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PubMed2001

Transglutaminase-mediated crosslinking of specific core histone subunits and cellular senescence.

We observed that the transglutaminase (tTGase) level and activity increased in aged rats and senescent primary fibroblasts, suggesting that the tTGase-mediated macromolecule crosslinking may play a mechanistic role during aging. Although preliminary, our in vitro experiment suggests that the target of tTGase is core histones: H2A:H2B and H3:H4 are specifically crosslinked by tTGase. On the basis of these data, we postulate that the changes of DNA metabolism in association with cellular aging may be ascribed primarily to the crosslinking of core histone subunits. Further speculation awaits substantive data showing increased histone crosslinking in senescent cells and also what crosslinked histones in various DNA metabolisms may imply. At the moment, present data are sufficient to propose that tTGase is a senescence marker and it may be primarily responsible for the phenotypes associated with cellular senescence.

Aging↗
PubMed1997

SEN6, a locus for SV40-mediated immortalization of human cells, maps to 6q26-27.

Normal cells show a limited lifespan in culture and the phenotype of cellular senescence. Tumors and tumor cell lines have typically overcome this form of growth suppression and grow continuously as immortal cell lines in culture. We have exploited the DNA virus SV40 to study the mechanism by which human fibroblasts overcome senescence and become immortal. Multiple steps have now been identified, including inactivation of cellular growth suppressors through direct interaction with SV40 large T antigen and through mutation of a gene on chromosome 6 (designated SEN6). In this study, we sublocalize the site of SEN6 to 6q26-27 based on molecular genetic analysis. Twelve SV40-immortalized fibroblast cell lines share a deletion in this area based on assessment for loss of heterozygostiy (LOH) for seven informative markers on 6q. Two immortal cell lines (AR5 and HALneo) appeared to have retained separate single copies of chromosome 6 despite the fact that they are both derived from the same preimmortal SV40-transformant and should share the same mutated allele of SEN6 (Hubbard-Smith et al., 1992). Detailed analysis by polymerase chain reaction, restriction fragment length polymorphism and fluorescence in situ hybridization shows, however, that although they differ for 17 markers from the centromere to 6q26, they share AR5 derived sequences (eight markers) distal to 6q26 including the minimal deletion region, further supporting the assignment of SEN6 to this region. Since human tumors including non-Hodgkins lymphoma, mammary carcinoma and ovarian carcinoma show LOH in 6q26-27, inactivation of SEN6 may be responsible for immortalization of these tumors as well.

Cell Line, Transformed↗
PubMed2001

Telomerase expression prevents replicative senescence but does not fully reset mRNA expression patterns in Werner syndrome cell strains.

Reduced replicative capacity is a consistent characteristic of cells derived from patients with Werner syndrome. This premature senescence is phenotypically similar to replicative senescence observed in normal cell strains and includes altered cell morphology and gene expression patterns. Telomeres shorten with in vitro passaging of both WRN and normal cell strains; however, the rate of shortening has been reported to be faster in WRN cell strains, and the length of telomeres in senescent WRN cells appears to be longer than that observed in normal strains, leading to the suggestion that senescence in WRN cell strains may not be exclusively associated with telomere effects. We report here that the telomere restriction fragment length in senescent WRN fibroblasts cultures is within the size range observed for normal fibroblasts strains and that the expression of a telomerase transgene in WRN cell strains results in lengthened telomeres and replicative immortalization, thus indicating that telomere effects are the predominant trigger of premature senescence in WRN cells. Microarray analyses showed that mRNA expression patterns induced in senescent WRN cells appeared similar to those in normal strains and that hTERT expression could prevent the induction of most of these genes. However, substantial differences in expression were seen in comparisons of early-passage and telomerase-immortalized derivative lines, indicating that telomerase expression does not prevent the phenotypic drift, or destabilized genotype, resulting from the WRN defect.

Cells, Cultured↗
PubMed1993

Decreased expression of the platelet-derived growth factor beta-receptor in fibroblasts from a patient with Werner's syndrome.

The study of Werner's syndrome, a premature ageing syndrome, may provide insight into the processes of natural ageing. We investigated the reason for the reduced growth potential of fibroblasts in Werner's syndrome which is considered to be similar to that of fibroblasts in normal senescence. The responses to different isoforms of platelet-derived growth factor (PDGF) of fibroblasts from a patient with Werner's syndrome (W-cells) and from a normal subject (control cells) were assessed by [3H]thymidine incorporation assay, a 125I-PDGF-AB binding experiment, and Northern blot analysis with a PDGF beta-receptor specific cDNA probe. PDGF-stimulated increase in [3H]thymidine incorporation in W-cells was much lower than that in control cells, especially with PDGF-AB and -BB as mitogens. The specific binding of 125I-PDGF-AB to W-cells was correspondingly lower than that to control cells. Furthermore, Northern blot analysis demonstrated a decreased basal level, and lack of PDGF-AB-induced up-regulation of the PDGF beta-receptor transcript in W-cells. Decreased expression of the PDGF beta-receptor due to decrease in its synthesis may be a causative factor of the decreased mitogenic response of W-cells to PDGF.

Cell Division↗
PubMed2003

A DNA damage checkpoint response in telomere-initiated senescence.

Most human somatic cells can undergo only a limited number of population doublings in vitro. This exhaustion of proliferative potential, called senescence, can be triggered when telomeres--the ends of linear chromosomes-cannot fulfil their normal protective functions. Here we show that senescent human fibroblasts display molecular markers characteristic of cells bearing DNA double-strand breaks. These markers include nuclear foci of phosphorylated histone H2AX and their co-localization with DNA repair and DNA damage checkpoint factors such as 53BP1, MDC1 and NBS1. We also show that senescent cells contain activated forms of the DNA damage checkpoint kinases CHK1 and CHK2. Furthermore, by chromatin immunoprecipitation and whole-genome scanning approaches, we show that the chromosome ends of senescent cells directly contribute to the DNA damage response, and that uncapped telomeres directly associate with many, but not all, DNA damage response proteins. Finally, we show that inactivation of DNA damage checkpoint kinases in senescent cells can restore cell-cycle progression into S phase. Thus, we propose that telomere-initiated senescence reflects a DNA damage checkpoint response that is activated with a direct contribution from dysfunctional telomeres.

Adaptor Proteins, Signal Transducing↗
PubMed1989

Collagen synthesis and deposition in cultured fibroblasts from subcutaneous radiation-induced fibrosis. Modification as a function of cell aging.

Acute local gamma-irradiation of porcine skin induces, as in human skin, extensive mutilating sclerosis, characterized by continuous expansion of the fibrosis invading the adjacent muscle and by accumulation of extracellular matrix macromolecules. Collagen synthesis, content, and types were studied by measuring the incorporation of the radiolabeled precursor [3H]-proline into confluent primary cultures and subcultures of porcine fibroblasts obtained from normal and irradiated fibrotic dermis. A significant increase in collagen biosynthesis and deposition, with a preferential enhancement of type III collagen, was observed in primary cultures of fibrotic fibroblasts when compared to those of normal dermis. However, the percentage (36%) of neosynthesized collagen in relation to the total neosynthesized non-collagenous and collagenous proteins remained unchanged. In subcultures of normal cells, collagen synthesis and content remained constant until about the 10th passage and then progressively declined until death of the fibroblasts, at around the 15th passage. During the senescence phase, normal fibroblasts acquired the capacity to synthesize higher levels of non-collagenous proteins. On the contrary, collagen synthesis and content diminished in subcultures of fibrosis-induced fibroblasts from the second passage, and then progressively declined as a function of aging; particularly the ratio of type III to type I collagen returned to normal values from the second passage. Moreover, these fibroblasts did not become senescent, persisted in synthesizing low levels of collagen, and acquired later (around the 40th passage) a higher capacity to synthesize non-collagenous proteins. These results demonstrate that primary cultures of porcine fibroblasts obtained from normal or radiation-induced fibrotic dermis reproduce the in vivo situation. The results strongly suggest that: 1) the modification of collagen synthesis, content, and types observed in primary cultures of fibroblasts might be due to factors causing long-lasting changes in phenotypic expression and/in stimulation of the expansion of some fibroblast clones engaged in the accelerated synthesis of extracellular matrix macromolecules, such as collagen or non-collagenous proteins. 2) the rapid decrease in this active capacity to synthesize and accumulate collagen, observed in subcultures of fibrosis-induced fibroblasts, might be related to the loss of some activation factors or to the dedifferentiation of the cells. 3) the switch from collagen to non-collagenous protein synthesis during later subcultures of fibroblasts obtained from radiation-induced fibrosis, might be due to molecular modification at the transcriptional or DNA level.

Animals↗
PubMed1998

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↗
PubMed2002

Identification of telomere-dependent "senescence-like" arrest in mouse embryonic fibroblasts.

In contrast to human primary cells, mouse embryonic fibroblasts (MEF) do not show telomere shortening-mediated replicative senescence due to the fact that they have telomerase activity and show sufficiently long telomeres. Instead, it is now generally accepted that the "senescence-like" arrest that occurs in MEF after 5-10 divisions in culture is mediated by telomere-length-independent mechanisms generally referred to as stress. Using telomerase-deficient MEF Terc(-/-), we show here that telomere shortening to a critical length leads to a premature senescence-like arrest in MEF, as well as has a negative effect on spontaneous immortalization. Similarly, elimination of the telomere end-capping protein Ku86 also leads to a premature senescence-like arrest and has a negative effect on spontaneous immortalization. Both Terc(-/-) MEF with short telomeres and Ku86(-/-) MEF show dysfunctional telomeres, as indicated by similarly increased frequencies of end-to-end fusions. These results suggest that loss of telomere function is a general mechanism leading to cell arrest. These observations also indicate that telomere dysfunction is interfering with successful cell division and thus interferes with tumor formation. In summary, we have identified here two different ways to induce a telomere-dependent senescence-like arrest in MEF.

Animals↗
PubMed1999

Decreased expression and activity of the immediate-early growth response (Egr-1) gene product during cellular senescence.

Human diploid fibroblasts (HDFs) undergo a limited number of population doublings in culture before reaching the end of their proliferative life span, an event termed in vitro cellular senescence. Considerable evidence suggests that altered expression of key genes involved in the mitogenic response may be responsible for the inability of senescent cells to proliferate. Here we examined the expression and activity of the early growth response-1 (egr-1) gene, an "immediate-early" gene that is believed to link extracellular mitogenic signals to cell-cycle progression. We found that egr-1 was strongly downregulated in senescent HDFs at the level of mRNA, protein, and DNA binding activity. Decreased DNA binding activity of Egr-1 in vitro corresponded to decreased transcriptional activation in vivo. To further understand the mechanism of egr-1 downregulation, we examined the potential role of the serum response elements (SREs) present in the egr-1 promoter. Electrophoretic mobility shift studies using young and old cell nuclear extracts showed a marked decrease in serum response factor (SRF) binding activity to the SRE in old compared to young cells. Loss of SRF binding activity has been correlated with the loss of expression of another growth-related immediate-early gene (c-fos). These results suggest a common mechanism for the downregulation of c-fos, egr-1, and other SRE-dependent, mitogen-responsive genes during cellular senescence.

Cells, Cultured↗
PubMed2000

Subsenescent telomere lengths in fibroblasts immortalized by limiting amounts of telomerase.

Human fibroblasts expressing the catalytic component of human telomerase (hTERT) have been followed for 250-400 population doublings. As expected, telomerase activity declined in long term culture of stable transfectants. Surprisingly, however, clones with average telomere lengths several kilobases shorter than those of senescent parental cells continued to proliferate. Although the longest telomeres shortened, the size of the shortest telomeres was maintained. Cells with subsenescent telomere lengths proliferated for an additional 20 doublings after inhibiting telomerase activity with a dominant-negative hTERT mutant. These results indicate that, under conditions of limiting telomerase activity, cis-acting signals may recruit telomerase to act on the shortest telomeres, argue against the hypothesis that the mortality stage 1 mechanism of cellular senescence is regulated by telomere positional effects (in which subtelomeric loci silenced by long telomeres are expressed when telomeres become short), and suggest that catalytically active telomerase is not required to provide a protein-capping role at the end of very short telomeres.

Cell Line, Transformed↗
PubMed1990

Spontaneous abnormalities in normal fibroblasts from patients with Li-Fraumeni cancer syndrome: aneuploidy and immortalization.

Families of patients with the Li-Fraumeni cancer syndrome have an inherited pattern of sarcomas and various other types of cancers that follow a dominant mode of transmission, an early age of onset, and exhibit multiple primary tumors. As soft tissue sarcomas (including fibrosarcomas) are frequently observed with this syndrome, the in vitro growth characteristics of fibroblasts derived from skin biopsies of Li-Fraumeni syndrome patients were studied. Control fibroblasts maintained a normal morphology and eventually senesced in culture. Fibroblasts from seven of eight affected individuals developed changes in morphology, anchorage-independent growth, and chromosomal abnormalities. In a fashion similar to that of fibroblasts from normal donors they underwent a growth crisis during which their growth was slow, but they continued to grow past the point at which control samples had stopped dividing (35 population doublings). Fibroblasts from Li-Fraumeni cancer patients escape senescence, growing well beyond 35 population doublings with growth rates similar to early-passage cells. Patient fibroblasts maintain the morphology of a transformed cell but remain nontumorigenic in nude mice. These observations of the behavior of fibroblasts from patients with the Li-Fraumeni syndrome may have predictive value for the determination of gene carriers within these families who are at high risk of cancer.

Aneuploidy↗
PubMed1992

The effect of in vitro aging on human lung fibroblast beta-adrenergic receptor density, coupling and response.

Age-related changes in regulation of receptor response have been observed in several tissues and include regulation of beta-adrenergic receptor (beta-receptor) responses. The role of cellular aging in age-related changes in receptor response is not clear. We have examined the effect of aging in vitro on human fibroblast beta-receptor function. MRC-5 (embryonic lung) fibroblasts were aged by replication to produce cells of early, middle and late stages corresponding to the following cumulative population doublings: 15-20, 35-45 and greater than 50, respectively. Fibroblast membrane beta-receptor responses to isoproterenol (ISO, 0.1 mM) did not differ between the three stages. Adenylate cyclase responses to prostaglandin E1 (PGE1, 1 microM), guanosine triphosphate (GTP, 0.1 mM) and 5'-guanylimidodiphosphate (Gpp(NH)p, 0.1 mM) were also similar between the stages. Beta-receptor density (Bmax) was unaffected by in vitro aging. Beta-receptor agonist affinity, an indication of the capacity for beta-receptor coupling to the nucleotide binding protein (NS), was also unaffected by cell aging. These findings suggest that cellular aging in fibroblasts alone is not accompanied by changes in beta-receptor function.

Adenylyl Cyclases↗
PubMed1975

A rapid rise in cell variants during the senescence of populations of human fibroblasts.

An attempt has been made to measure the frequency of mutations throughout the lifespan of human fibroblast strain MRC-5. A novel procedure has been used which involves staining individual cells for high levels of glucose-6-phosphate dehydrogenase. Evidence is presented that this phenotype is due to mutation. The frequency of variants was scored from passage 16 until the final phase of senescence (passage 60). There is an exponential increase of stained cells throughout this period. The results are in agreement with the general error theory, which proposes that aging is due to a breakdown in the fidelity of information transfer between macromolecules.

Cell Line↗
PubMed2003

Human melanocyte senescence and melanoma susceptibility genes.

The molecular mechanisms and biology of cellular senescence in human melanocytes are discussed, including similarities to and differences from senescence in fibroblasts and other cell lineages. Special reference is made to the fact that the known melanoma susceptibility genes in the human, Inhibitor A of [cyclin-dependent] kinase 4-alternative reading frame (INK4A-ARF) and cyclin-dependent kinase 4, are involved in the regulation of cellular senescence, and possible reasons why this should be so. Based on the evidence including growth and survival kinetics of human and mouse melanocytes carrying germline deficiencies in the INK4A sequence, it is suggested that an 'M0' or p16/RB-dependent form of senescence may be particularly important in melanocytes. A speculative model is proposed, relating current concepts of early melanoma progression to the processes of cellular senescence and immortalization. This includes the suggestion that moles or nevi are senescent clones of melanocytes.

Animals↗
PubMed2004

Morphological adjustment of senescent cells by modulating caveolin-1 status.

Morphological change is one of the cardinal features of the senescent phenotype; for example, senescent human diploid cells have a flat large shape. However, the mechanisms underlying such senescence-related morphological alterations have not been well studied. To investigate this situation, we characterized the senescence-dependent changes of cellular structural determinants in terms of their levels and activities. These determinants included integrins, focal adhesion complexes, and small Rho GTPases, and special emphasis was placed on their relationships with caveolin-1 status. We observed that the expression integrin beta(1) and focal adhesion kinase (FAK) were increased and that the phosphorylations of FAK and paxillin, hallmarks of focal adhesion formation, were also increased in senescent human diploid fibroblast cells. Moreover, the Rho GTPases Rac1 and Cdc42 were found to be highly activated in senescent cells. In addition, focal adhesion complexes and Rho GTPases were up-regulated in the caveolin-rich membrane domain in the senescent cells. Activated Rac1 and Cdc42 directly interacted with caveolin-1 in senescent cells. Interestingly, caveolin-1 knock-out senescent cells, achieved by using small interfering RNA and antisense oligonucleotide, showed disrupted focal adhesion formation and actin stress fibers via the inactivation of FAK, which resulted in morphological adjustment to the young cell-like small spindle shape. Based on the results obtained, we propose that caveolin-1 plays an important role in senescence-associated morphological changes by regulating focal adhesion kinase activity and actin stress fiber formation in the senescent cells.

Caveolin 1↗
PubMed2001

Senescence and the healing rates of venous ulcers.

OBJECTIVE: Premature cellular senescence has been linked to venous hypertension and may contribute to delayed healing of venous ulcers. We hypothesized that the percentage of senescent cells in in vitro populations of fibroblasts isolated from venous ulcers is directly related to the clinical time-to-healing. METHODS: Biopsy specimens were obtained from ulcer margins and unaffected dermal tissue of the ipsilateral thigh of seven patients with active venous ulcers. Using explant culture techniques, we obtained populations of wound fibroblasts and normal fibroblasts. The percentage of senescence in these cell populations was determined with X-Gal (5-bromo-4-chloro-3-indolyl beta-D-galactoside), which was used as a stain for B-galactosidase, a biomarker for senescent dermal fibroblasts. The X-Gal stain is a peroxidase stain for B-galactosidase. All patients in the study were treated with compression dressings. On a weekly basis, digital images were taken until ulcers healed. Planimetric healing rates were calculated from these images, and an overall time-to-healing was recorded. All cytologic investigations were performed on first passage cells. RESULTS: The average starting ulcer size was 4.2 cm2. Five of the data points represented healed ulcers. The two remaining patients withdrew from the study to pursue other therapies after having been treated with compression dressings for a long time. Linear regression analysis of healed ulcers identified a relationship between percent of senescence and time-to-healing, which was statistically significant (R2 = 0.81, P =.037). High percentages of senescent cells also had a correlation with slowed planimetric healing, which was not statistically significant. CONCLUSIONS: This study demonstrates a clinical correlation between quantitative in vitro senescence and time-to-healing. A percentage of senescence that is greater than 15% in populations of cells isolated from venous ulcers may identify a "difficult to heal" ulcer. There is no good clinical indicator for determining the likelihood of ulcer healing, but these results indicate that senescence percentage may have potential in this regard.

Adult↗
PubMed1998

Protein kinase C-alpha levels are inversely associated with growth rate in cultured human dermal fibroblasts.

Human dermal fibroblasts are known to express the alpha, delta, epsilon, and zeta isoforms of protein kinase C (PKC). We asked whether the growth of human dermal fibroblasts correlates with expression of a particular PKC isoform. Of total PKC activity measured in the presence of calcium, a condition permissive for activation of all PKC isoforms, 75%) was contributed by PKC-alpha, suggesting that PKC-alpha is the dominant isoform in human dermal fibroblasts. We then further studied PKC-alpha under different culture conditions and in cultures derived from different aged donors. In both subconfluent and confluent cultures, total PKC activity and the level of PKC-alpha protein were consistently higher in slowly proliferating adult cells than in more rapidly proliferating newborn cells. Moreover, in newborn fibroblasts density strongly influenced these parameters. At subconfluent density, when cells were dividing exponentially, total PKC activity was 345+/-63 cpm/,ug protein; whereas at confluent density, when cells were growth arrested, it was 6-7 fold higher, 2334+/-50 cpm/ug protein. Immunoblot analysis using a specific monoclonal antibody against PKC-alpha exhibited a similar 6-7 fold increase in the level of PKC-alpha protein at confluent density. However, in adult cells, density had no influence on the already high total activity or level of PKC-alpha. To further determine whether the increases in the levels of total PKC activity and the alpha isoform correlate with the decreased growth rate, a characteristic of both adult donor-derived and confluent cells, total PKC activity and the level of PKC-alpha in subconfluent quiescent cells was compared to that in paired exponentially growing cells at the same density. Total PKC activity was 8836+/-71 cpm/microg protein in subconfluent quiescent cells versus 4415+/-175 cpm/microg protein in dividing cells. The level of PKC-alpha protein was also 2-3 fold higher in quiescent than in growing cultures. However, the amount of PKC-alpha mRNA in these two conditions was identical as determined by northern blot analysis. Taken together, these results suggest an inverse relationship between the levels of total PKC activity and PKC-alpha protein and fibroblast growth rate that is regulated at the post-transcriptional level.

Adult↗