the data indicate that aging is characterized by a stochastic accumulation of molecular damage and a progressive failure of maintenance and repair, and the genes involved in homeodynamic pathways are the most likely candidate virtual gerontogenes. Several approaches are being tried and tested to modulate aging in a wide variety of organisms, but with the ultimate aim of improving the quality of human life in old age. These approaches include gene therapy, hormonal supplementation, nutritional modulation, and intervention by antioxidants and other molecules. A recent approach is that of applying hormesis in aging research and therapy, which is based on the principle of stimulation of maintenance and repair pathways by repeated exposure to mild stress.
Abstract: Since aging is primarily the result of a failure of maintenance and repair mechanisms, various approaches are being developed in order to stimulate these pathways and modulate the process of aging. One such approach, termed hormesis, involves challenging cells and organisms by mild stress that often results in anti-aging and life prolonging effects. In a series of experimental studies, we have reported that repeated mild heat stress (RMHS) has anti-aging hormetic effects on growth and various cellular and biochemical characteristics of human skin fibroblasts undergoing aging in vitro. These beneficial effects of repeated challenge include the maintenance of stress protein profile, reduction in the accumulation of oxidatively and glycoxidatively damaged proteins, stimulation of the proteasomal activities for the degradation of abnormal proteins, improved cellular resistance to other stresses, and enhanced levels of cellular antioxidant ability. In order to elucidate the molecular mechanisms of hormetic effects of RMHS, we are now undertaking studies on signal transduction pathways, energy production and utilisation kinetics, and the proteomic analysis of patterns of proteins synthesised and their posttranslational modifications in various types of human cells undergoing cellular aging in vitro. Human applications of hormesis include early intervention and modulation of the aging process to prevent or delay the onset of age-related conditions, such as sarcopenia, Alzheimer's disease, Parkinson's disease, cataracts and osteoporosis.
Abstract: In a series of experimental studies, it was shown that repetitive mild heat stress has antiaging hormetic effects on growth and various other cellular and biochemical characteristics of human skin fibroblasts undergoing aging in vitro. We have reported the hormetic effects of repeated challenge at the levels of maintenance of stress protein profile; reduction in the accumulation of oxidatively and glycoxidatively damaged proteins; stimulation of the proteasomal activities for the degradation of abnormal proteins; improved cellular resistance to ethanol, hydrogen peroxide, and ultraviolet-B rays; and enhanced levels of various antioxidant enzymes. Detailed analysis of the signal transduction pathways to determine alterations in the phosphorylation and dephosphorylation states of ERK, JNK, and p38 MAP kinases as a measure of cellular responsiveness to mild and severe heat stress is in progress. Furthermore, comparative studies using nonaging immortal cell lines, such as SV40-transformed human fibroblasts, spontaneous osteosarcoma cells, and telomerase-immortalized human bone marrow cells are also in progress for establishing differences in normal and cancerous cells for their responsiveness to mild and severe stresses.
Abstract: The phenomenon of hormesis is represented by mild stress-induced stimulation of maintenance and repair pathways resulting in beneficial effects for the cells and organisms. Anti-aging and life-prolonging effects of a wide variety of the so-called stressors, such as pro-oxidants, aldehydes, calorie restriction, irradiation, heat shock, and hypergravity, have been reported. Molecular mechanisms of hormesis due to different stresses are yet to be elucidated, but there are indications that relatively small individual hormetic effects become biologically amplified resulting in the collective significant improvement of cellular and organismic functions and survival. Accepting that some important issues with respect to establishing the optimal hormetic conditions still need to be resolved by future research, hormesis appears to be a promising and effective approach for modulating aging, for preventing or delaying the onset of age-related diseases, and for improving quality of life in old age.
Abstract: Repeated mild heat shock (RMHS) has been shown to have several beneficial hormetic effects on human skin fibroblast undergoing aging in vitro. Because an age-related decline in proteasome activity is 1 of the reasons for the accumulation of abnormal proteins during aging, we have investigated the effects of RMHS on the 20S proteasome, which is the major proteolytic system involved in the removal of abnormal and oxidatively damaged proteins. Serially passaged human skin fibroblasts exposed to RMHS at 41 degrees C for 60 minutes twice a week had increased 3 proteasomal activities by 40% to 95% in early- and midpassage cultures. RMHS-treated cells also contained a 2-fold higher amount of the proteasome activator 11S, and the extent of the bound activator was double in early- and midpassage cells only. Furthermore, there was no difference in the content of the 19S proteasome regulator in the stressed and the unstressed cells. Therefore, RMHS-induced proteasome stimulation in early- and midpassage fibroblasts appears to be due to an induction and enhanced binding of 11S proteasome activators. In contrast to this, the proteasomal system in late-passage senescent cells appears to be less responsive to the stimulatory effects of mild heat shock.
Abstract: The phenomenon of hormesis is represented by mild stress-induced stimulation of maintenance and repair pathways, resulting in beneficial effects for cells and organisms. We have reported that repeated mild heat stress (RMHS) has anti-aging hormetic effects on growth and various cellular and biochemical characteristics of human skin fibroblasts undergoing aging in vitro. These effects of RMHS include the maintenance of the stress protein profile, reduction in the accumulation of oxidatively and glycoxidatively damaged proteins, stimulation of the activities of the proteasome and its 11S activator, improvement in cellular resistance to ethanol, hydrogen peroxide, and ultraviolet rays, and increased antioxidative activity of the cells. We have also reported that RMHS prolongs the lifespan of Drosophila. Others have reported anti-aging and life prolonging effects of a wide variety of so-called stressors, such as pro-oxidants, aldehydes, calorie restriction, irradiation, heat shock, and hypergravity. Although molecular mechanisms of hormesis are yet to be elucidated, there are indications that relatively small hormetic effects become biologically amplified, resulting in significant improvement of cellular and organic functions and survival. Hormesis, therefore, can be an effective approach for modulating aging, for preventing or delaying the onset of age-related diseases, and for improving the quality of life in old age.
Abstract: We assessed the impact of repeated episodes of a mild heat stress on lifespan, fecundity, heat stress resistance and Hsp70 expression in Drosophila melanogaster. There was a significant increase in lifespan of females repeatedly exposed to a mild heat stress when measured in both a pair and a group situation. There was no effect on fecundity when the flies were first exposed to the mild heat stress at an age later than 3 days old, but when it did occur on day 3, there was a significant effect on cumulative fecundity levels over 18 days. The negative fitness effect appears to be the result of a direct cessation or reduction of oviposition during the first bout of stress exposure, and is influenced by the age at which this first exposure occurs. The mild heat stress had no impact on egg viability. The mild heat stress exposures increased resistance to potentially lethal heat stress and levels of Hsp70 expression in heat-exposed flies were higher than those in controls.
Abstract: Repeated mild heat shock (RMHS) has anti-aging effects on growth and various other cellular and biochemical characteristics of human skin fibroblasts undergoing aging in vitro. In this study, we have tested whether RMHS can reduce the accumulation of heavily damaged proteins, such as oxidized and glycoxidized proteins involved in the development of many pathological consequences of aging. Cultured human skin fibroblasts were subjected to RMHS and were subsequently incubated either with glyoxal (0.1-1 mM) generating Nepsilon-carboxymethyl-lysine (CML), or with tert-butyl-hydroperoxide (t-BHP 10-700 microM) producing oxidized proteins. About 50% more carbonylated-proteins were produced in control cells treated with t-BHP than in cells previously exposed to RMHS. More dramatically, a treatment with 0.1 mM glyoxal for 48 h generated CML only in control cells. Such modulation of the level of damaged proteins is most likely related to the beneficial effects of hormesis resulting from exposure to mild stress.
Abstract: Repeated mild heat-shock (RMHS) treatment has anti-aging hormetic effects on human fibroblasts undergoing aging in vitro. Since heat and various other stresses induce the transcription and translation of heat-shock proteins (Hsp), it was investigated if RMHS treatment affected the basal levels of four major stress proteins Hsp27, 70, 90 and Hsc70. The basal levels of Hsp27, Hsc70, and Hsp70 increased significantly in late passage senescent cells, which is indicative of an adaptive response to cumulative intracellular stress during aging. RMHS increased the levels of these Hsp even in early passage young cells and were maintained high throughout their replicative lifespan. In comparison, the amount of Hsp90 decreased both with aging and RMHS treatment in vitro. However, whereas the difference in the levels of Hsp70 and Hsp90 was statistically significant, the levels of Hsp27 and Hsc70 were statistically similar in normal and RMHS-treated serially passaged cells. These alterations were accompanied by an improved functional and survival ability of the cells in terms of increased proteasomal activities, increased ability to decompose H(2)O(2), reduced accumulation of lipofuscin and enhanced resistance to ethanol, H(2)O(2) and UV-A radiation.
Abstract: Human bone marrow stromal cells (hMSCs) were stably transduced by a retroviral vector containing the gene for the catalytic subunit of human telomerase (hTERT). Transduced cells (hMSC-TERTs) had telomerase activity, and the mean telomere length was increased as compared with that of control cells. The transduced cells have now undergone more than 260 population doublings (PD) and continue to proliferate, whereas control cells underwent senescence-associated proliferation arrest after 26 PD. The cells maintained production of osteoblastic markers and differentiation potential during continuous subculturing, did not form tumors, and had a normal karyotype. When implanted subcutaneously in immunodeficient mice, the transduced cells formed more bone than did normal cells. These results suggest that ectopic expression of telomerase in hMSCs prevents senescence-associated impairment of osteoblast functions.
Abstract: Decreased bone formation is an important pathophysiological mechanism responsible for bone loss associated with aging and osteoporosis. Osteoblasts (OBs), originate from mesenchymal stem cells (MSCs) that are present in the bone marrow and form colonies (termed colony-forming units-fibroblastic [CFU-Fs]) when cultured in vitro. To examine the effect of aging and osteoporosis on the MSC population, we quantified the number of MSCs and their proliferative capacity in vitro. Fifty-one individuals were studied: 38 normal volunteers (23 young individuals [age, 22-44 years] and 15 old individuals [age, 66-74 years]) and 13 patients with osteoporosis (age, 58-83 years). Bone marrow was aspirated from iliac crest; mononuclear cells were enriched in MSCs by magnetic activated cell sorting (MACS) using STRO-1 antibody. Total CFU-F number, size distribution, cell density per CFU-F, number of alkaline phosphatase positive (ALP+) CFU-Fs, and the total ALP+ cells were determined. In addition, matrix mineralization as estimated by alizarin red S (AR-S) staining was quantified. No significant difference in colony-forming efficiency between young individuals (mean +/- SEM; 87 +/- 12 CFU-Fs/culture), old individuals (99 +/- 19 CFU-Fs/culture), and patients with osteoporosis (129 +/- 13 CFU-Fs/culture; p = 0.20) was found. Average CFU-F size and cell density per colony were similar in the three groups. Neither the percentage of ALP+ CFU-Fs (66 +/- 6%, 65 +/- 7%, and 72 +/- 4% for young individuals, old individuals, and patients with osteoporosis, respectively) nor the percentage of ALP+ cells per culture (34 +/- 5%, 40 +/- 6%, and 41 +/- 4%) differed between groups. Finally, mineralized matrix formation was similar in young individuals, old individuals, and patients with osteoporosis. Our study shows that the number and proliferative capacity of osteoprogenitor cells are maintained during aging and in patients with osteoporosis and that other mechanisms must be responsible for the defective osteoblast (OB) functions observed in these conditions.
Abstract: Adequate responses to various hormones, such as 1,25-dihydroxyvitamin D(3) (calcitriol) are a prerequisite for optimal osteoblast functions. We have previously characterized several human diploid osteoblastic cell lines that exhibit typical in vitro aging characteristics during long-term subculturing. In order to study in vitro age-related changes in osteoblast functions, we compared constitutive mRNA levels of osteoblast-specific genes in early-passage (< 50% lifespan completed) with those of late-passage cells (> 90% lifespan completed). We found a significant reduction in mRNA levels of alkaline phosphatase (AP: 68%), osteocalcin (OC: 67%), and collagen type I (ColI: 76%) in in vitro senescent late-passage cells compared to early-passage cells, suggesting an in vitro age-related impairment of osteoblast functions. We hypothesized that decreased osteoblast functions with in vitro aging is due to impaired responsiveness to calcitriol known to be important for the regulation of biological activities of the osteoblasts. Thus, we examined changes in vitamin D receptor (VDR) system and the osteoblastic responses to calcitriol treatment during in vitro osteoblast aging. We found no change in the amount of VDR at either steady state mRNA level or protein level with increasing in vitro osteoblast age and examination of VDR localization, nuclear translocation and DNA binding activity revealed no in vitro age-related changes. Furthermore, calcitriol (10(-8)M) treatment of early-passage osteoblastic cells inhibited their proliferation by 57 +/- 1% and stimulated steady state mRNA levels of AP (1.7 +/- 0.1-fold) and OC (1.8 +/- 0.2-fold). Similarly, calcitriol treatment increased mRNA levels of AP (1.7 +/- 0.2-fold) and OC (3.0 +/- 0.3-fold) in late-passage osteoblastic cells. Thus, in vitro senescent osteoblastic cells maintain their responsiveness to calcitriol and some of the observed in vitro age-related decreases in biological markers of osteoblast functions can be reverted by calcitriol treatment.
Abstract: Repeated mild heat shock (RMHS) has beneficial hormesis-like effects on various characteristics of human skin fibroblasts undergoing replicative senescence in vitro. We have tested whether RMHS could reduce the accumulation of oxidized and glycoxidized proteins, which is a major age-related change. Levels of carbonylated proteins, furosine, N(epsilon)-carboxymethyl-lysine-rich proteins and advanced glycation end products increased during serial passaging of fibroblasts in culture. However, the extent of accumulation of oxidized and glycoxidized proteins was significantly reduced in RMHS cells. The basal concentration of reduced glutathione was higher and that of oxidized glutathione was lower in RMHS cells. Whereas the basal level of heat shock protein HSP27 decreased in both RMHS and control cells during serial passaging, the increase of the basal level of HSP70 with increasing passage level was significantly higher in RMHS cells. These results show that the slower accumulation of damaged proteins in fibroblasts exposed to RMHS results partly from the increased ability of these cells to cope with oxidative stress, and to synthesize HSP responsible for protein capping and refolding.
Abstract: Biology of aging is well understood at a descriptive level. Based on these data, biogerontological research is now able to develop various possibilities for intervention. A promising approach for the identification of gerontogenes and gerontogenic processes is through the hormetic effects of mild stress on slowing down aging. Although there are several issues remaining to be resolved, specially with regard to the notion of mild stress, application of hormesis in aging research and therapy is a powerful new approach.
Abstract: Ageing is associated with a decrease in the ability of cells to cope with environmental challenges. This is due partly to the attenuation of a primordial stress response, the so-called heat shock (HS) response, which induces the expression of heat shock proteins (HSPs), composed of chaperones and proteases. The attenuation of the HS response during ageing may be responsible for the accumulation of damaged proteins as well as abnormal regulation of cell death. Maintenance of the HS response by repeated mild heat stress causes anti-ageing hormetic effects on cells and organisms. Here, we describe the molecular mechanism and the state of the HS response as well as the role of specific HSPs during ageing, and discuss the possibility of hormetic modulation of ageing and longevity by repeated mild stress.
Abstract: After a long period of collecting empirical data describing the changes in organisms, organs, tissues, cells, and macromolecules, biogerontological research is now able to develop various possibilities for intervention. Because aging is a stochastic and nondeterministic process characterized by a progressive failure of maintenance and repair, it is reasoned that gene involved in homeodynamic repair pathways are the most likely candidate gerontogenes. A promising approach for the identification of critical gerontogenic processes is through the hormesis-like positive effects of mild stress. Stimulation of various repair pathways by mild stress has significant effects on delaying the onset of various age-associated alterations in cells, tissues, and organisms.
Abstract: In order to understand the reasons for age-related impairment of the function of bone forming osteoblasts, we have examined the steady-state mRNA levels of the transcription factor CBFA1 and topoisomerase I during cellular aging of normal human trabecular osteoblasts, by the use of semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). There is a progressive and significant reduction of the CBFA1 steady-state mRNA level down to 50% during cellular aging of human osteoblasts. In comparison to the normal cells, human osteosarcoma cell lines SaOS-2 and KHOS/NP, and the SV40-transformed human lung fibroblast cell line MRC5V2 have 20 to 40% higher levels of CBFA1 mRNA. Similar levels of CBFA1 mRNA are detectable in normal human skin fibroblasts, and these cells also exhibit an age-related decline to the same extent. In addition, the expression of topoisomerase I is reduced by 40% in senescent osteoblasts, and the mRNA levels are significantly higher (40-70%) in transformed osteoblasts and fibroblasts. These changes in gene expression may be among the causes of impaired osteoblast functions, resulting in reduced bone formation during aging.
Abstract: We tested the ability of N(6)-furfuryladenine (kinetin) to protect against oxidative and glycoxidative protein damage generated in vitro by sugars and by an iron/ascorbate system. At 50 microM, kinetin was more efficient (82% inhibition) than adenine (49% inhibition) to inhibit the bovine serum albumin (BSA)-pentosidine formation in slow and fast glycation/glycoxidation models. Kinetin also inhibited the formation of BSA-carbonyls after oxidation significantly more than adenine did. However both compounds inhibited the advanced glycation end product (AGE) formation to the same extent (59-68% inhibition). At 200 microM, kinetin but not adenine, limited the aggregation of BSA during glycation. These data suggest that kinetin is a strong inhibitor of oxidative and glycoxidative protein-damage generated in vitro.
Abstract: A large number of studies have demonstrated that various kinds of DNA damage accumulate during aging and one of the causes for this could be a decrease in DNA repair capacity. However, the level of total genomic repair has not been strongly correlated with aging. DNA repair of certain kinds of damage is known to be closely connected to the transcription process; thus, we chose to investigate the level of gene-specific repair of UV-induced damage using in vitro aging of human diploid skin fibroblasts and trabecular osteoblasts as model systems for aging. We find that the total genomic repair is not significantly affected during cellular aging of cultures of both human skin fibroblasts and trabecular osteoblasts. Gene-specific repair was analyzed during cellular aging in the dihydrofolate reductase housekeeping gene, the p53 tumor suppressor gene, and the inactive region X(754). There was no clear difference in the capacity of young and old cells to repair UV-induced pyrimidine dimers in any of the analyzed genes. Thus, in vitro senescent cells can sustain the ability to repair externally induced damage.
Abstract: Intracellular and extracellular proteins are subject to a variety of spontaneous non-enzymatic modifications which affect their structure, function and stability. Protein oxidation and glycation are tightly linked and are implicated in the development of many pathological consequences of aging. Although multiple endogenous pathways in the cell can prevent the formation of oxidized and glycated proteins, and repair and degrade abnormal proteins, such abnormal proteins do accumulate during aging. The heat shock response involving the family of stress-proteins or the so-called heat shock proteins (HSP), represents the quickest and highly conserved response to proteotoxic insults. Since repeated mild heat stress is able to prevent the onset of various age-related changes during cellular aging in vitro, we suggest that treatments which increase HSP expression should reduce the extent of accumulation of abnormal proteins during aging. Such modulation of aging is an example of hormesis, which is characterized by the beneficial effects resulting from the cellular responses to mild repeated stress.
Abstract: Evolutionary theories of ageing and longevity argue against the existence of specific genes that cause ageing. However, genes whose altered activity influences ageing and longevity, may be termed gerontogenes. Several putative gerontogenes have been identified in various ageing systems, including the Drosophila, budding yeast, nematodes and cells in culture. Since ageing is characterized by a progressive failure of maintenance and repair, it is reasoned that genes involved in homeodynamic repair pathways are the most likely candidate gerontogenes. A promising approach for the identification of critical gerontogenic processes is hormesis-like positive effects of stress. Stimulation of various repair pathways by mild stress has significant effects on delaying the onset of various age-associated alterations in cells, tissues and organisms.
Abstract: Age-related bone loss is thought to be due to impaired osteoblast functions. Insulin-like growth factors (IGFs) have been shown to be important stimulators of bone formation and osteoblast activities in vitro and in vivo. We tested the hypothesis that in vitro osteoblast senescence is associated with changes in components of the IGF-system including IGF-I, IGF-II, IGF-binding proteins (IGFBPs) and IGFBP-specific proteases. We employed a human diploid osteoblast cell line obtained from trabecular bone explants and that exhibit typical characteristics of in vitro senescence during serial subculturing. Using a non-competitive reverse-transcriptase polymerase-chain reaction (RT-PCR) assay, we found that the constitutive level of IGF-I mRNA decreased progressively to 49.9 +/- 4.9% in old osteoblasts as compared to the levels found in the young cells. No age-related change was found in IGF-II steady-state mRNA levels. Changes in IGFBPs gene expression and protein production were assessed using Northern blot analysis and Western ligand blotting (WLB), respectively. IGFBP-3 mRNA levels decreased to 30% and protein production to 16% in aged osteoblasts as compared to levels found in young cells. We also found age-related decreases in mRNA levels of both IGFBP-4 and IGFBP-5 to 70% and 60% in aged osteoblasts, respectively, compared to young cells. While IGFBP-5 protein was not detected by WLB, IGFBP-4 protein production showed a biphasic change with 50% decrease in middle-aged cells and a subsequent increase in aged osteoblasts to levels similar to those in young osteoblasts. We found an age-related increase in the immunoreactive levels of IGFBP-4 protease, however, no detectable IGFBP-4 or IGFBP-3 protease activities in conditioned media from osteoblast cultures were observed. Our findings demonstrate that osteoblast aging is associated with impaired production of the stimulatory components of the IGF-system, that may be a mechanism contributing to age-related decline in osteoblast functions.
Abstract: N(6)-Furfuryladenine (kinetin) has been shown to have anti-ageing effects on several different systems including plants, human cells in culture, and fruitflies. Since most of the experimental data point toward kinetin acting as an antioxidant both in vitro and in vivo, and since much evidence supporting a causal role of oxidative damage in ageing is accumulating, we tested the antioxidant properties of kinetin directly. Using 8-oxo-2'deoxyguanosine (8-oxo-dG) in calf thymus DNA as a marker for oxidative damage, we demonstrate that kinetin significantly (P < 0.005) protects the DNA against oxidative damage mediated by the Fenton reaction. Kinetin inhibited 8-oxo-dG formation in a dose-dependent manner with a maximum of 50% protection observed at 100 microM kinetin.
Abstract: We have compared the telomere length, as assessed by Southern analysis, of telomere restriction fragments (TRFs) generated by RsaI/HinfI digestion of genomic DNA in: (i) in vitro cultured human trabecular osteoblasts undergoing cellular aging; and (ii) peripheral blood leukocytes (PBL) obtained from three groups of women: young (aged 20-26 years, n = 15), elderly (aged 48-85 years, n = 15) and osteoporotic (aged 52-81 years, n = 14). The mean TRF length in human osteoblasts undergoing aging in vitro decreased from an average of 9.32 kilobasepairs (kb) in middle-aged cells to an average of 7.80 kb in old cells. The rate of TRF shortening was about 100 bp per population doubling, which is similar to what has been reported for other cell types, such as human fibroblasts. Furthermore, there was a 30% decline in the total amount of telomeric DNA in senescent osteoblasts as compared with young cells. In the case of PBL, TRF length in the DNA extracted from young women was slightly longer (6.76 +/- 0.64 kb) than that from a group of elderly women (6.42 +/- 0.71 kb). A comparison of TRFs in the DNA extracted from the PBL from osteoporotic patients and from age-matched controls did not show any significant differences (6.47 +/- 0.94 versus 6.42 +/- 0.71 kb, respectively). Therefore, using TRF length as a marker for cellular aging in vitro and in vivo, our data comparing TRFs from osteoporotic patients and age-matched controls do not support the notion of the occurrence of a generalized premature cellular aging in osteoporotic patients.
Abstract: There are over 100 modified bases and their derivatives found in RNA and DNA. For some of them, data concerning their properties, synthesis and roles in cellular metabolism are available, but for others the knowledge of their functions and biosynthetic pathways is rather limited. We have analysed the chemical structure of modified nucleosides of DNA and RNA considering mainly their putative synthetic routes. On this basis we suggest, that in addition to enzymatic biosynthetic pathways well established for some odd bases, many rare nucleosides can be recognised as products of random chemical reactions. We identify them as primary or secondary products of the reaction of nucleic acids with hydroxyl radicals, the most active oxidising agent in the cell.
Abstract: Cellular senescence and limited proliferative capacity of normal diploid cells has a dominant phenotype over immortality of cancerous cells, suggesting its regulation by the expression of a set of genes. In order to isolate the genes that associate with senescence, we have employed a clonal system of conditional SV40 T antigen rat embryo fibroblast cell lines which undergo senescence upon T antigen inactivation. Construction of cDNA libraries from two conditional cell lines and application of differential screening and subtractive hybridization techniques have resulted in the cloning of eight senescence-induced genes (SGP-2/Apo J, alpha 1-procollagen, osteonectin, fibronectin, SM22, cytochrome C oxidase, GTP-alpha, and a novel gene) and a senescence-repressed gene (FRS-2). Three of these genes encode for extracellular matrix proteins, others are involved in the calcium-dependent signal transduction pathways, while the SGP-2/Apo J gene may have a cellular protective function. RNA analysis has shown that the senescence-associated genes are overexpressed in both normal rat embryonic fibroblasts and human osteoblasts cell cultures undergoing aging in vitro. In comparison, the expression of these genes in a rat fibroblast immortalized cell line (208F cells) was down-regulated after both its partial and its full transformation by ras oncogenes. Thus, cloning of senescence-associated genes opens up new ways to elucidate and/or to modulate aging and cancer.
Abstract: The effects of repetitive mild heat shock (30 min, 41 degrees C) on growth and various cellular and biochemical characteristics of human skin fibroblasts undergoing ageing in vitro were analysed. Human skin cells not only tolerated more than 30 repeated heat shocks throughout their replicative lifespan, but also maintained several characteristics of young cells until late in life. Whereas the growth rates, population doubling rates, and cumulative population doubling levels achieved in vitro remained unaffected, age-related changes in cellular morphology, cell size, cytoskeletal organisation, autofluorescence and neutral beta-galactosidase activity were significantly slowed down by repeated mild heat shock. These hormesis-like effects of stress-induced defence processes can be useful to elucidate the role of maintenance and repair mechanisms in ageing.
Abstract: Our survival and the physical quality of life depends upon an efficient functioning of various maintenance and repair processes. This complex network of the so-called longevity assurance processes is composed of several genes, termed vitagenes. The homeodynamic property of living systems is a function of such a vitagene network. Because aging is characterized by the failure of homeodynamics, a decreased efficiency and accuracy of the vitagene network can transmutate it into a gerontogene network. It is not clear how various components of the vitagene network operate and influence each other in a concordant or a discordant manner. Experimental strategies through which this transmutation of vitagenes into virtual gerontogenes may be elucidated include induction of molecular damage, antisense intervention, and genetic screening for varied efficiencies of the members of the vitagene family. A reversal of this approach by maintaining or recovering the activity of vitagenes will lead to a delay of aging, a decreased occurrence of age-related diseases, and a prolongation of a healthy life span.
Abstract: Throughout human history, search for means to prevent or slow down aging has followed three main lines--(1) removing waste products and cleansing impurities; (2) using products of plants and animals as medicine; and (3) compensating for decrease in various hormones, vitamins and other chemicals in the body. Even in modern times, immense popularity of various spas and water therapies is an example of the first type of anti-aging approach for which there are no real scientific basis. Some preliminary support from laboratory and/or clinical tests is available for various herbal and other medicinal plant products, such as ginseng, ginkgo biloba and garlic, as nutritional supplements. Replacement therapy, especially hormonal replacement therapy as an anti-aging treatment has been used and misused for quite some time.
Abstract: The proliferative capacity and cellular and biochemical characteristics of human trabecular bone osteoblasts were analysed throughout their replicative lifespan in vitro. Like several other cell types, human osteoblasts demonstrated a typical Hayflick phenomenon of cellular aging comprising a period of rapid proliferation until cumulative population doubling level (CPDL) 22 to 24, followed by a phase of slow growth and the final cessation of cell division at CPDL 32 to 34. Comparing young cells (less than 20% lifespan completed) and old cells (more than 90% lifespan completed) revealed a progressive increase in population doubling (PD) time, a decrease in attachment frequency, a decrease in the number of S-phase positive cells, a decrease in the rates of DNA, RNA and protein synthesis, an increase in the protein content per cell and an increased proportion of senescence-specific beta-galactosidase positive cells. While osteoblastic production of collagen type I decreased progressively during aging, alkaline phosphatase activity dropped rapidly after the first few passages and then remained constant during the rest of the proliferative lifespan, Significant morphological changes from thin and spindle-shaped early passage young cells to large, flattened and irregularly shaped late passage old cells full of intracellular debris were observed. In comparison, osteoblasts established from an osteoporotic bone sample showed a maximum CPDL of less than 5, had a longer PD time and exhibited abnormal senescent morphology. Thus, we have demonstrated for the first time that human osteoblasts, like several other diploid cell types, have a limited proliferative capacity in vitro and undergo aging and senescence as measured by various cellular and biochemical markers. In addition, preliminary studies show that cells from osteoporotic bone have a severely reduced proliferative capacity. This model of bone cell aging facilitates study of the molecular mechanisms of osteoblast senescence as well as factors related to osteoblast dysfunction in patients with osteoporosis.
Abstract: Kinetin, a cytokinin plant growth hormone, retards senescence in plants, delays aging in human cells in culture, slows down development of insects and prolongs their lifespan. We have now observed that the increased longevity of Kn-fed Zaprionus fruitflies was accompanied by an increase in the specific activity of catalase during developmental stages and in adult insects. In addition, the egg laying capacity of Kn-fed fruitflies was reduced drastically as compared with those kept on a normal diet. These results support the view that improved maintenance of the soma and prolongation of its life is achieved at the cost of decreased reproductive activity.
Abstract: Recently, we have detected kinetin (N6-furfuryladenine), a well known cytokinin plant hormone, in commercially available DNA, in freshly extracted cellular DNA and in plant cell extracts. We had suggested that the furfuryl moiety of kinetin originates from furfural which is one of the primary oxidation products of deoxyribose in DNA. Here we show that the human cell extracts treated with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine (PFBHA) give rise to oxime derivatives of various aldehydes present in the cell. Mass spectrometric analysis of silylated oximes showed several mass signals of different species, one of which was identified as furfural. Furthermore, detailed inspection of the mass spectra of DNA showed the mass signals of 165, 180, 189 and 206 m/z which correspond to cytosine-propenal, thymine-propenal, adenine-propenal and guanine-propenal, respectively. The presence of furfural, along with four base-propenals in the cell extract, as the primary oxidation products of deoxyribose, suggests that degradation of sugar residues in DNA is one of the major routes of cellular damage in addition to the modification of nucleic acid bases.
Abstract: Recently, we have reported the presence of kinetin (N6-furfuryladenine) in commercially available DNA, in freshly extracted cellular DNA and in plant cell extracts. We have also found that kinetin has electrochemical properties which can be used for monitoring the level of this modified base in DNA. Here, for the first time, we propose a mechanism for the formation of kinetin in DNA in vivo, based on the analyses of its mass spectra. Since hydroxy radical oxidation at the carbon 5' of the deoxyribose residue yields furfural, we propose that this aldehyde reacts with the amino group of adenine and, after intramolecular rearrangement, kinetin is formed in vivo. Thus kinetin is the first stable secondary DNA damage product known to date with very well defined cytokinin and anti-aging properties, linked to oxidative processes in the cell. These results also indicate that N6-furfuryladenine or kinetin is an important component of a new salvage pathway of hydroxy radicals constituting a 'free radical sink'. In this way, the cells can neutralize the harmful properties of hydroxyl radical reaction products, such as furfural, and respond to oxidative stress by inducing defence mechanisms of maintenance and repair.
Abstract: The highly complex nature of the process of ageing implicates both genetic and epigenetic causative factors. A progressive failure of maintenance underlines and typifies this process. The instability of the nuclear and the mitochondrial genomes is an important determinant of ageing. Infidelity and misregulation of genetic information transfer, loss of cellular proliferative capacity, altered cellular responsiveness and defective pathways of signal transduction are major aspects of the failure of homeostasis. These are also the basis of age-related diseases and impairments, such as osteoporosis, arthritis, immune deficiency, altered drug clearance and altered functioning of the brain. Studies directed towards understanding the mechanisms of interaction and inter-dependence of various genes involved in maintenance and repair networks are the most promising research strategies for identifying gerontogenes.
Abstract: Slowing down of bulk protein synthesis is one of the most commonly observed biochemical changes during aging. The implications and consequences of slower rates of protein synthesis are manifold, including a decrease in the availability of enzymes for the maintenance, repair, and normal metabolic functioning of the cell, an inefficient removal of inactive, abnormal, and damaged macromolecules in the cell, the inefficiency of the intracellular and intercellular signalling pathways, and a decrease in the production and secretion of hormones, antibodies, neurotransmitters, and the components of the extracellular matrix. Age-related changes in the activity, specificity, and stability of a large number of proteins have been reported. However, the molecular mechanisms responsible for such alterations are still poorly understood. Studies on various components of the protein synthetic machinery have revealed a decline in the efficiency and accuracy of ribosomes, an increase in the levels of rRNA and tRNA, and a decrease in the amounts and activities of elongation factors. Because posttranslational modifications of proteins determine their activity and stability, alterations in the extent and level of various modifications such as phosphorylation, methylation, ADP-ribosylation, oxidation, glycation, and conformational changes during aging are being studied. Changes in the regulation of protein synthesis, posttranslational modifications, and protein turnover are crucial determinants of age-related decline in the maintenance, repair, and survival of the organism.
Abstract: Normal diploid cells cultivated in vitro exhibit limited division potential while undergoing ageing during serial passaging. In contrast, cells that have been genetically transformed appear to have lost the regulatory mechanisms of limited growth potential and may continue to divide indefinitely. While cellular mortality is characterised by a progressive cessation of cell growth manifested in cell culture by senescence, immortalisation is the escape from senescence as a result of multiple mechanisms involving the inactivation of tumour suppressor genes, the elevated expression of oncogenes, as well as other genetic and epigenetic changes. The mechanisms governing mortality and immortality are fundamentally linked. The physiological and biochemical features which characterise cellular mortality are examined, herein in the search for markers and timing mechanisms of mortality. The genetic elements involved in the control of mortality and immortality are also discussed, and the fundamental similarities between the molecular and genetic aspects which govern the determination of the phenotypes manifesting the two processes are underlined.
Abstract: In contrast to the current view that kinetin (N6-furfuryladenine) is an unnatural and synthetic compound, we have detected it in commercially available DNA, in freshly extracted cellular DNA from human cells and in plant cell extracts by two independent methods. First, we discovered that N6-furfuryladenine has electrochemical properties that can be applied for monitoring this modified base by a HPLC/UV/EC method. Second, we have confirmed electrochemical assignments by mass-spectrometric analysis. A pathway of kinetin formation is proposed in which the formation of furfural by oxidative damage of the deoxyribose moiety of DNA is followed by its reaction with adenine residues to form N6-furfuryladenine. Since this modification can lead to mutations, the odd DNA base has to be removed by repair enzymes.
Abstract: We have examined the processes of differentiation and apoptosis in relation to cellular aging by using a culture system based on serial passaging of monolayer cultures of human keratinocytes in a serum-free low calcium medium. Differentiation was analyzed by cellular morphology and by the expression of keratinocyte transglutaminase. Keratinocyte cultures at all passages could be induced to differentiate into mature keratinocytes by raising the calcium concentration in the medium. Differentiation could also be induced in senescent cultures, but the process was significantly slower. Apoptosis was analyzed by cellular morphology and by the expression of tissue transglutaminase. Apoptotic cells constituted a very small proportion of the culture and no detectable change in the incidence of apoptosis occurred during serial passaging.
Abstract: The view that the life span of an organism is intrinsically limited and is largely species-specific necessarily involves certain notions of genetic elements of regulation. The term gerontogenes refers to any such genetic elements that are involved in the regulation of aging and life span. The existence of genes for programmed aging is generally discounted on the basis of evolutionary arguments against the notion of the adaptive nature of aging. It is suggested here that the concept of gerontogenes be linked with the idea of genes involved in homeostasis and longevity assurance, which is not contradictory to the nonadaptive nature of aging. Because these genes were not originally selected as real genes for aging, their involvement in aging is an emergent property making them virtual gerontogenes. Some experimental evidence is available that suggests that sets of genes involved in the maintenance and repair of various cellular functions are the primary candidates qualifying as virtual gerontogenes.
Abstract: A cytokinin plant growth hormone kinetin (Kn) retards senescence in plants and delays ageing in human cells in culture. We have now observed that Kn also slows down ageing and prolongs the lifespan of the fruitfly Zaprionus paravittiger when these insects are fed with 25-125 ppm Kn added in their diet medium. In addition, 25 ppm Kn also slows down the larval and the pupal stages of the developing insects. However, the anti-ageing effects of Kn are not merely due to the slowing-down of development, but are mainly due to a reduction in the age-specific death rates throughout the adult lifespan.
Abstract: Slowing down of the rate of protein synthesis during ageing is accompanied by alterations in the amounts and activities of elongation factors, eEF-1 and eEF-2. Since the activity of eEF-2 is regulated by phosphorylation, we have determined the changes in the activities of eEF-2-specific phosphorylating and dephosphorylating enzymes during ageing. Previously, we have reported an age-related increase in the activity of eEF-2 kinase (BBRC, 192, 1210, 1993). We have now compared the activities of a dephosphorylating enzyme protein phosphatase 2A (PP2A) in young and old liver extracts from freely-fed or calorie-restricted rats. The activity of PP2A remain unaltered during ageing. Furthermore, there was no change in the kinetics and extent of PP2A-dependent and PP2A-independent dephosphorylation of eEF-2 during ageing.
Abstract: Aging of human epidermal keratinocytes was studied by serial passaging them as a monolayer culture in a serum-free medium containing 0.09 M calcium. Under these conditions the cells were constantly kept in a proliferative mode and in an undifferentiated state. Aging characteristics in terms of growth and longevity, cumulative population doubling level, cell yield, protein content, and alterations in morphology and in macromolecular synthesis were studied. Furthermore, the induction of differentiation by raising the calcium concentration in the growth medium has facilitated the use of this system to address the issues of the relationship among aging, differentiation and apoptosis in keratinocytes in culture.
Abstract: Osteoblasts are the bone forming cells, which synthesize and secrete the components of the bone matrix. An imbalance between the breakdown and the build up of bone and a decline in osteoblast activity is considered to be the basis of age-related changes in bone structure and function, including the origin of osteoporosis. In order to determine whether the osteoblast activity decreases because of a decreased proliferative capacity or some other reason, we have examined several cellular and biochemical characteristics of human trabecular osteoblasts serially passaged in culture. We have studied growth and maximum lifespan in terms of cumulative population doubling level achieved in culture. We have also determined changes in morphology, protein content and the synthesis of proteins, DNA and RNA during aging of osteoblasts. Furthermore, changes in the cytoskeletal components actin and microtubuli have been observed along with a comparison of one dimensional and two dimensional gel electrophoretic protein pattern during aging.
Abstract: Kinetin (Kn) is a synthetic cytokinin plant growth hormone having some senescence-retarding effects in plants. Its effects on animal cells have been, until now, best considered as growth inhibitory and anti-tumorigenic. However, we have observed that an addition of 40-200 microM Kn in the culture medium of human diploid fibroblasts can both delay the onset and decrease the extent of many of the ageing characteristics that appear during serial passaging of normal cells in culture. Age-related changes that are affected by Kn include morphological alterations, growth rates, cell size, cytoskeletal organisation, macromolecular synthesis and the intensity of autofluorescence due to the oxidative damage product lipofuscin. These anti-ageing effects of Kn are achieved without any increase in the cell culture lifespan in terms of maximum proliferative capacity in vitro.
Abstract: The beneficial effects claimed for the use of garlic as a nutritional supplement include detoxification, antioxidation, antifungal activity, antibacterial activity, tumour suppression and, possibly, anti-ageing and rejuvenating effects. We have used the Hayflick system of cellular ageing in culture in order to test garlic for its anti-ageing effects on long-term growth characteristics, morphology and macromolecular synthesis of human skin fibroblasts. Our results show that an addition of garlic extract into the normal cell culture medium can support serial subculturing for over more than 55 population doublings in 475 days, and that this treatment has some youth-preserving, anti-ageing and beneficial effects on human fibroblasts in terms of maximum proliferative capacity and morphological characteristics. In comparison, similar or lesser doses of garlic extracts are growth inhibitory for cancerous cells that could not be grown over longer periods in the presence of garlic. To our knowledge, this is the first report of the effects of garlic on the long-term growth characteristics and macromolecular synthesis of normal human skin cells, the results of which have applications for both anti-ageing and anti-cancer research.
Abstract: DNA damage due to oxidative free radicals is considered to be a major cause of ageing and age-related diseases including cancer. Of more than 20 modifications formed in DNA by the action of hydroxyl radicals, 8-hydroxy-2'-deoxyguanosine (oh8dG) is potentially highly mutagenic and is known to occur most frequently. Using HPLC combined with electrochemical (HPLC/EC) detection of oh8dG, fivefold higher levels of oh8dG are detected in the DNA of cultured normal human skin fibroblasts as compared with SV40-transformed human fibroblasts MRC-5V2. In comparison, the levels of oh8dG were similar in the growth medium of both types of cells. Applications of this method range from studies on the genomic stability and instability of normal and cancerous cells to the clinical and laboratory testing of toxic substances and drugs.
Abstract: Phorbol esters modulate gene expression, reorganize the cytoskeleton and stimulate bulk protein synthesis and the steps of initiation and elongation. We have observed that a phorbol ester PMA stimulates protein synthesis and increases the amounts of active elongation factors, EF-1 alpha and EF-2 in cultured human fibroblasts MRC-5 undergoing ageing. Although bulk protein synthesis slows down during ageing, the cellular response to the stimulatory effects of PMA is higher in senescent cells. Similarly, despite the age-related decline in the amounts of active EF-1 alpha and EF-2, senescent cells exhibit a higher response to PMA. The results indicate an age-dependent increase of cellular responsiveness to PMA and provide evidence about both the integrity of the translational apparatus and the effectiveness of the signal transduction pathways during cellular ageing. In comparison, the effects of PMA on SV40-transformed MRC-5V2 cells were minimal.
Abstract: Elongation factors involved in polypeptide chain elongation are considered to be rate limiting for the slowing down of total protein synthesis during ageing. The activities of elongation factors are themselves regulated by various means, including phosphorylation. Here we have compared the activity of a protein kinase, called calcium- and calmodulin-dependent protein kinase III (CaM PK III), specific for the phosphorylation of elongation factor eEF-2, in cell-free extracts prepared from livers isolated from young and old male Fischer 344 rats maintained under freely fed or calorie-restricted dietary regimes. There was a significant increase of more than 70% in the activity of CaM PK III in 24 month old freely fed rat livers as compared with young animals. This age-related increase was found but to a lower extent (46%) in calorie-restricted rats of the same age. Therefore, slowing down of ageing in calorie-restricted animals is also reflected at the level of the regulation of the activity of protein elongation factor eEF-2 by CaM PK III.
Abstract: Phorbol esters induce drastic morphological alterations in cells of different origin by altering the conformation and the interrelationship of the elements of the cytoskeletal system. Treatment of early passage (young) and late passage (senescent) human fibroblasts MRC-5 with phorbol-12-myristate-13-acetate (PMA) results in the rearrangement of actin and tubulin filaments. PMA brings about the disorientation and diffusion of the heavily criss-crossed network of actin and microtubulin fibres characteristic of senescent cells suggesting thereby an increased sensitivity of senescent cells to phorbol esters. Since phorbol esters are known to be specific activators of protein kinase C (PKC), the PMA-induced modulation of the cytoskeleton patterns in ageing fibroblasts provides further support for the view that the effectiveness of the signalling mechanisms is retained during cellular ageing.
Abstract: 4-beta-phorbol-12-beta-myristate-13-alpha-acetate (PMA) alters cellular growth properties by modulating gene expression in a wide variety of cell types. Human diploid fibroblasts MRC-5 were treated with PMA at different phases of their lifespan in vitro and the alterations of their short-term growth characteristics and macromolecular synthesis in response to PMA were analysed. PMA stimulates DNA and RNA synthesis in both Phase II (young) and Phase III (senescent) MRC-5 cells. Treatment of senescent cells with various PMA concentrations results in a greater stimulation of DNA and RNA synthesis than that in young cells. Senescent cells are also more sensitive to the PMA-induced alterations of growth characteristics and higher concentrations of PMA become toxic for them. The age-related alterations of cellular responsiveness are also apparent in the gradual loss of responsiveness to serum, observed in parallel with the increased sensitivity to PMA. Furthermore, serum-induced stimulation of macromolecular synthesis is inhibited by PMA. Since it is known that serum and PMA elicit their effects via different signal transduction pathways, our results point to suggest the differential regulation of the signalling mechanisms during cellular ageing.
Abstract: Posttranslational modifications of proteins are involved in determining their activities, stability, and specificity of interaction. More than 140 major and minor modifications of proteins have been reported. Of these, only a few have been studied in relation to the aging of cells, tissues, and organisms. These include phosphorylation, methylation, ADP-ribosylation, oxidation, glycation, and deamidation. Several of these modifications occur on proteins involved in crucial cellular processes, such as DNA synthesis, protein synthesis, protein degradation, signal transduction, cytoskeletal organization, and the components of extracellular matrix. Some of the modifications are the markers of abnormal and altered proteins for rapid degradation. Others make them less susceptible to degradation by normal proteolytic enzymes, and hence these accumulate during aging.
Abstract: We show that kinetin, a non-natural product with strong cytokinin activity, is incorporated into prokaryotic and eukaryotic tRNAs in the exchange reaction catalysed by a putative tRNA-kinetin transglycosylase. We also show that kinetin is specifically incorporated into E. coli tRNA(Tyr) and most probably at position 37. To our knowledge, this is the first report of a nucleic acid base exchange reaction occurring at this position.
Abstract: The slowing down of protein synthesis is a change widely observed during the aging of organisms. It has also been claimed that a decline in the rate of protein synthesis occurs during cellular aging. However, the evidence in favour of this view is not clear-cut, and reliable estimates of rates of protein synthesis during cellular aging have yet to be made. Studies on various components of the protein synthetic machinery during cellular aging have revealed a decline in the efficiency and accuracy of ribosomes, an increase in the levels of rRNA and tRNA, and a decrease in the amounts and activities of elongation factors. Detailed studies on the structure and function of ribosomes, tRNA isoacceptor profiles, activities of aminoacyl-tRNA synthetases, levels and activities of initiation factors, rates of protein elongation, and the accuracy of protein synthesis will be needed before the molecular mechanisms of the regulation of protein synthesis during cellular aging can be understood.
Abstract: Dietary calorie restriction of rats has been previously shown to increase protein synthetic rates in liver and kidney cells during ageing. Here we have compared the activity and amounts of active elongation factors EF-1 alpha and EF-2 in cell-free extracts prepared from livers isolated from male Fischer 344 rats of different ages. Although there is some age-related decline in the catalytic activity and amounts of active EF-1 alpha during ageing, no differences between freely-fed and calorie-restricted animals were observed. In the case of EF-2, the amounts of ADP-ribosylatable EF-2 neither declined during ageing nor differed between freely-fed and calorie-restricted animals. Thus differences in the protein synthetic rates in calorie-restricted and freely-fed rats are not reflected at the level of protein elongation factors, and may involve some other mechanisms of regulation.
Abstract: The inability of normal cells to maintain themselves for ever is a reflection of homoeostatic imbalance and a progressive failure of maintenance. Ageing cells respond less to growth stimulants whereas they show increased sensitivity to toxic agents including antibiotics, phorbol esters, radiation and other physical stresses. No major quantitative and qualitative defects in the receptor systems have been detected that could explain the reasons for altered responsiveness during ageing. Random metabolic defects in the processes involved in maintaining homoeostasis may be critical for causing homoeostatic imbalance, cellular ageing and death.
Abstract: The capacity of cells and organisms to respond to external stimuli and to maintain stability in order to survive decreases progressively during ageing. The mitogenic and stimulatory effects of growth factors, hormones and other agents are reduced significantly during cellular ageing. The sensitivity of ageing cells to toxic agents including antibiotics, phorbol esters, radiations and heat shock increases. This failure of homeostasis during cellular ageing does not appear to be due to any quantitative and qualitative defects in the receptor systems. Instead, metabolic defects in the pathways of macromolecular synthesis may be the basis of altered cellular responsiveness during ageing.
Abstract: The elongation step is involved in the regulation of protein synthesis during the cell cycle, environmental stress, ageing and transformation. Using a diphtheria toxin-mediated assay for measuring the levels of ADP-ribosylatable elongation factor EF-2, we have observed an irreversible decrease of up to 64% in the amount of ADP-ribosylatable EF-2 in normal diploid human fibroblasts MRC-5 undergoing ageing in vitro. However, a similar decrease in low serum-associated G0/G1-arrested cells is reversible both in MRC-5 cells and in their SV40-transformed counterparts. Reduced levels of ADP-ribosylatable EF-2 could account for the slowing-down of protein synthesis during cell cycle arrest and during cellular ageing in culture.
Abstract: In eukaryotes, peptide chain elongation is mediated by elongation factors EF-1 and EF-2. EF-1 is composed of a nucleotide-binding protein EF-1 alpha, and a nucleotide exchange protein complex, EF-1 beta gamma, while EF-2 catalyses the translocation of peptidyl-tRNA on the ribosome. Elongation factors are highly conserved among different species and may be involved in functions other than protein synthesis, such as organization of the mitotic apparatus, signal transduction, developmental regulation, ageing and transformation. Yeast contains a third factor, EF-3, whose structure and function is not yet well understood.
Abstract: The amount of protein elongation factor EF-2 that can be inactivated by diphtheria toxin-mediated ADP-ribosylation, a measure of its active content, decreases by 45% and 66% in G1-arrested normal human fibroblasts and in HeLa cells respectively. On restimulation of cells with fresh serum, the amounts of ADP-ribosylatable EF-2 begin to increase within 4 h. Whereas the level of active EF-2 returns to normal (exponential phase of growth) in 20 h in the case of fibroblasts, only 47% recovery was observed for HeLa cells during this period. The apparent long half-lives of EF-2 mRNA and protein indicate possibilities of posttranslational ADP-ribosylation and de-ADP-ribosylation as the regulators of the amounts of active EF-2 during human cell cycle.
Abstract: The content of the elongation factor (EF-2) can be measured by diphtheria toxin-dependent ADP-ribosylation in cell-free extracts of samples prepared from small amounts of tissues and cells containing less than 100 micrograms of total protein. A 20 min in vitro assay, in which a radioactive ADP-ribosyl residue is transferred specifically and 1:1 stoichiometrically to EF-2, is sufficient to estimate the total amounts of ADP-ribosylatable active EF-2. The method is very useful for monitoring changing levels of EF-2 during various pathological and biological processes, including cell cycle, ageing, cancer and other diseases.
Abstract: A significant decline in amount of active elongation factor, EF-1 alpha, and in its catalytic activity was observed in cell-free extracts prepared from normal human diploid fibroblasts (MRC-5) and their SV40-transformed counterparts, after subjecting the cells to 60 min heat shock at different temperatures. Old MRC-5 cells which had become senescent on serial passaging were more sensitive to heat shock-related changes in activity and amounts of active EF-1 alpha than were rapidly proliferating normal and transformed cells.
Abstract: Stoichiometrically estimated amounts of active elongation factor, EF-1 alpha, remain constant in serially passaged Phase II cultures of human fibroblasts, MRC-5, but decrease by 45% towards the end (Phase III) of their lifespan. Catalytic activity of EF-1 alpha is also reduced by 35% in Phase III old cells. The SV40 transformed immortal cell line MRC-5V2 has 30% higher levels of active EF-1 alpha without significant increase in its catalytic activity. Low-serum-associated G1 arrest of normal and transformed cells reduces amounts of active EF-1 alpha by 35% and 20%, respectively. Catalytic activity, however, is reduced rapidly only in G1 arrested normal cells and not in transformed cells. Even though the cell cycle-related changes are reversible both in normal and transformed cells, the age-related decline in amounts of active EF-1 alpha and its activity are irreversible and, most probably, crucial.
Abstract: Human fetal lung fibroblasts grown in the presence of dansyl-paromomycin (DNS-Pm), a fluorescent derivative of the aminoglycoside antibiotic, paromomycin, probably accumulate DNS-Pm in the lysosomes. The intracellular concentration of DNS-Pm is proportional to the extracellular concentration and to the length of time cells are exposed to the compound. The accumulation of DNS-Pm by human fibroblasts continued to increase for several days, reaching a saturation after 7 days. The kinetic data are consistent with the establishment of a steady state in the cell between fluid-phase pinocytosis and exocytosis of DNS-Pm. About 80% of the intracellular DNS-Pm ws released in 24 hr when fresh medium without the analogue was added. The residual 20% remained within the cells, suggesting that it may be irreversibly bound to the lysosomes, endoplasmic reticulum, or ribosomes. The uptake of paromomycin by cells in culture may be a useful means to study error propagation during growth and lifespan of cells in vitro.
Abstract: Chick embryo fibroblasts are often used as a model of cellular ageing in vitro. It seems important to culture these cells in optimum conditions which may not be the same as those for mammalian cells. An incubation temperature of 40 degrees C and the use of chicken serum were found to be optimum for growth and longevity of chick embryo fibroblasts in culture.
Abstract: An exponential increase in autofluorescence (AF) during serial passaging of human diploid fibroblasts, strain MRC-5, was observed using a fluorescence-activated cell sorter. Skin fibroblasts from patients with premature-ageing syndromes had high levels of AF, whereas virus-transformed cell lines had negligible amounts of AF.
