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David Kipling

Publications and source records attributed to David Kipling.

23 records · Page 2Linked to original sources
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↗
PubMed2003

Extensive allelic variation and ultrashort telomeres in senescent human cells.

By imposing a limit on the proliferative lifespan of most somatic cells, telomere erosion represents an innate mechanism for tumor suppression and may contribute to age-related disease. A detailed understanding of the pathways that link shortened telomeres to replicative senescence has been severely hindered by the inability of current methods to analyze telomere dynamics in detail. Here we describe single telomere length analysis (STELA), a PCR-based approach that accurately measures the full spectrum of telomere lengths from individual chromosomes. STELA analysis of human XpYp telomeres in fibroblasts identifies several features of telomere biology. We observe bimodal distributions of telomeres in normal fibroblasts; these distributions result from inter-allelic differences of up to 6.5 kb, indicating that unexpectedly large-scale differences in zygotic telomere length are maintained throughout development. Most telomeres shorten in a gradual fashion consistent with simple losses through end replication, and the rates of erosion are independent of allele size. Superimposed on this are occasional, more substantial changes in length, which may be the consequence of additional mutational mechanisms. Notably, some alleles show almost complete loss of TTAGGG repeats at senescence.

Alleles↗
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↗