A retarded rate of DNA replication and normal level of DNA repair in Werner's syndrome fibroblasts in culture

We investigated the cloning efficiency, DNA repair, and the rate of DNA replication in the skin fibroblasts from patients with Werner's syndrome (WS) of an autosomal recessive premature aging disease. Five WS strains exhibited normal levels of sensitivity toward X-ray and UV killings and repair of X-ray induced single strand breaks of DNA (rejoining) and UV damage to DNA (unscheduled DNA synthesis). The sedimentation of newly synthesizing DNA in alkaline sucrose gradients demonstrated a characteristic feature that only the elongation rate of DNA chains, estimated by the molecular weight increase, was significantly slower during early passages in WS cells than in normal Hayflick Phase II fibroblasts. In addition, plating efficiencies as well as the replicative potentials of five WS strains were more limited than those of normal cells under the identical culture conditions. It seems therefore that at least in the WS cells tested, the slow rate of DNA replication may be more related to the shortened lifespan and enhanced cell death, as manifestation of premature senescence at the cellular level, than be the DNA repair ability.     

Decrease in the average size of replicons in a Werner syndrome cell line by Simian Virus 40 infection

We have measured the distance between replicon initiation sites as well as the rate of DNA chain elongation in Simian Virus 40 (SV40)-infected and uninfected Werner syndrome (WS) and normal cell lines by DNA fiber-autoradiography. There was no difference in the rate of chain elongation among these cell lines. On the other hand, the replicon center-to-center distance was clearly longer in WS fibroblasts than that in normal fibroblasts. SV40 infection changed the center-to-center distance in WS cells toward that in normal cells.     

Hypothesis: Werner syndrome and biological ageing: A molecular genetic hypothesis

Werner syndrome (WS) is an inherited disorder that produces somatic stunting, premature ageing and early onset of degenerative and neoplastic diseases. Cultured fibroblasts derived from subjects with WS are found to undergo premature replicative senescence and thus provide a cellular model system to study the disorder. Recently, several overexpressed gene sequences isolated from a WS fibroblast cDNA library have been shown to possess the capacity to inhibit DNA synthesis and disrupt many normal biochemical processes. Because a similar constellation of genes is overexpressed in WS and senescent normal fibroblasts, these data suggest the existence of a common molecular genetic pathway for replicative senescence in both types of cell. We propose that the primary defect in WS is a mutation in a gene for a trans-acting repressor protein that reduces its binding affinity for shared regulatory regions of several genes, including those that encode inhibitors of DNA synthesis (IDS). The mutant WS repressor triggers a sequence of premature expression of IDS and other genes, with resulting inhibition of DNA synthesis and early cellular senescence, events which occur much later in normal cells.     

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.     

Turnover of sulfated glycosaminoglycans in fibroblasts derived from patients with Werner''s syndrome

Fibroblasts derived from patients with Werner's syndrome (WS) were incubated with radioactive sulfate to study the incorporation of 35S into glycosaminoglycans (GAGs). The accumulation of cell-associated 35S radioactivity in the GAGs of WS fibroblasts was consistently higher than parallel accumulation in normal human fibroblasts, but was substantially less than in fibroblasts derived from patients with Hurler's syndrome (HS). However, when fibroblasts were labeled with 35SO4(2-), trypsinized to remove extracellular and pericellular radioactive GAGs, replated, and chased to follow the fate of the intracellular radioactivity, both WS and normal cells showed a rapid release of the intracellular 35S, while HS cells showed little or no loss of intracellular radioactivity. The radioactivity released from WS and normal cells was of low molecular weight (LMW), eluting from gel filtration columns at the same position as free sulfate. These results establish that WS cells degrade intracellular sulfated GAGs and argue against the hypothesis that a defect in GAG degradation pathways is the basis for the increased level of cell-associated GAGs. Other possible explanations for the increased cell-associated [35S]GAGs in WS cells as compared with normal cells were also considered: increased GAG sulfation; an increase in GAG chain length; an increased rate of GAG synthesis; and a decreased rate of shedding of cell surface proteoglycan into the medium. No difference between normal and WS fibroblasts in any of the above parameters was observed. These results strongly imply that the primary biochemical defect in WS fibroblasts does not involve sulfated GAG metabolism.     

Regulation of c-fos expression in senescing Werner syndrome fibroblasts differs from that observed in senescing fibroblasts from normal donors

The Werner syndrome (WS) is a segmental progeroid syndrome caused by a recessive mutation (WRN) mapped to 8p12. The replicative life spans of somatic cells cultured from WS patients are substantially reduced compared to age-matched controls. Certain molecular concomitants of the replicative decline of normal fibroblast cultures have recently been defined, and it appears that multiple changes in gene expression accompany normal cell senescence. If the mechanisms by which WS cells exit the cell cycle were entirely comparable, the molecular markers of senescence should be identical in normal and WS cells. We find that this is not the case. The constitutive expression of statin, a nuclear protein associated with the nonproliferating state, was comparably expressed in normal and WS senescent cells. Likewise, the steady state levels of p53, a protein known to be involved in the G1 checkpoint of the cell cycle, were similar in early-passage fibroblasts from normal and WS subjects. The levels of p53 were not increased in senescent fibroblasts, whether derived from normal or WS subjects. By contrast, the inducibility of mRNA and protein expression of the c-fos protooncogene is preserved in late-passage WS cells. This is in contrast to what is observed in late-passage fibroblasts from normal subjects. Additional genotypes will have to be examined, however, to determine the specificity of this new aspect of the WS phenotype. © 1995 Wiley-Liss, Inc.     

Homologous recombination is elevated in some Werner-like syndromes but not during normal in vitro or in vivo senescence of mammalian cells

Werner syndrome (WS) is a recessive genetic condition associated with markedly reduced replicative lifespans of cells in culture, high chromosomal instability in vivo and in vitro, and premature appearance of many characteristics of normal aging, including an increased incidence of cancer. We have monitored plasmid homologous recombination frequencies in diploid fibroblasts from 6 Werner or Werner-like syndrome patients, following transfection with a plasmid substrate containing 2 overlapping fragments of the TN5 Neor gene. Plasmid DNA recovered from these cells was then assayed for homologous recombination by (a) transformation of recA- bacteria to Ampr (indicating total viable plasmid) or Neor (indicating viable recombinant plasmid), and (b) by limited-cycle polymerase chain reaction (PCR) to co-amplify a recombinant fragment containing the overlap region, and a control region of the same plasmid, without bacterial transformation. Bacterial assay data indicated that recombination rates in 3 of the 6 WS strains were significantly elevated above normal controls; 4 of 6 appeared elevated by PCR assay. The highest-recombination WS strain showed evidence of reduced degradation of transfected plasmid DNA. For this small sample of WS strains, clinical severity of WS was not well correlated with recombination rate as determined by either assay (Pearson r = 0.78, not significant, for PCR assay); elevated recombination may, however, define a subset of WS at greatest risk for cancer and/or atherosclerosis. PCR assay of a hyperoxia-resistant HeLa cell line, displaying substantially increased chromosome breakage, indicated increased recombination between direct-repeat fragments. Nevertheless, elevated recombination in WS strains is unlikely to be secondary to impaired replicative capacity characteristic of WS cells, or to defective repair of chromosome damage which is increased in WS, since recombination in non-WS strains was unaffected by passage level or repeated UV irradiation.     

Werner''s syndrome: proliferation in vitro of clones of cells bearing chromosome translocations.

Each of several cultures of Werner's syndrome (WS) fibroblasts and lymphoblasts examined was found to be composed of one or several clones of cells with mutated chromosome complements. Two "sister" fibroblasts cell lines (FCLs) that were derived from a mixture of explants cut from the same WS skin biopsy were found to have completely different rearranged chromosome complements. Daily observation of the skin explants from which these two sister FCLs were derived revealed not only that no more than a few fibroblasts ever migrated from a given explant but also that fibroblasts migrated from only a few of the explants. Two of three lymphoblastoid cell lines (LCLs), each probably developed as an independent clone from a different cell from the same WS blood sample, were mosaic, comprised of cells having both normal and rearranged chromosome complements. The third LCL studied, although nonmosaic, had a rearranged chromosome complement, but one that was completely different from those in the other two lines. Based on the observations described, hypotheses have been formulated to explain both the preponderance in long-term WS cultures of clones with mutated chromosome complements and the abbreviated lifespan characteristic of WS fibroblast cultures.     

Life span elongation of Werner's syndrome fibroblasts by co-culture with origin-defective SV-40 DNA transformed cells

Summary Co-culturing with origin-defective SV40 DNA-injected transformants, we succeeded in activating the limited growth potential of late-phase-II fibroblasts from patients with Werner's syndrome (WS cells). Residual life spans of three WS cell lines increased 2.3–5.6 fold. Co-culture with normal IMR-90 cells showed variable effects on different WS cell lines.     

Tn10 transposition does not respond to environmental stress

The rate of DNA synthesis after gamma-irradiation was studied either by analysis of the steady-state distribution of daughter [3H]DNA in alkaline sucrose gradients or by direct assay of the amount of [3H]thymidine incorporated into DNA of fibroblasts derived from a normal donor (LCH882) and from Down's syndrome (LCH944), Werner's syndrome (WS1LE) and xeroderma pigmentosum (XP2LE) patients with chromosomal sensitivity to ionizing radiation. Doses of gamma-irradiation that markedly inhibited the rate of DNA synthesis in normal human cells caused almost no inhibition of DNA synthesis in the cells from the affected individuals. The radioresistant DNA synthesis in Down's syndrome cells was mainly due to a much lower inhibition of replicon initiation than that in normal cells; these cells were also more resistant to damage that inhibited replicon elongation. Our data suggest that radioresistant DNA synthesis may be an intrinsic feature of all genetic disorders showing increased radiosensitivity in terms of chromosome aberrations.     

Radioresistant DNA synthesis in cells of patients showing increased chromosomal sensitivity to ionizing radiation

The rate of DNA synthesis after γ-irradiation was studied either by analysis of the steady-state distribution of daughter [³H]DNA in alkaline sucrose gradients or by direct assay of the amount of [³H]thymidine incorporated into DNA of fibroblasts derived from a normal donor (LCH882) and from Down's syndrome (LCH944), Werner's syndrome (WS1LE) and xeroderma pigmentosum (XP2LE) patients with chromosomal sensitivity to ionizing radiation. Doses of γ-irradiation that markedly inhibited the rate of DNA synthesis in normal human cells caused almost no inhibition of DNA synthesis in the cells from the affected individuals. The radioresistant DNA synthesis in Down's syndrome cells was mainly due to a much lower inhibition of replicon initiation than that in normal cells; these cells were also more resistant to damage that inhibited replicon elongation. Our data suggest that radioresistant DNA synthesis may be an intrinsic feature of all genetic disorders showing increased radiosensitivity in terms of chromosome aberrations.     

Diverse gene sequences are overexpressed in werner syndrome fibroblasts undergoing premature replicative senescence.

Genes that play a role in the senescent arrest of cellular replication are likely to be overexpressed in human diploid fibroblasts (HDF) derived from subjects with Werner syndrome (WS) because these cells have a severely curtailed replicative life span. To identify some of these genes, a cDNA library was constructed from WS HDF after they had been serum depleted and repleted (5 days in medium containing 1% serum followed by 24 h in medium containing 20% serum). Differential screening of 7,500 colonies revealed 102 clones that hybridized preferentially with [32P]cDNA derived from RNA of WS cells compared with [32P]cDNA derived from normal HDF. Cross-hybridization and partial DNA sequence determination identified 18 independent gene sequences, 9 of them known and 9 unknown. The known genes included alpha 1(I) procollagen, alpha 2(I) procollagen, fibronectin, ferritin heavy chain, insulinlike growth factor-binding protein-3 (IGFBP-3), osteonectin, human tissue plasminogen activator inhibitor type I, thrombospondin, and alpha B-crystallin. The nine unknown clones included two novel gene sequences and seven additional sequences that contained both novel segments and the Alu class of repetitive short interspersed nuclear elements; five of these seven Alu+ clones also contained the long interpersed nuclear element I (KpnI) family of repetitive elements. Northern (RNA) analysis, using the 18 sequences as probes, showed higher levels of these mRNAs in WS HDF than in normal HDF. Five selected mRNAs studied in greater detail [alpha 1(I) procollagen, fibronectin, insulinlike growth factor-binding protein-3, WS3-10, and WS9-14] showed higher mRNA levels in both WS and late-passage normal HDF than in early-passage normal HDF at various intervals following serum depletion/repletion and after subculture and growth from sparse to high-density confluent arrest. These results indicate that senescence of both WS and normal HDF is accompanied by overexpression of similar sets of diverse genes which may play a role in the senescent arrest of cellular replication and in the genesis of WS, normal biological aging, and attendant diseases.     

Increase in DNA synthesis in Werner''s syndrome cells by hybridization with normal human diploid and HeLa cells

The dominance or recessiveness of the senescent phenotype in cells from patients with Werner's syndrome (WS cells) was investigated using cell fusion. The [³H]thymidine labeling index of normal human diploid fibroblast cell X WS cell heterodikaryons was considerably lower than that of normal homodikaryons, but was significantly higher than that of WS homodikaryons. The labeling index of WS cell X HeLa cell heterodikaryons was the same as that of HeLa homodikaryons. The labeling indices of heterodikaryons obtained by fusion between various strains of premature aging cells were as low as those of parental homodikaryons. These results indicate: (1) the senescent phenotype of WS cells appears to be partially recessive to the phenotype of normal cells and completely recessive to that of HeLa cells; (2) the marked inhibition of DNA synthesis in normal nuclei in heterodikaryons with WS cells could be due to ‘senescent factor(s)’ in WS cells; and (3) no complementation phenomenon was observed among genetically different premature aging cells, probably due to ‘senescent factor(s)’.     

The susceptibility of Werner's syndrome and other human skin fibroblasts to SV40-induced transformation and immortalization

In attempts to transform and immortalize human cell cultures, skin fibroblasts from normal donors of different ages, from patients with the premature ageing diseases Werner's syndrome (WS) and progeria (PR), and from donors with the cancer-prone diseases ataxia telangiectasia (AT), Bloom's syndrome (BS) and Fanconi's anaemia (FA), were infected with SV40 virus and their growth monitored thereafter. Lesch-Nyhan (LN) fibroblasts were also infected. SV40-infected cultures from two normal and from WS, AT and LN donors attained a spectrum of transformed properties, high mitotic activity at confluence, presence of T-antigen, anchorage independence and altered morphology. Most of these pretransformed cultures died in the crisis period. However, two cultures from the WS and LN patients survived the crisis period and have now been grown to more than 200 passages. For the LN culture the crisis period was at least 200 days. Both permanent lines retain the properties of pretransformed cells, but differ in their modal chromosome number and ability to grow in methionine-free medium. It can be concluded from these experiments that transformation by SV40 to permanent lines is a rare event in human skin fibroblasts, even when these cells were taken from patients predisposed to form cancers.     

Werner's syndrome protein is required for correct recovery after replication arrest and DNA damage induced in S-phase of cell cycle

Werner's syndrome (WS) is a rare autosomal recessive disorder that arises as a consequence of mutations in a gene coding for a protein that is a member of RecQ family of DNA helicases, WRN. The cellular function of WRN is still unclear, but on the basis of the cellular phenotypes of WS and of RecQ yeast mutants, its possible role in controlling recombination and/or in maintenance of genomic integrity during S-phase has been envisaged. With the use of two drugs, camptothecin and hydroxyurea, which produce replication-associated DNA damage and/or inhibit replication fork progression, we find that WS cells have a slower rate of repair associated with DNA damage induced in the S-phase and a reduced induction of RAD51 foci. As a consequence, WS cells undergo apoptotic cell death more than normal cells, even if they arrest and resume DNA synthesis at an apparently normal rate. Furthermore, we report that WS cells show a higher background level of DNA strand breaks and an elevated spontaneous induction of RAD51 foci. Our findings support the hypothesis that WRN could be involved in the correct resolution of recombinational intermediates that arise from replication arrest due to either DNA damage or replication fork collapse.     

Asymmetry of DNA replication fork progression in Werner's syndrome

Human aging is associated with accumulation of cells that have undergone replicative senescence. The rare premature aging Werner's syndrome (WS) provides a phenocopy of normal human aging and WS patient cells recapitulate the aging phenotype in culture as they rapidly lose the ability to proliferate or replicate their DNA. WS is associated with loss of functional WRN protein. Although the biochemical properties of WRN protein, which possesses both helicase and exonuclease activities, suggest an involvement in DNA metabolism, its action in cells is not clear. Here, we provide experimental evidence for a role of the WRN protein in DNA replication in normally proliferating cells. Most importantly, we demonstrate that in the absence of functional WRN protein, replication forks from origins of bidirectional replication fail to progress normally, resulting in marked asymmetry of bidirectional forks. We propose that WRN acts in normal DNA replication to prevent collapse of replication forks or to resolve DNA junctions at stalled replication forks, and that loss of this capacity may be a contributory factor in premature aging.     

Reduced 5-hydroxymethyluracil-DNA glycosylase activity in Werner's syndrome cells.

Werner's syndrome (WS) is an autosomal recessive disease marked by early symptoms of accelerated aging. There is evidence indicating accumulation of oxidized DNA bases to be a major factor in cellular aging. The first step of excision repair of such bases in human cells is their removal from DNA by glycosylases. 5-Hydroxymethyluracil (HMU)-DNA glycosylase excises HMU from DNA; another glycosylase removes many non-aromatic pyrimidine derivatives. Levels of glycosylases that excise oxidized pyrimidines from DNA were compared between confluent and proliferating populations of WS cells, age-matched controls, and young control cells. They were assayed by measurements of direct release of free bases from their respective DNA substrates. Specific activities of the glycosylase that releases various modified pyrimidines and of uracil-DNA glycosylase (which removes uracil from DNA) were essentially the same in all cell lines. Cell cycle variations of these enzymes also did not differ between WS and control cells. HMU-DNA glycosylase specific activity was reduced in WS cells. Reduction of HMU-DNA glycosylase has been described in senescent human WI-38 cells. Therefore, while neither WS nor senescent cells have overall deficiencies of DNA glycosylase activities, they both might have reduced excision of HMU from DNA. This indicates a possible role of HMU accumulation in the aging process.     

Indian medicinal plants as antiradicals and DNA cleavage protectors

Celastrus paniculatus L. (Celastraceae) (CP), Picrorhiza kurroa L. (Scrophulariaceae) (PK) and Withania somnifera L. (Solanaceae) (WS) are Indian medicinal plants having a remarkable reputation, as a factor of health care, among the indigenous medical practitioners. The plants exhibit varying degrees of therapeutic value some of which useful in the treatment of cognitive dysfunction, epilepsy, insomnia, rheumatism, gout, dyspepsia. In this work, we have investigated the free radical scavenging capacity of methanolic extracts from CP, PK, WS and the effect on DNA cleavage induced by H2O2 UV-photholysis. In addition, we investigated whether these plant extracts are capable of reducing the hydrogen peroxide-induced cytotoxicity and DNA damage in human non-immortalized fibroblasts. These extracts showed a dose-dependent free radical scavenging capacity and a protective effect on DNA cleavage; methanolic extracts from PK was more active than extracts from CP and WS. These results were confirmed by a significant protective effect on H2O2-induced cytoxicity and DNA damage in human non-immortalized fibroblasts. These antioxidant effects of active principle of CP, PK and WS may explain, at least in part, the reported anti-stress, immunomodulatory, cognition-facilitating, anti-inflammatory and antiaging effects produced by them in experimental animal and in clinical situations and may justify the further investigation of their other beneficial biological properties.