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.

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.

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 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.

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.

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.