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PubMed2006

Downstream molecular events in the altered profiles of lysophosphatidic acid-induced cAMP in senescent human diploid fibroblasts.

Lysophosphatidic acid (LPA) is a phospholipid growth factor that acts through G-protein-coupled receptors. Previously, we demonstrated an altered profile of LPA-dependent cAMP content during the aging process of human diploid fibroblasts (HDFs). In attempts to define the molecular events associated with the age-dependent changes in cAMP profiles, we determined the protein kinase A (PKA) activity, phosphorylation of cAMP-response element binding protein (CREB), and the protein expression of CRE-regulatory genes, c-fos and COX-2 in young and senescent HDFs. We observed in senescent cells, an increase in mRNA levels of the catalytic subunit a of PKA and of the major regulatory subunit Ialpha. Senescence-associated increase of cAMP after LPA treatment correlated well with increased CREB phosphorylation accompanying activation of PKA in senescent cells. In senescent cells, after LPA treatment, the expression of c-fos and COX-2 decreased initially, followed by an increase. In young HDFs, CREB phosphorylation decreased following LPA treatment, and both c-fos and COX-2 protein levels increased rapidly. CRE-luciferase assay revealed higher basal CRE-dependent gene expression in young HDFs compared to senescent HDFs. However, LPA-dependent slope of luciferase increased more rapidly in senescent cells than in young cells, presumably due to an increase of LPA-induced CREB phosphorylation. CRE-dependent luciferase activation was abrogated in the presence of inhibitors of PKC, MEK1, p38MAPK, and PKA, in both young and senescent HDFs. We conclude that these kinase are coactivators of the expression of CRE-responsive genes in LPA-induced HDFs and that their changed activities during the aging process contribute to the final expression level of CRE-responsive genes.

Catalytic Domain↗
PubMed1990

Senescent human diploid fibroblasts are able to support DNA synthesis and to express markers associated with proliferation.

The characteristic limited reproductive life-span of normal human fibroblasts in culture is due to a steadily decreasing fraction of cells able to proliferate in the standard rich growth media. We have observed that restricting the growth factor supply to old cells for variable lengths of time in culture increases the fraction of cells that can enter S-phase; although these cells do not go on to divide. Thus, it seems that there is a transient phase between the proliferating state and the irreversibly post-mitotic, senescent state. Perhaps a 'quiescent-G0' state, which can be maintained in the presence of growth factors, is a stage on the pathway to mortalization and senescence.

Antigens↗
PubMed2001

Translocational inefficiency of intracellular proteins in senescence of human diploid fibroblasts.

In order to investigate signal transduction pathways and related changes of actin cytoskeleton organization in cellular senescence, H-ras double mutants--V12S35, V12G37, and V12C40--were constitutively expressed in human foreskin fibroblast (HDF). Senescent HDF cells as well as the H-ras mutant expressers accumulated p-Erk1/2 in the cytoplasm with increased MEK activity and failed to translocate it to nuclei on EGF stimulation. Senescent HDF cells, V12S35 and V12G37 expressers, revealed a failure to export actin fiber from nucleus to cytoplasm and also to form stress fibers. Perinuclear expression of Rac1 was prominent in the HDF cells and V12C40 expresser; however, in the V12S35 expresser, translocation of Rac1 from perinucleus to nucleus and strong expression of RhoA were obvious. In summary, the H-ras double mutant expressers induced premature senescence through the MEK pathway, accompanied by nuclear accumulation of actin and Rac1 proteins, cytoplasmic retention of p-Erk1/2, and marked induction of RhoA expression, suggesting the translocational inefficiency of the intracellular proteins in the senescent HDF cells.

3T3 Cells↗
PubMed1998

Agents that cause DNA double strand breaks lead to p16INK4a enrichment and the premature senescence of normal fibroblasts.

The occurrence of DNA double strand breaks induces cell cycle arrest in mortal and immortal human cells. In normal, mortal fibroblasts this block to proliferation is permanent. It depends on the growth regulator p53 and a protein p53 induces, the cyclin dependent kinase inhibitor, p21. We show here that following DNA damage in mortal fibroblasts, the induction of p21 and p53 is to a large degree shortlived. By 8 days after a brief exposure to DNA strand breaking agents, bleomycin or actinomycin D, p53 protein is at baseline levels, while the p53 transactivation level is only slightly above its baseline. By this time the concentration of p21 protein, which goes up as high as 100-fold shortly after treatment, is down to just 2-4-fold over baseline levels. Following the drop in p21 concentration a large increase in the expression level of the tumor suppressor gene p16INK4a is observed. This scenario, where a transient increase in p21 is followed by a delayed induction of p16INK4a, also happens with the permanent arrest that occurs with cellular senescence. In fact, these cells treated with agents that cause DNA double strand breaks share a number of additional markers with senescent cells. Our findings indicate that these cells are very similar to senescent cells and that they have additional factor(s) beside p21 and p53 that maintain cell cycle arrest.

Base Sequence↗
PubMed2002

Peroxisome senescence in human fibroblasts.

The molecular mechanisms of peroxisome biogenesis have begun to emerge; in contrast, relatively little is known about how the organelle functions as cells age. In this report, we characterize age-related changes in peroxisomes of human cells. We show that aging compromises peroxisomal targeting signal 1 (PTS1) protein import, affecting in particular the critical antioxidant enzyme catalase. The number and appearance of peroxisomes are altered in these cells, and the organelles accumulate the PTS1-import receptor, Pex5p, on their membranes. Concomitantly, cells produce increasing amounts of the toxic metabolite hydrogen peroxide, and we present evidence that this increased load of reactive oxygen species may further reduce peroxisomal protein import and exacerbate the effects of aging.

Aging↗
PubMed2003

Aging-related attenuation of EGF receptor signaling is mediated in part by increased protein tyrosine phosphatase activity.

As fibroblasts near senescence, their responsiveness to external signals diminishes. This well-documented phenomenon likely underlies physiological deterioration and limited tissue regeneration in aging individuals. Understanding the underlying molecular mechanisms would provide opportunities to ameliorate these situations. A key stimulus for human dermal fibroblasts are ligands for the epidermal growth factor receptor (EGFR). We have shown earlier that EGFR expression decreases by about half in near senescent fibroblasts (Shiraha et al., 2000, J. Biol. Chem. 275 (25), 19343-19351). However, as the cell responses are nearly absent near senescence, other aging-related signal attenuation changes must also occur. Herein, we show that EGFR signaling as determined by receptor autophosphorylation is diminished over 80%, with a corresponding decrease in the phosphorylation of the immediate postreceptor adaptor Shc. Interestingly, we found that this was due at least in part to increased dephosphorylation of EGFR. The global cell phosphotyrosine phosphatase activity increased some threefold in near senescent cells. An initial survey of EGFR-associated protein tyrosine phosphatases (PTPases) showed that SHP-1 (PTPIC, HCP, SHPTP-1) and PTPIB levels are increased in parallel in these cells. Concomitantly, we also discovered an increase in expression of receptor protein tyrosine phosphatase alpha (RPTPalpha). Last, inhibition of protein tyrosine phosphatases by sodium orthovanadate in near senescent cells resulted in increased EGFR phosphorylation. These data support a model in which, near senescence, dermal fibroblasts become resistant to EGFR-mediated stimuli by a combination of receptor downregulation and increased signal attenuation.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Premature senescence of skin fibroblasts from insulin-dependent diabetic patients with kidney disease.

Diabetic glomerulopathy develops in a subset only of patients with insulin-dependent diabetes (IDDM) and early, in its course, is characterized by cell hypertrophy and by excessive extracellular matrix production. These observations suggest that an alteration in the control of cell growth processes may contribute to its pathogenesis and be related to the susceptibility to kidney disease. We therefore investigated whether the development of diabetic nephropathy is associated with abnormalities of cell growth and morphology. Cultured skin fibroblasts from 14 IDDM patients with nephropathy (DN) were compared with those of 10 IDDM patients without nephropathy (D) and of 14 control non-diabetic subjects (C). Cell volume (in arbitrary units) and total protein content (microgram/10, 000 cells) were increased in serially passaged skin fibroblasts of IDDM patients with nephropathy (DN = 809.5 +/- 33.1 and 1.93 +/- 0.38 vs. D = 764.4 +/- 31.5 and 1.5 +/- 0.37, P = 0.005 and P = 0.03, respectively; vs. C = 756.2 +/- 36.3 and 1.5 +/- 0.38, P = 0.0006 and P = 0.03, respectively). These hypertrophic cells had a tendency to a slower duplication rate and exhibited a dissociation of the DNA and cytoplasmic cell-cycles, resulting in a higher proportion of tetraploid cells (DN = 25 +/- 15% vs. D = 6 +/- 4%, P = 0.005; and vs. C = 10 +/- 8%, P = 0.04). The frequency of terminally differentiated post-mitotic fibrocytes, cells specialized for extracellular matrix production, was higher in patients with nephropathy compared to that of patients without nephropathy and normal controls (DN = 34 +/- 14% vs. D = 21 +/- 10%, P = 0.02; and vs. C = 19 +/- 12%, P = 0.008). That early differentiation was a specific feature of cells derived from patients with diabetic nephropathy was confirmed by the study of cell life-span which demonstrated that these cells aged prematurely (log rank test, chi 2 = 10,012; P = 0.0067). We conclude that an acceleration of cell aging is a peculiar feature of diabetic kidney disease and may contribute to its pathological tissue changes.

Adult↗
PubMed1996

RAR beta 2-mediated growth inhibition in HeLa cells.

Retinoic acid inhibits the growth of a variety of normal and transformed cells in vitro and in vivo. How retinoic acid inhibits cell growth is poorly understood but involves interactions between the ligand and a series of nuclear and cytoplasmic receptors. The nuclear receptors for retinoic acid are of two types, the RARs and the RXRs. Each can function as a ligand-inducible transcription enhancing factor. In previous studies, we have demonstrated that an isoform of one RAR, RAR beta 2, is transcriptionally up-regulated in senescent human dermal fibroblasts and senescent human mammary epithelial cells. Moreover, we have also shown that RAR beta 2 can inhibit oncogene-induced focus formation, in primary rat embryo fibroblasts, as effectively as the tumor suppressor gene p53. Here, we extend our studies of retinoid-regulated signal transduction pathways that inhibit cell proliferation by demonstrating that HeLa cells expressing an RAR beta 2 construct are growth inhibited by greater than 50% when compared to the parent cell lines. The RAR beta 2-expressing cell lines are inhibited further by the addition of exogenous all-trans-retinoic acid. Finally, soft agar assays show that the RAR beta 2-expressing cell lines also demonstrate an inhibition of growth in soft agar, when compared to the parent growth cell lines, and are inhibited further in the presence of added all-trans-retinoic acid. These data definitively show that RAR beta 2 can inhibit cell proliferation in an established tumor cell line and provide more strength to the notion that this isoform is an effective growth inhibitor in vitro and, most likely, in vivo.

Base Sequence↗
PubMed1986

An altered response in the induction of cell membrane (Na + K)ATPase by thyroid hormone is characteristic of senescence in cultured human fibroblasts.

There have been numerous investigations of thyroid function during senescence in humans. However, very little information is available on thyroid hormone action at the cell level during senescence. Therefore, we have investigated thyroid hormone induction of cell membrane (Na + K)ATPase during human senescence using three experimental fibroblast cell culture systems: (1) cells from premature aging syndrome, progeria; (2) aging in vitro; and (3) early passage cells from aged patients. In all cases senescence is associated with a dramatic alteration from the normal dose-dependent thyroid hormone induction of (Na + K)ATPase. Senescent cells depleted of thyroid hormones demonstrated an elevated activity of (Na + K)ATPase, while non-senescing cells exhibit the characteristic basal enzyme activities in the hypothyroid state. These results indicate that human senescence is associated with extreme alterations in thyroid hormone regulation of (Na + K)ATPase; and may suggest a more general change in thyroid hormone action at senescence. These changes may be associated with important alterations in cell metabolism and intracellular ionic environment during senescence.

Adolescent↗
PubMed1986

C-ras expression decreases during in vitro senescence in human fibroblasts.

Expression of the c-ras oncogene was determined in growing early and late passage human (IMR-90) fibroblasts using northern blot analysis of total cellular RNA. It was found that late passage cells demonstrated lower levels of c-ras by about four fold when compared to levels found in early passage cells. Southern blot analysis of genomic DNA from early and late passage fibroblasts digested with either SacI or BamHI showed somewhat increased hybridization levels in early passage cells compared to late passage cells. Data is discussed in relation to a previous report of c-ras expression and gene amplification.

Cell Cycle↗
PubMed1978

Nuclear area changes in senescing human diploid fibroblasts.

The pattern of mean nuclear area changes was determined in human fibroblast cell strains from male donors of three different ages (8,40 and 84 years) during their in vitro lifespan. There was a statistically significant increase in mean nuclear area of cells of all three strains during their aging in vitro. A gradual increase of the subpopulation of cells with larger nuclei was also observed. Evidence for a positive correlation between aging in vitro, as reflected by mean nuclear areas at the tenth doubling from the end of in vitro doubling activity, and donor age is presented.

Adult↗
PubMed1987

Lack of recovery from oxygen-induced damage to colony formation and DNA synthesis in senescent human diploid fibroblasts.

The colony formation and DNA synthesis of cultured human fibroblasts were inhibited under hyperbaric oxygen. When maintained in a growth-arrested state for a few days after oxygen exposure, early passage diploid and SV-40-transformed cells repaired the oxygen-induced damage, while late passage diploid cells did not. In early passage cells to which cycloheximide, a protein synthesis inhibitor, was administered, no recovery from the damage to colony formation was found, indicating that de novo protein synthesis is necessary to the repair process. The repair function of the late passage cells against oxidative damage seems to be impaired. Oxidative stress may be involved in cellular ageing.

Cell Division↗
PubMed1991

Longitudinal study of in vivo wound repair and in vitro cellular senescence of dermal fibroblasts.

A longitudinal study was performed to confirm the inverse relationship between in vivo age and in vitro proliferative capacity previously observed in a cross-sectional study, and to investigate the relationship between the growth of dermal fibroblasts in vitro and a physiological function (i.e., wound repair) that is known to decline with age in vivo. Fibroblast cultures were generated from skin punch biopsies from 12 male hamsters beginning at 1 month of age and at 6-months interval thereafter until the natural death of the animal. All cultures from all individuals exhibited finite proliferative capacity, and an inverse relationship was observed between donor age and maximum in vitro proliferative capacity. In addition, a direct correlation between the in vitro proliferative capacity of the dermal fibroblasts in vitro and the repair efficiency of the biopsy site was observed. However, these changes in the in vitro proliferative capacity and in vivo wound repair efficiency were not progressive beyond 12-18 months of age and were not indicative at any age of an individual's ultimate lifespan. This study provides evidence that in vitro proliferative capacity of dermal fibroblasts and in vivo wound repair may be comparable phenomena that share a common mechanism. However, the nonprogressive nature and the lack of correlation between these phenomena and the individual's ultimate lifespan indicate that their use as biological markers of aging is limited to animals younger than the mean lifespan of the species.

Aging↗