Prevention of Accelerated Cell Agingin the Werner Syndrome

TERENCE DAVIS, MICHELE F. HAUGHTON, CHRISTOPHER J. JONES,AND DAVID KIPLING

Department of Pathology, School of Medicine, Cardiff University, Heath Park,Cardiff CF14 4XN, UK

ABSTRACT: In the Werner syndrome (WS) fibroblasts have an increasedlife span and growth rate when treated with the p38 inhibitor SB203580.Additionally, the cellular morphology reverts to that seen in young nor-mal fibroblasts. The p38 pathway is activated in young WS cells, as-sociated with high levels of p21WAF1 leading to cell cycle arrest, and issuppressed by SB203580. As these changes are also seen in telomerizedWS cells, these data show that the growth problems seen in WS cells, andperhaps the accelerated in vivo aging, are due to a telomere-independentpremature senescence mechanism. The suppression of this mechanism bySB203580 treatment suggests a route whereby WS may be amenable totherapeutic intervention.

KEYWORDS: actin stress fibers; p21WAF1; p38 MAPK; p53; SB203580;senescence; telomeres; telomerase; Werner syndrome

INTRODUCTION

The Werner syndrome (WS) is a rare genetic disorder in which patients showthe premature onset of many clinical features of old age.1,2 The median lifeexpectancy is 47 years, with myocardial infarction or mesenchymal neoplasmsas the major causes of death. With some exceptions, such as the absence ofcentral nervous system degeneration, WS provides a stunning mimicry of nor-mal aging and is widely used as a model disease to investigate the mechanismsunderlying normal human aging.1

The molecular mechanism of WS appears to be related to accelerated cellaging. Cultured cells from normal individuals divide a limited number of timesbefore they enter a growth-arrest state termed replicative cellular senescence.3

This is postulated to contribute to normal human aging and is accelerated inWS, the fibroblasts from WS patients having a dramatically reduced cellular

Address for correspondence: Terence Davis, Department of Pathology, Henry Wellcome Building,School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, Wales, UK. Voice: +44-29-2074 3398; fax: +44-29-2074 4276.

e-mail: davist2@cardiff.ac.uk

Ann. N.Y. Acad. Sci. 1067: 243–247 (2006). C© 2006 New York Academy of Sciences.doi: 10.1196/annals.1354.031

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life span.4 This shortened life span is argued as a cause of the accelerated agingseen in WS individuals,1,2 and the pathways leading to replicative senescenceappear to be conserved in old WS fibroblasts.5,6

However, WS fibroblasts show several characteristics of cells growing underconditions of stress (e.g., slow growth rates and an elongated cell cycle) andWS may not be simply accelerated normal cell aging. Cells growing understressful conditions are often enlarged with prominent F-actin stress fibers,7

and many young WS cells resemble fibroblasts that have undergone stress-induced premature senescence (SIPS). SIPS results from the activation of p38MAPK and the use of the p38 selective inhibitor SB203580 essentially preventsthe accelerated aging seen in primary WS cells (FIG. 1A), showing that theaccelerated aging is due to activation of an SB203580-suppressible pathway.p38 is activated in young WS cells with associated high levels of p21WAF1 andphospho-HSP27; thus young WS cells have actin stress fibers and show cellcycle arrest.4 SB203580 treatment reduces the activated p38, p21WAF1, andphospho-HSP27 levels. In contrast, control young MRC5 cells have low levelsof activated p38 that increase as the cells age, and the p21WAF1 level is low inyoung and high in senescent MRC5 cells.4,8

Interestingly, SB203580-treated cells have stress fibers when they reach M1despite low levels of phospho-HSP27.4 However, normal and WS cells haveelevated phospho-cofilin at M1 (FIG. 1E), which is known to induce stressfiber formation in fibroblasts.9 This shows that several independent pathwaysare involved in stress fiber formation in fibroblasts. Phospho-cofilin is alsoelevated in young WS cells and appears to be suppressed with drug treatment.Thus, SB203580-treated young WS cells have low levels of both phospho-HSP27 and phospho-cofilin associated with low levels of F-actin stress fibers.

WS cells can be immortalized using ectopic expression of the catalytic sub-unit of human telomerase;10 however, the telomerized WS cells retain theslow growth seen in primary WS cells. The growth rate for a typical telom-erized WS culture (AG03141.hTERT clone 8) is 0.31 ± 0.01 PD/day (FIG.1B).4 In the presence of SB203580 this rate significantly increases to 0.53 ±0.02 PD/day, a rate not significantly different from that of telomerized HCA2cells (0.55 ± 0.07 PD/day; P > 0.65). Upon drug removal the growth rate ofAG03141.hTERT clone 8 reverts to that seen in untreated cells (0.30±0.05PD/day). By contrast, SB203580 treatment has no significant effect on thegrowth rate of telomerized HCA2 cells [0.58 PD/day (FIG. 1B)].

In addition to the slow-growth phenotype, the altered morphology seen inprimary WS cells is still apparent in telomerized WS cells, and SB203580treatment is successful in reverting this phenotype (FIG. 1D) to that seen intelomerized normal cells. The altered morphology of WS cells is associatedwith high levels of F-actin stress fibers4 that are still apparent in the telomerizedcells (FIG. 1C). As with the primary cells the level of stress fibers in SB203580-treated telomerized cells is low. By contrast, telomerized HCA2 cells have amorphology resembling that of young primary HCA2 cells with few stressfibers and this morphology is not altered by SB203580 treatment (not shown).

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FIGURE 1. Growth of cells in the presence or absence of SB203580. All methods areas described.4 (A) Replicative life span of WS (AG03141, AG05229, AG12795) and MRC5cells. (B) Growth rates of telomerized WS and HCA2 cells. For growth rates: light gray bars= control; black bars = SB-treated; dark gray bar = growth rate after removal of SB203580,statistical significance is a two-tailed t-test. (C) Phalloidin staining and (D) phase-contrastof telomerized WS cells grown in the absence (left panel) and presence (right panel) ofSB203580. Bar = 50 �m in (C) and 100 �m in (D). Immunoblot analysis of primary WSand MRC5 cells (Y = young, M1 = senescent) (E) and telomerized WS cells (F). Antibod-ies used for immunoblots: phospho-MKK3/6(S189/S207), phospho-p38(T180/Y182), p38,phospho-MK2(T334), phospho-HSP27(S82), HSP27, phospho-cofilin(S3), cofilin (cellsignaling), p21 (6B6, Becton Dickinson).

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That the p38 MAP kinase pathway is still activated despite the telomerizedWS cells being immortalized is shown by the presence of phosphorylatedforms of p38 and its activating kinase MKK3/6 (FIG. 1F). p38 activity leads tothe activation of the downstream kinase MK2 (MAPKAP-K2), which in turnphosphorylates HSP27, leading to stress fiber formation. In addition, there is ahigh level of p21WAF1 that should induce a degree of cell cycle arrest. Despitethe high p21WAF1 levels in the telomerized cells, this was a growing culture. Asthe immunoblot analysis measures an average level of p21WAF1 for the wholecell population, it is possible that some of the cells had a high p21WAF1 leveland were growth-arrested, while the rest had low p21WAF1 levels and werethus growing. This would provide an explanation for the slow growth rate ofthe culture. As expected, SB203580 treatment prevents the phosphorylationof MK2 and HSP27, inhibits stress fiber formation, and suppresses the levelof p21WAF1. As is the case in young primary WS cells, SB203580 preventsthe activation of p38 in telomerized WS cells, and in addition reduces theactivation of MKK3/6 (FIG. 1F). These data suggest that the inducing signal intelomerized WS cells (and young WS cells) is distinct from the short-telomere-induced signal seen in old WS and MRC5 primary cells.4

CONCLUSIONS

Primary SB203580-treated WS fibroblasts have an increased replicativelife span and growth rate compared to untreated cells. p38 is activated in youngcells associated with high levels of p21WAF1 that are suppressed in drug-treatedcells. Moreover, p38 activation is still apparent in telomerized WS cells. Thesedata using telomerized WS cells show that the growth problems are still presentdespite immortalization and telomere maintenance, and thus are most probablynot due to major defects in telomere metabolism in WS cells.11 Therefore, theshortened replicative life span seen in WS cells is due to the activation of atelomere-independent stress-induced pathway. As activation of this pathway isstill apparent in hTERT-immortalized cells, these telomerized WS cells maybe useful for investigating the pathways that lead to premature growth arrestin WS cells.

ACKNOWLEDGMENT

This work was funded by the BBSRC’s Experimental Research on AgingInitiative.

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