SA beta Gal staining: biomarker or delusion.
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Animals↗
Biomedical subjects
V J Cristofalo
Publications and source records attributed to V J Cristofalo.
Lack of Elk-1 phosphorylation and dysregulation of the extracellular regulated kinase signaling pathway in senescent human fibroblast.
Replicative senescence is characterized by numerous phenotypic alterations including the loss of proliferative capacity in response to mitogens and numerous changes in gene expression including impaired serum inducibility of the immediate-early genes c-fos and erg-1. Transcription of c-fos in response to mitogens depends on the activation of a multiprotein complex formed on the c-fos serum response element (SRE), which includes the transcription factors SRF (serum response factor) and TCF (ternary complex factor). Our data indicate that at least two defects are responsible for the decreased c-fos transcription in senescent cells, one caused by diminished DNA binding activity of the SRF and another resulting from impaired activation of the TCF, Elk-1. In nuclei isolated from serum stimulated senescent cells the activating phosphorylation of p62(TCF)/Elk-1, which is catalyzed by the members of the extracellular-regulated kinase (ERK) family was strikingly diminished and correlated with a decrease in the abundance of activated ERK proteins. In contrast, in total cell lysates ERK phosphorylation and ERK activity (normalized to total protein) reached similar levels following stimulation of early- and late-passage cells. Interestingly, senescent cells consistently exhibited higher ERK protein abundance. Thus, the proportion of phosphorylated (active) ERK molecules in stimulated senescent cells was lower than in early passage cells. The accumulation of unphosphorylated ERK molecules in senescent cells correlated with the diminished abundance of phosphorylated (active) MEK. These data indicate that in senescent cells there is a general dysregulation in the ERK signaling pathway, which results in the accumulation of inactive ERK molecules, decreased abundance of active ERK in the nucleus of senescent cells, and subsequent lack of activation of the transcription factor TCF(Elk-1). These impairments, together with the impaired DNA binding activity of SRF, could potentially account for the lack of c-fos expression in senescent cells and for multiple other molecular changes dependent upon this pathway.
Blotting, Western↗
Is increased arachidonic acid release a cause or a consequence of replicative senescence?
Arachidonic acid (AA) has been related to both stimulation and inhibition of cellular proliferation. During replicative senescence of human fibroblasts, increased levels of AA have been thought to play a causal role in the limited proliferative capacity of the cells. To clarify the role of AA in the proliferation of normal fibroblasts and in cellular senescence, we examined uptake from and release of AA into the culture media and its effects on DNA synthesis. Our results indicate that some aspects of AA metabolism in normal human fibroblasts aged in culture are significantly different in comparison to early passage cells. Particularly, AA release following different mitogenic stimulation is higher in senescent than in young cells. Notwithstanding this significant difference, AA, at the concentration used, has no inhibitory effect on fibroblast DNA synthesis. Moreover AA and prostaglandins are responsible for the proliferative block in neither senescent cells nor mediate ceramide inhibition of DNA synthesis. So our results suggest that the increasing AA release is not causal, but rather the result of in vitro aging.
Aging↗
Loss of HuR is linked to reduced expression of proliferative genes during replicative senescence.
Cellular aging is accompanied by alterations in gene expression patterns. Here, using two models of replicative senescence, we describe the influence of the RNA-binding protein HuR in regulating the expression of several genes whose expression decreases during senescence. We demonstrate that HuR levels, HuR binding to target mRNAs encoding proliferative genes, and the half-lives of such mRNAs are lower in senescent cells. Importantly, overexpression of HuR in senescent cells restored a "younger" phenotype, while a reduction in HuR expression accentuated the senescent phenotype. Our studies highlight a critical role for HuR during the process of replicative senescence.
Aged↗
Regulation of EPC-1/PEDF in normal human fibroblasts is posttranscriptional.
The EPC-1 (early population doubling level cDNA-1) gene, also known as pigment epithelium-derived factor, encodes a protein belonging to the serine protease inhibitor (serpin) superfamily that has been reported to inhibit angiogenesis and proliferation of several cell types. We have previously reported that the EPC-1 mRNA and the secreted EPC-1 protein are expressed at levels more than 100-fold higher in early passage, G(0), WI-38 cells compared to either proliferating or senescent WI-38 fibroblasts. To examine the molecular mechanisms that regulate changes in EPC-1 gene expression in WI-38 cells, we isolated and characterized the human EPC-1 gene and determined the mRNA cap site. Transcriptional assays showed no change in the transcription rates of EPC-1 between young proliferating, quiescent, and senescent WI-38 cells. These results suggest posttranscriptional regulation of the EPC-1 gene. Reverse transcriptase polymerase chain reaction measurements (of hnRNA) indicate regulation at the hnRNA level. The regulation of the EPC-1 gene at the level of hnRNA can explain the observed slow increase in the steady-state EPC-1 mRNA levels when cells become quiescent. The reduction of EPC-1 mRNA levels that occurs when cells exit G(0) and are induced to proliferate can be accounted for by a reduction of the EPC-1 mRNA stability in stimulated cells as compared to quiescent cells.
Animals↗
Mitogen-independent phosphorylation of S6K1 and decreased ribosomal S6 phosphorylation in senescent human fibroblasts.
The p70 ribosomal S6 kinase (S6K1) is rapidly activated following growth factor stimulation of quiescent fibroblasts and inhibition of this enzyme results in a G(1) arrest. Phosphorylation of the ribosomal S6 protein by S6K1 regulates the translation of both ribosomal proteins and initiation factors, leading to an increase in protein synthesis. We have examined the activation of S6K1 in human fibroblasts following mitogen stimulation. In early passage fibroblasts S6K1 is activated following serum stimulation as evidenced by increased kinase activity and site-specific phosphorylation. In contrast, site-specific phosphorylation of S6K1 at Thr421/Ser424 is diminished in senescent fibroblast cultures. A second phosphorylation site within S6K1 (Ser411) is phosphorylated even in the absence of serum stimulation and the enzyme shows increased phosphorylation as judged by decreased electrophoretic mobility. Inhibitor studies indicate that this phosphorylation is dependent upon the mammalian target of rapamycin, PI 3-kinase, and the MAPK pathway. In order to understand the consequences of the altered phosphorylation of the S6K1, we examined the phosphorylation state of the ribosomal S6 protein. In early passage fibroblasts the ribosomal S6 protein is phosphorylated upon serum stimulation while the phosphorylation of the ribosomal S6 protein is drastically reduced in senescent fibroblasts. These results suggest that the intracellular regulators of S6K1 are altered during replicative senescence leading to a deregulation of the enzyme and a loss of ribosomal S6 phosphorylation.
Blood Proteins↗
Is beta-galactosidase staining a marker of senescence in vitro and in vivo?
Cytochemically detectable beta-galactosidase (beta-gal) at pH 6.0 has been reported to increase during the replicative senescence of fibroblast cultures and has been used widely as a marker of cellular senescence in vivo and in vitro. In this study, we have characterized changes in senescence-associated (SA) beta-gal staining in early and late passage cultures, cultures established from donors of different ages, virally immortalized cells, and tissue slices obtained from donors of different ages. The effects of different culture conditions were also examined. While we confirm the previous report that SA beta-gal staining increased in low-density cultures of proliferatively senescent cells, we were unable to demonstrate that it is a specific marker for aging in vitro. Cultures established from donors of different ages stained for SA beta-gal activity as a function of in vitro replicative age, not donor age. We also failed to observe any differences in SA beta-gal staining in skin cells in situ as a marker of aging in vivo. The level of cytochemically detectable SA beta-gal was elevated in confluent nontransformed fibroblast cultures, in immortal fibroblast cultures that had reached a high cell density, and in low-density, young, normal cultures oxidatively challenged by treatment with H2O2. Although we clearly demonstrate that SA beta-gal staining in cells is increased under a variety of different conditions, the interpretation of increased staining remains unclear, as does the question of whether the same mechanisms are responsible for the increased SA beta-gal staining observed in senescent cells and changes observed in cells under other conditions.
Adolescent↗
Extension of cell life-span and telomere length in animals cloned from senescent somatic cells.
The potential of cloning depends in part on whether the procedure can reverse cellular aging and restore somatic cells to a phenotypically youthful state. Here, we report the birth of six healthy cloned calves derived from populations of senescent donor somatic cells. Nuclear transfer extended the replicative life-span of senescent cells (zero to four population doublings remaining) to greater than 90 population doublings. Early population doubling level complementary DNA-1 (EPC-1, an age-dependent gene) expression in cells from the cloned animals was 3.5- to 5-fold higher than that in cells from age-matched (5 to 10 months old) controls. Southern blot and flow cytometric analyses indicated that the telomeres were also extended beyond those of newborn (<2 weeks old) and age-matched control animals. The ability to regenerate animals and cells may have important implications for medicine and the study of mammalian aging.
Animals↗
Effects of donor age on the expression of a marker of replicative senescence (EPC-1) in human dermal fibroblasts.
EPC-1 (early population doubling level cDNA-1) is a quiescence-specific gene expressed at high levels by early passage WI-38 fibroblasts under conditions of either density-dependent growth arrest or serum deprivation. Late passage WI-38 cells lose the ability to express EPC-1 under all conditions tested. The decline in EPC-1 mRNA is gradual during the replicative life span and correlates inversely with the population doubling level (PDL) of the cells. The objective of this study was to determine whether the decline in EPC-7 mRNA abundance observed during proliferative senescence also occurs in cultures derived from donors of different ages. To address this question, we examined the abundance of EPC-1 mRNA in 28 skin fibroblast lines established from healthy donors of different ages ranging from 12 fetal weeks to 94 years. EPC-1 expression was measured, under conditions of growth arrest, prior to the end of the replicative life span of the cultures. Despite some variability in steady-state transcript levels among the cell lines, EPC-1 expression was significantly lower in cells derived from the fetal donor group (12-20 gestational weeks) than in cells derived from adult donors. An in vitro age-dependent decline in EPC-1 expression was observed in all the skin lines examined, independent of donor age; however, no significant difference was observed between the young adult donor group (17-33 years) and the old adult donor group (78-94 years). Thus, expression of EPC-1 is linked to the replicative age of the cells and whether the cells are derived from fetal skin or adult skin. In adults, EPC-1 expression is independent of donor age.
Adolescent↗
Differences in electron transport potential, antioxidant defenses, and oxidant generation in young and senescent fetal lung fibroblasts (WI-38).
The activities and mRNA abundances of enzymes that regulate the rate of electron flow through the electron transport chain (ETC), including NADH dehydrogenase, succinate dehydrogenase, and cytochrome c oxidase, were examined in young and senescent fetal lung fibroblasts (WI-38). We also determined the activities and mRNA abundances of antioxidant defenses including superoxide dismutase, catalase, and glutathione peroxidase. We confirmed our previous report of a senescence-related increase in the abundance of ND4, a mitochondrially encoded subunit of NADH dehydrogenase. The activities of cytochrome c oxidase and NADH dehydrogenase were also elevated in senescent cultures. No differences were observed in the mRNA abundances of COX-1, a mitochondrially encoded subunit of cytochrome c oxidase or of nuclearly encoded subunits of various electron transport components (SD, COX-4, and ND 51). Lucigenin-detected chemiluminescence and H2O2 generation were both elevated in senescent cells. Catalase activity was also elevated in senescent fibroblasts. However, no differences in catalase mRNA abundance were observed. A small decrease in GSH peroxidase (GPx) mRNA abundance was observed in senescent cells. No other changes in the activities or mRNA abundances of any of the antioxidant defenses were observed in early and late passage cultures. The relationships between oxidant generation, mitochondrial enzyme activities, and antioxidant defense observed during proliferative senescence are dissimilar to those detected between fetal and postnatal fibroblasts as well as those found between fibroblast lines obtained from young and old individuals. The relevance of the differences between these models is discussed.
Acridines↗
Cell culture aging: insights for cell aging in vivo?
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Animals↗
Relationship between donor age and the replicative lifespan of human cells in culture: a reevaluation.
Normal human diploid fibroblasts have a finite replicative lifespan in vitro, which has been postulated to be a cellular manifestation of aging in vivo. Several studies have shown an inverse relationship between donor age and fibroblast culture replicative lifespan; however, in all cases, the correlation was weak, and, with few exceptions, the health status of the donors was unknown. We have determined the replicative lifespans of 124 skin fibroblast cell lines established from donors of different ages as part of the Baltimore Longitudinal Study of Aging. All of the donors were medically examined and were declared "healthy," according to Baltimore Longitudinal Study of Aging protocols, at the time the biopsies were taken. Both long- and short-lived cell lines were observed in all age groups, but no significant correlation between the proliferative potential of the cell lines and donor age was found. A comparison of multiple cell lines established from the same donors at different ages also failed to reveal any significant trends between proliferative potential and donor age. The rate of [3H]thymidine incorporation and the initial rates of growth during the first few subcultivations were examined in a subset of cell lines and were found to be significantly greater in fetal lines than in postnatal lines. Cell lines established from adults did not vary significantly either in initial growth rate or in [3H]thymidine incorporation. These results clearly indicate that, if health status and biopsy conditions are controlled, the replicative lifespan of fibroblasts in culture does not correlate with donor age.
Adolescent↗
Senescent WI-38 fibroblasts overexpress LPC-1, a putative transmembrane shock protein.
We have recently reported the isolation of cDNAs for a number of genes that are differentially expressed between nonproliferating early (young) and late (senescent) population doubling level (PDL) WI-38 human, fetal lung-derived, fibroblast-like cells. We now demonstrate that one of these isolates, LPC-1 (Late PDL cDNA-1), derives from an approximately 2.9-kb mRNA species that is expressed at a two- to fivefold higher level in serum-starved, confluent, senescent versus similarly treated young WI-38 cells. Nucleotide sequence analysis of this cDNA confirms its identity with that of a cDNA encoding a marker (p63) for the rough endoplasmic recticulum and a related swine hepatic cardiogenic shock protein. We show that LPC-1 expression in early PDL WI-38 cells is strictly cell cycle-regulated and its expression peaks 9-12 h after serum stimulation of G0 cultures. The steady state levels of LPC-1 transcript in early PDL cells preceeding and following its peak expression are low, reflecting basal levels seen in G0 upon removal of serum. Late PDL cells, however, seem to have lost this tight cell cycle regulation seen in early PDL cells and inappropriately express high levels of the transcript after serum stimulation. Specific antiserum detects a protein of approximately 63 kDa by Western analysis and elicits intense cytoplasmic staining of senescent fibroblasts by immunohistochemistry. Related genomic sequences are found in all mammalian species examined as well as in the chicken. These findings are consistent with the hypothesis that senescent WI-38 cells exhibit a state of growth arrest fundamentally distinct from that of quiescent (G0) young cells.
Animals↗
A phosphatidylinositol 3-kinase inhibitor induces a senescent-like growth arrest in human diploid fibroblasts.
The signal transduction cascade initiated by the activation of phosphoinositide 3-kinase (PI-3 kinase) is implicated in mitogenic and antiapoptotic signaling generated by growth factors in a variety of cell types. We have examined the consequences of an inhibition of this pathway in human diploid fibroblasts. We find that a specific PI-3 kinase inhibitor (LY294002) causes growth arrest in these cells accompanied by changes in gene expression that are similar to those seen during cellular senescence. A second inhibitor, PD58029, which is specific for the mitogen-activated protein kinase kinase 1 (MEK-1), also induces a growth arrest but does not induce the same spectrum of gene expression. The pattern of gene expression in the presence the MEK-1 inhibitor is similar to that seen during growth arrest induced by serum starvation. The specific phenotypic changes seen following inhibition of PI-3 kinase are: an increase in beta-galactosidase activity; a decrease in EPC-1 gene expression; and a dramatic increase in collagenase gene expression. Thus, growth arrest with a PI-3 kinase inhibitor induces a senescent-like phenotype that is not seen when cells are growth arrested by either serum starvation or a MEK-1 inhibitor.
Cell Division↗
Antioxidants stimulate transcriptional activation of the c-fos gene by multiple pathways in human fetal lung fibroblasts (WI-38).
We have examined the effects of three structurally distinct antioxidants (N-acetylcysteine [NAC], Trolox C [a water-soluble vitamin E derivative], and nordihydroguaiaretic acid [NGA]) on the expression of the c-fos gene over a 2-hour period. Determination of cellular glutathione concentration (the primary determinant of the cellular redox state) over the same time-course verifies that all the compounds studied cause an increase in cellular reduction potential. The level of c-fos messenger RNA increased rapidly in response to micromolar concentrations of these compounds, reaching a peak in 30-60 minutes. Induction of c-fos expression by these antioxidants is at least partly due to an increase in transcription, as determined by nuclear run-on assay. Down regulation of protein kinase C (PKC) by pretreatment for 24 hours with 500 nm PMA prevents induction by subsequent stimulation with either PMA or NGA. NAC induction of c-fos is unaffected by PMA pretreatment, while Trolox C superinduced c-fos following PMA pretreatment. None of these treatments stimulated translocation of PKC-alpha from the cytosol to the membrane. These results suggest that increasing the intracellular reducing potential induces c-fos expression through multiple pathways.
Acetylcysteine↗
Enhanced proliferation of human fibroblasts, in the presence of dexamethasone, is accompanied by changes in p21Waf1/Cip1/Sdi1 and the insulin-like growth factor type 1 receptor.
The addition of dexamethasone (dex) to human fibroblast cultures has been found to elicit enhanced proliferation. This enhancement is manifested by an increase in the initial growth rate, saturation density, and proliferative life span of WI-38 fibroblast cultures grown in the presence of dex. We examined the acute effects of dex on a number of growth-related genes in WI-38 cells. Our results show a decrease in the level of the cyclin-dependent kinase inhibitor p21Waf1/Cip1/sdi1 in response to dex. In addition, the level of the insulin-like growth factor type 1 receptor (IGF-1R) is increased in dex-treated cells. These changes are correlated with changes in the activity of the p21waf1/Cip1/Sdi1 and IGF-1R promoters. The results presented in this report suggest that dex may delay growth arrest in response to contact inhibition, as well as during cellular senescence. Thus, dex may act at multiple levels to enhance cellular proliferation in WI-38 cells: first, to decrease the level of an inhibitor of cell-cycle progression, and second, to increase the sensitivity of WI-38 cells to the proliferative effects of IGF-1. These acute effects may cooperate with other, as yet uncharacterized effects, to result in the enhanced proliferation seen in the presence of dex.
Cell Division↗
Age-dependent modifications of gene expression in human fibroblasts.
Normal human fibroblasts exhibit a limited proliferative potential in culture. When populations are serially subcultured, they grow well initially, but ultimately reach a stage when they are no longer able to proliferate in response to mitogenic stimuli. This state is designated "replicative senescence". In addition to failure to proliferate, numerous morphological and physiological changes characterize the senescent phenotype. Both stochastic and genetic mechanisms have been postulated as causal effectors of the aging process. However, the pathway leading to cellular senescence is likely to be complex with numerous changes. In this article we provide an overview of cell and molecular changes that occur during cell aging, with special emphasis on signal transduction pathways and cell cycle proteins that are likely to play key roles in determining the limited replicative life span and the changes that occur.
Animals↗