Werner Syndrome Fibroblasts Research Articles

Telomere-based proliferative lifespan barriers in Werner-syndrome fibroblasts involve both p53-dependent and p53-independent mechanisms.

Werner-syndrome fibroblasts have a reduced in vitro life span before entering replicative senescence. Although this has been thought to be causal in the accelerated ageing of this disease, controversy remains as to whether Werner syndrome is showing the acceleration of a normal cellular ageing mechanism or the occurrence of a novel Werner-syndrome-specific process. Here, we analyse the signalling pathways responsible for senescence in Werner-syndrome fibroblasts. Cultured Werner-syndrome (AG05229) fibroblasts senesced after approximately 20 population doublings with most of the cells having a 2N content of DNA. This was associated with hypophosphorylated pRb and high levels of p16(Ink4a) and p21(Waf1). Senescent AG05229 cells re-entered the cell cycle following microinjection of a p53-neutralizing antibody. Similarly, production of the human papilloma virus 16 E6 oncoprotein in presenescent AG05229 cells resulted in senescence being bypassed and extended cellular life span. Werner-syndrome fibroblasts expressing E6 did not proliferate indefinitely but reached a second proliferative lifespan barrier, termed M(int), that could be bypassed by forced production of telomerase in post-M1 E6-producing cells. The conclusions from these studies are that: (1) replicative senescence in Werner-syndrome fibroblasts is a telomere-induced p53-dependent event; and (2) the intermediate lifespan barrier M(int) is also a telomere-induced event, although it appears to be independent of p53. Werner-syndrome fibroblasts resemble normal human fibroblasts for both these proliferative lifespan barriers, with the strong similarity between the signalling pathway linking telomeres to cell-cycle arrest in Werner-syndrome and normal fibroblasts providing further support for the defect in Werner syndrome causing the acceleration of a normal ageing mechanism.

A lack of telomeric non-reciprocal recombination (TENOR) may account for the premature proliferation blockade of Werner's syndrome fibroblasts.

Cell lineages survive as long as their descendants replicate. In human diploid fibroblasts (HDF) there is a telomere-driven clock that determines the limited replicative life span of their cell branches. Werner's syndrome fibroblasts (WF) prematurely limit their replicative life span compared to HDF on account of a defective RecQ helicase, WRNp. Here, we introduce the concept of telomeric non-reciprocal recombination (TENOR) and suggest that its dysregulation in WF is responsible for their premature senescence. We postulate that TENOR functions at the level of four-strand replisomes which are specific for and initiated at fused chromosome ends. Consequently, healthy replicatively senescent fibroblasts can undergo one further division mediated by a TENOR event. Thereby, one of the two daughter cells re-acquires proliferation capacity that initiates a new proliferating cell branch from the formerly replicatively senescent cell. We argue that the unique mechanisms of action of WRNp support this concept, and we apply it to WF. If it is stipulated that WF are defective for TENOR because of their defective WRNp, a simple model emerges as to how they become prematurely senescent.

1P-0222 Cdc42, a RhoGTPase, restores defective choleterol efflux in Werner syndrome fibroblasts

1P-0222 Cdc42, a RhoGTPase, restores defective choleterol efflux in Werner syndrome fibroblasts

WRN helicase accelerates the transcription of ribosomal RNA as a component of an RNA polymerase I-associated complex.

Werner syndrome (WS) is a rare autosomal recessive genetic disorder causing premature aging. The gene (WRN) responsible for WS encodes a protein homologous to the RecQ-type helicase. WRN has a nucleolar localization signal and shows intranuclear trafficking between the nucleolus and the nucleoplasm. WRN is recruited into the nucleolus when rRNA transcription is reactivated in quiescent cells. Inhibition of mRNA transcription with alpha-amanitin has no effect on nucleolar localization of WRN whereas inhibition of rRNA transcription with actinomycin D releases WRN from nucleoli, suggesting that nucleolar WRN is closely related to rRNA transcription by RNA polymerase I (RPI). A possible function of WRN on rRNA transcription through interaction with RPI is supported by the results described here showing that WRN is co-immunoprecipitated with an RPI subunit, RPA40. Here we show that WS fibroblasts are characterized by a decreased level of rRNA transcription compared with wild-type cells, and that the decreased level of rRNA transcription in WS fibroblasts recovers when wild-type WRN is exogenously expressed. By contrast, exogenously expressed mutant-type WRN lacking an ability to migrate into the nucleolus fails to stimulate rRNA transcription. These results suggest that WRN promotes rRNA transcription as a component of an RPI-associated complex in the nucleolus.

A model for the phenotypic presentation of Werner's syndrome

Werner's syndrome (WS) is a valuable model of accelerated ageing and results from mutations in a recQ helicase ( wrn). WS fibroblasts show a mutator phenotype, replication fork stalling, increased rates of mean telomeric loss and accelerated cellular senescence. Senescence has been proposed as a candidate mechanism for the ageing of mitotic tissue. However, some mitotic tissues (such as the immune system) seem unaffected in WS. Is this evidence against a role for cell senescence in ageing? Two experiments resolve this paradox (i) the demonstration that the abbreviated replicative lifespan of WS fibroblasts can be corrected by the ectopic expression of telomerase and (ii) the demonstration that T cells derived from WS patients have the mutator phenotype characteristic of the disease but show no reduction in replicative potential. Since T cells can upregulate telomerase naturally these findings are consistent with a model in which the only wrn-mediated deletions that have a significant effect on replicative lifespan are those at or near the telomere. These data are thus supportive of a role for senescence in the ageing of the immune system. Emerging data on divisional counting mechanisms have the potential to produce many other apparent WS ‘paradoxes’. Accordingly, we propose a general model for the phenotypic presentation of WS, which includes a modification of the Olovnikov model of telomere erosion. Somewhat unexpectedly, this predicts that accelerated senescence should not be observed in all telomerase-negative WS cell types.

The Saccharomyces cerevisiae WRN homolog Sgs1p participates in telomere maintenance in cells lacking telomerase.

Werner syndrome (WS) is marked by early onset of features resembling aging, and is caused by loss of the RecQ family DNA helicase WRN. Precisely how loss of WRN leads to the phenotypes of WS is unknown. Cultured WS fibroblasts shorten their telomeres at an increased rate per population doubling and the premature senescence this loss induces can be bypassed by telomerase. Here we show that WRN co-localizes with telomeric factors in telomerase-independent immortalized human cells, and further that the budding yeast RecQ family helicase Sgs1p influences telomere metabolism in yeast cells lacking telomerase. Telomerase-deficient sgs1 mutants show increased rates of growth arrest in the G2/M phase of the cell cycle as telomeres shorten. In addition, telomerase-deficient sgs1 mutants have a defect in their ability to generate survivors of senescence that amplify telomeric TG1-3 repeats, and SGS1 functions in parallel with the recombination gene RAD51 to generate survivors. Our findings indicate that Sgs1p and WRN function in telomere maintenance, and suggest that telomere defects contribute to the pathogenesis of WS and perhaps other RecQ helicase diseases.

Accelerated methylation of ribosomal RNA genes during the cellular senescence of Werner syndrome fibroblasts.

Ribosomal DNA (rDNA) metabolism has been implicated in cellular and organismal aging. The role of rDNA in premature and normal human aging was investigated by measuring rDNA gene copy number, the level of rDNA methylation, and rRNA expression during the in vitro senescence of primary fibroblasts from normal (young and old) donors and from Werner syndrome (WS) patients. In comparison to their normal counterparts, WS fibroblasts grew slowly and reached senescence after fewer doublings. The rDNA copy number did not change significantly throughout the life span of both normal and WS fibroblasts. However, in senescent WS and normal old fibroblasts, we detected rDNA species with unusually slow electrophoretic mobility. Cellular aging in Saccharomyces cerevisiae is accompanied by the formation and accumulation of rDNA circles. Our analysis revealed that the rDNA species observed in this study were longer, linear rDNA molecules attributable to the inhibition of ECO:RI cleavage by methylation. Furthermore, isoschizomeric restriction analysis confirmed that in vitro senescence of fibroblasts is accompanied by significant increases in cytosine methylation within rDNA genes. This increased methylation is maximal during the abbreviated life span of WS fibroblasts. Despite increased methylation of rDNA in senescent cells, the steady-state levels of 28S rRNA remained constant over the life span of both normal and WS fibroblasts.

Overexpression of mRNAs of TGFβ-1 and related genes in fibroblasts of Werner syndrome patients

We analyzed mRNAs that were up- or down-regulated in fibroblasts from Werner syndrome (WS) patients compared with those from normal individuals. The mRNAs from normal and WS cells were first screened by differential display, and those mRNAs that were apparently up- or down-regulated were selected except for mRNAs related to extra-cellular matrix (ECM) proteins that are already known to be up-regulated in WS fibroblasts. Then, the expression levels of these mRNAs were semiquantified by northern blot analysis, and six up-regulated and two down-regulated mRNAs were identified in WS cell lines. Among the six up-regulated mRNAs were three mRNAs that coded TGFβ-1 and two proteins, their expressions of which were increased by TGFβ-1. These results together with the fact that TGFβ-1 up-regulates the expression of ECM proteins strongly suggest that TGFβ-1 has a key role in accelerated cellular senescence of fibroblasts of WS patients.

Telomerase prevents the accelerated cell ageing of Werner syndrome fibroblasts.

Werner syndrome (WS) is a rare disorder inherited in an autosomal recessive manner and characterized by accelerated ageing1. WS fibroblasts display an accelerated rate of senescence in vitro2, which has been linked to this progeroid phenotype. The senescence of normal human fibroblasts is triggered by telomere shortening3,4,5, whereas the premature senescence of WS fibroblasts has been assumed6,7 to reflect the accumulation of DNA damage. Here we show that forced expression of telomerase in WS confers extended cellular lifespan and probable immortality.

Spectral karyotyping of Werner syndrome fibroblast cultures

Fibroblast cultures from two Werner syndrome patients were analyzed by spectral karyotyping. There were multiple, pseudodiploid clones in both cultures, mostly marked by random balanced reciprocal translocations. One of the cultures contained a clone with three-way exchanges involving chromosomes 2, 3, and 16. Duplication-deficiencies were exceptional, as were completely normal metaphases. The most frequent breakpoint occurred at 16q22 which corresponds to FRA16B, possibly reflecting difficulties of WS cells in replicating AT-rich repetitive DNA structures. Both cultures ceased proliferation after eight in vitro passages, but a single clone with exceptional growth potential emerged in one of the senescing cultures. Due to its identical translocations, the derivation of this near tetraploid clone (with tetrasomy for all autosomes except chromosomes 4 and 6) could be traced to the most prevalent pseudodiploid clone of the parental mass culture. Our study confirms the existence of variegated translocation mosaicism as the cytogenetic hallmark of WS fibroblast cultures and suggests that tetraploidization in combination with certain chromosome rearrangements and selective chromosome dosage may overcome the severely limited in vitro lifespan of WS fibroblasts.

Cell fusion corrects the 4-nitroquinoline 1-oxide sensitivity of Werner syndrome fibroblast cell lines

Cell fusion corrects the 4-nitroquinoline 1-oxide sensitivity of Werner syndrome fibroblast cell lines

Cell fusion corrects the 4-nitroquinoline 1-oxide sensitivity of Werner syndrome fibroblast cell lines.

We have shown that Werner syndrome (WRN) fibroblast cell lines are unusually sensitive to the DNA-damaging agent 4-nitroquinoline 1-oxide (4NQO), though not to gamma radiation or to hydrogen peroxide. The fusion of 4NQO-sensitive WRN and 4NQO-resistant control fibroblast cell lines generated proliferating WRN x control cell hybrids that expressed WRN protein and were 4NQO-resistant. These results establish the recessive nature of 4NQO sensitivity in WRN cell lines and provide a cellular assay for WRN protein function.

P53-mediated apoptosis is attenuated in Werner syndrome cells.

The WRN DNA helicase is a member of the DExH-containing DNA helicase superfamily that includes XPB, XPD, and BLM. Mutations in WRN are found in patients with the premature aging and cancer susceptibility syndrome known as Werner syndrome (WS). p53 binds to the WRN protein in vivo and in vitro through its carboxyl terminus. WS fibroblasts have an attenuated p53- mediated apoptotic response, and this deficiency can be rescued by expression of wild-type WRN. These data support the hypothesis that p53 can induce apoptosis through the modulation of specific DExH-containing DNA helicases and may have implications for the cancer predisposition observed in WS patients.

A comparative analysis of the proteins between the fibroblasts from Werner's syndrome patients and age-matched normal individuals using two-dimensional gel electrophoresis

Werner's syndrome (WS) is an autosomal recessive disorder causing symptoms of premature aging. The fibroblasts of WS patients have a shorter life-span than normal fibroblasts. We analyzed the fibroblast proteins from three WS patients and from three age-matched normal individuals using two-dimensional gel electrophoresis and image processing. The expressions of 12 proteins were shown to be augmented or suppressed in WS fibroblasts compared with normal fibroblasts: 11 of 12 spots on the electrophoresis gel of WS fibroblasts were denser than the corresponding spots of normal individual fibroblasts, while the remaining one spot was fainter in WS fibroblasts than in normal fibroblasts. The abundance of these proteins were compared to those of the corresponding proteins from normal fibroblasts at various cell passages in vitro reported in the TMIG-2DPAGE database. The result shows that the change in the protein patterns in in vitro aging did not necessarily correspond to the change in WS fibroblasts, except for three proteins abundant in WS fibroblasts, which increased their abundance during in vitro aging. These results suggest that the premature aging process of WS fibroblasts shares only part of the in vitro aging process of normal fibroblasts.

Cycling Werner's Syndrome Fibroblasts Display Calcium-Dependent Potassium Currents

Werner's Syndrome (WS) fibroblasts undergo premature senescence. Two hypotheses have been proposed to explain this phenomenon: (i) the phenotype is due to the overexpression of senescence-specific proteins in every cell in the population. Such proteins are known to suppress calcium-dependent potassium currents. (ii) The WS mutation greatly increases the proportion of cells that stop cycling at each generation and become senescent. If hypothesis (i) is correct, such currents should be suppressed in all WS fibroblasts; whereas hypothesis (ii) predicts that they will be retained in the cycling fraction of the population. To distinguish between these hypotheses whole-cell patch-clamp currents were recorded from cycling cells. Slowly activating outward calcium-dependent potassium currents were detected in both cycling WS and control fibroblasts. These findings support hypothesis (ii): the premature senescence of WS fibroblasts is due to an increased rate of transition from cycling to senescence in the total cell population.

Altered Expression and Regulation of the α5β1 Integrin–Fibronectin Receptor Lead to Reduced Amounts of Functional α5β1 Heterodimer on the Plasma Membrane of Senescent Human Diploid Fibroblasts

Previously, we reported that fibronectin (FN) mRNA was overexpressed in normal late-passage (old) and prematurely senescent Werner syndrome (WS) fibroblasts when compared to normal early-passage (young) cells (Muranoet al. Mol. Cell. Biol.11, 3905–3914, 1991). Therefore, we investigated the expression and function of the α5β1 FN receptor (FNR), a member of the integrin family, in young and senescent normal and WS cells. Levels of the α5 polypeptide, a unique subunit of the α5β1 FNR, were reduced in old cells, so that old cells produced fewer α5β1 heterodimers on the plasma membrane. The reduced levels of α5 polypeptide might be due to deficient translation and/or nonfunctional α5 mRNA since increased mRNA levels and unchanged polypeptide turnover were found in these cells. Moreover, the α5 polypeptides on the senescent cell surface were less accessible to monoclonal antibody, suggesting sequestration of this subunit, which might affect receptor–ligand binding. In contrast, β1 subunit, a common subunit for the β1 integrin subfamily, showed relatively stable levels during cellular aging, but underwent slower intracellular processing. Old cells exhibited reduced attachment to FN, which might be in part mediated by the α5β1 FNR. More importantly, old cells were deficient in response to FN-induced DNA synthesis and cell proliferation. This induction was pronounced in young cells, however, and could be completely inhibited by α5-specific monoclonal antibody, indicating mediation by α5β1 FNR. WS cells behaved like normal old cells in the above assays. Our results indicate that reduction of α5β1 FNR expression and its mediated effects are associated with the senescent phenotype of fibroblasts. These findings provide new insight into the mechanism(s) of replicative senescence in human fibroblasts.

DNA Repair Fine Structure in Werner's Syndrome Cell Lines

Werner's syndrome (WS) is a human segmental progerioid disorder with an autosomal recessive pattern of inheritance. Patients with WS exhibit a number of symptoms resembling a premature aging phenotype. We have examined the fine structure of the DNA repair of UV-induced cyclobutane pyrimidine dimers in Epstein–Barr virus (EBV)-transformed WS lymphoblastoid cell lines and in a primary WS fibroblast cell line. The repair was measured at the level of the gene and also in the general genome. Gene-specific and strand-specific DNA repair was measured in the actively transcribed genes dihydrofolate reductase (DHFR), c-myc,and p53, and in the transcriptionally inactive regions, δ globin and the X-linked 754 domain. Both gene-specific repair and strand-specific repair were deficient in the transformed WS lymphoblastoid cell lines compared to normal controls. In normal cells, repair in the transcribed strand was 25 (4 h), 43 (8 h), and 72% (24 h); in the WS cells on average, repair in the transcribed strand was 18 (4 h), 27 (8 h), and 44% (24 h). However, in the primary WS fibroblast cell line, we found a pattern of preferential gene repair which was similar to that in normal human cells. In contrast to cells from patients with the gene-specific repair deficient disease Cockayne's syndrome, which show greatly delayed RNA synthesis recovery after UV irradiation, the WS cells had normal recovery of RNA synthesis. The DNA repair results differ for the different cell types, and our findings thus do not establish a general DNA repair phenotype for WS cells. The fibroblasts had proficient repair, but in the WS lymphoblasts we find a deficiency in DNA repair which could contribute to the reported hypermutability in these cells. The lymphoblasts are, however, transformed cells, and it raises the concern that biological findings in transformed cells may not reflect the situation in primary cells.

Altered Regulation of Fibronectin Gene Expression in Werner Syndrome Fibroblasts

Fibronectin (FN) production was quantified in the extracellular medium and in extracts of human diploid fibroblasts (HDF) by enzyme-linked immunosorbent assay and by immunoprecipitation of [35S]methionine biosynthetically labeled proteins. FN output was increased in normal late passage (old) HDF and in prematurely senescent Werner syndrome (WS) fibroblasts, compared to normal early passage (young) HDF. Output was maximal when cells were preconfluent, exceeding the level found at confluence and postconfluence. For all cell types the highest proportion of newly synthesized FN was found in the extracellular compartment. Both young and old HDF exposed to regular growth medium containing 15% fetal bovine serum (FBS) secreted twice as much FN as when exposed to serum-free medium, and cognate mRNA levels were commensurate with protein output. WS fibroblasts secreted 1.6-fold more FN than old HDF when both cell types were exposed to medium containing 15% FBS. Immunofluorescent analysis revealed greater association of FN with WS and old HDF than with young HDF. Furthermore, WS cells exposed to 15% FBS contained 3.4-fold more FN mRNA and produced seven times more FN protein compared to WS cells exposed to serum-free medium. Thus, WS fibroblasts secrete more FN than old normal and young normal HDF in the presence of 15% FBS due to augmentation of FN mRNA levels and enhanced efficiency of FN mRNA translation and/or post-translational processing.

Activation of fibroblast proliferation by Werner's syndrome fibroblast-conditioned medium

The effects of Werner's syndrome (WS) fibroblast-conditioned medium on cell proliferation and collagen production of normal fibroblasts were studied using four WS fibroblast strains. The conditioned medium from the WS fibroblasts brought about activation of normal fibroblast proliferation, whereas, that from late passage normal fibroblasts and fibroblasts from aged donors' skin did not. Collagen and non-collagenous protein synthesis in normal fibroblasts were increased by the addition of conditioned medium from the WS fibroblasts, but the relative rates of collagen synthesis and non-collagenous protein synthesis were unaltered. These results suggest that activation of fibroblast proliferation by conditioned medium is one of the characteristics of WS fibroblasts.

The expression of proliferation-dependent antigens during the lifespan of normal and progeroid human fibroblasts in culture

Normal human fibroblasts display a limited lifespan in culture, which is due to a steadily decreasing fraction of cells that are able to proliferate. Using antibodies that react with antigens present in proliferating cells only, in an indirect immunofluorescence assay, we have estimated the fraction of proliferating cells in cultures of normal human fibroblasts. Furthermore, we have estimated the rate of decline in the fraction of proliferating cells during the process of cellular ageing by application of the assay to normal human fibroblasts throughout their lifespan in culture. Werner's Syndrome is an autosomal recessive disease in which individuals display symptoms of ageing prematurely. Werner's Syndrome fibroblasts display a reduced lifespan in culture compared with normal human fibroblasts. Like normal human fibroblasts, the growth of Werner's Syndrome fibroblasts is characterised by a decreasing fraction of cells reacting with the proliferation-associated antibodies throughout their lifespan in culture. However, the rate of loss of proliferating cells in Werner's Syndrome fibroblasts during the process of cellular ageing is accelerated 5- to 6-fold compared with the rate determined for normal human fibroblasts.