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PubMed1993

Spontaneous immortalization of mouse fibroblasts involves structural changes in senescence inducing protein, mortalin.

Mortalin, a novel member of mouse heat shock protein 70 (hsp70) family, is seen to distinguish the cellular mortal and immortal phenotypes by virtue of its cytosolic and perinuclear distribution, respectively. We report here that the cytosolic and perinuclear forms of mortalin from CD1-ICR mouse embryonic fibroblasts and NIH 3T3 cells, respectively, differ by two amino acids, can be distinguished on two-dimensional SDS-polyacrylamide gel. The perinuclear mortalins from RS-4 and Balb/c 3T3 cells harbor the same two amino acids as that of NIH 3T3 cells. However, these when analyzed with C-MEF mortalin did not exhibit the mobility shift equivalent to C-MEF and NIH 3T3 mortalins. The data indicate that the perinuclear mortalin from different immortal fibroblasts are not identical and implicate the possibility of additional structural changes in mortalin during immortalization. Such differences may also contribute to the differential in vitro growth characteristics of these immortal cells.

3T3 Cells↗
PubMed1995

Increased expression of p21Sdi1 in adrenocortical cells when they are placed in culture.

Expression of the cell cycle inhibitor p21Sdi1/WAF1/Cip1 was investigated in a differentiated cell type, the adrenocortical cell, at different stages of culture, from the preparation of cells from the adrenal gland to senescence after long-term growth. In bovine adrenocortical cells, expression of SDI1 was much higher in culture than in vivo. Elevation of SDI1 mRNA, accompanied by elevation of the level of a protein reacting with anti-p21Sdi1 antibodies, was observed as early as 3 h after the start of the tissue dissociation procedure used to prepare cells for culture. This level of expression was then maintained during plating and subsequent long-term growth in culture. Growth and quiescence in bovine adrenocortical cells can be modulated by inclusion or removal of FGF from the culture medium. In these cells SDI1 mRNA was not increased by long-term mitotic quiescence resulting from FGF deprivation. In cultured fetal human adrenocortical cells, SDI1 mRNA was also detected at all stages of the culture life span, including 2 days after isolation of cells from the adrenal gland and plating in culture. Mid-life-span cells had higher SDI1 mRNA than senescent human fibroblasts. Clones of human adrenocortical cells nearing senescence in culture had somewhat higher SDI1 mRNA than early passage cells. Thus, SDI1 expression in adrenocortical cells is not associated with mitotic quiescence either in vivo or in vitro, yet isolation of the cells and culturing them exerts a powerful inductive influence on its expression.

Adrenal Cortex↗
PubMed2001

Gene profile of replicative senescence is different from progeria or elderly donor.

In vitro cellular senescence of human diploid fibroblast has been a good model for aging research, which shows similar phenotypes to in vivo aging. Gene expression profiling would provide an insight to understand the mechanism of senescence. Using cDNA microarray containing 384 known genes, we compared the expression profiles of three different types of aging models: replicative senescence, fibroblasts from progeria or from elderly donor. Although all of them showed senescence phenotypes, distinct sets of genes were altered in each group. Pairwise plots or cluster analysis of activation fold of gene expression revealed closer relationships between fibroblasts from progeria or from old individual, but not between replicative senescence fibroblasts and either models. Differential expression pattern of several genes were confirmed by RT-PCR. We suggest that the replicative senescence model might behave differently to other types of aging models due to the distinct gene expression.

Aged↗
PubMed2002

Cell cycle arrest by human cytomegalovirus 86-kDa IE2 protein resembles premature senescence.

Primary human embryo lung fibroblasts and adult diploid fibroblasts infected by the human cytomegalovirus (HCMV) display beta-galactosidase (beta-Gal) activity at neutral pH (senescence-associated beta-Gal [SA-beta-Gal] activity) and overexpression of the plasminogen activator inhibitor type 1 (PAI-1) gene, two widely recognized markers of the process designated premature cell senescence. This activity is higher when cells are serum starved for 48 h before infection, a process that speeds and facilitates HCMV infection but that is insufficient by itself to induce senescence. Fibroblasts infected by HCMV do not incorporate bromodeoxyuridine, a prerequisite for the formal definition of senescence. At the molecular level, cells infected by HCMV, beside the accumulation of large amounts of the cell cycle regulators p53 and pRb, the latter in its hyperphosphorylated form, display a strong induction of the cyclin-dependent kinase inhibitor (cdki) p16(INK4a), a direct effector of the senescence phenotype in fibroblasts, and a decrease of the cdki p21(CIP1/WAF). Finally, a replicative senescence state in the early phases of infection significantly increased the number of cells permissive to virus infection and enhanced HCMV replication. HCMV infection assays carried out in the presence of phosphonoformic acid, which inhibits the virus DNA polymerase and the expression of downstream genes, indicated that immediate-early and/or early (alpha) genes are sufficient for the induction of SA-beta-Gal activity. When baculovirus vectors expressing HCMV IE1-72 or IE2-86 proteins were inoculated into fibroblasts, the increase of p16(INK4a) (observed predominantly with IE2-86) was similar to that observed with the whole virus, as was the induction of SA-beta-Gal activity, suggesting that the viral IE2 gene leads infected cells into senescence. Altogether our results demonstrate for the first time that HCMV, after arresting the cell cycle and inhibiting apoptosis, triggers the cellular senescence program, probably through the p16(INK4a) and p53 pathways.

Cell Cycle↗
PubMed2002

UVB-induced premature senescence of human diploid skin fibroblasts.

In this work, we show that repeated stresses with UVB (290-320 nm) induce stress-induced premature senescence (SIPS) of skin human diploid fibroblasts (HDFs). HDFs at early cumulative population doublings were exposed three or five times to increasing subcytotoxic doses of UVB with one stress per day. After 2 days of recovery, several biomarkers of replicative senescence were established. First, there was an increase in the proportion of cells positive for senescence-associated beta-galactosidase activity. Second, there was a loss of replicative potential as assessed by a very low level of [3H]-thymidine incorporation. Third, the steady-state level of the mRNA of three senescence-associated genes, i.e. fibronectin, osteonectin and SM22, was increased in HDFs at 72 h after three and five exposures to UVB. In conclusion, these results suggest that it is possible to induce SIPS in HDFs after repeated exposures to subcytotoxic doses of UVB. This model could be used to test whether HDFs in UVB-induced premature senescence are able to promote epithelial cell growth and tumorigenesis in skin, as shown recently with HDFs in H(2)O(2)-induced premature senescence.

Cell Line↗
PubMed2002

Differential response of cardiac fibroblasts from young adult and senescent rats to ANG II.

The intracardiac ANG II-forming pathway is activated in the senescent myocardium, raising the possibility of enhanced ANG II effects on cardiac fibroblasts. This study established an in vitro model of cultured cardiac fibroblasts from aged rats to examine if the response of these cells to ANG II is modified in the aged heart. Levels of mRNA encoding renin, angiotensinogen, and the AT(1) receptor subtype in cardiac fibroblasts from young adult and senescent rats were quantified by RT-PCR, net collagen production by a hydroxyproline-based assay, and transforming growth factor (TGF)-beta levels using a commercial kit. In cardiac fibroblasts from young adult rats, ANG II significantly enhanced AT(1) mRNA levels, net collagen production, and TGF-beta production. In fibroblasts from the aged myocardium, ANG II downregulated AT(1) mRNA expression, had a less pronounced effect on net collagen production, and had no effect on TGF-beta production. Such age-related modification of the response of cardiac fibroblasts to ANG II may counteract the effects of augmented intracardiac ANG II production in the senescent heart, limiting fibrogenesis.

Aging↗
PubMed1996

Human fibroblast commitment to a senescence-like state in response to histone deacetylase inhibitors is cell cycle dependent.

Human diploid fibroblasts (HDF) complete a limited number of cell divisions before entering a growth arrest state that is termed replicative senescence. Two histone deacetylase inhibitors, sodium butyrate and trichostatin A, dramatically reduce the HDF proliferative life span in a manner that is dependent on one or more cell doublings in the presence of these agents. Cells arrested and subsequently released from histone deacetylase inhibitors display markers of senescence and exhibit a persistent G1 block but remain competent to initiate a round of DNA synthesis in response to simian virus 40 T antigen. Average telomere length in prematurely arrested cells is greater than in senescent cells, reflecting a lower number of population doublings completed by the former. Taken together, these results support the view that one component of HDF senescence mimics a cell cycle-dependent drift in differentiation state and that propagation of HDF in histone deacetylase inhibitors accentuates this component.

Antigens, Viral, Tumor↗
PubMed2001

TRADD domain of Epstein-Barr virus transforming protein LMP1 is essential for inducing immortalization and suppressing senescence of primary rodent fibroblasts.

Mutation analysis of latent membrane protein 1 (LMP1) in Epstein-Barr virus (EBV)-induced B-cell immortalization revealed two transformation effector sites, TES1 and TES2. TES2 mediates the interaction with tumor necrosis factor receptor-associated death domain protein (TRADD) and plays a key role in transactivating NF-kappa B and AP-1. Recombinant EBV containing LMP1 with TES2 deleted induces a limited proliferation of B cells. The present study shows that a mutant with an LMP1 site-specific mutation at TES2, LMP1(TRADD), initially stimulates cell growth and significantly extends the life span of MEF. However, it is not sufficient for the immortalization of MEF, and MEF-LMP1(TRADD) cells eventually enter growth arrest. Further analysis reveals that although LMP1(TRADD) promotes cell growth, it does not prevent the eventual onset of senescence and the expression of tumor suppressor p16(Ink4a).

Animals↗
PubMed1979

Premature senescence in cultured skin fibroblasts from subjects with cystic fibrosis.

Cultured skin fibroblasts from subjects with cystic fibrosis exhibited normal population doubling times in early passages. After about 13 cumulative population doublings, cystic fibrosis lines doubled more slowly than controls and ceased doubling after about 19 weekly passages. Control lines continued doubling for 27 passages. The premature senescence noted in cells from subjects with cystic fibrosis reconciles controversial observations of cell doubling reported in the literature. Data presented here demonstrate that experiments with cystic fibrosis cells in late passage may generate misleading results since differences from control lines may be ascribed to generalized senile changes rather than to specific results of the cystic fibrosis genotype.

Adolescent↗
PubMed1990

Glycosylases that excise modified DNA pyrimidines in young and senescent human WI-38 fibroblasts.

Cellular DNA is continuously subject to damages by both endogenous and exogenous oxidizing agents. Excision repair in human cells is initiated by DNA glycosylases which remove oxidized bases from DNA. 5-Hydroxymethyluracil-DNA glycosylase excises 5-hydroxymethyluracil from DNA. A different enzyme has glycosylic activity against many ring-saturated DNA pyrimidines. Levels of these enzymes were examined in WI-38 fibroblasts of different culture ages. All glycosylases were assayed by measurements of direct release of modified free bases from their respective DNA substrates. Levels of 5-hydroxymethyluracil-DNA glycosylase were reduced in aging cells. Specific activities of the glycosylase that releases ring-saturated pyrimidines and of uracil-DNA glycosylase were not substantially altered in senescent cells. Therefore, although aging cells might have reduced excision of DNA 5-hydroxymethyluracil, there is no overall age-dependent decrease of DNA glycosylase activities.

Cell Line↗
PubMed2005

Probing tumor phenotypes using stable and regulated synthetic microRNA precursors.

RNA interference is a powerful method for suppressing gene expression in mammalian cells. Stable knock-down can be achieved by continuous expression of synthetic short hairpin RNAs, typically from RNA polymerase III promoters. But primary microRNA transcripts, which are endogenous triggers of RNA interference, are normally synthesized by RNA polymerase II. Here we show that RNA polymerase II promoters expressing rationally designed primary microRNA-based short hairpin RNAs produce potent, stable and regulatable gene knock-down in cultured cells and in animals, even when present at a single copy in the genome. Most notably, by tightly regulating Trp53 knock-down using tetracycline-based systems, we show that cultured mouse fibroblasts can be switched between proliferative and senescent states and that tumors induced by Trp53 suppression and cooperating oncogenes regress upon re-expression of Trp53. In practice, this primary microRNA-based short hairpin RNA vector system is markedly similar to cDNA overexpression systems and is a powerful tool for studying gene function in cells and animals.

Animals↗
PubMed1992

An altered repertoire of fos/jun (AP-1) at the onset of replicative senescence.

With multiple divisions in culture, normal diploid cells suffer a loss of growth potential that leads to replicative senescence and a finite replicative capacity. Using quantitative RT-PCR, we have monitored mRNA expression levels of c-fos, c-jun, JunB, c-myc, p53, H-ras, and histone H4 during the replicative senescence of human fibroblasts. The earliest and the largest changes in gene expression occurred in c-fos and junB at mid-senescence prior to the first slowing in cell growth rates. The basal level of c-fos mRNA decreased to one-ninth that of the early-passage levels, while junB declined to one-third and c-jun expression remained constant. The decline in the basal c-fos mRNA level in mid-senescence should lead to an increase in Jun/Jun AP-1 homodimers at the expense of Fos/Jun heterodimers and may trigger a cascade of further changes in c-myc, p53, and H-ras expression in late-passage senescent fibroblasts.

Base Sequence↗
PubMed1994

Expression of growth arrest-specific (gas) genes in senescent murine cells.

The growth arrest-specific (gas) genes were initially identified on the basis of their preferential expression in mouse fibroblasts during quiescence, followed by down-regulation upon reentry into the cell cycle. We here report studies on the expression of these genes in murine fibroblasts undergoing replicative senescence in vitro. Our results indicate a different behavior between senescent and GO-arrested quiescent fibroblasts. Expression of the gas1 and 6 genes was dramatically reduced in senescent cells. Only basal levels of gas2, 3, and 5 genes were detected in senescent fibroblasts, and they were independent of the growing conditions of the cultures. Down-regulation of the gas1 gene expression in senescent cells was apparently due to reduced transcription of the gas1 gene. This correlates with an altered pattern of factors that bind to the promoter region of the gas1 gene, as measured by band shift assay with nuclear extracts of senescent fibroblasts.

Animals↗
PubMed1994

Decline of protein kinase C activation in response to growth stimulation during senescence of IMR-90 human diploid fibroblasts.

We have studied the activation of protein kinase C (PKC) during senescence of human IMR-90 fibroblasts by analyzing the phosphorylation of its in vivo substrate MARCKS (myristoylated alanine-rich C kinase substrate). It was found that the extent of TPA-induced phosphorylation of MARCKS was not significantly different between young and old IMR-90 fibroblasts. In contrast, the increase of MARCKS phosphorylation after serum stimulation was 4.5-fold in young fibroblasts as compared to 1.8-fold in old fibroblasts. Analysis of PKC by Western blotting showed that the levels of PKC were not changed during senescence of IMR-90 fibroblasts. However, the generation of diacylglycerol in response to serum stimulation declined in old fibroblasts. These results suggest that the efficiency of signal transduction mediated by diacylglycerol generation and PKC activation during the mitogenic response is age-dependent in human IMR-90 fibroblasts.

Blotting, Western↗
PubMed2004

Cisplatin-induced premature senescence with concomitant reduction of gap junctions in human fibroblasts.

To examine the role of gap junctions in cell senescence, the changes of gap junctions in cisplatin-induced premature senescence of primary cultured fibroblasts were studied and compared with the replicative senescent human fibroblasts. Dye transfer assay for gap junction function and immunofluorescent staining for connexin 43 protein distribution were done respectively. Furthermore, cytofluorimetry and DAPI fluorescence staining were performed for cell cycle and apoptosis analysis. p53 gene expression level was detected with indirect immunofluorescence. We found that cisplatin (10 mM) treatment could block cell growth cycle at G1 and induced premature senescence. The premature senescence changes included high frequency of apoptosis, elevation of p53 expression, loss of membranous gap junctions and reduction of dye-transfer capacity. These changes were comparable to the changes of replicative senescence of human fibroblasts. It was also concluded that cisplatin could induce premature senescence concomitant with inhibition of gap junctions in the fibroblasts. Loss of functional gap junctions from the cell membrane may account for the reduced intercellular communication in the premature senescent fibroblasts. The cell system we used may provide a model useful for the study of the gap junction thus promoting agents against premature senescence.

Apoptosis↗
PubMed1985

Membrane glycoprotein changes during the senescence of normal human diploid fibroblasts in culture.

The plasma membrane is intimately involved in a variety of cellular phenomena and may play an important role in the aging of human cells in culture. Significant differences in human diploid fibroblast surface glycoproteins were observed with in vitro aging. Senescent cells bound more concanavalin A (Con A) than young cells and exhibited two distinct classes of binding sites. Cell surfaces of senescent cells incorporated less labelled mannose and more labelled fucose and glucosamine than young cell surfaces. Membranes prepared from older cells were also less effective than membrane preparations from young cells in incorporating mannose from GDP-mannose into a group of oligolipid intermediates, required for the synthesis of glycoproteins with asparagine-linked oligosaccharides. These results demonstrate novel quantitative changes in membrane structure and lectin reactivity in aging human diploid fibroblasts, which must reflect the fundamental physiological modifications in the cell that occur during senescence.

Cell Membrane↗
PubMed2003

Telomere length and the expression of natural telomeric genes in human fibroblasts.

Progressive telomere shortening occurs with division of normal human cells, and eventually leads to replicative senescence. The mechanism by which the shortened telomeres cause growth arrest is largely unknown. Transcriptional silencing of genes adjacent to telomeres, also called telomere position effect, has been hypothesized as a possible mechanism of telomere-mediated senescence. However, there is no report regarding telomere position effect on natural telomeric genes in human cells. To address whether the expression of natural telomeric genes is regulated by telomere length, we combined quantitative RT-PCR with quantitative fluorescence in situ hybridization to comparatively analyze the expression of 34 telomeric genes and telomere length of their 24 corresponding chromosome ends in young and senescent human fibroblasts. We have demonstrated here that telomere length alone is not sufficient to determine the expression status of natural telomeric genes. An extended analysis of a tandem of eight telomeric genes on a single chromosome end revealed a discontinuous pattern of changed expression during telomere shortening and some of the changes are senescence-specific rather than non-dividing-related. These results suggest that the expression of natural telomeric genes may be influenced by alteration of local heterochromatin structure.

Cells, Cultured↗