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Christopher J Jones

Publications and source records attributed to Christopher J Jones.

13 recordsLinked to original sources
PubMed2006

Prevention of accelerated cell aging in the Werner syndrome.

In the Werner syndrome (WS) fibroblasts have an increased life span and growth rate when treated with the p38 inhibitor SB203580. Additionally, the cellular morphology reverts to that seen in young normal fibroblasts. The p38 pathway is activated in young WS cells, associated with high levels of p21(WAF1) leading to cell cycle arrest, and is suppressed by SB203580. As these changes are also seen in telomerized WS cells, these data show that the growth problems seen in WS cells, and perhaps the accelerated in vivo aging, are due to a telomere-independent premature senescence mechanism. The suppression of this mechanism by SB203580 treatment suggests a route whereby WS may be amenable to therapeutic intervention.

Actins↗
PubMed2005

Characterisation of novel mutations in Cockayne syndrome type A and xeroderma pigmentosum group C subjects.

We report that a subject with Cockayne syndrome type A (CS3BE) was a compound heterozygote for mutations in CKN1, the gene encoding the CSA protein (MIM 216400). CS3BE displayed a novel missense mutation (A160V) and a previously described nonsense mutation (E13X). Although residing between the second and third WD-40 repeats characteristic of the CSA protein, A160 is completely conserved in all species that possess a CKN1 homologue. We also describe a mutation in a previously uncharacterised xeroderma pigmentosum group C subject (XP8CA) in the XPC gene (MIM 278720). XP8CA was homozygous for a 2 bp TG deletion in codon 547 resulting in premature termination at codon 572. Immunoblotting of XP8CA extracts confirmed the absence of full-length XPC protein that was present in unaffected cell lines.

Cell Line↗
PubMed2005

Prevention of accelerated cell aging in Werner syndrome using a p38 mitogen-activated protein kinase inhibitor.

We investigated the role of p38 mitogen-activated protein kinase (MAPK) signalling in the accelerated aging of Werner Syndrome (WS) fibroblasts by use of SB203580, a cytokine-suppressive anti-inflammatory drug that targets p38 activity. SB203580 treatment reverts the aged morphology of young WS fibroblasts to that seen in young normal fibroblasts. In addition, SB203580 increases the life span and growth rate of WS fibroblasts to within the normal range. In young WS cells, p38 is activated coincident with an up-regulation of p21(WAF1), and a reduction in the levels of both activated p38 and p21(WAF1) are seen following treatment with SB203580. As these effects are not seen in young normal cells, our data suggest that the abbreviated replicative life span of WS cells is due to a stress-induced, p38-mediated growth arrest that is independent of telomere erosion. With some p38 inhibitors already in clinical trials, our data suggest a potential route to drug intervention in a premature aging syndrome.

Actins↗
PubMed2004

Telomere erosion triggers growth arrest but not cell death in human cancer cells retaining wild-type p53: implications for antitelomerase therapy.

Telomerase activity in tumours is often associated with p53 mutation. Many antitelomerase therapies take advantage of the inability of cells expressing mutant p53 to undergo replicative senescence, since this allows telomere erosion to continue until 'crisis', hence providing the desired cytotoxic effect. However, some tumour types, including breast, melanomas and thyroid, retain wild-type p53 function and the effectiveness of antitelomerase therapies in such tumour cells have not been adequately addressed. To explore this, we made use of two thyroid cancer cell lines K1 and K2, which retain wt p53. Telomere erosion induced by the expression of a dominant-negative (DN) hTERT resulted in delayed onset of growth arrest in K1 and K2 cells, reminiscent of replicative senescence, with low levels of BrdU labelling and apoptosis, associated with high p21(WAF1) and senescence-associated beta galactosidase expression. In contrast, abrogation of p53 function by the expression of HPV16 E6 in K1 and K2 cells either at the same time as DNhTERT or just prior to the onset of senescence allowed cells to continue growing until 'crisis'. Likewise, microinjection of a p53 neutralizing antibody into 'senescent' K1 DNhTERT cells permitted re-entry into the cell cycle. We conclude that thyroid tumour cells with wild-type p53 retain an intact p53-mediated growth arrest response to telomere erosion. This raises the intriguing question of why, therefore, p53 mutation is not selected for in such cancers, and also calls into question the therapeutic value of telomerase inhibitors in such cases.

Apoptosis↗
PubMed2004

Normal telomere erosion rates at the single cell level in Werner syndrome fibroblast cells.

The aim of this study was to investigate whether the accelerated replicative senescence seen in Werner syndrome (WS) fibroblasts is due to accelerated telomere loss per cell division. Using single telomere length analysis (STELA) we show that the mean rate of telomere shortening in WS bulk cultures ranges between that of normal fibroblasts [99 bp/population doubling (PD)] and four times that of normal (355 bp/PD). The telomere erosion rate in the fastest eroding strain slows in the later stages of culture to that observed in normal fibroblasts, and appears to be correlated with a reduction in the heterogeneity of the telomere-length distributions. Telomere erosion rates in clones of WS cells are much reduced compared with bulk cultures, as are the variances of the telomere-length distributions. The overall lack of length heterogeneity and the normal erosion rates of the clonal populations are consistent with simple end-replication losses as the major contributor to telomere erosion in WS cells. We propose that telomere dynamics at the single cell level in WS fibroblasts are not significantly different from those in normal fibroblasts, and suggest that the accelerated replicative decline seen in WS fibroblasts does not result from accelerated telomere erosion.

Cells, Cultured↗
PubMed2004

Fibroblast clones from patients with Hutchinson-Gilford progeria can senesce despite the presence of telomerase.

Hutchinson-Gilford progeria (HGP) is a genetic disorder in which individuals prematurely display features of ageing. Mutations in LMNA (lamin A) have recently been shown to underlie HGP, although how such mutations lead to the complex phenotype seen in the disease remains unclear. HGP is often associated with the premature replicative senescence of dermal fibroblasts. Normally dermal fibroblast senescence is initiated by erosion of chromosomal ends (telomeres) resulting from sustained cell division. Since ectopic expression of telomerase reproducibly immortalises human dermal fibroblasts, it is of interest to determine whether HGP fibroblasts immortalise via the same route, and at the same frequency. Three strains of HGP fibroblasts (AGO6917A, AGO6297B and AGO8466) were infected with a retroviral vector expressing the catalytic subunit of telomerase (hTERT). Here we report that fibroblast clones derived from HGP donors frequently fail to immortalise with telomerase. Of the 15 independently isolated clones from the three donors, five failed to immortalise despite the restoration of telomerase activity and the stabilisation of telomere length. In contrast, out of four clones isolated from a culture of hTERT transduced control fibroblasts, no failures to immortalise were detected. This suggests a novel cellular phenotype in HGP, one whereby the HGP mutation confers resistance to 'telomerisation'.

Cells, Cultured↗
PubMed2004

Investigation of the signaling pathways involved in the proliferative life span barriers in werner syndrome fibroblasts.

Werner syndrome (WS) fibroblasts enter replicative senescence after a reduced in vitro life span. Although this has been postulated as causal in the accelerated aging seen in this disease, controversy remains as to whether WS is showing the acceleration of a normal cellular aging mechanism or, instead, the occurrence of a novel WS-specific process. To address this, we analyzed the signaling pathways involved in senescence in WS fibroblasts. Cultured WS fibroblasts underwent senescence after approximately 20 population doublings, with the majority of the cells having a 2N DNA content. This was associated with high levels of the CdkIs p16 and p21. Senescent WS cells reentered the cell cycle after microinjection of a p53-neutralizing antibody. Similarly, presenescent WS fibroblasts expressing the E6 and/or E7 oncoproteins bypassed M1 and ultimately reached a second proliferative life span barrier, which strongly resembled the second life span barriers found in normal cells for growth dynamics, cellular morphology, and expression of p16 and p21. The strong similarity between the signaling pathways triggering cell cycle arrest in WS and normal fibroblasts provides support for the defect in WS causing the acceleration of a normal aging mechanism and validates the use of WS as a model for some aspects of human aging.

Aging↗
PubMed2003

A P53-dependent, telomere-independent proliferative life span barrier in human astrocytes consistent with the molecular genetics of glioma development.

An in vitro model, based on normal (primary) human astrocytes (NHAs), was used to investigate the nature of the selection pressures for events that occur during the progression of astrocyte-derived tumors and, in particular, the potential role of proliferative life span barriers (PLBs). As with fibroblasts, NHAs senesced with elevated p21(WAF1) and senescence-associated beta-galactosidase activities. Unlike fibroblasts, replicative senescence (M1) occurred much earlier, after approximately 20 pd and was not bypassed by hTERT expression. Abrogation of p53 function, by expression of human papillomavirus type 16 E6, led to an extension of life span, implying that replicative senescence in NHAs was p53-dependent but telomere-independent. human papillomavirus type16 E6 expression promoted additional growth of up to 12 pd, until a second telomere-independent PLB (termed M(INT)) was imposed associated with elevated p16(INK4A) levels. A proportion of cells escaped from M(INT) lost p16(INK4A) expression and achieved approximately an additional 25 pd until a crisis-like third PLB (M2) was reached. Expression of hTERT in post-M(INT) cells allowed these cells to become immortal and bypass this third PLB. The in vitro PLBs appear, in order of occurrence, dependent upon p53, p16(INK4A), and telomere erosion, a situation that mirrors an equivalent order of mutational events during tumor progression in vivo. This study describes a model that provides a plausible explanation for the selective pressures driving mutational events in this tumor type and provides direct evidence of a p53-dependent, telomere-independent PLB.

Astrocytes↗
PubMed2003

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.

Aging↗
PubMed2003

An analysis of replicative senescence in dermal fibroblasts derived from chronic leg wounds predicts that telomerase therapy would fail to reverse their disease-specific cellular and proteolytic phenotype.

The accumulation of senescent fibroblasts within tissues has been suggested to play an important role in mediating impaired dermal wound healing, which is a major clinical problem in the aged population. The concept that replicative senescence in wound fibroblasts results in reduced proliferation and the failure of refractory wounds to respond to treatment has therefore been proposed. However, in the chronic wounds of aged patients the precise relationship between the observed alteration in cellular responses with aging and replicative senescence remains to be determined. Using assays to assess cellular proliferation, senescence-associated staining beta-galactosidase, telomere length, and extracellular matrix reorganizational ability, chronic wound fibroblasts demonstrated no evidence of senescence. Furthermore, analysis of in vitro senesced fibroblasts demonstrated cellular responses that were distinct and, in many cases, diametrically opposed from those exhibited by chronic wound fibroblasts. Forced expression of telomerase within senescent fibroblasts reversed the senescent cellular phenotype, inhibiting extracellular matrix reorganizational ability, attachment, and matrix metalloproteinase production and thus produced cells with impaired key wound healing properties. It would appear therefore that the distinct phenotype of chronic wound fibroblasts is not simply due to the aging process, mediated through replicative senescence, but instead reflects disease-specific cellular alterations of the fibroblasts themselves.

Cell Adhesion↗
PubMed2002

In vivo effects of meloxicam and aspirin on blood, gastric mucosal, and synovial fluid prostanoid synthesis in dogs.

OBJECTIVE: To evaluate in vivo activity in dogs of meloxicam or aspirin, previously shown in vitro to be a selective cyclooxygenase-2 (COX-2) inhibitor (COX-1 sparing drug), or a nonselective COX inhibitor, respectively. ANIMALS: 12 male dogs with unilateral osteoarthritis of the stifle joint. PROCEDURE: Each dog was treated in a crossover design with aspirin or meloxicam for 21 days. Prostaglandin E2 (PGE2) concentrations were measured at days 0 (baseline), 7, and 21 of each treatment period in lipopolysaccharide (LPS)-stimulated blood, synovial fluid collected by arthrocentesis, and endoscopic gastric mucosal biopsy specimens. Thromboxane B2 (TXB2) was evaluated in blood on days 0, 7, and 21 of each treatment period. RESULTS: Aspirin administration significantly suppressed PGE2 concentrations in blood, gastric mucosa, synovial fluid, and suppressed TXB2 concentration in blood at days 7 and 21. Meloxicam administration significantly suppressed PGE2 concentrations in blood and synovial fluid at days 7 and 21, but had no effect on concentrations of TXB2 in blood or PGE2 in gastric mucosa. Suppression of LPS-stimulated PGE2 concentrations in blood and synovial fluid by aspirin and meloxicam administration is consistent with activity against the COX-2 isoenzyme. Suppression of concentrations of PGE2 in the gastric mucosa and TXB2 in blood by aspirin administration is consistent with activity against COX-1. Meloxicam, in contrast, had a minimal effect on functions mediated by COX-1. CONCLUSIONS AND CLINICAL RELEVANCE: Meloxicam acts in vivo in dogs as a COX-1 sparing drug on target tissues by sparing gastric PGE2 synthesis while retaining antiprostaglandin effects within inflamed joints.

Animals↗
PubMedDate not supplied

Replicative senescence in sheep fibroblasts is a p53 dependent process.

Studies on telomere and telomerase biology are fundamental to the understanding of human ageing, and age-related diseases such as cancer. However, human studies are hampered by the lack of fully reflective animal model systems. Here we describe basic studies of telomere length and telomerase activity in sheep tissues and cells. Terminal restriction fragment lengths from sheep tissues ranged from 9 to 23 kb, with telomerase activity present in testis but suppressed in somatic tissues. Sheep fibroblasts had a finite lifespan in culture, after which the cells entered senescence. During in vitro growth the mean terminal restriction fragment lengths decreased in size at a rate of 210 and 350 bp per population doubling (PD). Senescent skin fibroblasts had increased levels of p53 and p21WAF1 compared to young cells. Incubation of senescent cells with siRNA duplexes specific for p53 suppressed p53 expression and allowed the cells to re-enter the cell cycle. Five PDs beyond senescence the siRNA-treated cells reached a second proliferative barrier. This study shows that telomere biology in sheep is similar to that in humans, with senescence in sheep GM03550 fibroblasts being a telomere-driven, p53-(p21WAF1)-dependent process. Therefore sheep may represent an alternative model system for studying telomere biology, replicative senescence, and by implication human ageing.

Animals↗