Can we say that senescent cells cause ageing?

Joseph Bird, Elizabeth L. Ostler, Richard G.A. Faragher*

School of Pharmacy and Biomolecular Sciences, University of Brighton Sciences, Cockcroft Building, Lewes Road,

Moulsecoomb, Brighton, East Sussex BN2 4GJ, UK

Abstract

Replicative senescence, the irreversible loss of proliferative capacity, is a common feature of somatic cells derived from many different

species. The molecular mechanisms controlling senescence in mammals, and especially in humans, have now been substantively elucidated.

However, to date, attempts to link the senescence of cells with the ageing of the organisms they comprise has not met with any similar degree

of success, largely due to a lack of systematic investigation and the absence of the necessary biochemical tools. This review will summarise

current data linking replicative senescence and organismal ageing. It will also suggest some essential tests of the cell senescence hypothesis

and some necessary ground work which must be carried out before such tests can be fruitfully performed. It will not discuss the detailed

molecular ‘clockwork’ controlling the decision to exit the cell cycle irreversibly because this is covered by other authors in this special issue.

q 2003 Published by Elsevier Inc.

Keywords: Senescence; Marker; Werner’s syndrome; Telomerase

1. Introduction

For the purposes of this article, it is sufficient to say that

senescence is a cyclin-dependent kinase inhibitor-mediated

block to further replication leading to indefinite cell cycle

arrest (usually at the G1–S phase transition). This block to

replication (at least in vitro) can be produced by the

activation of either telomere-dependent or independent

pathways. Classic telomere-driven senescence arises as a

consequence of a p53 and p21waf mediated cell cycle arrest

as a result of progressive telomeric attrition. This telomere

shortening can occur either as a result of end-replication loss

alone or end-replication loss accompanied by additional

terminal sequence loss as a consequence of oxidative

damage. The best evidence in support of a telomere-

dependent senescence mechanism is the observation that

ectopic expression of the catalytic subunit of telomerase

(hTERT) in presenescent human fibroblasts, retinal pig-

mented epithelial cells, mesothelial cells and other cell

types leads to immortalisation (Bodnar et al., 1998; Dickson

et al., 2000; Rheinwald et al., 2002). In contrast, evidence

for telomere-independent senescence mechanisms is

provided by a growing body of data which demonstrates

that the prevention of telomeric attrition does not always

bypass senescence. In some human cell types, such as

thyroid follicular epithelial cells, pancreatic islet b cells and

bladder uroepithelial cells, ectopic expression of hTERT

alone is insufficient for immortalisation and senescence

results from hypophosphorylation of the retinoblastoma

protein (pRB) as a consequence of overexpression of

p16INK4A (an inhibitor of CDK 4-cyclin D and CDK 6-

cyclin E kinase pairs). The validity of telomere-independent

senescence as an in vivo source of senescent cells is

currently unclear. However, p16INK4A can be upregulated

by a wide variety of stimuli and thus it is likely that at least

some cells could become senescent in vivo as a result of this

pathway. An important point to stress is that neither of these

mechanisms appears to be responsible for rodent cell

senescence. Rodent fibroblasts undergo a well-characterised

senescence under standard tissue culture conditions and

evidence exists for similar cells in rodent tissue. One

plausible cause of rodent cell senescence appears to be a

telomere-independent but p53-dependent mechanism result-

ing from the action of the p19arf gene product, possibly as a

result of chronic oxidative stress in culture (Parrinello et al.,

2003). It should be noted all these mechanisms require

ongoing cell turnover to produce the senescent state.

In addition to the pathways outlined above, there is one

further route by which senescent cells may be produced. A

number of studies have shown that cells can be induced to

enter a state very similar to ‘normal’ senescence as a result

of exposure to a fairly wide range of environmental stimuli

(including ectopic expression of RAS-V12 and treatment

with ceramide) in the absence of any significant cell

0531-5565/$ - see front matter q 2003 Published by Elsevier Inc.

doi:10.1016/j.exger.2003.09.011

Experimental Gerontology 38 (2003) 1319–1326

www.elsevier.com/locate/expgero

* Corresponding author. Tel.: þ44-1273-642-124; fax: þ44-273-679-

333.

E-mail address: rgaf@brighton.ac.uk (R.G.A. Faragher).

division. It is sometimes considered that to be a ‘true’

senescence mechanism a molecular pathway capable of

generating permanent arrest must also be capable of

counting divisional time. However, whilst this property is

clearly present in telomere-driven systems, and has the

potential to be present in some telomere-independent

systems, the contribution made by senescent cells to the

ageing of tissue is essentially independent of divisional

counting. This is because all senescent states, however, they

are generated, appear to be associated with global

transcriptional regearing of the arrested cell resulting in a

phenotype that is profoundly different from that of the

growing counterpart. It is precisely through this altered

phenotype and its potential to affect the tissue microbalance

that senescent cells are proposed to exert their ‘ageing’

effects (Fig. 1). Thus, it is to this altered phenotype we must

now turn.

2. The senescent cell phenotype

Senescent cells remain metabolically active and can be

maintained in culture for periods that appear to be

dependent on the baseline apoptotic frequency of the cell

type in question. This results in post-mitotic survival times

ranging from years (human fibroblasts) to weeks (human

vascular endothelium). The most distinctive feature of

senescent cells is, of course, their inability to divide in

response to a mitotic stimulus (instead they frequently

demonstrate G2 arrest on restimulation without division). In

addition, senescent cells display marked alterations in both

their morphology and overall metabolic profile. A very wide

range of changes have been reported (Stanulis-Preager,

1987) including increased cell, nuclear and lysosomal size,

with increased mitochondrial mass and an altered cytoske-

leton associated with decreased filamin expression (Far-

agher and Kipling, 1998, 1999; Kipling, 2001). These

cytoskeletal changes occur in conjunction with both a

decline in migration rate and in the ability to invade and

contract collagen. The downstream transcriptional changes

that occur as a result of the onset of the senescent state are

also both broad in degree and diverse in kind (Table 1).

However, it is important to note that different cell types

do not exhibit a consistent set of changes. Rather, the

mRNA expression patterns displayed by senescent cells

vary widely from one cell lineage to another (Shelton et al.,

1999). In general, the gene profile of senescent human

fibroblasts shows a deficit in the transcription of genes

important in the early and mid G1 response and an increase

in the transcription of genes associated with the inflamma-

tory response such as IL-15 and IL-1b (Funk et al., 2000).

This transcriptional profile overlaps substantially with the

gene expression pattern observed when growth competent

fibroblasts are activated during wound healing and suggests

a shift toward a primarily catabolic phenotype. However, a

shift towards an inflammatory expression profile is not

universal. Retinal pigmented epithelial cells do not show

Fig. 1. Simple schematic comparing (a) the cell senescence hypothesis of

ageing (right hand flow) with (b) the dysdifferentiation hypothesis of ageing

(left hand flow). The cell senescence hypothesis postulates that in the

normal course of life there is cell loss. That loss is balanced by cell division

which is actively monitored. One or more “replicometers” act to trigger

permanent cell cycle exit (senescence) in individual cells (see text). Cell

cycle exit is associated with a broad alteration in gene expression leading to

an altered phenotype that affects the microenvironment in which the cell

resides and ultimately the entire tissue. In the dysdifferentiation model

chronic oxidative stress leads to a regearing of gene expression generating

an altered cellular phenotype which contributes to tissue ageing. The two

models have many essential similarities.

Table 1

A selection of genes which display altered transcription with the onset of

the senescent state

Repressed at onset of senescence Upregulated at onset of senescence

c-Fos Collagenase

Cyclins A and D Gas 1

IGF1 TIMP-2

TGFb PAI 2

Interleukin 6 Fibronectin

Id 1, 2 and 4 Interleukin 1a and b

a1 (III)-Procollagen Interleukin 15

EPC1 ICAM-1

Microphthalmia-associated

transcription factor (Mitf)

MMP3

MMP10

IGF-BP2, 3 and 5

Ws3.10

J. Bird et al. / Experimental Gerontology 38 (2003) 1319–13261320

upregulation of key inflammatory mediators although the

expression of matrix and structural proteins is down-

regulated in a similar manner to that seen in dermal

fibroblasts or vascular endothelial cells.

3. Senescence and ageing tissues

Currently, there is little information directly linking the

accumulation of senescent cells in tissue to the changes

associated with those tissues as they age. However, this

results principally from the limited number of studies that

have attempted to demonstrate the existence of senescent

cells in vivo rather than either a failure to detect any

senescent cells following systematic investigation or the

regular demonstration that such cells do occur in vivo but in

patterns that are inconsistent with the development of age-

associated pathology.

Studies designed to detect senescent cells are rare

because such morphometric analysis is both time consum-

ing and extremely difficult to perform. This is due to an

almost total lack of reliable markers for the senescent state

in vivo that do not also detect quiescent cells. Much interest

was generated by the demonstration of senescence-associ-

ated b-galactosidase (SAb) activity in senescent cells some

years ago. Because SAb activity can be detected using

catalytic histochemical techniques, this offered the great

potential advantage that it could be used to demonstrate the

existence of such cells in tissue. Unfortunately, success in

this area has been limited. An initial report (Dimri et al.,

1995) demonstrated positive SAb staining in dermal

fibroblasts in nine donors aged from 70 to 90 years and no

significant staining in 10 donors aged 20–39. No quanti-

tative morphometric assessments or sequential pattern

analyses were performed. A more recent study (Severino

et al., 2000) observed no difference in the frequency of SAb

positive material in a sample of 51 donors whose ages

ranged from 40 to 89 years. This study was in fact unable to

demonstrate any cellular localisation of SAb activity at all

with X-gal deposition being limited to the lumen of eccrine

or sebaceous glands. The possibility was raised that this

stained material was in fact microbial in origin. However, it

is unclear if a standard positive control for b-galactosidase

(incubation at pH 5.0) was performed on the tissue sections

in question. Thus, all that can be concluded at the current

time is that the SAb technique is far from robust. Indeed, it

is ironic that the marker is now very rarely deployed in the

role for which it showed most initial promise but instead is

used frequently for in vitro demonstrations of senescence

where its advantages are greatly outweighed by its

disadvantages (compared to the unambiguous demon-

stration of the senescent state by label-exclusion during

long 3H-thymidine labels) (Severino et al., 2000). Despite

the lack of unambiguous markers, a small number of

studies have attempted to address the critical question of

the existence of senescent cells in tissue and these will be

considered below.

The possibility that the tissue used to produce cultures of

human fibroblasts might contain a significant non-prolif-

erative fraction appeared relatively early in the history of the

field. Hayflick and Moorhead’s observation that cultures of

fibroblasts derived from foetal biopsies had a greater

proliferative capacity than those of adults implied the

existence of a fraction of cells incapable of division, but was

also consistent with an overall reduction in mean prolif-

erative capacity in the adult biopsy without any of the cells

in the population having undergone senescence (Hayflick

and Moorhead, 1961). A more extensive analysis was

performed by Schneider and Mitsui on the in vitro growth

capacity of cultures of fibroblasts derived from ‘young’

(21 – 36-year-old) and ‘old’ (62 – 92-year-old) donors

(Schneider and Mitsui, 1976). This comparative study

demonstrated statistically significant declines in average

fibroblast migration rate, in vitro replicative lifespan and

growth rate and saturation density at confluence. None of

these differences, however, was as large as those found

between ‘young’ (,20PD) and ‘old’ (.40PD) embryonic

human fibroblasts. It was also demonstrated that these

differences could not be explained by simple differences in

cellularity (number of cells per unit tissue) between old and

young skin. Taken together, these studies were consistent

with (i) the presence of elevated numbers of senescent cells

in the older tissue biopsies compared to the younger ones

and (ii) a functional deficit in the cell population as a

consequence of the altered behaviour of a fraction of its

members.

Several cohort studies and a number of less formal

analyses have compared donor age with the residual in vitro

replicative capacity of cells from a tissue of interest

(typically the dermal layer of the skin). The most widely

quoted study, by Martin and co-workers, used fibroblast

cultures derived from an unselected cohort of 100 subjects

with an age range from foetal to 90 years and obtained a

regression line with a slope of 0.2PD per year of donor life

(Martin et al., 1970). A similar study conducted more

recently used fibroblast cultures derived from the Baltimore

Longitudinal Survey on ageing. This demonstrated no

statistically significant decline in the replicative potential

of fibroblast derived from healthy donors (as did an earlier

study by Goldstein and colleagues (Goldstein et al., 1978)).

It is possible to invoke reasons of experimental technique,

such as differential selection of biopsy areas between the

two studies and different degrees of tissue autolysis, as

reasons for this failure to observe a decline in growth

capacity. However, central to the cell senescence hypothesis

of ageing is the notion that senescent cells, through their

altered phenotype, act as causal agents of age-related

degeneration. This implies that any cohort selected on the

basis of an absence of age-related disease is also selected on

the basis of the absence of all of its causal agents, including

senescent cells. It would be close to direct disproof of

J. Bird et al. / Experimental Gerontology 38 (2003) 1319–1326 1321

the theory that senescent cells play a causal role in ageing if

the mitotic tissues of the elderly were composed almost

exclusively of senescent cells but showed no diminution in

physiological function.

The immune system undergoes an age-related decline

resulting in an increased susceptibility to infective and

neoplastic diseases (Pawelec et al., 2002). Reported

changes with age include a decrease in the number of

circulating T-cells, an alteration in the distribution of

memory and naıve T-cell subsets and a decrease in their

capacity for activation and clonal expansion. T-cells from

ageing individuals exhibit a reduction in population growth,

more rapid entry into senescence in vitro and apoptosis. One

possible explanation of these observations is that they result

from progressive waves of antigen-driven cell proliferation

followed by selective apoptosis throughout the lifespan.

Such a situation can be produced artificially in vitro where

repeated stimulation of a population of T-cells with the same

antigen drives the responding T-cells into an irreversible

state of senescence. This is accompanied by increased stress

protein production, apoptosis resistance, critically short

telomeres and no capacity to upregulate telomerase

(Valenzuela and Effros, 2002). Perhaps the best evidence

at the current time that the immune system may contain

senescent cells is the frequent observation of a decline in

mean telomere length in peripheral blood cells with

increasing age (Weng et al., 1995). Although human

T-lymphocytes appear to show telomere-driven senescence,

they are telomerase positive for a significant portion of their

replicative lives and thus the link between telomere

shortening and divisional history is broken. It follows that

qualitative observations of telomeric loss probably provide

an underestimation of cell turnover. A recent report has

demonstrated that the decline in telomerase activity parallels

the loss of CD28 expression in both the CD4 and CD8

subsets. Flow-scytometric detection of loss of CD28 activity

may thus provide a better indicator of the number of

senescent T cells present in the immune system.

Human skin has a reduced repair capacity with advancing

age. Ageing skin demonstrates increased fragility, reduced

amounts of types I and III collagen and clumping of the

collagen present (Fligiel et al., 2003). This occurs con-

comitantly with an increase in the production of MMP-1

(collagenase) and a reduction in TIMP expression. Addition-

ally, a decline in collagen synthesis is apparent with age and

may be linked to the presence of degraded collagen (Fligiel

et al., 2003). Elastin gene expression also declines with age.

It could be envisaged that the presence of degraded collagen

fibrils in the extracellular matrix, as a consequence of the

overproduction of active collagenase, might produce a

damage-repair response in adjacent cells, including cell

turnover as an important component. Taken together, these

observations are not inconsistent with a causal role for and

the presence of senescent cells in the ageing of skin.

Some of the features of aged human skin can be

recapitulated in a reconstituted skin model seeded with

senescent (but not early passage) human fibroblasts. In

particular, increased fragility and intermittent splitting of

the dermal–epidermal junction (blistering) can be observed.

This can be reversed if the skin model is reconstituted using

fibroblasts immortalised by the ectopic expression of

telomerase (Funk et al., 2000). These studies demonstrate

that the presence of senescent cells can have deleterious

effects on a tissue in vivo. However, they do not provide

more than the most basic information on the frequency of

senescent cells that must be present in a tissue for such

effects to be manifest (blistering occurs when a fraction of

senescent cells equivalent to that present in BJ fibroblasts at

60PD is present).

Articular cartilage also demonstrates an age-related

decline in the integrity of the extracellular matrix.

Cartilage collagen has a very long half-life and thus

retains any damage accumulated through the lifespan of

the organism. In humans, degradation of collagen first

becomes apparent after 40 years of age and increases

thereafter. This occurs despite an age-related accumu-

lation of advanced glycation end products which confer

resistance to MMP-mediated attack. This is accompanied

by a decline in chondrocyte mean telomere length

(suggesting ongoing cell turnover) and an increase in

the proportion of senescent cells in the tissue (Martin and

Buckwalter, 2003). Cultured chondrocytes from older

subjects display an altered phenotype which resembles

that of patients with degenerative diseases traditionally

associated with ageing (e.g. osteoarthritis). This implies

that the senescent phenotype of these cells may be

associated with the predispostion toward tissue failure

and disease seen with articular ageing.

Among the best evidence for the existence of

senescent cells currently available is a study by Wolf

and co-workers (Li et al., 1997) using the lens epithelium

of mice as a target tissue. These researchers elegantly

combined long bromodeoxyuridine labelling studies in

vivo with Smith-Whitney colony size analysis in vitro.

These experiments demonstrated (i) that the number of

mitotic cells in the proliferative region of lens epithelium

declined smoothly with the age of the animal; (ii) that

this decline in mitotic index was associated with an

increase in the number of cells showing the functional

criteria of senescence as measured by colony size

analysis; (iii) that calorie restriction both lengthened the

life spans of the animals and reduced the rate at which

senescent cells appeared. This work probably represents

the best possible demonstration of the presence and

accumulation of senescent cells with age in tissue given

the current technical limitations surrounding their

visualisation.

Overall, these studies suggest (i) that senescent cells are

probably present in normal tissue; (ii) that their altered

phenotype can exert effects; (iii) that the clinical presen-

tation of at least some aged tissues is not inconsistent with

the effects that could be exerted by senescent cells.

J. Bird et al. / Experimental Gerontology 38 (2003) 1319–13261322

This is a long way from an explicit link between the

accumulation of senescent cells with their altered phenotype

and the biochemical and metabolic changes associated with

ageing. The biggest problem that must be overcome is the

lack of any robust marker for the senescent state. Such a

marker would enable an accurate assessment of the

frequency and distribution of senescent cells in tissues of

known age and donor pathology to be made. It would also

allow the impact of individual senescent cells on either their

immediate neighbours or the functional capacity of the

tissue in which they reside to be determined. This is a

specialised case of a generalised and as yet unsolved

problem for all current mechanistic hypotheses in gerontol-

ogy. At the time of writing, we have no clear idea of the

precise number of senescent cells or aberrant mitochondria

or oxidised protein that are required to induce a physio-

logical deficit in any tissue. Unless quantitative measure-

ments are employed to determine the degree of loss of

function required to produce an ageing phenotype, as a field

we lay ourselves open to the charge of being long on

mechanistic generalisations and short on facts.

4. Werner’s syndrome

One way in which the question, “How many senescent

cells cause problems?” may be addressed is to consider

Werner’s syndrome (WS, MCK227700). This is a seg-

mental progeroid syndrome in which patients display a

series of symptoms highly reminiscent of the normal ageing

process. These include graying of the hair, pattern baldness,

tight skin, bilateral cataracts, type II diabetes mellitus,

hypogonadism, osteoporosis, arteriosclerosis and athero-

sclerosis (Salk, 1982). The limbs show poor muscular

development and ulcerative lesions often develop over

pressure points. The patients present with a shorter stature

than normal as a result of the failure of the teenage growth

burst. Death occurs at an average age of 47, usually as a

result of cancer or arteriosclerosis.

WS is caused by a variety of loss of function mutations in

a gene coding for a member of the RecQ helicase family

(wrn). The Werner (WRN) protein and its principal

transcription factors (SP1 and AP2) have a tissue- and

age-specific (post-pubertal) expression in normal individ-

uals (Motonaga et al., 2002) which shows some correlation

with the tissue distribution and age of onset of the

pathologies associated with the disease. WRN interacts

with a wide variety of proteins involved in DNA replication

and repair (Hickson, 2003). These include FEN-1 (DNase

IV), proliferating cell nuclear antigen (PCNA), replication

protein A (RPA), Ku70/80 and DNA-dependent protein

kinase (DNA-PK), p53, DNA polymerase d, RAD 51,

WHIP, topoisomerase 1, p21Cip-1/WAF1, Ubc9, telomeric

repeat binding factor (TRF2) and Bloom protein (BLM).

Through Ubc9, indirect interaction is enabled with SUMO-1

(a ubiquitin-like protein which functions by extending

the half-life of interacting proteins), whilst BLM allows

association with a variety of proteins such as topoisomerase

III and ATM kinase. Through these interactions, the cellular

pathways involving WRN include DNA replication, recom-

bination, transcription, repair (e.g. recombinational

repair, non-homologouos end joining, long patch base

excision repair) and apoptosis. However, its principal

function appears to be the processing of replication

forks that have stalled as a result of adducted DNA

(Rodrıguez-Lopez, 2002).

Two striking cellular phenotypes arise as a result of

loss of wrn. The first of these is the extremely poor

replicative potential of cultured fibroblasts. Literature

comparisons of the lifespans of all WS cell strains

published to 1984 with those of published normal controls

showed that 90% of WS cultures have an in vitro lifespan

of less than 20 population doublings (Tollefsbol and

Cohen, 1984). The cause of this limited replicative

capacity is a greatly increased rate of decline in the

mitotic fraction of WS fibroblasts as measured using

either the expression of endogenous proliferation markers

such as pKi67 or short bromodeoxyuridine pulse labels

(Faragher et al., 1993; Kill et al., 1994).

The second distinct property of WS cells is a mutator

phenotype that is most readily demonstrated by selection

experiments designed to detect loss of function mutations at

the HPRT locus by treatment with 6-thioguanine (6-TG).

WS cultures produce a significantly higher fraction of 6-TG

resistant colonies than wild type controls, most of which

show large deletions at the HPRT locus which probably

result from replication fork stalling. A hyperrecombino-

genic phenotype has also been demonstrated in fibroblasts

using plasmids containing overlapping fragments of the

neomycin gene. So, how do these cellular manifestations of

the disease relate to one another, to the clinical presentation

of the patient and, most importantly, to the ageing process in

general? Three possibilities suggest themselves as answers

to this question, (i) that WS is of no real value in

understanding normal ageing because the pathology of

WS sheds light only on that pathology and on nothing else;

(ii) that the key feature of WS is the mutator phenotype

which suggests that WS may inform significantly on the

relationship between normal ageing and genetic instability

or (iii) that the premature replicative senescence seen in

some WS cell types is the central causal mechanism

underlying the pathology. WS can provide valuable data on

the extent to which senescent cells play a role in the

development of normal aged pathology only if this latter

possibility is the correct one.

In accordance with this logic, ectopic expression of

hTERT was forced in WS fibroblasts in order to determine if

their premature senescence resulted exclusively from some

telomere-independent pathway (such as global genomic

instability) or was due to telomere-driven senescence. WS

fibroblasts were found to immortalise normally following

the reintroduction of telomerase, an observation consistent

J. Bird et al. / Experimental Gerontology 38 (2003) 1319–1326 1323

with the hypothesis that such cells do indeed use telomeric

attrition to monitor divisional history (Wyllie et al., 2000).

This being said, two interpretations of this experiment are

possible, (i) that WS is associated with an increased rate of

telomeric loss (possibly as a result of deletions at or near the

telomere caused by the mutator phenotype); or (ii) that the

mutator phenotype causes an increased rate of loss of cells

from the mitotic pool due to the presence of a fraction of

irreversibly arrested S phase cells (Poot et al., 1992) and this

in turn requires the residual telomere-driven population to

cycle more frequently. This would result in an apparent

acceleration of telomere-dependent senescence due to the

additional turnover required from the remaining mitotic

fraction. The existing data on the telomere dynamics of WS

fibroblasts are insufficiently precise to distinguish between

these two interpretations. Although WS fibroblasts have

previously been reported to senesce in culture with telomere

lengths longer than normal diploid fibroblasts, Choi et al.

(2001) have recently shown that the WS telomere restriction

fragment length is within the size range observed for normal

controls. Unfortunately, these studies measured mean

terminal restriction fragment length, not true telomere

length and this is known to mask population heterogeneity

in telomere length at senescence. New techniques such as

STELA (Baird et al., 2003) do not suffer from this

disadvantage and will probably have the required precision

to resolve this question.

Regardless of whether WS fibroblasts display an

increased rate of telomere-driven senescence or an

increased rate of loss of cells from the mitotic pool, one

would expect the disease to be marked by the over

production of senescent cells in vivo. It should thus affect

all tissues that show any significant degree of cell turnover.

In fact, the disease affects some mitotic tissues very severely

whilst others remain essentially normal. For example, the

dermal layer of the skin is severely affected but the immune

system appears to be clinically unaffected (Miller, 2000;

Goto et al., 1985). Mass cultures of T-cells derived from WS

patients display no lifespan deficit compared to those taken

from normal controls (James, et al., 2000). However, such

cells do display elevated mutation rates at the HPRT locus

following selection with 6-TG (Fukuchi et al., 1990). This

observation of significant markers of global genomic

damage in cells from both clinically affected tissues (dermal

fibroblasts) and those which are apparently normal (T-cells)

suggests that genomic instability per se is not the primary

driver of mitotic tissue ageing in WS. In contrast the

appearance of premature replicative senescence in an

affected tissue but not in an unaffected one is consistent

with a causal role for senescent cells in the development of

the disease pathology. It also suggests an answer to the

question of how many senescent cells are required to cause a

physiological deficit. A reduction of the overall fibroblast

divisional capacity within the dermal layer to 20 population

doublings or less would be expected to produce the skin of a

WS patient.

Once senescent, WS fibroblasts display an altered

phenotype that is essentially identical to that of senescent

normal fibroblasts, (e.g. the expression of stromelysins 1

and 2, collagenase, cathepsin O, ICAM-1, IL-6, monocyte

chemoattractant protein-1 and IGF-BPs 2 and 5 are all

increased) (Choi et al., 2001; Lecka-Czernik et al., 1996). It

is to be expected that cells from other tissues would behave

likewise. Given that the phenotypes of senescent WS and

normal cells are so similar, the key question remains why

are all mitotic tissues not equally affected? A principal

factor is probably the ameliorative effect of endogenous

telomerase expression in balancing the premature removal

of cells from the mitotic pool. Such endogenous expression

is present in T-cells for at least part of their replicative

lifespan but is absent from dermal fibroblasts (Bodnar et al.,

1996). The importance of this difference was independently

recognised by two groups (Ostler et al., 2002; Johnson et al.,

2001) both of which considered the emergence of pathology

in WS essentially as the result of premature replicative

senescence resulting from accelerated telomeric loss. Taken

at face value, this would imply that all telomerase negative

tissues with any significant degree of cell turnover should

show pathology. However, Ostler et al. (2002) qualified this

with some important caveats, which may be summarised as:

(i) Truly post-mitotic cells will be unaffected by wrn

mutations, since the phenotypic impact of the disease is

based on the generation of senescent cells and WRN

only appears to be used during S phase. This

emphasises the limits of the replicative senescence

hypothesis which is formulated to explain the ageing of

mitotic tissue.

(ii) Cell types that normally show telomere-independent

senescence should be unaffected by mutations in wrn.

It follows that if such cells comprise a majority within a

given tissue, that tissue itself will be unaffected.

(iii) Cell types that show small telomeric end-replication

losses (such as fibroblasts) should be severely affected

in WS. However, the amount of DNA lost as a result of

the end-replication problem varies between cell types

(as a result of variations in the length of the 30

overhang). A priori loss of wrn should impose a fairly

fixed additional rate of telomeric loss (based on a

loosely fixed frequency of replication fork stalls in

responses to adducts). Thus, as normal telomeric loss

rate increases, the additional loss caused by a mutation

in wrn probably becomes progressively less significant.

The replicative capacity of tissues comprised of cells

with a high endogenous telomeric loss rate is therefore

likely to be only marginally decreased by loss of the

WS helicase. One would therefore expect to see an

absence or markedly lessened severity of the disease

phenotype in such tissues.

It can be seen that the initial postulate that the premature

senescence of WS fibroblasts results from accelerated

J. Bird et al. / Experimental Gerontology 38 (2003) 1319–13261324

telomeric attrition leads to the prediction that at least some

tissues composed of telomerase negative mitotic cell types

would not be expected to show a phenotype in the WS

patient.

Interestingly, an identical prediction emerges if it is

proposed that wrn mutations do not affect the rate of

telomeric loss but do cause an increased rate of loss of cells

from the mitotic pool. This alternative postulate leads to the

predictions:

(i) That post-mitotic cell populations will be unaffected.

(ii) That both telomere-driven and telomere-independent

mitotic cell types will be affected but with the

following caveats:

(a) hTERT will have a protective effect on the

proliferative capacity of the mitotic pool in cell

populations which show telomere-dependent

senescence.

(b) Any cell type which shows a high intrinsic rate of

exit from the mitotic pool will be less affected

than those which have intrinsic exit rates equal to

or lower than those shown by dermal fibroblasts in

culture.

This latter distinction is potentially important. Mitotic

cell populations are frequently assumed to show a

uniform rate of exit from the growth fraction over

divisional time (as measured in population doublings).

However, it has been shown that the rate at which the

division competent fraction of a cell culture is lost

(measured as decline in the labelling index per

population doubling) varies significantly between differ-

ent cell types (Thomas et al., 1997; Kalashnik et al.,

2000; Kill et al., 1994). The normal exit rate of human

dermal fibroblasts from the mitotic pool is approximately

0.79 ^ 0.13% PD21 as measured by pKi67 staining.

However, the presence of a wrn mutation increases this

exit rate to 4.85 ^ 0.67% PD21 (Kill et al., 1994). This

increase in the exit rate is of the order of ,4% per

population doubling, a figure provocatively similar to the

fraction of cells previously shown to irreversibly arrest in

S phase in some WS cell types (Poot et al., 1992). The

increased exit rate seen in fibroblasts probably represents

a combination of the telomere-independent exit caused

by wrn and an additional telomere-dependent component

resulting from extra proliferation required from the

reduced mitotic fraction to generate the population

doubling. An additional exit rate of this size might be

expected to have only marginal effects on some cell

types given their endogenous exit rates. For example,

HUVEC show a normal exit rate of 4.43 ^ 0.31% PD21

(Kalashnik et al., 2000). Presence of a wrn mutation

would be predicted to only increase this loss rate to

,8% or less resulting in a maximum estimated decrease

in the proliferative capacity of a HUVEC culture from

,15 to ,7.5 population doublings. Given that 4%

probably represents an overestimate of the additional exit

rate, it is highly likely that the difference between normal

and WS tissue under these circumstances would be

extremely difficult to detect.

In vitro experimcnts, however elegantly designed, will

always leave a significant element of uncertainty as to their

in vivo validity. It is our contention that, until now, this lack

of knowledge was something of a necessary evil because it

was almost impossible to detect senescent cells in tissue.

This is no longer the case. The field has the capacity to

generate effective markers for the senescent state and,

through WS, at least some idea of the likely frequencies at

which they would need to be present to produce physio-

logical deficits. Currently, the technology exists to make

effective markers for the senescent state. Only the

application of such markers will allow the question, ‘Can

we say that senescent cells cause ageing?’ to be unambigu-

ously answered.

Acknowledgements

The authors gratefully acknowledge the financial support

of the BBSRC Experimental Research on Ageing (ERA)

Special Initiative. Thanks are due to Katherine Sainsbury

for keyboarding the manuscript.

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