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PubMed1980

Senescence of cultured human diploid fibroblasts. Are mutations responsible?

Two predictions of the error/mutation hypothesis of cellular senescence (Orgel, '73) namely,a) exponential accumulation of somatic mutations during the replicative lifespan and b) shortening of culture lifespan upon treatment with mutagens have been examined experimentally in a strain of cultured human diploid fibroblasts. Our studies show that as cells traverse the replicative lifespan (from 10 to 75 mean population doublings (MPD); total lifespan congruent to 95 MPD), no rapid and exponential increase occurs in the accumulation of mutations measured by the frequencies of Thgr(thioguanine resistance) and Dipr (diphtheria toxin resistance) mutants. Furthermore, repeated cycles of treatment (from 1- to 14-times) of human fibroblasts with two mutagens, ethyl methane sulfonate (EMS) and N-methyl-N' nitro-nitrosoguanidine, which led to a marked increase in the mutation frequency for the Dipr marker (congruent to 100-fold), failed to shorten the lifespan of cultured fibroblasts. On the contrary, repeated mutagen treatment (12 times with EMS) prolonged the lifespan of one replicative culture (110 MPD versus 94--98 MPD). These results strongly indicate that mutations are unlikely to be the primary event in cellular senescence and suggest instead that senescence is probably controlled by one or more (specific) gene(s) whose expression can be modified by mutations.

Cell Line↗
PubMed2000

Accumulation of single-strand breaks is the major cause of telomere shortening in human fibroblasts.

Telomere shortening triggers replicative senescence in human fibroblasts. The inability of DNA polymerases to replicate a linear DNA molecule completely (the end replication problem) is one cause of telomere shortening. Other possible causes are the formation of single-stranded overhangs at the end of telomeres and the preferential vulnerability of telomeres to oxidative stress. To elucidate the relative importance of these possibilities, amount and distribution of telomeric single-strand breaks, length of the G-rich overhang, and telomere shortening rate in human MRC-5 fibroblasts were measured. Treatment of nonproliferating cells with hydrogen peroxide increases the sensitivity to S1 nuclease in telomeres preferentially and accelerates their shortening by a corresponding amount as soon as the cells proliferate. A reduction of the activity of intracellular peroxides using the spin trap alpha-phenyl-t-butyl-nitrone reduces the telomere shortening rate and increases the replicative life span. The length of the telomeric single-stranded overhang is independent of DNA damaging stresses, but single-strand breaks accumulate randomly all along the telomere after alkylation. The telomere shortening rate and the rate of replicative aging can be either accelerated or decelerated by a modification of the amount of oxidative stress. Quantitatively, stress-mediated telomere damage contributes most to telomere shortening under standard conditions.

Alkylation↗
PubMed2003

Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts.

Most mammalian cells do not divide indefinitely, owing to a process termed replicative senescence. In human cells, replicative senescence is caused by telomere shortening, but murine cells senesce despite having long stable telomeres. Here, we show that the phenotypes of senescent human fibroblasts and mouse embryonic fibroblasts (MEFs) differ under standard culture conditions, which include 20% oxygen. MEFs did not senesce in physiological (3%) oxygen levels, but underwent a spontaneous event that allowed indefinite proliferation in 20% oxygen. The proliferation and cytogenetic profiles of DNA repair-deficient MEFs suggested that DNA damage limits MEF proliferation in 20% oxygen. Indeed, MEFs accumulated more DNA damage in 20% oxygen than 3% oxygen, and more damage than human fibroblasts in 20% oxygen. Our results identify oxygen sensitivity as a critical difference between mouse and human cells, explaining their proliferative differences in culture, and possibly their different rates of cancer and ageing.

Animals↗
PubMed1994

Prohibitin expression during cellular senescence of human diploid fibroblasts.

Prohibitin is an evolutionarily conserved gene postulated to possess tumor suppressor activity and to contribute to the limited lifespan of human diploid fibroblast-like cells. Prohibitin mRNA and protein expression and its ability to become post-translationally modified were determined in human diploid fibroblast-like cells of different in vitro ages. The expression of prohibitin mRNA and protein changes little with increasing in vitro age; however, its protein product is post-synthetically modified in younger but not older cells. These results suggest that prohibitin is similar to the retinoblastoma gene product whose anti-proliferative activity remains active in older cells because it is not post-synthetically modified.

Cell Cycle↗
PubMed1990

Novel monoclonal antibodies identify antigenic determinants unique to cellular senescence.

Normal human diploid fibroblasts exhibit a limited lifespan in vitro and are used as a model to study in vivo aging. Monoclonal antibodies were generated against partially purified surface membranes from human diploid fibroblasts at the end of their lifespan (senescent). Three hybridomas were isolated that secreted antibodies reacting to cellular determinants expressed specifically on senescent human fibroblasts of different origin, including neonatal foreskin, embryonic lung, and adult skin punch biopsy, but not expressed on matched young cells. The antibodies did not bind to immortal human cells and normal young cells made reversibly nondividing, indicating the antigens are not expressed in cells that are not senescent. The antibodies identified senescent cells in a mixed cell population and expression of the senescent cell antigens correlated strongly with the cells inability to synthesize DNA at the onset of senescence. The antigens appeared to be cell surface or extracellular matrix associated, and the epitopes were destroyed by mild trypsin treatment. Western analysis indicated all three antibodies reacted with fibronectin. Though the antigenic determinants on the fibronectin molecule were not accessible in the intact young cell, the epitopes were present in fibronectin extracted from both senescent and young cells, as well as purified human plasma fibronectin. These antibodies and the senescent specific expression of the antigens provide powerful tools to investigate the mechanisms leading to in vitro senescence. This may enable us to investigate directly the relationship between cellular aging and aging of the individual.

Aging↗
PubMedDate not supplied

The pathway of cell senescence: WI-38 cells arrest in late G1 and are unable to traverse the cell cycle from a true G0 state.

Senescent human diploid fibroblasts have an undefined arrest state partially characterized by the differential expression of cell cycle-regulated genes and a failure to complete the mitogen-stimulated cascade of signalling events that lead to DNA synthesis. We present evidence that this arrest state precludes the entry of senescent fibroblasts into a normally reversible G0 or quiescent state. Both nuclear association kinetics and quinacrine dihydrochloride nuclear fluorescence show chromatin condensation patterns consistent with arrest in late G1 and exclusion of senescent cells from the G0 phase of the cell cycle. Steady-state thymidine kinase mRNA levels indicate that some of the signalling cascades initiated from a functional G0 state may be intact in senescent cells, at least qualitatively, and that this expression may represent an abortive attempt to complete pathways required for DNA replication. Taken together, the evidence suggests that growth arrest in senescent cells likely occurs in a physiologic state fundamentally distinct from that of the G0, quiescent state that is achieved by nonproliferating young cells. A full response to serum or growth factor addition, leading from quiescence to DNA synthesis, may require cells to initiate this traverse from a true G0 state. If so, senescent cells would be excluded from this pathway.

Biomarkers↗
PubMed2002

Replication and/or separation of some human telomeres is delayed beyond S-phase in pre-senescent cells.

Cultured primary human cells, which lack telomerase, enter a state of replicative senescence after a characteristic number of population doublings. During this process telomeres shorten to a critical length of approximately 5-7 kb. The mechanistic relationship between advanced cell passage, cellular senescence and telomeric function has yet to be fully elucidated. In the study described here, we investigated the relationship between changes in telomeric replication timing and/or sister chromatid separation at telomeric regions and advanced cell passage. Using fluorescence in situ hybridization, we analyzed the appearance of double hybridization signals (doublets), which indicate that the region of interest has replicated and the replicated products have separated sufficiently to be resolved as two distinct signals. The results showed that the replication and separation of several telomeric regions occurs during the second half of S-phase and that a delay in replication and/or separation of sister chromatids at these regions occurs in pre-senescent human fibroblasts. Surprisingly, in a significant percentage of pre-senescent cells, several telomeric regions did not hybridize as doublets even in metaphase chromosomes. This delay was not associated with extensive changes in methylation levels at subtelomeric regions and was circumvented in human fibroblasts expressing ectopic telomerase. We propose that incomplete replication and/or separation of telomeric regions in metaphase may be associated with proliferative arrest of senescent cells. This cell growth arrest may result from the activation of a mitotic checkpoint, or from chromosomal instability consequent to progression in the cell cycle despite failure to replicate and/or separate these regions completely.

Cell Cycle↗
PubMed1991

Decline of poly(ADP-ribosyl)ation during in vitro senescence in human diploid fibroblasts.

Poly(ADP-ribose) polymerase activity was determined at various times during the in vitro life span of two human diploid fibroblast-like cell lines of different donor ages. The cell lines differed in their ability to transfer ADP-ribose, with cells from an embryonic donor exhibiting 2 to 3 times the activity found in cells obtained from a newborn donor. The activity in both cell lines decreased by 30-60% as the cells moved through their in vitro life spans. The decline could not be attributed to increases in glycohydrolase or the leakage of polymerase from older cell preparations. Enzyme activation with DNase I indicated that similar levels of enzyme were present in both cell lines at all in vitro ages. These results indicate that although poly(ADP-ribosyl)ation is inversely related to donor age as well as in vitro age the decrease is in response to other factors which change with increasing age.

Cell Line↗
PubMed1980

Changes in DNA alkali-sensitive sites during senescence and establishment of fibroblasts in vitro.

DNA molecular weight was studied in human embryonic and mouse newborn lung fibroblasts in vitro at different passages of the culture using alkaline and neutral sucrose gradient techniques. Reduction of molecular weight of single-stranded DNA due to alkaline-sensitive sites appeared spontaneously during the growth decline of the mouse cells. These changes disappeared when the mouse fibroblasts became a permanent cell line. At the end of phase II of the human fibroblasts, the molecular weight of single-stranded DNA also decreased, followed by the restitution of some high molecular weight DNA in the ultimate passages. When treated with 1 mM caffeine, the mouse fibroblasts during growth crisis did not survive, while cells of the established line resisted. Thus it might be possible that a DNA repair process was involved in the recovery of the mouse fibroblasts. Furthermore, results favor the hypothesis that the cells that become established are not present in the primary culture but originate in vitro.

Animals↗
PubMed1980

Alterations in prostaglandin synthesis during senescence of human lung fibroblasts.

Prostaglandin (PG) synthesis by human embryo lung fibroblasts in culture was determined with increasing population doubling. These cultures synthesized PGE2, PGF2 alpha, PGI2 (prostacyclin) and TXA2 (thromboxane A2) in response to fetal bovine serum or ascorbic acid. Changes in prostaglandin synthesis by these cells during aging were observed in response to serum or ascorbic acid. These included a shift from the synthesis of PGI2 and PGE2 to TXA2 and PGF2 alpha and finally to PGF2 alpha alone. We also observed a marked increase during aging in the release of precursor fatty acid from lipid stores upon stimulation with ascorbic acid. A parallel decrease in the ability to convert the released fatty acid to prostaglandins was detected.

Cell Survival↗
PubMed2005

Overexpression of proteasome beta5 assembled subunit increases the amount of proteasome and confers ameliorated response to oxidative stress and higher survival rates.

The proteasome is the major cellular proteolytic machinery responsible for the degradation of both normal and damaged proteins. Proteasomes play a fundamental role in retaining cellular homeostasis. Alterations of proteasome function have been recorded in various biological phenomena including aging. We have recently shown that the decrease in proteasome activity in senescent human fibroblasts relates to the down-regulation of beta-type subunits. In this study we have followed our preliminary observation by developing and further characterizing a number of different human cell lines overexpressing the beta5 subunit. Stable overexpression of the beta5 subunit in WI38/T and HL60 cells resulted in elevated levels of other beta-type subunits and increased levels of all three proteasome activities. Immunoprecipitation experiments have shown increased levels of assembled proteasomes in stable clones. Analysis by gel filtration has revealed that the recorded higher level of proteasome assembly is directly linked to the efficient integration of "free" (not integrated) alpha-type subunits identified to accumulate in vector-transfected cells. In support we have also found low proteasome maturation protein levels in beta5 transfectants, thus revealing an increased rate/level of proteasome assembly in these cells as opposed to vector-transfected cells. Functional studies have shown that beta5-overexpressing cell lines confer enhanced survival following treatment with various oxidants. Moreover, we demonstrate that this increased rate of survival is due to higher degradation rates following oxidative stress. Finally, because oxidation is considered to be a major factor that contributes to aging and senescence, we have overexpressed the beta5 subunit in primary IMR90 human fibroblasts and observed a delay of senescence by 4-5 population doublings. In summary, these data demonstrate the phenotypic effects following genetic up-regulation of the proteasome and provide insights toward a better understanding of proteasome regulation.

Catalytic Domain↗
PubMed2002

A novel transcriptional inhibitory element differentially regulates the cyclin D1 gene in senescent cells.

Senescent human diploid fibroblasts are unable to initiate DNA synthesis following mitogenic stimulation and adopt a unique gene expression profile distinct from young or quiescent cells. In this study, a novel transcriptional regulatory element was identified in the 5'-untranslated region of the cyclin D1 gene. We show that this element differentially suppresses cyclin D1 expression in young versus senescent fibroblasts. Electrophoretic mobility shift assays revealed abundant complexes forming with young cell nuclear extracts compared with senescent cell nuclear extracts. Binding was maintained in young quiescent cells, showing that loss of this activity was specific to senescent cells and not an effect of cell cycle arrest. Site-directed mutagenesis within this cyclin D1 inhibitory element (DIE) abolished binding activity and selectively increased cyclin D1 promoter activity in young but not in senescent cells. Sequences with homology to the DIE were found in the 5'-untranslated regions of other genes known to be up-regulated during cellular aging, suggesting that protein(s) that bind the DIE might be responsible for the coordinate increase in transcription of many genes during cellular aging. This study provides evidence that loss of transcriptional repressor activity contributes to the up-regulation of cyclin D1, and possibly additional age-regulated genes, during cellular senescence.

5' Untranslated Regions↗
PubMed2004

The peptide methionine sulfoxide reductases, MsrA and MsrB (hCBS-1), are downregulated during replicative senescence of human WI-38 fibroblasts.

In contrast to other oxidative modifications of amino acids, methionine sulfoxide can be enzymatically reduced back to methionine in proteins by the peptide methionine sulfoxide reductase system, composed of MsrA and MsrB. The expression of MsrA and one member of the MsrB family, hCBS-1, was analyzed during replicative senescence of WI-38 human fibroblasts. Gene expression decreased for both enzymes in senescent cells compared to young cells, and this decline was associated with an alteration in catalytic activity and the accumulation of oxidized proteins during senescence. These results suggest that downregulation of MsrA and hCBS-1 can alter the ability of senescent cells to cope with oxidative stress, hence contributing to the age-related accumulation of oxidative damage.

Cell Line↗
PubMed2001

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↗
PubMed1985

On the importance of deoxyribonucleotide pools in the senescence of cultured human diploid fibroblasts.

Previous studies have indicated that ribonucleotide reductase may participate in a mechanism of cellular senescence, which involves modifications in deoxyribonucleotide pools, the products of the reductase reaction. Since very little information about the levels of these pools in senescing cells is currently available, an analysis of deoxyribonucleotide levels was carried out in young, old and very old normal human diploid fibroblasts, as well as in a variant human fibroblast strain with an altered replicative life span. These studies indicated that there are marked age-related perturbations in these pools which imply that they may be fundamentally important in a process determining the nonproliferative or senescent state.

Cell Line↗