Werner Syndrome Fibroblasts Research Articles

Decreased Type VI Collagen Gene Expression in Cultured Werner's Syndrome Fibroblasts

Gene expression of collagens VI, I, and III in Werner's syndrome was studied by measuring messenger RNA (mRNA) and protein production levels in four fibroblast strains from patients with Werner's syndrome and comparing them with age-matched healthy subjects. Levels of type VI collagen mRNA were decreased in all Werner's syndrome fibroblast strains and the decreases were in parallel in all three chains (alpha 1, alpha 2, and alpha 3) of type VI collagen. A coordinate increase of the alpha 1(I) and alpha 1(III) collagen mRNA levels was observed in three of the four Werner's syndrome fibroblast strains. However, no qualitative abnormality of these mRNA transcripts in Werner's syndrome fibroblasts were found by Northern blot analysis. Changes in type VI and type I collagen mRNA correlated well with production levels of corresponding proteins, as determined by immunologic assays. These data suggest that there are changes in expression of multiple connective tissue constituents in Werner's syndrome fibroblasts.

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.

Analysis of the primary structure of insulin-like growth factor binding protein-3 cDNA from Werner syndrome fibroblasts.

The mRNA encoding insulin-like growth factor binding protein-3 (IGFBP-3) is equally overexpressed in late-passage (old) normal human diploid fibroblasts (HDF) and in HDF derived from individuals with the premature aging disorder Werner syndrome (WS), relative to early-passage (young) normal HDF. However, the accumulation of IGFBP-3 protein in medium conditioned by WS cells is substantially less than in medium of old cells. In an attempt to understand this disparity between mRNA levels and protein output, we determined the nucleotide sequence of IGFBP-3 cDNA isolated from a WS cDNA library derived from mRNA of WS HDF, and compared it to three published normal IGFBP-3 DNA sequences. In the open reading frame, our results differed from one of the three sequences by a glycine substitution for alanine at residue 32. Minor differences in the 3'-untranslated region between the WS cDNA sequence and all three of the normal DNA sequences were also detected as 12 individual base substitutions and one adenine insertion. Thus, dampened accumulation of IGFBP-3 in medium conditioned by WS cells is not due to significant alterations in the sequence of the cognate mRNA.

Isolation and characterization of gene sequences overexpressed in werner syndrome fibroblasts during premature replicative senescence

Isolation and characterization of gene sequences overexpressed in werner syndrome fibroblasts during premature replicative senescence

Nucleotide sequence analysis of human hypoxanthine phosphoribosyltransferase ( HPRT) gene deletions

We have determined the nucleotide sequences of 10 intragenic human HPRT gene deletion junctions isolated from thioguanine-resistant PSV811 Werner syndrome fibroblasts or from HL60 myeloid leukemia cells. Deletion junctions were located by fine structure blot hybridization mapping and then amplified with flanking oligonucleotide primer pairs for DNA sequence analysis. The junction region sequences from these 10 HPRT mutants contained 13 deletions ranging in size from 57 bp to 19.3 kb. Three DNA inversions of 711, 368, and 20 bp were associated with tandem deletions in two mutants. Each mutant contained the deletion of one or more HPRT exon, thus explaining the thioguanine-resistant cellular phenotype. Deletion junction and donor nucleotide sequence alignments suggest that all of these HPRT gene rearrangements were generated by the nonhomologous recombination of donor DNA duplexes that share little nucleotide sequence dentity. This result is surprising, given the potential for homologous recombination between copies of repeated DNA sequences that constitute approximately a third of the human HPRT locus. No difference in deletion structure or complexity was observed between deletions isolated from Werner syndrome or from HL60 mutants. This suggests that the Werner syndrome deletion mutator uses deletion mutagenesis pathway(s) that are similar or identical to those used in other human somatic cells.

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.

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.

Reduced levels of mitogen-mediated protein phosphorylation in Werner's syndrome fibroblast

Reduced levels of mitogen-mediated protein phosphorylation in Werner's syndrome fibroblast

Increased Collagen Synthesis Accompanying Elevated m-RNA Levels in Cultured Werner's Syndrome Fibroblasts

Although Werner's syndrome (WS) is a premature aging disease and its fibroblasts typically grow poorly in culture, WS may cause abnormalities in connective tissue metabolism that are seldom seen in normal aging, such as scleroderma-like skin. In a preliminary report, we described increased collagen synthesis in fibroblasts derived from two WS patients. The present study was undertaken to determine the degree of the regulation of collagen gene expression in dermal fibroblasts from two other patients. Overproduction of collagenase sensitive protein was observed in WS fibroblasts. Collagen m-RNA levels, that were determined by hybridization of RNA blots with specific cDNA were about 2 times greater than those in the control cells. These results suggest that control of collagen synthesis in WS fibroblasts is altered at the transcriptional level.

Levels of DNA methylation in diploid and SV40 transformed human fibroblasts

Total 5-methylcytosine (5mC) has been measured in the DNA of cultured human cells by HPLC chromatography. In agreement with other reports, the level of 5mC declines continuously during the serial passaging of diploid fibroblasts, strain MRC-5. On the contrary, fibroblasts infected with SV40, which have acquired most of the properties of transformed cells, maintain a constant level of 5mC, prior to dying out in the crisis period. MRC-5 and Werner's syndrome fibroblasts fully transformed and immortalised by SV40, also maintain a constant level of 5mC. These results indicate that the ability of cells to maintain DNA methylation is a necessary but not a sufficient step for immortalisation.

Mitogen-mediated protein phosphorylation in Werner's syndrome fibroblasts.

DNA synthesis of WF-1 fibroblasts derived from a patient with Werner's syndrome was stimulated by fetal calf serum and adult human serum but not by various mitogens including epidermal growth factor, platelet-derived growth factor (PDGF), fibroblast growth factor, insulin and 12-O-tetradecanoylphorbol-13-acetate (TPA). To clarify the cause of nonresponsiveness to these mitogens, we compared the rate of protein phosphorylation in normal fibroblasts HF-O and Werner's WF-1 cells. PDGF and TPA enhanced the phosphorylation of a Mr 80 K protein, which is known to be a substrate for protein kinase C, both in HF-O and WF-1 cells. This indicates that the pathway involving PDGF receptor, phosphatidylinositol turnover and protein kinase C activation is operational in WF-1 cells. Several species of phosphoproteins of Mr 250 K, 135 K, 110 K, 78 K and 42 K were detected in normal HF-O cells by immunoprecipitation using an anti-phosphotyrosine antibody. The same species of phosphoproteins were detected in Werner's WF-1 cells at passage 6, but only when treated with various mitogens and were not detected in WF-1 cells at passage 10 even after the PDGF- or TPA-treatment. These results suggest that the reduction of phosphorylation of these target proteins may be in part responsible for the diminished mitogenic responsiveness of Werner's fibroblasts.

Reduced sensitivity to catecholamine in Werner's syndrome fibroblasts

The β-adrenergic receptor-coupled adenylate cyclase system has been investigated in normal and Werner's syndrome fibroblasts. The basal levels of cAMP in Werner and normal control cells were similar, whereas the isoproterenol-induced increase in cAMP levels was far less for Werner cells than for control cells. In the broken cell preparations isoproterenol stimulated the adenylate cyclase of only control cells, not of Werner cells, although NaF or prostaglandin E 1 stimulated the enzyme of both cells to the same extent. The β-adrenergic receptor concentrations analyzed with hydrophilic radioligand were nearly equal in Werner and in control cells. A reduction of functional activity of the β-adrenergic receptor in Werner cells is thus suggested.

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 35SO 4 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: ( a) increased GAG sulfation; ( b) an increase in GAG chain length; ( c) an increased rate of GAG synthesis; and ( d) 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.

Diminished response of Werner's syndrome fibroblasts to growth factors PDGF and FGF.

Patients with Werner's syndrome, an autosomal recessive disorder, undergo an accelerated aging process that leads to premature death. Fibroblasts from such patients typically grow poorly in culture. Here it is shown that fibroblasts from a patient with Werner's syndrome have a markedly attenuated mitogenic response to platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF). In contrast, they have a full mitogenic response to fetal bovine serum. Both PDGF binding and receptor numbers per cell are unaltered. The Werner's syndrome cells express high constitutive levels of collagenase in vitro. Although PDGF enhances collagenase expression through increased levels of hybridizable collagenase messenger RNA in normal skin fibroblasts, no induction of collagenase occurs in the Werner's syndrome fibroblasts. Moreover, the failure to respond to this agonist effect of PDGF is not restored by fetal bovine serum. The data suggest that failure of one or more PDGF-mediated pathways in Werner's syndrome cells may contribute to the phenotypic expression of the disorder.

Elevated spontaneous mutation rate in SV40-transformed werner syndrome fibroblast cell lines

Spontaneous mutation rates of the cells from patients with Werner syndrome were examined, and we found that the spontaneous mutation rates at the hypoxanthine-guanine phosphoribosyltransferase locus in SV40-transformed Werner syndrome cell lines were markedly elevated, compared to those in SV40-transformed normal control cell lines. Our results suggest that Werner syndrome is a mutation mutant.

Glycosaminoglycan synthesis in untransformed and transformed Werner syndrome fibroblasts: a preliminary report.

Glycosaminoglycan (GAG) synthesis was studied in untransformed and transformed normal and Werner syndrome (WS) fibroblasts, because WS manifests pleiotropic abnormalities in connective tissue. Continuous labelling of cells with [3H] glucosamine and [35S] sulfate for 48 hours revealed enhanced synthesis of cellular GAG, more rapid transfer of these into the pericellular fraction, and more accumulation of GAG in the medium in cultures of untransformed WS fibroblasts compared with cultures of normal diploid cells. Total GAG in the 24 hour medium from confluent cultures was composed of 80-90% hyaluronic acid (HA) and 10-20% sulfated GAG (S-GAGs) in both untransformed normal and WS fibroblasts, whereas it was approximately 50% each HA and S-GAG in transformed normal and WS cells. The proportional enhancement of [35S] GAG synthesis in response to exogenous beta-D-xylopyranosides was similar in normal and WS cells, although transformed cells demonstrated only approximately one-half the enhancement observed in non-transformed cells. Thus, the overall activity of GAG synthesis is not grossly altered by the WS gene mutation. Enhanced synthesis and accumulation of HA and dermatan sulfate (DS) in the medium was characteristic of untransformed WS fibroblasts, but appeared to be normalized in an SV40-transformed WS cell line (PSV811), as in transformed normal cells (WI38CT-1). We need more experiments to determine whether aberrant GAG metabolism in WS cells is a direct or indirect expression of the primary gene defect.

Cell surface changes in senescent and Werner's syndrome fibroblasts: their role in cell proliferation.

Cell surface is known to participate in the regulation of cell proliferation through interaction with adjacent cell surfaces or the extracellular matrix, or both. A clinical survey of the Werner syndrome suggests some disorders in glycosaminoglycan metabolism. Also, the skin fibroblasts derived from the patients with WS have a reduced proliferation capacity. We here examined, in vitro and in vivo, alterations of the cell-surface properties of WS cells and aging human fibroblasts. Cell-surface negative charges, examined by electrophoretic mobility of dispersed single cells in buffer, were seen to decline steadily as a function of cumulative population doublings. A strict linear relationship was found between electrophoretic mobility (micron/sec/V/cm) and number of cells harvested at each passage in all cell lines examined. The slope of this line in cells from donors of different ages indicated that WS fibroblasts resemble cells from much older normal controls. The same conclusion was drawn from our previous study of Con A-mediated red cell adsorption, which was confirmed as reflecting an alteration of cell-surface coat negative charge. Electrophoretic mobility after treatment of cell surface with degradative enzymes showed that the cell-surface negative charges were attributable to sialic acid, chondroitin sulphates, hyaluronic acid, and heparan sulphate. Two-dimensional electrophoresis of 3H-glucosamine incorporated glycosaminoglycans (GAGs) revealed that heparan sulphate was the main component of GAGs on the fibroblast cell surface and that the relative amount of heparan sulphate among GAGs on the cell surface increased in vitro with the number of passages. Growth kinetics of fibroblasts on sheets of fixed cells treated with a fixative (glutaraldehyde) and degradative enzymes were examined to elucidate the role of cell-surface GAGs in the regulation of cell proliferation. Cell growth was inhibited 40% when the fibroblasts were cultured on the fixed sheets of late passage cells. Treatment of the fixed cell sheets with heparitinase or nitrous acid resulted in complete recovery from the growth inhibition. Cell growth on sheets of fixed cells derived from young, middle, and senescent fibroblasts showed that the surface of the senescent cells had the greatest inhibitory effect. These inhibitory effects of fixed cell sheets correlated well with both the amount of heparan sulphate relative to the total GAGs on the surface and to the saturation density of cell growth at each passage. These findings strongly suggest that heparan sulphate, or its complex, on the cell surface is involved in the regulation of cell proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)

Histone H1 in G1 arrested, senescent, and Werner syndrome fibroblasts.

Histone H1 content and synthesis were examined in normal, Werner-syndrome, and transformed fibroblasts. Analysis of 3H-lysine incorporation indicated that senescent cells, but not G1-arrested young cells, had a lower ratio of molar synthesis of H1 histone to nucleosome histones than did growing young cells or gamma-ray-transformed cells. Furthermore, a biochemical study of histone H1 content plotted as a function of DNA synthesis activity and an immunocytological study using antiserum against histone H1 revealed that senescent cells had a lower histone H1 content than did young cultures at all stages of cell proliferation. Werner syndrome skin fibroblasts at early passage, however, had amounts of histone H1 comparable to those of age-matched normal control fibroblasts. We conclude that a decline, with increasing passage number, in content and synthesis of H1 histone relative to nucleosomal histones (Mitsui et al., 1980) was not simply due to passage-related accumulation of G1-arrested cells, but actually reflected age specific changes of cultured human fibroblasts. The depletion of histone H1 in the chromatin of senescent cells is a possible cause of DNA strand breakage or relaxation of gene repression.

Abnormal fibroblast aging and DNA replication in the Werner syndrome.

Cell and DNA replicative potentials were studied in 10 strains of skin fibroblasts from unrelated patients with the Werner syndrome (WS) and in a progeric CRL 1277 strain. The lifespans of all WS strains and of CRL 1277 cells are greatly abbreviated in vitro, due to large fractions of non-cycling cells and the basic process of progressive clonal attenuation during Phase II. In some WS strains, the population-doubling rate per day and the cloning efficiency fluctuated concurrently in random fashion during the cellular aging process, indicating alternating successions of adaptively well and poorly growing clones, probably resulting from various chromosome translocations. No detectable defect in excision repair was found in WS or CRL 1277 cells. However, the rate of increase in the molecular weight of pulse-chased DNA involving overall rates of chain elongation and replicon fusion was retarded in WS and CRL 1277 cells. Also, pulse-labelled DNA in WS fibroblasts was less enriched in the nuclear matrix and was more slowly chased out than in normal cells. These results led us to postulate a misfiring or delayed initiation due to the sticky attachment of replicating DNA to the nuclear matrix in the replisomes of WS fibroblasts. A suggested model for abnormal DNA replication is presented and discussed to explain the loss of DNA and the chromosome abnormalities in WS cells. The abnormal DNA-synthetic profiles so derived appeared to be normalized in SV40-transformed PSV811 (WS) cells as were in gamma ray-transformed wild-type WI38CT-1 cells.

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

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.