Michele Fleur Haughton

Telomerase prevents the accelerated cell ageing of Werner syndrome fibroblasts.

Werner syndrome (WS) is a rare disorder inherited in an autosomal recessive manner and characterized by accelerated ageing. WS fibroblasts display an accelerated rate of senescence in vitro, which has been linked to this progeroid phenotype. The senescence of normal human fibroblasts is triggered by telomere shortening, whereas the premature senescence of WS fibroblasts has been assumed to reflect the accumulation of DNA damage. Here we show that forced expression of telomerase in WS confers extended cellular lifespan and probable immortality.

Control of Replicative Life Span in Human Cells: Barriers to Clonal Expansion Intermediate Between M1 Senescence and M2 Crisis

ABSTRACT The accumulation of genetic abnormalities in a developing tumor is driven, at least in part, by the need to overcome inherent restraints on the replicative life span of human cells, two of which—senescence (M1) and crisis (M2)—have been well characterized. Here we describe additional barriers to clonal expansion (Mint) intermediate between M1 and M2, revealed by abrogation of tumor-suppressor gene (TSG) pathways by individual human papillomavirus type 16 (HPV16) proteins. In human fibroblasts, abrogation of p53 function by HPVE6 allowed escape from M1, followed up to 20 population doublings (PD) later by a second viable proliferation arrest state, MintE6, closely resembling M1. This occurred despite abrogation of p21WAF1 induction but was associated with and potentially mediated by a further ∼3-fold increase in p16INK4a expression compared to its level at M1. Expression of HPVE7, which targets pRb (and p21WAF1), also permitted clonal expansion, but this was limited predominantly by increasing cell death, resulting in a MintE7 phenotype similar to M2 but occurring after fewer PD. This was associated with, and at least partly due to, an increase in nuclear p53 content and activity, not seen in younger cells expressing E7. In a different cell type, thyroid epithelium, E7 also allowed clonal expansion terminating in a similar state to MintE7 in fibroblasts. In contrast, however, there was no evidence for a p53-regulated pathway; E6 was without effect, and the increases in p21WAF1 expression at M1 and MintE7 were p53 independent. These data provide a model for clonal evolution by successive TSG inactivation and suggest that cell type diversity in life span regulation may determine the pattern of gene mutation in the corresponding tumors.

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 ∼20 population doublings with most of the cells having a 2N content of DNA. This was associated with hypophosphorylated pRb and high levels of p16Ink4a and p21Waf1. 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 Mint, 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 Mint 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.

Telomerase activity and telomere length in thyroid neoplasia: biological and clinical implications

Despite several recent studies, the biological status and clinical relevance of telomerase expression in tumours derived from the thyroid follicular cell remain controversial. This study has analysed a series of normal, benign, and malignant thyroid samples using two novel approaches: the use of purified epithelial cell fractions to eliminate false‐positives due to telomerase‐positive infiltrating lymphocytes; and the simultaneous measurement of telomere length to provide a clearer interpretation of telomere dynamics in thyroid neoplasia. The data obtained support the prediction that the epithelial component of non‐neoplastic thyroid and of follicular adenomas is telomerase‐negative, any positive results being explicable by lymphocyte infiltration. In contrast, many malignant tumours, both follicular and papillary, were telomerase‐positive. However, serial dilution of extracts indicated a wide spectrum of activity in these cancers, possibly related to variation in the proportion of telomerase‐positive cells. Furthermore, an unexpectedly high proportion were telomerase‐negative, a finding which was not explicable by technical problems such as TRAP (telomeric repeat amplification protocol) assay sensitivity. Many of these apparently telomerase‐negative tumours had abnormally long telomeres. Correlation of telomerase and telomere length data suggests that thyroid cancers fall into three biological groups: telomerase‐positive lesions, consistent with the conventional model of telomere erosion followed by telomerase reactivation; telomerase‐negative tumours, which maintain telomere length by a mechanism independent of telomerase; and telomerase‐negative tumours which are still undergoing telomere erosion and may therefore be composed of mortal cancer cells. From a clinical standpoint, it is concluded that telomerase detection on unfractionated tissue, such as fine needle aspirates, is of no value as a marker of malignancy in follicular lesions, due to both low sensitivity and specificity. Copyright

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.

Prevention of accelerated cell aging in the Werner syndrome

Abstract:  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 p21WAF1 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.

Opposing effects of mutant ras oncoprotein on human fibroblast and epithelial cell proliferation: implications for models of human tumorigenesis

Using microinjection of recombinant protein to directly control ‘expression’ levels, we have compared the proliferative response to ras oncogene activation in two normal cell types – fibroblast and thyroid epithelial cell – which give rise to human tumours with very low and high frequencies of ras mutation respectively. A concentration-dependent stimulation of DNA synthesis was observed in thyrocytes, matched by an almost perfectly reciprocal inhibition in fibroblasts. A concentration-dependent induction of the cyclin-dependent kinase (CDK) inhibitor p21WAF1 was observed in both cell types, but p16Ink4a was induced by ras only in fibroblasts. This difference could not account for the fibroblast specificity of the growth-inhibitory response, however, since proliferation of p16-deficient fibroblasts was also inhibited by mutant ras. We conclude that the striking contrast in proliferative response to ras between fibroblasts and thyroid epithelial cells cannot readily be explained by differential induction of either of the two key CDK inhibitors, p16Ink4a and p21WAF1, but is consistent with a differential ability of p21WAF1 to antagonize ras-induced mitogenic signals in the two cell types. Such tissue-specific differences provide an attractive explanation for the observed specificity of ras mutation for particular human tumour types, and emphasize the inappropriateness of fibroblasts as a model for ras-induced tumorigenesis.

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 p21WAF1 and senescence-associated β 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.

Mutant p53 can delay growth arrest and loss of CDK2 activity in senescing human fibroblasts without reducing p21WAF1 expression

Functional wild-type p53 is required for human diploid fibroblasts (HDF) to enter an irreversible growth arrest known as replicative senescence. Experimentally, abrogation of p53 function by expression of human papillomavirus type 16 E6 or disruption of a key downstream effector p21 by homologous recombination both extended HDF life span. However, although sufficient to extend life span, p21 down-regulation is not necessary, because expression of a dominant-negative mutant p53 (143(ala)) extends life span without apparently decreasing p21 expression. Given the importance of p53 in cellular senescence and the general assumption that p21 may be the sole mediator of its action in this process, we have investigated how abrogation of p53 function can overcome senescence without lowering expression of p21. We have found up-regulated levels of the cyclin-dependent kinase 2 (cdk2) protein in HDF expressing 143(ala) mutant p53 as compared to senescent controls, together with an increase in p21-free cdk2 which, in conjunction with cyclin E, is able to form an active kinase which can phosphorylate the retinoblastoma protein. However, forced overexpression of cdk2 in near-senescent HDF failed to restore cdk2-associated kinase activity. Our data suggest that p53-mediated senescence depends on factor(s) other than p21 which modulate formation of cyclin E-cdk2 complexes.