Replicative senescence in sheep fibroblasts is a p53 dependent process

Replicative senescence in sheep fibroblasts is a p53 dependent process

Terence Davisa, Julia W. Skinnera, Richard G.A. Faragherb,Christopher J. Jonesa, David Kiplinga,*

aDepartment of Pathology, School of Medicine, University of Cardiff, Heath Park, Cardiff CF14 4XN, Wales, UKbSchool of Pharmacy & Biomolecular Sciences, University of Brighton, Lewes Road, Brighton BN2 4GJ, England, UK

Received 19 July 2004; received in revised form 6 September 2004; accepted 13 September 2004

Available online 5 October 2004

Abstract

Studies on telomere and telomerase biology are fundamental to the understanding of human ageing, and age-related diseases such as cancer.

However, human studies are hampered by the lack of fully reflective animal model systems. Here we describe basic studies of telomere length

and telomerase activity in sheep tissues and cells. Terminal restriction fragment lengths from sheep tissues ranged from 9 to 23 kb, with

telomerase activity present in testis but suppressed in somatic tissues. Sheep fibroblasts had a finite lifespan in culture, after which the cells

entered senescence. During in vitro growth the mean terminal restriction fragment lengths decreased in size at a rate of 210 and 350 bp per

population doubling (PD). Senescent skin fibroblasts had increased levels of p53 and p21WAF1 compared to young cells. Incubation of senescent

cells with siRNA duplexes specific for p53 suppressed p53 expression and allowed the cells to re-enter the cell cycle. Five PDs beyond

senescence the siRNA-treated cells reached a second proliferative barrier. This study shows that telomere biology in sheep is similar to that in

humans, with senescence in sheep GM03550 fibroblasts being a telomere-driven, p53-(p21WAF1)-dependent process. Therefore sheep may

represent an alternative model system for studying telomere biology, replicative senescence, and by implication human ageing.

q 2004 Elsevier Inc. All rights reserved.

Keywords: Ageing; Animal models; Cellular immortalisation; p21WAF1; Proliferative lifespan barriers; Telomerase

1. Introduction

Normal human fibroblasts have a limited replicative

lifespan in culture and undergo only a finite number of

population doublings (PDs) before they enter an irreversible

non-dividing state termed mortality stage 1 (M1) or

senescence (Hayflick and Moorhead, 1961; Shay and

Wright, 2000). At M1 the cells have an enlarged

morphology, a low BrdU labelling index and a high staining

level for the SAb-gal activity (Dimri et al., 1995). This

replicative senescence is thought to result from the

progressive loss of telomeric repeats at the ends of

chromosomes due to the inability of the DNA replication

machinery to efficiently duplicate the 5 0 ends of linear

chromosomes, the so-called ‘end replication problem’

(Olovnikov, 1971, 1973; Blackburn, 1991). This has led to

0531-5565/$ - see front matter q 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.exger.2004.09.004

* Corresponding author. Tel.: C44 29 2074 4847; fax: C44 29 2074

4276.

E-mail address: [email protected] (D. Kipling).

the telomeric loss hypothesis as a mitotic clock mechanism

in fibroblasts (Harley et al., 1990; Hastie et al., 1990;

Allsopp et al., 1992; Vaziri et al., 1994), a hypothesis that

proposes that when telomeres shorten to a critical limit they

lose their function (e.g. end protection), resulting in

chromosome fusion. The telomere signal is transduced via

the p53 tumour suppressor protein that in turn activates the

cyclin-dependent kinase inhibitor p21WAF1 and shuts down

the cell cycle (Vaziri and Benchimol, 1996). Telomere

shortening is believed to play an important role in cellular

senescence in vivo and has been observed in ageing normal

somatic tissues (Lindsey et al., 1991; Vaziri et al., 1994;

Chang and Harley, 1995). In addition, telomerase activity is

associated with the ability of cells to proliferate indefinitely

and in carcinogenic progression (Campisi, 2000), and has

been shown to immortalise human fibroblasts in culture

(Bodnar et al., 1998; Vaziri and Benchimol, 1998). The

resultant ‘telomerised’ cells do not show telomere erosion.

These data indicate that studies on telomere and telomerase

biology are fundamental to the understanding of ageing

Experimental Gerontology 40 (2005) 17–26

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T. Davis et al. / Experimental Gerontology 40 (2005) 17–2618

and age-related diseases such as cancer. However, human in

vivo studies have been hampered by the lack of a suitable

fully reflective animal model.

The most used animal model for human biology is the

mouse. However, humans and mice show considerable

differences with respect to the biology of their telomeres. In

the laboratory maintained mouse (Mus musculus) telomeres

are long (30 kb to greater than 200 kb) and hyper-variable

(Kipling and Cooke, 1990; Starling et al., 1990; Prowse and

Greider, 1995; Zijlmans et al., 1997). In most human tissues

telomerase activity is not readily detectable (Kim et al.,

1994), whereas telomerase activity is found in many mouse

tissues (Greenberg et al., 1998). In addition, mouse

fibroblasts have a short replicative lifespan in vitro and a

very high spontaneous immortalisation frequency, compared

to the long lifespan and low immortalisation frequency seen

in human fibroblasts (Todaro and Green, 1963; Aaronson and

Todaro, 1968; Blasco et al., 1997; Kim et al., 2002; Wright

and Shay, 2002a). Recently, some but not all wild-derived

mouse strains (including some colonies of M. spretus) have

been shown to have telomeres similar in length to that seen in

humans (Coviello-McLaughlin and Prowse, 1997; Kipling,

1997; Hemann and Greider, 2000); however, telomerase

activity is present in several tissues from these strains. In

addition, although the telomeres from Mus spretus fibro-

blasts show progressive shortening with ongoing cell

division, the cells eventually up-regulate telomerase activity

leading to immortalisation (Prowse and Greider, 1995;

Coviello-McLaughlin and Prowse, 1997). These obser-

vations suggest that the mouse does not show telomere-

dependent replicative senescence, and is therefore not a fully

reflective animal model for studying human senescence.

There have been a number of studies of telomere biology

in other mammals that have addressed the possibility of

developing an animal model for human senescence. Similar

telomere sizes and attrition rates to human are observed in

horse, donkey, cow and Indian muntjac fibroblasts (Betts

et al., 2001; Zou et al., 2002; Argyle et al., 2003). No

significant telomerase level was found in cow fibroblasts

(Betts et al., 2001) or various donkey tissues (Argyle et al.,

2003). In dogs, the telomeres in tissues are similar in length

to that seen in humans, and telomere shortening is seen with

age in vivo in some breeds and in fibroblasts grown in vitro,

with telomerase activity limited to the germ line and

immortal cells (Nasir et al., 2001; Argyle and Nasir, 2003).

However, telomerase activity has been reported in several

normal tissues (liver, lymph node, ovary, and thymus) in

dogs (Carioto et al., 2001; Yazawa et al., 2003). Ectopic

expression of hTERT is effective in immortalising fibro-

blasts derived from various primates, Indian muntjac and

sheep (Zou et al., 2002; Steinert et al., 2002; Cui et al.,

2003). Although these studies suggest that aspects of

telomere biology are conserved in various mammals, they

are limited in the scope of their investigations.

One of the best-characterised animal models with respect

to replicative senescence is the sheep (Ovis aries). Sheep

fibroblasts have a defined in vitro life span, telomeres in

sheep fibroblasts have been reported to shorten at rates

comparable to that seen in humans, and ectopic expression

of hTERT effectively immortalises the cells (Cui et al.,

2002, 2003; Clark et al., 2003). However, there is limited

data on the telomere lengths and the distribution of

telomerase activity in sheep tissues, and the events down-

stream of telomere erosion have not been examined. In this

work we have analysed the rate of telomeric erosion in two

fibroblast types, measured telomere lengths and telomerase

distribution in several sheep tissues, and used siRNA

techniques to examine the possible roles of p53 and

p21WAF1 in the senescence of sheep fibroblasts. The results

indicate that replicative senescence in some sheep fibro-

blasts is a similar process to that seen in humans, thus

validating the sheep as a model to study the contribution of

replicative senescence to organismal ageing in human cells.

2. Materials and methods

2.1. Cell lines and cell culture

GM03550 cells are sheep dermal fibroblasts from a

4-month-old Southdown ewe obtained from the Coriell Cell

Repository. Bar-1 cells are lung tissue fibroblasts from a

6-month-old lamb obtained from a local abattoir. Bar-1 cells

were grown in Earle’s modification, and GM03550 cells in

Dulbecco’s modification, of Eagle’s medium with 10%

foetal calf serum at 37 8C in an atmosphere of 20% oxygen

containing 5% CO2. Cells were seeded at approximately 106

per T75 vessel and serially passaged every 3–5 days as

necessary. Population doublings were calculated from the

formula PDZ logðNt KN0Þ=log 2, where Nt is number

counted and N0 is number seeded.

2.2. BrdU incorporation assays

The number of cells undergoing DNA synthesis was

measured by incubation of cells on cover slips in 10 mM

BrdU for 1 h, following which BrdU incorporation was

detected by immunoperoxidase, essentially as described in

(Bond et al., 1999). All assays were done in duplicate.

2.3. Detection of SAb-gal activity

Endogenous activity was assessed histochemically

essentially as described (Bond et al., 1999). The proportion

of b-galatosidase positive cells was assessed in a total count

of 500 cells.

2.4. Detection of telomerase activity

Telomerase present in whole cell extracts was detected

using the TRAP assay essentially as described (Kim and

Wu, 1997). The TRAP assay is a two-stage protocol in

T. Davis et al. / Experimental Gerontology 40 (2005) 17–26 19

which telomerase adds TTAGGG repeats to a primer. In the

second stage extension products are detected by PCR. The

cell line 293 provided a telomerase-positive control.

Reaction products were separated on non-denaturing 10%

polyacrylamide gels and visualized by Sybr Gold staining

and fluorimaging on a STORM system using blue

fluorescence mode (AP Biotech).

2.5. Telomere length determination

Incubation of genomic DNA with restriction endonu-

cleases HinfI and RsaI leaves a TRF resistant to enzymatic

digestion containing telomeric and subtelomeric DNA.

Separation of these on gels and hybridization with a

TTAGGG-specific probe produces a smear representing a

distribution of telomeric sequences of all the chromosomes

from a population of cells. DNA was isolated from cultured

cells and TRF length was determined as described

(Ouellette et al., 2000) prior to phosphorimaging (STORM).

2.6. Production of MRC5 cells infected with the HPV E6

oncoprotein

MRC5 cells are foetal lung fibroblasts obtained from the

European Collection of Cell Culture (Salisbury, UK). These

were grown in standard DMEM until the cells were almost

senescent (BrdU labelling index of !2%), infected with

amphotropic viruses containing the HPV16 E6 oncoprotein,

selected with neomycin, and passaged until the cells reached

the proliferative barrier called Mint (all procedures and the

Mint state are described in Bond et al., 1999). The Mint state

was reached approximately 15 PDs beyond M1 (data not

shown), similar to that seen in human HCA2 cells (Bond

et al., 1999).

2.7. Western blotting

Protein samples were prepared and separated on sodium

dodecyl sulphate-polyacrylamide electrophoresis gels, elec-

troblotted to Immobilon-P polyvinylidene difluoride mem-

brane (Millipore), and probed with antibodies as described

(Davis et al., 2003). Antibodies used: a mouse monoclonal

anti-p21WAF1 that recognises the human epitope aa 58–77

(6B6, Becton Dickinson); a rabbit polyclonal anti-p21WAF1

that recognises the human epitope aa 146–164 (C-19, Santa

Cruz); a mouse monoclonal anti-p53 that recognises the

human epitope aa 156–214 (Pab240, Santa Cruz). After use

the filter was stained with India ink, and quantification of the

specific signal and the amount of protein loaded was

performed by using a Bio-Rad imaging densitometer with

Molecular Analyst software.

2.8. siRNA treatment of sheep GM03550 cells

siRNA sequences were designed from the coding

sequence for the Ovis aries p53 gene accession X81705

(Dequiedt et al., 1995): oligonucleotide sp53.1, 5 0-GAU-

GUUGUCACCUGGCUGG (bases 271–289); oligonucleo-

tide sp53.2, 5 0-UGAAGCGCCCCAAAUGCCA (bases

303–321); oligonucleotide sp53.3, 5 0-GACCUACCCUGG-

CAACUAC (bases 393–411). All oligonucleotides were

duplexes tailed with dTdT on each strand, at least 143 bp

from the start of the coding sequence and immediately

downstream of an AA dinucleotide, designed by reference

to the Dharmacon literature, and synthesised by Dharmacon

Research Inc. (Colorado, USA). Each oligonucleotide was

scanned through GenBank using the BLASTn programme

to insure that it was 100% complementary only to the p53

gene. A scrambled siRNA oligonucleotide (sequence 5 0-

CAGUCGCGUUUGCGACUGG) was used as a control.

All siRNA transfections were done using 10,000 senescent

GM03550 cells per well of 12 well microtitre dishes, seeded

in standard DMEM containing 10% foetal calf serum 24 h

prior to the addition of siRNA. Immediately before the

addition of siRNA the medium was replaced with DMEM

containing 10% foetal calf serum but without antibiotics.

For each transfection the following cocktail was made. Tube

A: 7 ml of 20 mM siRNA duplex was mixed with 125 ml

Opti-MEM (Gibco). Tube B: 7 ml of Oligofectamine reagent

(Invitrogen) was mixed with 36 ml Opti-MEM and incu-

bated for 7–10 min at room temperature. Solutions A and B

were combined and incubated for 20–25 min at room

temperature. A further 25 ml Opti-MEM was added to make

a final volume of 200 ml and the mix was added to each well

containing 0.8 ml of medium (final concentration of siRNA

duplex is 140 nM). The medium was replaced 24 h later

with DMEM containing antibiotics. DNA synthesis was

measured at 24-h intervals using a 2-h BrdU incorporation

assay. A mock assay without siRNA was used as a control.

To determine the long-term effects of siRNA treatment on

cell growth, microtitre dishes were transfected as above

with scrambled siRNA or oligonucleotide sp53.1. This was

repeated at intervals of 72 h over a period of 12 days (a total

of five transfections). Mock-transfected cells were used as a

control. DNA synthesis was measured at 72-h intervals

using a 2-h BrdU incorporation assay. Dishes were also

examined visually at the same intervals. To collect cells for

Western blot analysis 60 mm dishes seeded with 200,000

senescent GM03550 cells were used. To each dish 3.2 ml

siRNA cocktail were added to 6.8 ml DMEM. This was

repeated every 72 h for 12 days and the cells collected after

15 days.

3. Results

3.1. Growth characteristics of sheep fibroblasts

Sheep dermal and lung fibroblasts were continually

grown and passaged for up to 300 days. The dermal

(GM03550) fibroblasts grew rapidly for the first 20 PDs,

after which the growth rate slowed and the cells finally

Fig. 2. Telomeres show progressive erosion in sheep fibroblasts grown in

vitro: TRF profiles for; (A) GM03550 cells; (B) Bar-1 cells, at various PDs.

The marker is l DNA cut with HindIII (sizes in kb).

Fig. 3. Analysis of TRF lengths and telomerase activity in sheep cells and

tissues: (A) TRF lengths; Bar-1 fibroblasts (lane 1), Testis (lane 2), Lung

animal 1 (lane 3), Lung animal 2 (lane 4). The marker is l DNA cut with

HindIII (sizes in kb); (B) TRAP assay: human HCA2 fibroblasts

(lanes 1, 2), telomerised HCA2 fibroblasts (lanes 3, 4), sheep testis

(lanes 5, 6), sheep lung animal 1 (lanes 7, 8), sheep lung animal 2 (lanes 9,

10), sheep Bar-1 lung fibroblasts (lanes 11, 12), 293 cells (lane 13, 14),

sheep GM03550 dermal fibroblasts (lanes 15, 16). Samples were heat

treated (C) at 85 8C for 10 min to destroy the telomerase activity.

Fig. 1. Growth dynamics of sheep fibroblasts: (A) Growth curve of

population doublings against days in culture: Bar-1 cells (–C–), GM03550

cells (–B–); (B) Phase contrast of GM03550 cells at 3 PDs (upper panel)

and 31 PDs (lower panel). Bar is 100 mm.


ceased dividing after a total of 32 PDs (Fig. 1A). The cells at

low PDs were small and regular in morphology with

numerous mitoses visible (Fig. 1B), had a BrdU labelling

index of 34G2.1%, and a low level of SAb-gal activity (not

shown). At M1, however, they became flattened and

enlarged with the typical appearance of senescent cells

(Fig. 1B). The cells at M1 had a BrdU labelling index of 2G0.6% and were O95% positive for SAb-gal activity (not

shown). The lung fibroblasts (Bar-1) grew more slowly than

dermal fibroblasts, but had a greater proliferative life span

and ceased dividing after 62 PDs (Fig. 1A). These cells at

M1 were similar in appearance to the dermal cells (not

shown).

3.2. Telomere attrition occurs during in vitro ageing

of sheep fibroblasts

Telomere lengths were measured in these cells by

Southern blot analysis at several stages throughout their

growth. The TRF size distribution in the dermal cells had a

mean length of approximately 16 kb at 3 PDs and the

distribution declined progressively in length to a mean of

approximately 7 kb at 30 PDs (Fig. 2A). This is an average

rate of decline of 330 bp/PD. The TRFs of the lung cells

declined in length from approximately 19 kb at 11 PDs to

8 kb at 62 PDs (Fig. 2B), an average rate of decline of

210 bp/PD. Thus both dermal and lung sheep fibroblasts

show telomere erosion with ongoing cell division.

3.3. Mean TRF lengths and telomerase activity in normal

sheep tissues

Terminal restriction fragment lengths were examined in

sheep lung fibroblasts, testis, lung tissue (two separate

animals), and dermal fibroblasts by Southern blot hybrid-

isation. The mean TRFs of the samples ranged between 18

and 21 kb and the shortest telomeric length detectable was

approximately 8 kb (Figs. 2A and 3A). There was no

significant difference in mean TRF length in the samples

used. No telomerase activity (as determined by TRAP

assay) was found in sheep lung fibroblasts, dermal

fibroblasts, or in lung tissue taken from two individuals

(Fig. 3B). Telomerase activity was, however, found in sheep

testis (Fig. 3B).

3.4. siRNA knockdown of p53 allows senescent sheep

fibroblasts to re-enter the cell cycle

Sheep cells at M1 (BrdU labelling index of !2%) were

incubated in the presence of siRNA specific for the sheep

p53 gene. In order to assess the ability of the various

siRNAs to abrogate p53 function, a pilot experiment was


undertaken that involved incubating sheep GM03550 cells

with each of the three siRNAs, and measuring the effects

using a 2-h BrdU pulse at 24-h intervals subsequent to

addition of siRNA. The results for each of the three siRNAs

are shown in Fig. 4A. All three siRNAs enabled the

senescent cells to re-initiate DNA synthesis compared to

scrambled siRNA- or mock-transfected controls. siRNA

sp53.1 had the best response in terms of the level of BrdU

labelling achieved (6.4G1.1% of the cells) and persistence

of the effects (Fig. 4A). sp53.3 showed a good initial

response but the effects did not persist, and sp53.2 showed

an intermediate response. Using the sp53.1 and sp53.2

siRNAs mitotic cells could be clearly seen 72 h after

transfection (not shown). sp53.1 was thus chosen for the

following experiments.

Fig. 4. Time course of siRNA induced DNA synthesis in senescent

GM03550 cells: (A) cells were transfected with mock (–;–), scrambled

siRNA (–,–), sp53.1 (–C–), sp53.2 (–%–), sp53.3 (–6–) and BrdU

incorporation measured daily for 3 days; (B) 15-day time course of cells

transfected with sp53.1 (same symbols as in A); Phase contrast and BrdU

incorporation of cells after 15 days (five transfections); scrambled siRNA

(C,E), sp53.1 (D,F). Bars are 100 mm, arrows indicate dividing cells.

Senescent GM03550 cells were transfected with sp53.1

or scrambled siRNA at 72-h intervals. The BrdU labelling

index was assessed 72 h subsequent to each transfection. As

can be clearly seen from Fig. 4B, there was a low level of

BrdU labelling in the mock-treated and scrambled siRNA-

transfected cells throughout the experiment, indicative of

the low level of cell division in these cells. With sp53.1,

however, there was a steady increase in the level of BrdU

incorporation, with DNA synthesis occurring in O16% of

the cells after 15 days (five siRNA-transfections). A visual

analysis of these cells is shown in Fig. 4C–F. The scrambled

RNA- or mock-treated cells were large and irregular in

morphology (Fig. 4C) and showed a low level of BrdU

incorporation (Fig. 4E). The cells maintained this appear-

ance throughout the experiment and were essentially the

same as cells at M1. In the sp53.1-treated cells, the cellular

morphology had noticeably altered 6 days after the first

siRNA-transfection, and cells with a youthful morphology

were clearly visible (not shown). After 15 days, these

young-looking cells had formed colonies (Fig. 4D and F)

that in some cases were quite large suggesting a significant

level of cell division. A final cell count showed that the

scrambled siRNA-treated cells managed only a small

increase in cell numbers (!0.2 PDs), whereas the sp53.1-

treated cells managed O2 PDs after 15 days, demonstrating

that the cells not only could synthesise DNA, but that these

cells actively re-entered the cell cycle.

3.5. Expression of cell cycle proteins in normal

and siRNA-treated GM03550 cells

Senescent cells were siRNA-transfected with sp53.1 at

3-day intervals for 12 days (five transfections) and cells

harvested after 15 days. Protein samples were loaded onto

12% acrylamide gels and probed with the antibody Pab240

that is known to detect sheep p53 (Albaric et al., 2001). p53

protein could be readily detected in young sheep cells

(approximately 8 PDs) and p53 increased in amount in cells

at senescence (Fig. 5). With siRNA-treated cells the level of

p53 was reduced to undetectable levels, showing that the

siRNA was successful in abrogating p53 production. The

lack of p53 seen in siRNA-treated cells was not due to lack

of total protein loaded (Fig. 5). Both young and senescent

control MRC5 cells had a similar pattern of p53 expression

to sheep cells (Fig. 5). That the indicated protein is indeed

p53 is shown in E6-infected MRC5 cells at Mint, as the

levels of p53 are much reduced compared to uninfected

cells. Human cells infected with E6 have reduced levels of

p53 due its abrogation and subsequent degradation (Bond

et al., 1999).

In human cells p53 up-regulates p21WAF1 when the cells

reach senescence (Alcorta et al., 1996; Bond et al., 1999;

Stein et al., 1999). Thus we probed the immunoblot with

antibodies to detect p21WAF1 (Fig. 5). Using two different

anti-p21WAF1 antibodies that react to different non-overlap-

ping epitopes, the level of p21WAF1 was seen to be low in

Fig. 5. Immunoblot analysis of MRC5 and GM03550 fibroblasts: lysates

were prepared from cycling cells (young), senescent cells (M1), E6-

infected cells (E6), and siRNA-treated cells (siRNA). Expression levels

were compared for p53 or p21WAF1 as indicated. 6B6 and C-19 are the

mono and polyclonal anti p21WAF1 antibodies, respectively. Approximately

equal amounts of protein lysate (15 mg top, 11 mg bottom) were loaded per

lane as verified by India Ink staining.

Fig. 6. Extension of proliferative lifespan in senescent GM03550 cells using

sp53.1: (A) Bar chart showing additional PDs in siRNA-treated cells: no

treatment (lane 1), scrambled siRNA-treated cells (lane 2), sp53.1-treated

cells (lanes 3 and 4); (B) Phase contrast picture of sp53.1-treated cells at

Mint. Bar is 100 mm.


young MRC5 cells and up-regulated at M1 (Fig. 5). In E6-

infected MRC5 cells at Mint the level of p21WAF1 was low as

expected, due to the abrogation of p53. In the sheep two

proteins, with estimated Mw of 18 and 21 kDa that were

assumed to be p21WAF1, were detected at high levels in cells

at M1 (Fig. 5). The putative p21WAF1 was detectable at a

low level in young and siRNA-treated cells when the

immunoblots were exposed for a longer period (Fig. 5),

suggesting that these proteins are up-regulated at M1, and

down-regulated when p53 is abrogated. Interestingly, only

the 21-kDa protein was detected in siRNA-treated cells.

3.6. Continuous abrogation of p53 allows sheep cells to

reach a second proliferative life span barrier, Mint

Abrogation of p53 allows human cells to bypass M1 and

reach a second proliferative barrier, Mint, after some 10–20

PDs (Bond et al., 1999; Davis et al., 2003, 2004). Thus we

used siRNA abrogation of p53 to determine whether sheep

cells would reach this second proliferative barrier. Pre-

senescent GM03550 cells were treated with sp53.1 at 3-day

intervals until the cells ceased proliferating. Mock- or

scrambled siRNA-treated GM03550 cells divided continu-

ously for 17 days during which they achieved 4.6 and 5.3

PDs, respectively (Fig. 6A). Thereafter no further division

could be seen and the cells had a morphology resembling

that at M1 (not shown). When pre-senescent cells were

treated with sp53.1, however, the cells continued to divide

for up to 90 days before entering a state where no further

growth was seen despite continued siRNA transfection.

During this growth period the majority of the cells had

a small morphology resembling young cells and many

colonies were seen (not shown). The siRNA-treated cells

achieved up to 9.5 PDs (Fig. 6A), an extension of

proliferative life span beyond M1 of between 4 and 5

PDs. At growth arrest the cell morphology was large and

highly irregular with the cells being highly vacuolated

(Fig. 6B). In addition, many multinucleate cells were

present. These cells resembled human cells at Mint (Bond

et al., 1999; Davis et al., 2003, 2004) rather than sheep

cells at M1.

4. Discussion

It has recently been reported that Black Welsh and Finn

Dorset sheep foetal fibroblasts have limited in vitro

replicative life spans ranging from 35 to 113 PDs, and

show telomeric erosion at rates of 70–300 bp per cell

division (Clark et al., 2003; Cui et al., 2003). We have

shown that dermal fibroblasts from 4-month-old Southdown

sheep and lamb lung fibroblasts from an unknown strain,

have limited replicative capacities of 32 and 62 PDs, and

show telomeric erosion with rates of 210–330 bp/PD. Thus

both neonatal and foetal fibroblasts have similar life spans

and telomeric erosion rates. These rates and variability of

telomere erosion compare to an average for human

fibroblasts of 20–147 bp/PD (Huffman et al., 2000; Baird

et al., 2003). Thus the average rates of telomere erosion in

primary sheep fibroblasts are somewhat higher than that

found in human fibroblasts, although the range of pro-

liferative life span is similar (Smith and Whitney, 1980;

Huffman et al., 2000; Baird et al., 2003). In addition we

have shown that telomerase activity is repressed in two

somatic tissues (skin and lung), but active in sheep testis,

with the telomeres in tissues similar in length to that seen in

humans.

In contrast, telomeres in the laboratory-maintained

mouse are long and hyper variable, with telomerase being

widespread in tissues (Kipling and Cooke, 1990; Starling

et al., 1990; Kim et al., 1994; Prowse and Greider, 1995;


Zijlmans et al., 1997). Even in those mouse strains that have

human sized telomeres and show telomere erosion with

ongoing cell division, telomerase activity is eventually up-

regulated in cultured fibroblasts leading to immortalisation,

and telomerase activity is present in several tissues (Prowse

and Greider, 1995; Coviello-McLaughlin and Prowse, 1997;

Hemann and Greider, 2000). However, similar TRF sizes

and telomere attrition rates are observed in horse, donkey,

cow, Indian muntjac, and primate fibroblasts (Kakuo et al.,

1999; Betts et al., 2001; Nasir et al., 2001; Zou et al., 2002;

Argyle et al., 2003), and no significant telomerase level was

found in various donkey tissues (Argyle et al., 2003).

Telomerase activity, however, is associated with tumour

progression in horses and is present in equine sarcomas

(Argyle et al., 2003). In dogs while similar telomere lengths

to human are found in tissues, telomere shortening is seen

with age in vivo in some cases, but not in others, and the

telomerase distribution in dog tissues is disputed with

detectable activity being found in both normal and

neoplastic tissues (Carioto et al., 2001; Nasir et al., 2001;

McKevitt et al., 2002; Argyle and Nasir, 2003; Yazawa

et al., 2003). It should be noted here that the tissues found

positive for telomerase in dogs (liver, lymph node, ovary,

thymus) were not examined in the present study, so the

telomerase status of these tissues is unknown in sheep. Thus

it appears that in large, relatively long-lived mammals, the

telomere lengths and telomerase distribution appears to be

similar to that seen in humans, whereas in the smaller

shorter-lived mammals the situation is somewhat different.

Chicken has also been proposed as a possible model for

human cellular senescence, as chicken telomeres are similar

in length to human and chicken fibroblasts show telomere

erosion with PD with the cells reaching senescence after 32

PDs (Venkatesan and Price, 1998). However, the distri-

bution of telomerase activity is similar to that seen in mouse.

In human fibroblasts the telomere signal is transduced via

the tumour suppressor protein p53 that acts by up-regulating

p21WAF1 (Alcorta et al., 1996; Vaziri and Benchimol, 1996;

Stein et al., 1999). Abrogation of p53, with an associated

decrease in p21WAF1 levels, allows human cells to bypass

M1, with the cells eventually reaching an intermediate life

span barrier termed Mint (Bond et al., 1999; Davis et al.,

2003). Similarly, siRNA-induced p53 knockdown, with an

associated decrease in p21WAF1, allowed sheep fibroblasts

to avoid M1, and continue cycling until reaching a similar

life span barrier. It is interesting that almost 100% p53

knockdown was observed, yet the level of resumed mitosis

appeared to be low. This may be explained in two ways.

Firstly, abrogation of p53 allowed the cells to expand into

colonies. Some of these colonies were very large and the

cells may have been approaching Mint and thus had a slow

growth rate, with Mint being reached after only 5 PDs for the

culture (Fig. 6). However as these cells were still being

treated with siRNA, the p53 level would still be low.

Secondly, it is possible that some the cells had been

senescent for some time and were thus difficult to get back

into the cell cycle, possibly because of a build up of p16

levels, or because they had undergone secondary changes

(e.g. they were seen to become multi-nucleate) that

prevented such a re-entry. Although the mechanism for

this effect is not clear, it is observed routinely in human

fibroblasts that show a clear p53-dependent senescence, e.g.

BJ fibroblasts, in which microinjection of anti-p53 anti-

bodies only work on newly senescent cells, as opposed to

deep senescent cells (Gire and Wynford-Thomas, 1998).

The identification of p21WAF1 in the sheep is not

absolute, although the observed doublet on the immunoblots

behaves in a way that mimics the behaviour of p21WAF1 in

human cells (Fig. 5). In addition, the p21WAF1 doublet was

detected with two antibodies that recognise different

epitopes on the human protein (see experimental pro-

cedures). Although the p21WAF1 gene has not been cloned

from sheep, these epitopes are 100 and 95% conserved in cat

and dog p21WAF1 (Protein Accession numbers BAA23168

and AAC27627, respectively), and the antibody C-19 is

known to detect mouse and rat p21WAF1. This strongly

suggests that these bands do indeed correspond to p21WAF1.

Off-target effects have been reported for siRNA induced

gene knockdown (Jackson et al., 2003). However, our data

suggest that the bypass of senescence is directly related to

p53 knockdown, because three different siRNAs had a

similar effect on senescent cells (Fig. 4A). Different siRNAs

for the same gene have been shown to affect different

subsets of such off-target genes (Jackson et al., 2003).

Further support for a role of p53 in cell proliferation arrest in

sheep comes from the observation that levels of p53 protein

are inversely related to cellular growth rates in sheep

ovarian epithelial cells (Murdoch and Van Kirk, 2002).

The intermediate barrier seen when p53 was continu-

ously abrogated was reached only 5 PDs after M1 compared

to 10–20 PDs for human cells (Bond et al., 1999; Davis

et al., 2003, 2004). This may reflect the greater telomere

erosion rate seen in sheep GM03550 cells resulting in a

lower replicative potential. Alternatively, it is possible that

not all of the siRNA-treated cells were successfully

transfected, and the transfected cells actually achieved

more than 5 PDs. As sheep fibroblasts are similar to human

in that they show telomere erosion and can be immortalised

by the ectopic expression of hTERT (Bodnar et al., 1998;

Vaziri and Benchimol, 1998; Cui et al., 2002, 2003; Clark

et al., 2003), it appears overall that replicative senescence in

some sheep fibroblasts resembles senescence in human

fibroblasts in that it is a telomere-driven p53-(p21WAF1)-

dependent event.

The involvement of p53 has been implicated in

replicative senescence in several other animal systems,

and p53 is required for replicative senescence in mouse

embryonic fibroblasts (Wright and Shay, 2002b; Dirac and

Bernards, 2003). In Japanese macaques certain p53

mutations allow fibroblasts to by-pass M1 and continue

cycling, although they appear to reach a condition

resembling crisis (M2) with considerable cell death


occurring, rather than Mint (Shimizu and Ishida, 2002;

Shimizu et al., 2003). Interestingly, fibroblasts from

macaques have TRFs slightly larger than humans (Kakuo

et al., 1999), can be immortalised using hTERT (Steinert

et al., 2002), and show p53-dependent senescence (Shimizu

and Ishida, 2002; Shimizu et al., 2003). Thus macaque

fibroblasts strongly resemble human cells in these respects.

However, the seemingly high level of p53 mutations found

in these cells and their ability to up-regulate telomerase and

immortalise (Shimizu et al., 2003), suggest that macaque

cells may resemble mouse fibroblasts in their ability to

spontaneously by-pass senescence. Thus macaque may not

be a good model for studying replicative senescence in

humans. The transcriptional activity of p53 has been shown

to increase in chicken embryonic fibroblasts as the cells

became senescent, suggesting that the role of p53 in

replicative senescence extends beyond the mammals (Kim

et al., 2002). A possible role for p53 in replicative

senescence in other animals is as yet unknown.

The conclusions from this work are that telomere and

telomerase biology and the involvement of p53 (and

p21WAF1) in replicative senescence in some strains of

sheep fibroblasts strongly resemble that seen in human cells.

In addition, although data from long-term culture of sheep

fibroblasts are limited, the spontaneous immortalisation

frequency appears to be low (this work, Cui et al., 2002,

2003; Clark et al., 2003). Animals such as the macaque or

dog, appear to share some of the characteristics of both

human and mouse, and would thus be less suitable as animal

models, and data from other animals are at present limited.

As techniques exist for manipulation in sheep in vivo, such

as reproductive cloning (Wilmut et al., 1997), sheep

represent a plausible alternative model system for studying

telomere biology and replicative senescence.

Recent data have indicated that replicative senescence

may not be induced by telomere length per se, but due to

alterations in telomeric structure (Chin et al., 1999;

Blackburn, 2000; Masutomi et al., 2003). Telomeres form

terminal loops (T loop) stabilised by proteins such as TRF2

(van Steensel et al., 1998; Griffith et al., 1999). This loop

structure probably provides telomere capping functions, and

its opening exposes the single strand overhang triggering

senescence (van Steensel et al., 1998; Chin et al., 1999;

Saretzki et al., 1999; Blackburn, 2000; von Zglinicki, 2001;

Karlseder et al., 2002). However, even in the cases where

uncapping is observed, the telomeres appear on average to

be shorter (Proctor and Kirkwood, 2003). Alternatively, it

has been proposed that replicative senescence is due to the

erosion of the telomeric single strand overhang (Masutomi

et al., 2003; Stewart et al., 2003), though this has been

disputed (Keys et al., 2004). Still, in any given cell culture

absolute telomere length is the best predictor of replicative

senescence (Hemann et al., 2001), senescent fibroblasts

have shorter than average telomere length (Martens et al.,

2000; Serra and von Zglinicki, 2002), senescent

hTERT expressing fibroblasts have short telomeres

(Martin-Ruiz et al., 2004), and a specific group of

chromosomes with the shortest telomeres appears to be

responsible for replicative senescence (Zou et al., 2004).

Overall, the data are consistent with the concept that shorter

telomeres are a trigger for the processes leading to

replicative senescence in fibroblasts, although the mechan-

ism may involve exposure of the single strand overhang,

disrupting the T loop (Martens et al., 2000; Proctor and

Kirkwood, 2003; von Zglinicki, 2003; Sharpless and

DePinho, 2004). Therefore, irrespective of the inducing

mechanism, the study of an animal that shows telomeric

shortening associated with replicative senescence, should

provide a valuable model with which to study human

ageing.

Acknowledgements

We would like to thank members or our laboratories for

their discussions. This work was supported by the BBSRC

Science of Ageing and Experimental Research on Ageing

initiatives.

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Replicative senescence in sheep fibroblasts is a p53 dependent process