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PubMed2004

Hormetic mechanisms of anti-aging and rejuvenating effects of repeated mild heat stress on human fibroblasts in vitro.

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.

Cellular Senescence↗

PubMed2004

Cell separation of hepatocytes and fibroblasts through surface-modified polyurethane membranes.

The separation of fibroblast cells (L929 cells) and hepatocytes was investigated by using unmodified and surface-modified polyurethane (PU) foaming membranes (pore size of 12 microm) by the incorporation of various functional groups. L929 cells permeated more readily than hepatocytes, and very few populations of hepatocytes (<5%) permeated through the membranes. This result was thought to be due to the smaller cell size of the L929 cells (5-10 microm) relative to the hepatocytes (15-30 microm). The larger hepatocytes were thought to plug the pores of the membranes. A good cell separation between L929 cells and hepatocytes was achieved when the cell mixture permeated through the negatively charged PU membranes. The negatively charged membranes were thought to enhance the permeation of L929 cells because of the electrostatic repulsion between the membranes and negatively charged cells. On the other hand, the hepatocytes were unable to permeate through the membranes because of the sieve effect caused by their large cell size. The separation of hepatocytes isolated from mice at different ages was also accomplished by permeating the cell mixture through unmodified and surface-modified PU membranes.

Animals↗

PubMed1992

The regulation of intracellular calcium ion and pH in young and old fibroblast cells (WI-38).

The cytosolic free calcium concentration ([Ca2+]i) and the cytoplasmic pH (pHi) in young and old cells of normal human diploid cell line, WI-38, were measured with the fluorescent probes, fura-2 and BCECF, respectively. The basal concentrations of cytosolic calcium in young and old cells were approximately the same (40-50 nM). In order to investigate the difference in the regulation of calcium mobilization between young and old cells, both cell lines were stimulated with bradykinin (BK, 0.5 microM), and [Ca2+]i was monitored by fluorescent imaging. The [Ca2+]i increased within 10 sec following bradykinin exposure. The peak values for young and old cells were about 10 and 8 times higher than the basal level. After the peak, the recovery rate in young cells was slower than in old cells. This difference between young and old cells may be due to the change of the influx of extracellular calcium, as this difference was not observed in the calcium free medium. These data suggest that calcium influx may be reduced during cell aging. Intracellular pH in old cells was found to be more acidic than that of young cells in media with either 0.1% or 10% serum. This finding indicates that cell aging might induce acidification which in turn causes an inhibition of cell growth. The relationship between acidification and the activity of calcium channel in old cells is also discussed.

Bradykinin↗

PubMed2006

Preferential reduction of the alpha-2-6-sialylation from cell surface N-glycans of human diploid fibroblastic cells by in vitro aging.

Human diploid fibroblastic cell line, TIG-3, has a finite life span of about 80 population doubling levels (PDL), and is used for in vitro aging studies. Young cells (PDL 23) grew to higher cell densities at a higher growth rate than aged cells (PDL 77). When the electrophoretic mobility of cells was determined, the negative surface charge of the aged cells decreased significantly when compared to that of young cells. Lectin blot analysis of membrane glycoproteins showed that the alpha-2-6-sialylation but not the alpha-2-3-sialylation of N-glycans decreases markedly in the aged cells when compared to the young cells. In support of this observation, the cDNA microarray assay and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the gene expression of the alpha-2,6-sialyltransferase I (ST6Gal I), which transfers sialic acid to galactose residues of N-glycans, decreases in the aged cells. These results indicate that the concordant decrease of the alpha-2,6-sialylation of N-glycans with the ST6Gal I gene expression is induced in TIG-3 cells by in vitro aging.

Cell Line, Tumor↗

PubMed1994

A theory that may explain the Hayflick limit--a means to delete one copy of a repeating sequence during each cell cycle in certain human cells such as fibroblasts.

A model that may explain the limited division potential of certain cells such as human fibroblasts in culture is presented. The central postulate of this theory is that there exists, prior to certain key exons that code for materials needed for cell division, a unique sequence of specific repeating segments of DNA. One copy of such repeating segments is deleted during each cell cycle in cells that are not protected from such deletion through methylation of their cytosine residues. According to this theory, the means through which such repeated sequences are removed, one per cycle, is through the sequential action of enzymes that act much as bacterial restriction enzymes do--namely to produce scissions in both strands of DNA in areas that correspond to the DNA base sequence recognition specificities of such enzymes. After the first scission early in a replicative cycle, that enzyme becomes inhibited, but the cleavage of the first site exposes the closest site in the repetitive element to the action of a second restriction enzyme after which that enzyme also becomes inhibited. Then repair occurs, regenerating the original first site. Through this sequential activation and inhibition of two different restriction enzymes, only one copy of the repeating sequence is deleted during each cell cycle. In effect, the repeating sequence operates as a precise counter of the numbers of cell doubling that have occurred since the cells involved differentiated during development.

Base Sequence↗

PubMedDate not supplied

Increased susceptibility of late passage human diploid fibroblasts to oxidative stress.

Three strains of human diploid fibroblasts, TIG-3, TIG-7, and MRC-5, were serially cultivated. The susceptibility of early-passage and late-passage cells at 20-30 and 60-70 population doubling levels, respectively, to hydrogen peroxide, the superoxide radical (exposure to the hypoxanthine-xanthine oxidase system), or linoleic acid hydroperoxide was examined for lactate dehydrogenase release. The susceptibility of late-passage cells to such oxidative stress was considerably enhanced compared with early-passage cells. The concentration of reduced glutathione in late-passage cells was lower by 24-44% on a per-cell-number basis and by 86.0-94.5% on a per-protein-quantity basis than in early-passage cells. In addition, the activity of catalase in late-passage cells was lower by 19-46% compared with early-passage cells. There was, however, no difference between the mRNA levels of catalase in early-passage and late-passage cells. The activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase, and glutathione peroxidase in late-passage cells were all higher than in early-passage cells. These results suggest that late-passage cells are more susceptible to oxidative stress than early-passage cells presumably because of decreases in cellular reduced glutathione concentration and catalase activity, and that their primary defense against oxidative stress is reduced glutathione.

Catalase↗

PubMed1993

The effect of food chemicals on cell aging of human diploid cells in in vitro culture.

The potency of food chemicals to induce cell aging was evaluated in human diploid fibroblast cells HAIN-55 having a finite replicative potential by using in vitro aging markers, i.e., decreases of maximum proliferative potential (lifespan) of cells, saturation density in monolayer culture (SD), plating efficiency (PE) and mitotic index (MI), and an increase of cells with polyploid karyotypes. By treatment twice with low concentration of genotoxic chemicals aflatoxin B1, allylisothiocyanate or trans-cinnamaldehyde (severe clastogenic flavoring agent; Kasamaki et al., 1982), lifespan (expressed by the number of cumulative cell population doubling (CPD)) of the treated cells was reduced by 8-12 CPDs accompanied by change of the other aging markers. By successive treatment (29 or 25 times) with non-genotoxic chemical aspartame (N-L-aspartyl-L-phenylalanine) or L-canavanine (structural analogue of L-arginine), lifespan of the treated cells was also slightly shortened (by 2-6 CPDs) compared with the untreated control cells. In the process of cell aging, Mitochondrial activity (MTT activity) decreased almost in parallel with the decrease of SD and MI. On the basis of these results, a variety of genotoxic and non-genotoxic chemicals were examined by using MTT activity as the aging marker for their effects on the aging of HAIN-55 cells and bovine artery endothelial cells which also had a finite replicative potential. The results showed that seven genotoxic and nine non-genotoxic chemicals promoted cell aging.

Aflatoxin B1↗

PubMed2005

Distinct fibroblast growth factor (FGF)/FGF receptor signaling pairs initiate diverse cellular responses in the oligodendrocyte lineage.

Fibroblast growth factors (FGFs) have been implicated in numerous cellular processes, including proliferation, migration, differentiation, and survival. Whereas FGF-2, the prototypic ligand in a family of 22 members, activates all four tyrosine kinase FGF receptors (FGFR1-FGFR4), other members demonstrate a higher degree of selectivity. Oligodendrocytes (OLs), the myelin-producing cells of the CNS, are highly influenced by FGF-2 at all stages of their development. However, how other FGFs and their cognate receptors orchestrate the development of OLs is essentially undefined. Using a combination of specific FGF ligands and receptor blocking antibodies, we now show that FGF-8 and FGF-17 target OL progenitors, inhibiting their terminal differentiation via the activation of FGFR3, whereas FGF-9 specifically targets differentiated OLs, triggering increases in process growth via FGFR2 signaling; FGF-18 targets both OL progenitors and OLs via activation of both FGFR2 and FGFR3. These events are highly correlated with changes in FGF receptor expression from FGFR3 to FGFR2 as OL progenitors differentiate into mature OLs. In addition, we demonstrate that, although activation of FGFR1 by FGF-2 leads to proliferation of OL progenitors, it produces deleterious effects on differentiated OLs (i.e., aberrant reentry into cell cycle and down-regulation of myelin proteins with a loss of myelin membrane). These data suggest that ligand availability, coupled with changes in FGF receptor expression, yield a changing repertoire of ligand-receptor signaling complexes that contribute critically to the regulation of both normal OL development and potential OL/myelin pathogenesis.

Animals↗

PubMed1974

Dominance of the senescent phenotype in heterokaryons between replicative and post-replicative human fibroblast-like cells.

In heterokaryons between senescent and young diploid fibroblast-like cells, dominance of the former with respect to nuclear DNA synthesis (incorporation of [(3)H]thymidine) was demonstrated. For identification of the respective partners, double-layer autoradiography was used after the old cells were labeled with [(3)H]methionine and the young cells were labeled with [(14)C]thymidine. Synchrony of nuclear labeling (i.e., all nuclei in a cell labeled with [(3)H]thymidine) was observed in the majority of di- and polykaryons during the second and third of three 24-hr periods of labeling with [(3)H]thymidine. The results are compatible with either terminal differentiation or error theories of clonal senescence.

Adolescent↗

PubMed1999

Microarray analysis of replicative senescence.

BACKGROUND: Limited replicative capacity is a defining characteristic of most normal human cells and culminates in senescence, an arrested state in which cells remain viable but display an altered pattern of gene and protein expression. To survey widely the alterations in gene expression, we have developed a DNA microarray analysis system that contains genes previously reported to be involved in aging, as well as those involved in many of the major biochemical signaling pathways. RESULTS: Senescence-associated gene expression was assessed in three cell types: dermal fibroblasts, retinal pigment epithelial cells, and vascular endothelial cells. Fibroblasts demonstrated a strong inflammatory-type response, but shared limited overlap in senescent gene expression patterns with the other two cell types. The characteristics of the senescence response were highly cell-type specific. A comparison of early- and late-passage cells stimulated with serum showed specific deficits in the early and mid G1 response of senescent cells. Several genes that are constitutively overexpressed in senescent fibroblasts are regulated during the cell cycle in early-passage cells, suggesting that senescent cells are locked in an activated state that mimics the early remodeling phase of wound repair. CONCLUSIONS: Replicative senescence triggers mRNA expression patterns that vary widely and cell lineage strongly influences these patterns. In fibroblasts, the senescent state mimics inflammatory wound repair processes and, as such, senescent cells may contribute to chronic wound pathologies.

Blood Physiological Phenomena↗

PubMed2003

Control of the replicative life span of human fibroblasts by p16 and the polycomb protein Bmi-1.

The polycomb protein Bmi-1 represses the INK4a locus, which encodes the tumor suppressors p16 and p14(ARF). Here we report that Bmi-1 is downregulated when WI-38 human fibroblasts undergo replicative senescence, but not quiescence, and extends replicative life span when overexpressed. Life span extension by Bmi-1 required the pRb, but not p53, tumor suppressor protein. Deletion analysis showed that the RING finger and helix-turn-helix domains of Bmi-1 were required for life span extension and suppression of p16. Furthermore, a RING finger deletion mutant exhibited dominant negative activity, inducing p16 and premature senescence. Interestingly, presenescent cultures of some, but not all, human fibroblasts contained growth-arrested cells expressing high levels of p16 and apparently arrested by a p53- and telomere-independent mechanism. Bmi-1 selectively extended the life span of these cultures. Low O(2) concentrations had no effect on p16 levels or life span extension by Bmi-1 but reduced expression of the p53 target, p21. We propose that some human fibroblast strains are more sensitive to stress-induced senescence and have both p16-dependent and p53/telomere-dependent pathways of senescence. Our data suggest that Bmi-1 extends the replicative life span of human fibroblasts by suppressing the p16-dependent senescence pathway.

Cell Division↗

PubMed1995

Identification of the active region of the DNA synthesis inhibitory gene p21Sdi1/CIP1/WAF1.

The cloning of the negative growth regulatory gene, p21Sdi1, has led to the convergence of the fields of cellular senescence, cell cycle regulation and tumor suppression. This gene was first cloned as an inhibitor of DNA synthesis that was overexpressed in terminally non-dividing senescent human fibroblasts (SD11) and later as a p53 transactivated gene (WAF1) and a Cdk-interacting protein (CIP1, p21) that inhibited cyclin-dependent kinase activity. To identify the active region(s) of p21Sdi1, cDNA constructs encoding various deleted forms of the protein were analyzed. Amino acids 22-71 were found to be the minimal region required for DNA synthesis inhibition. Amino acids 49-71 were involved in binding to Cdk2, and constructs deleted in this region expressed proteins that were unable to inhibit Cdk2 kinase activity in vitro. The latter stretch of amino acids shared sequence similarity with amino acids 60-76 of the p27Kip1 protein, another Cdk inhibitor. Point mutations made in p21Sdi1 in this region confirmed that amino acids common to both proteins were involved in DNA synthesis inhibition. Additionally, a chimeric protein, in which amino acids 49-65 of p21Sdi1 were substituted with amino acids 60-76 of p27Kip1, had almost the same DNA synthesis inhibitory activity as the wild-type protein. The results indicate that the region of sequence similarity between p21Sdi1 and p27Kip1 encodes an inhibitory motif characteristic of this family of Cdk inhibitors.

Amino Acid Sequence↗

PubMed1998

Endogenous beta-galactosidase activity in continuously nonproliferating cells.

Cytochemically detectable activity of endogenous beta-galactosidase was found at pH 6.0 in Swiss 3T3 cells after long-term incubation in low serum or in the presence of heparin concentrations known to reversibly inhibit cell proliferation. A high percentage of beta-galactosidase-positive cells were detected in U937 and HL60 cultures at the late stage of macrophage-like differentiation induced by TPA. Interestingly, a small number of beta-galactosidase-positive cells were found even in the growing Swiss 3T3 cultures. These positive cells expressed morphological features similar to those of senescent cells. Thus, the activity of beta-galactosidase at pH 6.0 cannot be considered an exclusive marker of senescent cells since it is expressed in other types of nonproliferating cells.

3T3 Cells↗

PubMed1999

A natural extracellular factor that induces Hsp72, inhibits apoptosis, and restores stress resistance in aged human cells.

Experiments with cultured cells showed that most cellular stress resistance components are specialized for certain types of damage. For example, superoxide dismutase protects from oxidative damage; DNA repair enzymes guard against mutagens and other DNA-damaging agents. On the other hand, the major inducible heat shock protein Hsp72 protects cells from a large variety of stresses and thus represents a generalized repair/stress resistance component. Hsp72 not only refolds damaged proteins but also interferes with programmed cell death signaling pathways, thus providing cells with time to repair the damage, hence its universality as a stress protector. In the present study we demonstrate the occurrence in murine and human ascites fluids (AF) of a natural nontoxic extracellular factor (ascites Hsp72-inducing factor, AHIF) capable of activating Hsp72 expression in different types of cells via a pathway distinct from the heat shock response pathway. AHIF is unique in that it is the first physiological factor capable of inducing synthesis of Hsp72 not only in young cells but, remarkably, also in aged human cells that largely have lost the ability to express Hsp72 in response to stresses, a manifestation at the cellular level of a progressive impairment in the ability to adapt to environmental changes which characterizes aging. Pretreatment of aged human cells with AF triggers Hsp72 expression at levels seen in young stressed cells and protects cells from a variety of otherwise lethal stressful treatments such as heat shock, TNF, UV irradiation, etoposide, and menadione. Activation of Hsp72 expression is essential for antiapoptotic action of AHIF because specific inhibition of Hsp72 expression by antisense RNA abolishes the cytoprotective effect of AF. In view of an important link between stress resistance and longevity in different organisms, the abilities of AHIF make it a unique candidate for the role of a systemic regulator of the aging process. While a cell-autonomous stress response diminishes with aging, aged cells retain the ability to respond to an extracellular factor which induces the expression of Hsp72. This finding opens up exciting possibilities for using AF factor to restore stress resistance to old cells and organisms and the possibility of interfering with the aging process. The ability to induce stress resistance in young cells and to restore it in aged cells could serve as a basis for developing effective antiapoptotic therapies.

Adenoviridae↗

PubMed1991

Changes in fibronectin mRNA splicing with in vitro passage.

Senescent human fibroblasts produce larger fibronectin molecules with altered binding properties. To determine if this change could involve alternative splicing of fibronectin precursor mRNA, we developed an approach using reverse transcription and the polymerase chain reaction to study fibronectin mRNA splicing at each of the three alternatively spliced regions. Two of the three regions showed changes with in vitro passage incorporation of the ED-A region increased 8 fold.

Base Sequence↗

PubMed1989

Approach to the isolation of antiproliferative genes.

Poly(A) RNAs from normal rat liver and senescent human fibroblasts appear to have more antiproliferative activity than RNAs from regenerating rat liver and early passage human fibroblasts. We have screened two rat liver and one human liver library by differential hybridization and isolated four candidate cDNAs for this antiproliferative activity; one is fibronectin and three others do not match to any sequence in the mammalian portion of the GENBANK database. We are currently testing the antiproliferative nature of these cDNAs by microinjection of hybrid-selected RNA, and we describe an alternative strategy for cloning such genes based on construction of a cDNA library in an RNA expression vector.

Animals↗

PubMedDate not supplied

Oxidants and antioxidants in proliferative senescence.

In terms of the amount of experimental research it has generated the free radical theory of ageing is one of the most popular hypotheses to explain this ubiquitous phenomenon. From the theory two postulates were derived: either cellular defence mechanisms against free radical-dependent oxidants deteriorate during ageing of cells, or essential, unrepairable damages are imparted to the cell by oxidants regardless of the activity of antioxidant defence systems. The many reports dealing with a putative breakdown in antioxidant defence systems failed to positively support this postulate. However, a minor depletion in cellular glutathione by exposure to a model lipophilic peroxide led to a significant decrement in DNA and protein synthesis. In other words, the glutathione redox cycle is intrinsically fallible with respect to defending the cellular DNA replication system against this model lipophilic peroxide. Interestingly, after ageing in culture cells a partial uncoupling of the NADPH-producing and -consuming systems tends to take place. Experiments involving the addition of antioxidants to the culture medium have failed to significantly extend the lifespan of cultured diploid somatic cells. The level of antioxidants appears to be a modulator rather than a primary determinant of cellular ageing in culture. Several lines of evidence suggest that DNA damages accumulate during ageing of the organism, but no oxidant-related DNA damage has been pinpointed in the cultured cell system. Human mutants with defects in antioxidant enzymes have not shown conclusive signs of accelerated ageing. Cells from patients with Werner's syndrome (progeria of the adult), on the other hand, do not suffer from a defect in their antioxidant defence system, nor do they accumulate more than normal amounts of autofluorescent products resulting from lipid peroxidation. The recent finding that Werner's syndrome constitutes a mutator phenotype may prompt the comparison of oxidant- and ageing-related mutation spectra in order to investigate a mutational theory of ageing as a new derivative from the free radical hypothesis.

Animals↗

PubMed2005

Expression of TARSH gene in MEFs senescence and its potential implication in human lung cancer.

To reveal the molecular mechanism of cellular senescence, we have surveyed the genes that are specifically upregulated via MEFs senescence by suppression subtractive hybridization method. We show here that mTARSH was induced particularly in the relative early phase of MEFs cellular senescence. Further structural analysis of mTARSH disclosed five splicing variants shared a common reading frame whose diversity was derived from the SH3-binding motif cluster in the middle of the gene. We also show that mTARSH mRNA predominantly expressed in lung and that conspicuous expression of TARSH was drastically declined in all several lung cancer cell lines we tested. Thus, TARSH presumably represents a trigger gene for evoking cellular senescence, which has also been suggested to be involved in the prevention of tumorigenesis.

Aging↗