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Results for “Senescent fibroblasts”

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PubMed1998

Fibroblasts cultured from distal lower extremities in patients with venous reflux display cellular characteristics of senescence.

PURPOSE: Venous reflux precedes the development of venous ulcers. Our earlier work showed that the fibroblasts that are cultured from these wounds display more characteristics of senescence. We evaluated fibroblast senescence in patients with venous reflux but without ulcers to further investigate the role of venous reflux in the predisposition to venous ulcers. METHODS: Fibroblasts that were isolated from skin biopsy specimens of the "gaiter" area (distal) and of the ipsilateral thigh of the same patient (proximal) were compared. Twelve patients with venous reflux (9 patients in clinical, etiologic, anatomic, and pathologic classification 4; 3 patients in classification 5) with an average venous filling index of 5.45 mL/s and 4 patients without venous reflux were enrolled in the study. The growth rates, the response to basic fibroblast growth factor (b-FGF), and the senescence markers (beta-galactosidase activity at a pH level of 6, unstimulated fibroblasts fibronectin protein, and messenger RNA levels) were determined for each cell population. RESULTS: The number of senescence-associated beta-galactosidase positive cells (8.3% +/- 1.9% vs 2.2% +/- 0.8%; P =.008) and the level of cellular fibronectin protein (455.7 +/- 80 vs 210 +/- 51; P =.04) and messenger RNA (16.8 +/- 6.8 vs 13.5 +/- 5.7; P =.042) were significantly higher in the distal fibroblasts as compared with the proximal fibroblast cultures. The growth rates of the distal fibroblasts were lower when compared with the proximal fibroblasts (15,746 +/- 4287 cells/day vs 29,550 +/- 5035 cells/day; P <.002) but were not different in the presence of b-FGF (41,717 +/- 9542 cells/day vs 47,030 +/- 6133 cells/day; P =.53). In the patients without venous reflux, no site differences were noted in the growth rates or the senescence markers between the proximal and distal fibroblasts. CONCLUSION: Distal fibroblasts that are isolated from patients with venous reflux display more senescence characteristics than do proximal fibroblasts and have significantly lower growth rates. Despite senescence, b-FGF restored the distal-fibroblasts growth rate to that of the stimulated proximal fibroblasts, which proposes a therapeutic role for b-FGF. These changes precede ulcer formation and suggest a mechanism that is focal and intrinsically related to venous reflux.

Adult↗

PubMed2005

Cellular replicative capacity correlates primarily with species body mass not longevity.

Although the limited replicative capacity of human fibroblasts in culture is frequently used as a model for aging, a question of major interest is whether the relationship between in vitro fibroblast proliferative capacity and species longevity is primary or secondary to a relationship with species body size. In this report we establish that body mass is the primary correlative of proliferative potential rather than species life-span.

Adolescent↗

PubMed1999

Histone variants of H2A and H3 families are regulated during in vitro aging in the same manner as during differentiation.

In a previous communication, we showed that the H2A.1/H2A.2 histone variant ratio decreases in a linear manner during the in vitro aging of human diploid fibroblasts. This ratio is known to decrease in the same manner in progressive stages of development and in the process of differentiation, and is thus considered to be a biochemical marker for differentiation. A detailed analysis of the synthesis of H2A and H3 histone variants as a function of cumulative population doublings in the same in vitro cell system is presented in this study. Quantitative analysis of these variants in the G0 phase, synchronized fibroblasts has shown that their relative amount in chromatin, as well as their biosynthesis rate, change during in vitro aging of human diploid fibroblasts, revealing both up-and down-regulation of certain variants as a function of cumulative population doublings. Furthermore, we show by morphometric studies employing the seven distinct fibroblast morphotypes, as described by the Bayreuther classification, that this regulation is attributable to the replicative sub-populations. These results reveal that histone variants of the H2A and H3 families are regulated during in vitro aging in the same manner as that during differentiation.

Cell Differentiation↗

PubMed1975

The use of arrested populations of human diploid fibroblasts for the study of senescence in vitro.

It has been shown that human diploid cells from various donor ages can be arrested in an essentially nonmitotic state by reducing the serum concentration of the incubation medium from 10 to 0.5 percent. Cells incubated at this serum level maintained the population distribution that was present when the cells reached confluency. The population, which has 90 percent of the cells in the G1 phase of the division cycle, was not static and exhibited a low level of mitotic activity with prolonged interdivision times. These cells also exhibited a greatly reduced (30 percent) protein content which occurred within the first 7 days of cultivation with 0.5 percent serum. Cells arrested by incubation with low serum medium and subsequently recovered to a proliferative state underwent an equivalent or greater number of population doublings with a concomitant extension of in vitro calendar time when compared to growth controls. This indicated that the number of mitotic events and not the length of time in culture was the primary determinant of in vitro lifespan. The ultimate passage level achieved by experimental cells as compared to controls was different in the three cell strains studied. A gradient of attained passage number according to donor age was established with cells from younger donors reaching a significantly greater number than those from older donors. The use of arrested cell populations for the study of cellular senescence offers a unique opportunity to have an in vitro system which may more closely approximate those in vivo tissues which normally do not exhibit a rapid rate of proliferation. By the use of this system numerous biochemical parameters can be investigated at various cell ages without the interference of proliferative processes.

Adult↗

PubMed2005

Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins.

Sir2 is a NAD+-dependent protein deacetylase that extends lifespan in yeast and worms. This study examines seven human proteins homologous to Sir2 (SIRT1 through SIRT7) for cellular localization, expression profiles, protein deacetylation activity, and effects on human cell lifespan. We found that: 1) three nuclear SIRT proteins (SIRT1, SIRT6, and SIRT7) show different subnuclear localizations: SIRT6 and SIRT7 are associated with heterochromatic regions and nucleoli, respectively, where yeast Sir2 functions; 2) SIRT3, SIRT4, and SIRT5 are localized in mitochondria, an organelle that links aging and energy metabolism; 3) cellular p53 is a major in vivo substrate of SIRT1 deacetylase, but not the other six SIRT proteins; 4) SIRT1, but not the other two nuclear SIRT proteins, shows an in vitro deacetylase activity on histone H4 and p53 peptides; and 5) overexpression of any one of the seven SIRT proteins does not extend cellular replicative lifespan in normal human fibroblasts or prostate epithelial cells. This study supports the notion that multiple human SIRT proteins have evolutionarily conserved and nonconserved functions at different cellular locations and reveals that the lifespan of normal human cells, in contrast to that of lower eukaryotes, cannot be manipulated by increased expression of a single SIRT protein.

Cell Line↗

PubMed2006

Hypothesis: cell volume limits cell divisions.

Mammalian somatic cells and also cells of the yeast Saccharomyces cerevisiae are capable of undergoing a limited number of divisions. Reaching the division limit is referred to, apparently not very fortunately, as replicative aging. A common feature of S. cerevisiae cells and fibroblasts approaching the limit of cell divisions in vitro is attaining giant volumes. In yeast cells this phenomenon is an inevitable consequence of budding so it is not causally related to aging. Therefore, reaching a critically large cell volume may underlie the limit of cell divisions. A similar phenomenon may limit the number of cell divisions of cultured mammalian cells. The term replicative (generative) aging may be therefore illegitimate.

Animals↗

PubMed2005

Gerontomodulatory and youth-preserving effects of zeatin on human skin fibroblasts undergoing aging in vitro.

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.

Cellular Senescence↗

PubMed1994

Identification of a defect in the phospholipase D/diacylglycerol pathway in cellular senescence.

Normal cells become senescent in culture after a limited number of population doublings becoming unable to respond to mitogens. This raises the possibility of defects in mitogenic signaling pathways in cellular senescence. In contrast to young human diploid fibroblasts (HDF), their senescent counterparts failed to undergo protein kinase C translocation in response to serum stimulation. On the other hand, phorbol 12-myristate 13-acetate was equally active in inducing protein kinase C translocation in young and senescent HDF. This suggested a defect in generation of the endogenous activator of protein kinase C, diacylglycerol. Stimulation of young HDF with serum resulted in 3-4-fold generation of diacylglycerol (DAG). In contrast, senescent cells displayed insignificant DAG formation in response to serum. The mechanism of DAG generation was investigated next. In young HDF, serum induced a 5-fold activation of the phospholipase D (PLD) pathway as measured by the incorporation of exogenous ethanol into phosphatidylethanol, which is a measure of the transphosphatidylation reaction of PLD. In contrast, PLD in senescent cells was not activated by serum. Since senescent cells demonstrate significant elevations in the level of endogenous ceramide, the impact of ceramide on the PLD/DAG pathway was also investigated. A soluble analog of ceramide, C6-ceramide, was found to inhibit serum-stimulated DAG accumulation and PLD activation in young cells. These data demonstrate for the first time a defect in PLD activation in cellular senescence and suggest that ceramide may be responsible for the inhibition of this pathway.

Cells, Cultured↗

PubMed1998

Accelerated aging of dermal fibroblast-like cells from the senescence-accelerated mouse (SAM): acceleration of changes in DNA ploidy associated with in vitro cellular aging.

Accelerated changes in the DNA ploidy associated with in vitro aging were examined in fibroblast-like cells isolated from the dorsal dermis of newborn SAMP11 (accelerated senescence-prone, short-lived) mice, and were compared to changes observed in cell lines from SAMR1 (accelerated senescence-resistant, long-lived) mice. Flow cytometric analysis of the DNA content in confluent cells and chromosome analysis in mitoses revealed that the diploid cells were being replaced with tetraploid cells until a growth crisis; thereafter, hypotetraploid cells became predominant, accompanied by immortalization. The number of mitoses decreased as the crisis ensued, then increased. Although these changes were observed in the cell lines from both strains of mice, the changes occurred more rapidly and at earlier population doublings in the cell lines from the SAMP11 mice. These results suggest that the cell lines from SAMP11 mice might have higher susceptibility to factors that cause polyploidization, including oxidative stress.

Aging↗

PubMed1998

Thiamine deficiency decreases steady-state transketolase and pyruvate dehydrogenase but not alpha-ketoglutarate dehydrogenase mRNA levels in three human cell types.

Reductions in the levels and activities of enzymes that utilize thiamine diphosphate (ThDP) as a cofactor are thought to be responsible for the tissue damage suffered during thiamine deficiency. Although loss of cofactor can account in part for loss of enzyme activity, thiamine and its phosphorylated derivatives may also regulate the expression of the genes encoding these proteins. To examine this possibility, steady-state mRNA levels for three ThDP-dependent enzymes were measured in human fibroblasts, lymphoblasts and neuroblastoma cells cultured under conditions of thiamine sufficiency and deficiency. In all three cell types, the mRNA levels of transketolase and the E1beta subunit of pyruvate dehydrogenase complex were lower in thiamine-deficient cultures. In contrast, mRNA levels for a ThDP-binding subunit of alpha-ketoglutarate dehydrogenase, the E1 subunit did not differ. These results indicate that thiamine or a thiamine metabolite regulates the expression in humans of some, but not all, genes encoding ThDP-utilizing enzymes.

Blotting, Northern↗

PubMed1987

Cyclic activity of the receptors of cobalamin bound to transcobalamin II.

The activity of receptors specific for human transcobalamin II-Cobalamin (TC II-Cbl) were measured in virus-transformed lymphoblasts, hepatocytes (hepatoma) and diploid fibro lasts. In all three types of human cells the receptor activity increased as cells went from a resting phase to the most actively dividing phase. Receptor activity declined as cell division slowed. The changes in activity of lymphoblasts and hepatocytes were produced by changes in receptor number and not by changes in affinity between receptors and TC II-Cbl. The basis of the change in fibroblasts was not clear. The Cbl-dependent methionine synthetase activity of fibroblasts, in contrast, tended to be greatest when the cultures were confluent and replication had slowed. As the fibroblasts became senescent the receptor activity for TC II-Cbl declined and the fluctuations with the phase of the cell were blunted. However, the release of apo TC II from the cells was maintained. These observations must be taken into consideration when the respective cells are used as models. Even more important are the implications of the observations of the changes in receptor activity for TC II-Cbl for the regulation of the entry of Cbl into cells.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1996

Reverse transcriptase inhibitors suppress telomerase function and induce senescence-like processes in cultured mouse fibroblasts.

Spontaneous transformation of mouse embryonic fibroblasts in the presence of the reverse transcriptase inhibitors azidothymidine and carbovir led to the formation of telomerase-free clones. After prolonged cultivation of fibroblasts in the presence of carbovir, resistant cells with a very high level of telomerase activity were obtained. Azidothymidine and carbovir, but not dideoxycytidine, induced senescence-like processes in cultures of immortal mouse fibroblasts. After long-term incubation, cell proliferation gradually decreased, their morphology becoming similar to that of the senescent ones. The process was reversible: after inhibitor removal, the cells, including the giant ones, entered mitoses. All these data suggest that reverse transcriptase inhibitors block telomerase function in mouse cells.

3T3 Cells↗

PubMed2002

Modulation of the expression of p16INK4a and p14ARF by hnRNP A1 and A2 RNA binding proteins: implications for cellular senescence.

Cellular senescence is a terminal growth phase characteristic of normal human diploid fibroblasts. Altered gene expression during cellular senescence is numerous compared to that of younger proliferative cells in culture. We have previously reported that the levels and activities of hnRNP A1 and A2 RNA binding proteins are decreased in senescent human fibroblasts. Both proteins are multifunctional and may influence the expression of mRNA isoforms during development. In this study, we tested whether overexpression of either protein could modulate the mRNA isoforms of the INK4a locus, specifically p14(ARF) and p16(INK4a). Both INK4a mRNA isoforms have been shown to be growth suppressors and deletions of this locus allow cells to escape cellular senescence. We have found that increasing the ratio of either hnRNP A1 or A2 over that of splicing factor SF2/ASF results in the preferential generation of the p14(ARF) isoform. Overexpression of A1 or A2 RNA binding proteins also appear to increase the steady state mRNA levels of both isoforms, suggesting that in addition to alternative splicing, A1 and A2 may effect p14(ARF) and p16(INK4a) mRNA stability. A constitutive decrease in the ratio of hnRNP A1 or A2 to SF2/ASF in senescent fibroblasts is typically accompanied by an increase in the level of p16(INK4a) isoform. Our studies suggest that hnRNP A1 and A2 may exert an important role during replicative senescence by altering expression of cell cycle regulatory proteins through mRNA metabolism.

Cell Cycle Proteins↗

PubMed1996

[Role of oxidative stress in aging].

In this article is presented a synthesis of the complex relationships between oxidative stress ageing. Free radicals are produced in a continous way but they are also quickly removed by very efficient defence systems. The studies trying to increase the antioxidants uptake in the diet or to produce transgenic animals overexpressing antioxidant enzymes did not lead to increased life span in mammals. Only transgenic drosophils for SOD and catalase lived longer than the parents wild type. At the cellular level, oxidative stresses speed up the passage of fibroblasts from young to old morphotypes. This shift increases with stress intensity but is also influenced by the quantity of energy available to the cell. Thermodynamics of open systems very well account for the experimental observations of cell response to stresses, oxidative stress being only one of the challenges faced by living cells. Oxidative stresses must be considered in relationship with the level of defence and turnover systems as affecting continuously various cellular functions with sometimes excessive production in pathological situations. In this case, a global response of the cell is observed which is the result of complex mechanisms linked to cell activation and changes in gene transcription.

Aging↗

PubMed1991

Response of Müller cells to growth factors alters with time in culture.

We have developed an explant culture technique, using the retinae of newborn guinea pigs, that reliably yields cultures of Müller cells showing uniform morphology and phenotype. Since the guinea pig retina is avascular and lacks astrocytes, Müller cells are the only glial cell-type and the only vimentin-positive population present. Virtually all passaged cells (greater than 98%) contain vimentin-positive intermediate filaments and no glial fibrillary acidic protein (GFAP) has been detected using a range of GFAP antibodies known to label astrocytes in the guinea pig optic nerve. Most vimentin-positive cells were also labeled with an antibody to carbonic anhydrase II, an enzyme which in the retina is specific for Müller cells. Proliferating Müller cells were identified within the inner nuclear layer of retinal fragments as early as 2 days in culture using bromodeoxyuridine (BrdU) and vimentin double labeling. Cultured Müller cells change their growth characteristics with successive passaging. The length of the cell cycle increases from 25.4 h for cells at first passage, to 66.7 h for cells at fourth passage. Altered responses to mitogens were also observed with passaging. First-passage cultures responded to basic fibroblast growth factor (bFGF) but not to several other factors tested including interleukin-2 (IL-2). In contrast, older cultures were highly responsive to IL-2 but showed a minimal response to bFGF. The altered responsiveness to mitogens observed in vitro may be relevant to changes in growth control of Müller cells in the developing and mature retina. The guinea pig retina provides an ideal mammalian tissue for generating Müller cell cultures that are free of astrocytes, endothelial cells, and pericytes, the most frequent contaminants of retinal glial cultures. The monolayers obtained show a high degree of homogeneity and are well suited for studies of Müller cell function.

Animals↗

PubMed1998

The nature of gerontogenes and vitagenes. Antiaging effects of repeated heat shock on human fibroblasts.

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.

Aging↗

PubMedDate not supplied

Physiological oxygen tension is relevant to MHC-1 expression, spontaneous transformation, and interferon response of in vitro aging murine fibroblasts.

Working from the hypothesis that modest deviations from physiological oxygen tension will influence cell characteristics important for infections/immunity and tumor development, cells were studied at three oxygen tensions during in vitro aging. Primary mouse embryo fibroblasts were established and subsequently passaged at 3, 6, and 18 kPa oxygen tension (6 representing normal tissue tension and 18 being the conventionally tension at in vitro cultures). The growth rate was slightly higher at 6 than 3 and 18 kPa. All cultures eventually stopped growing and subsequently transformed to nonmalignant cells with unlimited growth capacity. Cells kept at 3 kPa reached the highest number of cell doublings before crisis. Stimulation with PolyI:C resulted in detectable interferon response only at the high oxygen tension, and after transformation none of the cultures responded with interferon production. Expression of the major histocompatibility complex H-2K was elevated above and below physiological oxygen tension, indicating regulatory processes optimizing MHC expression at about physiological oxygen tension.

Animals↗

PubMed1998

Telomerase activity during spontaneous immortalization of Li-Fraumeni syndrome skin fibroblasts.

Li-Fraumeni Syndrome (LFS) is characterized by heterozygous germline mutations in the p53 gene. Accompanied by genomic instability and loss or mutation of the remaining wild type p53 allele, a low frequency of spontaneous immortalization in LFS fibroblasts occurs. It is believed that the loss of p53 wild type function contributes to immortalization of these LFS fibroblasts, but it is not clear if this is sufficient. Because stabilization of telomere length is also thought to be a necessary step in immortalization, telomerase activity, expression of the telomerase RNA component (hTR) and telomere length were anlaysed at various passages during the spontaneous immortalization of LFS skin fibroblasts. One LFS strain which immortalized, MDAH087 (087), had no detectable telomerase activity whereas another LFS strain which immortalized, MDAH041 (041), had detectable telomerase activity. In preimmortal cells from both strains, hTR was not detected by in situ hybridization. Immortal 087 cells remained negative for hTR, while immortal 041 cells demonstrated strong hTR in situ hybridization signals. 087 cells had long and heterogenous telomeres whereas telomeres of 041 cells had short, stable telomere lengths. Tumorigenicity studies in nude mice with ras-transformed 087 and 041 cells resulted in both cell lines giving rise to tumors and retaining telomerase status. Overall these results suggest that strain specificity may be important in telomerase re-activation and that both abrogation of p53 function and a mechanism to maintain telomeres are necessary for immortalization.

Animals↗