Proteasome inhibitors induce changes in chromatin structure characteristic of senescent human fibroblasts

Inhibitors of proteasome induced premature senescence in normal human fibroblasts. Besides morphological alteration and expression of senescence marker genes, these cells manifested senescence-associated heterochromatic foci under staining of the nuclei with DAPI similar to normally senescent cells. These results suggest that declining ability in protein degradation may be involved in the formation of heterochromatic foci in senescent fibroblasts.     

Detachment-associated changes in lipid rafts of senescent human fibroblasts

We characterized the effects of in vitro cellular aging on constituents of lipid rafts in human diploid fibroblasts, TIG-1. Cholesterol recovery from lipid rafts of senescent cells was decreased by the detaching treatment, while the decrease was far less obvious in young cells. A probe that binds selectively to cholesterol in lipid rafts revealed that the amount of lipid rafts on the cell surface decreased in senescent cells upon cell detachment. Accompanying this change was the release of the raft-associated molecules caveolin and Fyn from lipid rafts upon cell detachment, suggesting a detachment-associated disorganization of lipid rafts in senescent cells. In addition, our observations showing differential sensitivities of lipid rafts from young and senescent cells to detaching treatment indicate a caution in how to detach cells. Particular attention needs to be paid to interpreting the results when lipid rafts are prepared from mechanically detached cells under detergent-free conditions.     

Attempted hybridizations with senescent human fibroblasts

If the aging of diploid human fibroblasts reflects stable genetic or epigenetic changes which are few in number and different in different cells, then complementation could occur in hybrids between individual senescent cells. However neither pairs of aged fibroblasts nor even pairs of young and senescent fibroblasts produce viable hybrids under conditions known to promote cell fusion.     

Induction of apoptosis in human replicative senescent fibroblasts

Cellular senescence is an irreversible growth phase characteristic of normal cells. We have found that human senescent fibroblasts can be induced to undergo programmed cell death (apoptosis) by ceramide, TNF-alpha, or okadaic acid. The most profound effects were induced by TNF-alpha and okadaic acid treatment. In the present study, we also evaluated the contribution of lysosomal activation as a possible mechanism underlying the induction of apoptosis. Four lysosomal enzyme activities were measured: beta-galactosidase, alpha-galactosidase A, beta-glucoronidase, and acid phosphatase. Using an in situ assay, we have found that the activity of beta-galactosidase, which is also a biochemical marker of senescence, is induced in young proliferating fibroblasts following exposure to all three apoptotic inducing agents. The other enzymes were not significantly induced in young fibroblasts following exposure to agents that induce apoptosis. During replicative senescence, three of the four lysosomal enzymes tested (beta-galactosidase, alpha-galactosidase A, and beta-glucoronidase) are constitutively expressed at high levels. TNF-alpha was the only agent that induced lysosomal activity in senescent fibroblasts, of which only alpha-galactosidase A activity was induced. Our studies show that senescent fibroblasts can be induced to undergo apoptosis in a signal-dependent manner. However, the lysosomal enzymes examined do not appear to be correlated with apoptotic induction.     

Up-regulation of PDCD4 in senescent human diploid fibroblasts

Programmed cell death 4 (PDCD4) has a common MI domain sharing with death associated protein 5 (DAP5) and a component of eukaryotic translation initiation factor (eIF4G) complex and it might also work as a tumor suppressor. We could find that the message and product of Pdcd4 gene were up-regulated in senescent human diploid fibroblasts. In yeast two hybrid analysis, the C-terminal region of PDCD4 interacted with ribosomal protein S13 (RPS13), ribosomal protein L5 (RPL5), and TI-227H. In in vitro binding assay, RPS13, a component of 40S ribosome was stably bound to PDCD4. We also found that PDCD4 was localized to polysome fractions. We could pull out eIF4G with GST-PDCD4, but eIF4E did not interact with PDCD4. From these results, we could assume that PDCD4 might regulate the eIF4G-dependent translation through direct interactions with eIF4G and RPS13 in senescent fibroblasts.     

Actin in young and senescent fibroblasts.

Long-term exposure of guinea pigs to a diet rich in maize oil caused an increase in adipose-tissue lipoprotein lipase activity. A similar diet rich in beef tallow had no such effect, and neither diet affected the enzyme activity in heart, lung, diaphragm or skeletal muscle.     

DNA-binding proteins in young and senescent normal human fibroblasts

DNA-binding proteins (DBP) from normal human diploid cells, strain WI38, were isolated by DNA-cellulose chromatography using undenatured calf thymus DNA. The DBP in the 0.15 M NaCl eluate were fractionated by polyacrylamide gel electrophoresis. Comparisons of the amounts of the DBP in different cell populations were made by labelling the cells with either ³H- or ¹⁴C-amino acid precursors for 40 h prior to pooling the cells for co-isolation of their DBP. When WI38 cells in the replicative and stationary phases were compared, five proteins, P5b (87 000 D), P6a (50 000 D), P8 (33 000 D), P9 (28 000 D) and P10 (25 000 D) were labelled to a greater extent in the replicating cells and two proteins, P5c (72 000 D) and P12 (18 000 D) were labelled to a greater extent in the stationary phase cells. In addition, several high molecular weight DBP, partially characterized as collagen and protocollagen, were preferentially labelled in the stationary phase cells. Stationary phase senescent WI38 cells at or near the end of their in vitro lifespan characteristically showed an increased proportion of protein component P8 (33 000 D) relative to stationary phase WI38 cells at early population doubling levels. Further characterization of WI38-P8 showed that it binds preferentially to single-stranded DNA and amounts to greater than 1% of the total soluble protein in young cells in growth phase. Thus WI38-P8 appears to be comparable to the P8 protein studied by Tsai & Green [27] in mouse 3T6 and human SB cells. The component which is increased in senescent or terminal phase non-dividing cell populations is judged to be the P8 protein by its position in SDS-gels and its preferential binding to single-stranded DNA.     

Origins of G1 arrest in senescent human fibroblasts

Human diploid fibroblasts have a finite proliferative lifespan in culture, at the end of which they are ararrested with G₁ phase DNA contents. Upon serum stimulation, senescent cells are deficient in carrying out a subset of early signal transduction events such as activation of protein kinase C and induction of c-fos. Later in G₁, they uniformly fail to express late G₁ genes whose products are required for DNA synthesis, implying that they are unable to pass the R point. Failure to pass the R point may occur because senescent cells are unable to phosphorylate the retinoblastoma protein, owing to the accumulation of inactive complexes of cyclin E/Cdk2 and possibly cyclin D/Cdk4. Senescent cells contain high amounts of p21, a potent cyclin-dependent kinase inhibitor whose levels are also elevated in cells arrested in G₁ following DNA damage, suggesting that both arrests might share a common mechanism. Cell aging is accompanied by a progressive shortening of chromosomal telomeres, which could be perceived by the cells as a form of DNA damage that gives rise to the signals that inactivate the cell cycle machinery.     

Ciliary abnormalities in senescent human fibroblasts impair proliferative capacity

Somatic cells senesce in culture after a finite number of divisions indefinitely arresting their proliferation. DNA damage and senescence increase the cellular number of centrosomes, the 2 microtubule organizing centers that ensure bipolar mitotic spindles. Centrosomes also provide the basal body from which primary cilia extend to sense and transduce various extracellular signals, notably Hedgehog. Primary cilium formation is facilitated by cellular quiescence a temporary cell cycle exit, but the impact of senescence on cilia is unknown. We found that senescent human fibroblasts have increased frequency and length of primary cilia. Levels of the negative ciliary regulator CP110 were reduced in senescent cells, as were levels of key elements of the Hedgehog pathway. Hedgehog inhibition reduced proliferation in young cells with increased cilium length accompanying cell cycle arrest suggesting a regulatory function for Hedgehog in primary ciliation. Depletion of CP110 in young cell populations increased ciliation frequencies and reduced cell proliferation. These data suggest that primary cilia are potentially novel determinants of the reduced cellular proliferation that initiates senescence.     

Degradation of endocytosed proteins is unaltered in senescent human fibroblasts

We compared the abilities of young and senescent fibroblasts to take up and degrade [3H]ribonuclease A (native and oxidized), [3H]ribonuclease4-13, [3H]hemoglobin, [3H]glyceraldehyde-3-phosphate dehydrogenase, [3H]beta-galactosidase, [3H]glycogen phosphorylase, and [125I]serum albumin. The endocytic uptake of these proteins ranged from fluid-phase to predominantly absorptive. Intralysosomal degradation rates of the different endocytosed proteins varied by an order of magnitude, but in no case was there a difference between cultures of young and senescent fibroblasts.     

Galectin-3 expression and subcellular localization in senescent human fibroblasts

Galectin-3 is a galactose/lactose-binding protein (M(r) approximately 30,000), identified as a required factor in the splicing of pre-mRNA. In the LG1 strain of human diploid fibroblasts, galectin-3 could be found in both the nucleus and the cytoplasm of young, proliferating cells. In contrast, the protein was predominantly cytoplasmic in senescent LG1 cells that have lost replicative competence through in vitro culture. Incubation of young cells with leptomycin B, a drug that disrupts the interaction between the leucine-rich nuclear export signal and its receptor, resulted in the accumulation of galectin-3 inside the nucleus. In senescent cells, galectin-3 staining remained cytoplasmic even in the presence of the drug, thus suggesting that the observed localization in the cytoplasm was due to a lack of nuclear import. In heterodikaryons derived from fusion of young and senescent LG1 cells, the predominant phenotype was galectin-3 in both nuclei. These results suggest that senescent LG1 cells might lack a factor(s) specifically required for galectin-3 nuclear import.     

Patterns of peptide synthesis in senescent and presenescent human fibroblasts

Peptide production in senescent and presenescent human foreskin fibroblasts was measured using 2-dimensional polyacrylamide gel electrophoresis. This procedure permits the visualization of a cohort of the major peptides being produced. Among this cohort of over 500 peptides only two were found to differ in relative amount in that more was being produced in senescent cells. This difference was confirmed by measurements of the relative intensity of the peptide spot. This difference was senescent cell-specific and not due to the differences in rate of growth of senescent and non-senescent cells.     

Re-modelling of nuclear architecture in quiescent and senescent human fibroblasts

Spatial organisation of the genome within the nucleus can play a role in maintaining the expressed or silent state of some genes [1]. There are distinct addresses for specific chromosomes, which have different functional characteristics, within the nuclei of dividing populations of human cells [2]. Here, we demonstrate that this level of nuclear architecture is altered in cells that have become either quiescent or senescent. Upon cell cycle exit, a gene-poor human chromosome moves from a location at the nuclear periphery to a more internal site in the nucleus, and changes its associations with nuclear substructures. The chromosome moves back toward the edge of the nucleus at a distinctive time after re-entry into the cell cycle. There is a 2-4 hour period at the beginning of G1 when the spatial organisation of these human chromosomes is established. Lastly, these experiments provide evidence that temporal control of DNA replication can be independent of spatial chromosome organisation. We conclude that the sub-nuclear organisation of chromosomes in quiescent or senescent mammalian somatic cells is fundamentally different from that in proliferating cells and that the spatial organisation of the genome is plastic.     

Reduction of UV-induced cell death in the human senescent fibroblasts

We studied mechanisms by which senescent cells acquire resistance to UV-induced cellular insults. Human primary foreskin fibroblast culture was used since it undergoes cellular senescence in vitro after a limited number of passages. Senescence was induced by a brief treatment of the early passage cells with 100 microM of H2O2 for 1 h, and subsequent culture for 3 weeks. Hydrogen peroxide-treated cells showed an enhancement of senescence-associated beta-galactosidase activity. In the senescent cells, DNA fragmentation in response to UV-irradiation was found to decrease significantly compared with that in the young cells. The SAPK/JNK activation by UV irradiation was reduced in both non-treated senescent cells and the hydrogen peroxide-induced senescent cells, suggesting that a reduced DNA fragmentation by UV-irradiation in the senescent cells is closely related to the decreased SAPK/JNK activity. Since a cell cycle inhibitor, p21Waf1, has been implicated in protecting cells against apoptotic cell death, we determined p21Waf1 to assess whether its elevation has any impact on the reduction of UV-induced activation of SAPK/JNK in the senescent cells. The expression of p21Waf1 increased in both the nontreated and the hydrogen peroxide-treated senescent cells. Our study also revealed that the blockage of SAPK/JNK activation in the senescent cells was closely related to the increased level of p21Waf1. Our observation might provide clues about molecular mechanism of resistance to DNA fragmentation and the consequent cell death by UV-irradiation.     

Proteasome response to interferon-gamma is altered in senescent human fibroblasts

We have investigated immunoproteasomes in human fibroblasts during replicative senescence. Unlike levels of constitutive proteasome catalytic subunits and 26S proteasome regulatory subunits, levels of immunosubunits did not decrease dramatically in senescent cells. However, the induction of immunosubunits by interferon-gamma (IFN-gamma) was lost in senescent cells. In contrast, levels of the 11S proteasome regulator, PA28, were increased by IFN-gamma even in senescent cells, and both immunosubunits and PA28 increased with the reversible growth arrest in confluent cell cultures. The results highlight differences in the mechanisms of regulation of immunoproteasomes compared to constitutive proteasomes and in the irreversible growth arrest of senescent cells compared to reversible contact-induced growth arrest.     

Fc gamma-receptor-mediated changes in the plasma membrane potential induce prostaglandin release from human fibroblasts

Binding of aggregated human immunoglobulin G (IgG) on diploid human fibroblasts leads to a rapid depolarization of the cells within 1-2 min. We resolved this membrane potential change into its plasma membrane and mitochondrial membrane components by measuring the transmembrane distribution of the lipophilic tritium-labelled cation tetraphenylphosphonium, [3H]Ph4P+. The responsibility of the plasma membrane for the membrane potential change, induced by binding of IgGs, is demonstrated. The IgG-induced membrane depolarization leads to the induction of prostaglandin E2 synthesis. Aggregated immunoglobulins (IgG) are specifically bound via the Fc portion because only binding of Fc fragments, in contrast to (Fab')2 fragments, leads to a stimulation of prostaglandin E2 synthesis comparable to that mediated by IgGs. Depolarization of the plasma membrane by short incubation of the fibroblasts in high-K+ buffer (5 min) results in a stimulation of prostaglandin E2 synthesis comparable to that mediated by either aggregated human IgGs or Fc fragments. Our previous results on Fc gamma-receptor-mediated antigen-IgG-antibody complex internalization showed that a maximum uptake of these complexes could be detected 60-90 min after binding. Therefore, we conclude that not internalisation but binding of aggregated IgGs to the Fc gamma receptors on human fibroblasts is the stimulus for plasma membrane depolarization leading to an enhanced prostaglandin E2 release.     

Heterogeneity of dimer excision in young and senescent human dermal fibroblasts

We have examined the relationship between nucleotide excision of the main UV-induced photoproduct, the cyclobutane pyrimidine dimer and in vitro cellular senescence. An in situ semiquantitative immunocytochemical assay has demonstrated that, following a UV-C dose of 15 J m-2, young human dermal fibroblasts maintained in a high level of serum are more efficient than senescent fibroblasts in the removal of dimers. However, in G0-arrested cultures (serum-starved), young fibroblasts are compromised in their ability to remove dimers and are significantly less efficient than senescent cells in this process. Supplementation of the culture medium with 0.1 mm deoxyribonucleosides enhances the removal of dimers in both young and senescent fibroblasts in proliferating or serum-starved cells. These data indicate that overall there is a modest but significant reduction in nucleotide excision of dimer photoproducts in cells as they age in vitro. In addition, G0-arrested young cells exhibit reduced removal of dimers, although this can be complemented by deoxyribonucleoside addition. In addition, this in situ assay has revealed heterogeneity in both susceptibility to UV-C-induced damage and excision. Overall, we provide evidence of reduced UV-induced damage excision in senescent compared with young fibroblasts, and demonstrate modulation of these processes in young and senescent cells under specific growth conditions.