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D Kipling

Publications and source records attributed to D Kipling.

At least 19 recordsLinked to original sources
PubMed2004

The role of replicative senescence in cancer and human ageing: utility (or otherwise) of murine models.

Replicative senescence has the potential both to act as an anti-tumour mechanism, and to contribute to age-related changes in tissue function. Studies on human cells have revealed much, both about the nature of cell division counters, some of which utilize the gradual erosion of chromosomal telomeres, and the downstream signalling pathways that initiate and maintain growth arrest in senescence. A powerful test of the hypothesis that senescence is linked to either ageing or tumour prevention now requires a suitable animal model system. Here we overview the current understanding of replicative senescence in human cells, and address to what extent the senescence of murine cells in culture mirrors this phenomenon. We also discuss whether examples of telomere-independent senescence, such as those seen in mouse embryonic fibroblasts (MEFs) and several human cells types, should be viewed not as a consequence of "inadequate growth conditions", but rather as a powerful potential model system to dissect the selective pressures that occur in the early stages of tumour development, ones that we speculate lead to the observed high frequency of abrogation of p16INK4a function in human cancer.

Aging↗
PubMed2001

Telomeres, replicative senescence and human ageing.

Ageing concerns the extracellular environment and cells that are either post-mitotic or capable of division during life. Primary human cells have a finite division capacity in culture before they enter a state of viable cell cycle arrest termed senescence. Cell division occurs during life in many tissues, either as part of normal tissue function or in response to tissue damage. The accumulation of cells at the end of their replicative lifespan in the elderly might contribute to aged tissue either because of a reduced ability to undergo proliferation or because of the known altered gene-expression patterns of senescent cells. This has been illustrated experimentally using a transgenic telomerase-negative mouse, which shows some premature ageing phenotypes. The mechanism whereby cells count divisions uses the gradual erosion of the ends of chromosomes (telomeres) with cell division caused by the repression of the telomere-maintenance enzyme telomerase in most human cells. Telomere erosion ultimately triggers replicative senescence in many cell types; this can be prevented experimentally by forcibly expressing telomerase. This extends the lifespan of normal human cells and those from progeroid syndromes such as Werner's. Telomere-driven senescence did not evolve to cause ageing, but is instead a by-product of a system devised to provide a tumour-suppression function, a concept that fits well with evolutionary arguments regarding trade-offs between somatic maintenance and reproduction. Work in the future will focus on the development of new animal models to critically address the quantitative significance of this ageing mechanism.

Aging↗
PubMed2001

Human telomerase reverse transcriptase-immortalized MRC-5 and HCA2 human fibroblasts are fully permissive for human cytomegalovirus.

MRC-5 cells are a well-characterized human diploid fibroblast cell line approved for vaccine production and favoured for the routine propagation of human cytomegalovirus (HCMV). Ectopic expression of telomerase in fibroblasts is capable of overcoming replicative senescence induced by telomere shortening. Following delivery of the hTERT gene to MRC-5 cells using a retrovirus vector three clones were generated that (i) expressed functional telomerase activity, (ii) exhibited telomere extension and (iii) were sustained for >100 population doublings. Immortalized MRC-5-hTERT and also HCA2-hTERT human fibroblasts were both fully permissive for HCMV as determined by plaque assay, studies of virus growth kinetics and measurement of virus yields. Furthermore, telomerase-immortalized HCA2 cells proved capable of supporting the stable maintenance of an EBV-based episomal vector with efficient transgene expression when driven by the HCMV immediate early promoter. An indicator cell line suitable for the efficient detection of HCMV infection was also generated using an episome containing a reporter gene (lacZ) under the control of the HCMV beta-2.7 early promoter. Telomerase immortalization of human fibroblasts will thus facilitate the growth and detection of HCMV and also the generation of helper cell lines for the propagation of HCMV deletion mutants. Immortalization of fibroblasts by telomerase does not affect cell morphology or growth characteristics. The MRC-5-hTERT clones may therefore be suitable for additional applications in virology, cell biology, vaccine production and biotechnology.

Cells, Cultured↗
PubMed2000

Evidence for a telomere-independent "clock" limiting RAS oncogene-driven proliferation of human thyroid epithelial cells.

An initiating role for RAS oncogene mutation in several epithelial cancers is supported by its high incidence in early-stage tumors and its ability to induce proliferation in the corresponding normal cells in vitro. Using retroviral transduction of thyroid epithelial cells as a model we ask here: (i) how mutant RAS can induce long-term proliferation in an epithelial cell in contrast to the premature senescence observed in fibroblasts; and (ii) what is the "clock" which eventually triggers spontaneous growth arrest even in epithelial clones generated by mutant RAS. The early response to RAS activation in thyroid epithelial cells showed two features not seen in fibroblasts: (i) a marked decrease in expression of the cyclin-dependent kinase inhibitor (CDKI) p27(kip1) and (ii) the absence of any induction of p21(waf1). When proliferation eventually ceased (after up to 20 population doublings) this occurred despite undiminished expression of mutant RAS and was tightly correlated with a return to the initial high level of p27(kip1) expression, together with the de novo appearance of p16(ink4a). Importantly, neither the CDKI changes nor the proliferative life span of RAS-induced epithelial clones was altered by induction of telomerase activity through forced expression of the catalytic subunit, hTERT, at levels sufficient to immortalize human fibroblasts. These data provide a basis for cell-type differences in sensitivity to RAS-induced proliferation which may explain the corresponding tumor-type specificity of RAS mutation. They also show for the first time in a primary human cell model that a telomere-independent mechanism can limit not only physiological but also oncogene-driven proliferation, pointing therefore to a tumour suppressor mechanism additional, or alternative, to the telomere clock.

Catalytic Domain↗
PubMed1998

How might replicative senescence contribute to human ageing?

Cell senescence is the limited ability of primary human cells to divide when cultured in vitro. This eventual cessation of division is accompanied by a specific set of changes in cell physiology, morphology, and gene expression. Such changes in phenotype have the potential to contribute to human ageing and age-related diseases. Until now, senescence has largely been studied as an in vitro phenomenon, but recent data have for the first time directly demonstrated the presence of senescent cells in aged human tissues. Although a direct causal link between the ageing of whole organisms and the senescence of cells in culture remains elusive, a large body of data is consistent with cell senescence contributing to a variety of pathological changes seen in the aged. This review considers the in vitro phenotype of cellular senescence and speculates on the various possible routes whereby the presence of senescent cells in old bodies may affect different tissue systems.

Aging↗
PubMed1997

A gene spans the pseudoautosomal boundary in mice.

The X and Y chromosomes of the mouse, like those of other mammals, are heteromorphic over most of their length, but at the distal ends of the chromosomes is a region of sequence identity, the pseudoautosomal region (PAR), where the chromosomes pair and recombine during male meiosis. The point at which the PAR diverges into X- and Y-specific sequences is called the pseudoautosomal boundary. We have completed a genomic walk from the X-specific Amelogenin gene to the PAR. Analysis of this region revealed that the pseudoautosomal boundary of mice is located within an intron of a transcribed gene that encodes a novel RING finger protein. The first three of the exons of the gene are located on the X chromosome whereas the 3' exons of the gene are located on both X and Y chromosomes. This unusual arrangement may indicate that the gene is in a state of transition from pseudoautosomal to X-unique and provides evidence for a process of attrition of the pseudoautosomal region on the Y chromosome.

Amelogenin↗
PubMed1997

Centromeres, CENP-B and Tigger too.

The highly conserved centromere-associated protein CENP-B is a common feature of mammalian centromeres. Binding sites for CENP-B, so-called 'CENP-B boxes', are present in the otherwise unrelated centromeric satellite DNAs of humans, Mus musculus, Mus caroli, ferrets, giant pandas, tree shrews and gerbils, suggesting a role for CENP-B in centromere function. However, CENP-B and its binding sites are not detected at the centromeres of mammalian Y chromosomes and few, if any, binding sites seem present on African green monkey chromosomes. There is extensive sequence similarity between CENP-B and transposase proteins encoded by the pogo superfamily of transposable elements, which includes the human Tigger elements. Intriguingly, Tigger 2 has an almost perfect match to the CENP-B-binding site within its terminal inverted repeat. Comparison of the amino acid sequence of CENP-B with related proteins raises the possibility that CENP-B might share the ability to cause single-stranded DNA breaks. Such nicks could promote recombination, as has been suggested for the Charcot-Marie-Tooth disease duplication where a recombination hotspot exists close to a mariner-like element. We suggest that by promoting nicks adjacent to CENP-B boxes, CENP-B might facilitate the evolution and maintenance of satellite sequence arrays, rather than have a direct role in centromere function.

Amino Acid Sequence↗
PubMed1997

Telomere structure and telomerase expression during mouse development and tumorigenesis.

Mouse telomeres are on average longer than those of man, raising questions regarding the link between telomere loss and replicative senescence in mice and the requirement for telomerase activity for mouse cell immortalisation. However, the emerging data on telomerase activity during tumorigenesis in the mouse must be interpreted in the context of the very different structure of mouse telomeres. It will be argued here that the evidence for a casual link between telomere loss and replicative senescence is weak in the mouse, with the observed upregulation of telomerase activity in mouse tumours perhaps instead reflecting co-ordinated regulatory changes in tumour cells. Its absence would be consistent with evolutionary considerations, which hypothesise that such a link is an additional layer of control against tumour formation that has evolved in man. The very different genomic substrates for telomerase in humans and mice mean that the initial phenotype of a telomerase knock-out mouse does not necessarily critically address the existence of a link between telomerase and tumorigenesis in man.

Animals↗
PubMed1997

Comparison of the human and mouse genes encoding the telomeric protein, TRF1: chromosomal localization, expression and conserved protein domains.

Mammalian chromosome ends contain long arrays of TTAGGG repeats that are complexed to a telomere specific protein, the TTAGGG repeat binding factor, TRF1. Here we describe the characterization of genes encoding the human and mouse TRF1 proteins, hTRF1 and mTRF1. The mTRF1 cDNA was isolated based on sequence similarity to the hTRF1 cDNA and the mTRF1 mRNA was shown to be ubiquitously expressed as a single 1.9 kb polyadenylated transcript in mouse somatic tissues. High levels of a 2.1 kb transcript were found in testes. In vitro translation of the mTRF1 cDNA resulted in a 56 kDa protein that binds to TTAGGG repeat arrays. mTRF1 displayed the same sequence specificity as hTRF1, preferring arrays of TTAGGG repeats as a binding substrate over TTAGGC and TTGGGG repeats. Expression of an epitope-tagged version of mTRF1 showed that the protein is located at the ends of murine metaphase chromosomes. In agreement, conceptual translation indicated that mTRF1 and hTRF1 are similarly-sized proteins with nearly identical C-terminal Myb-related DNA binding motifs. In addition, comparison of the predicted mTRF1 and hTRF1 amino acid sequences showed that the acidic nature of the N-terminus of TRF1 is conserved and revealed a highly conserved novel domain of approximately 200 amino acids in the middle of the proteins. However, other regions of the proteins are poorly conserved (<35% identity) and the overall level of identity of the mTRF1 and hTRF1 amino acid sequences is only 67%. The TRF1 genes are not syntenic; the hTRF1 gene localized to human chromosome 8 band q13 while the mTRF1 gene localized to mouse chromosome 17 band E3. The data indicate that the genes for mammalian telomeric proteins evolve rapidly.

Amino Acid Sequence↗
PubMed1997

Mammalian telomerase: catalytic subunit and knockout mice.

For the second time this year random cDNA sequencing, in combination with data from unicellular eukaryotes, has made a significant contribution to the analysis of human telomerase. Two groups have reported mammalian homologues of the Tetrahymena p80 telomerase-associated protein, in both cases the key breakthrough being mammalian cDNA clones with database matches to Tetrahymena p80. This has now been joined by the sequence of a candidate for the human telomerase catalytic subunit. The discovery that its message abundance closely follows telomerase activity could make a major impact on the utility of telomerase as a diagnostic marker for human malignancy. In addition, Blasco et al . report the phenotype of a transgenic mouse deleted for the mTR gene, which encodes the essential RNA component of telomerase. Interestingly tumour formation is unaffected in these mice, strengthening the argument that telomerase expression in mouse tumourigenesis is an innocent bystander rather than a necessary event. However, fundamental differences between the genomic organisation of mouse and human telomeres mean that the mouse is not a straightforward model to critically test the role of telomere loss and telomerase in human malignancy.

Amino Acid Sequence↗
PubMed1997

Progeroid syndromes: probing the molecular basis of aging?

A valid method of studying age related degenerative pathologies is to study human genetic diseases that appear to accelerate many, though not necessarily all, features of the aging process. Such diseases are described as progeroid syndromes because of their possible relevance to many aspects of aging and age related disease. This article describes the recent progress made at the cellular and molecular levels in understanding the pathogenesis of one of the best characterised of these disorders, Werner's syndrome. These observations are related to some of the less well characterised progeroid syndromes within the context of the cell senescence hypothesis of aging, a theory formulated to explain the aging of regenerative tissue in normal individuals.

Aging↗