1 Department of Pathology, University of Wales College of Medicine, Heath Park,
Cardiff, CF14 4XN, UK
2 School of Pharmacy and Biomolecular Sciences, University of Brighton,
Cockcroft Building, Lewes Road, Brighton, BN2 4GJ, UK
* Author for correspondence (e-mail: kiplingd{at}cardiff.ac.uk)
Accepted 18 December 2002
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Summary |
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Key words: Ageing, Cellular senescence, Oncoprotein, Microinjection, Telomerase
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Introduction |
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The underlying genetic defect in WS is a recessive loss-of-function
mutation in the WRN gene. WRN encodes a member of the RecQ
helicase family (Yu et al.,
1996) which, unlike other members of the RecQ family, also shows
3' to 5' exonuclease activity
(Huang et al., 1998
). WRNp is
a nuclear protein that is found predominantly in the nucleoli but that
relocates to replication foci at S phase of the cell cycle, and to sites of
DNA damage in response to DNA-damaging agents such as 4-nitroquinoline 1-oxide
(Gray et al., 1998
;
Marciniak et al., 1998
;
Shiratori et al., 1999
). It
interacts with a range of other proteins, including topoisomerase I, Ku 86/70,
replication protein A, p53 and DNA polymerase
(Shen and Loeb, 2001
). WRNp
has been implicated in DNA replication, transcription, DNA recombination and
DNA repair (Bohr et al., 2002
).
Although WS cells do show a chromosome instability phenotype
(Hoehn et al., 1975
;
Salk et al., 1981
;
Fukuchi et al., 1989
), WS is
not a dramatic DNA-repair-defect syndrome when compared with diseases such as
xeroderma pigmentosum. Although WRNp does play a role in DNA repair, loss of
expression has only a mild impact on the ability of WS cells to undergo DNA
repair (Bohr et al., 2001
).
Normal human fibroblasts have a finite capacity to divide, after which they
enter a state of permanent cell-cycle arrest termed replicative senescence
(also known as mortality stage 1 or M1). Cultures of such cells cease to
expand as a result of a progressive decline in the growth fraction. The
kinetics of this process have long been recognized to be consistent with the
operation of one or more molecular mechanisms capable of acting as a cell
division `counting' system. In human dermal fibroblasts, it is now known that
this counting mechanism is based on the activation of p53 as a result of the
progressive erosion of chromosomal telomeres
(Bodnar et al., 1998;
Vaziri and Benchimol, 1998
;
Wright and Shay, 2002
).
WS fibroblast cultures display a dramatic reduction in replicative life
span (Martin et al., 1970)
owing to an increased rate of decline in the culture growth fraction compared
with normal controls (Faragher et al.,
1993
). Loss of the WRN helicase has the potential to produce many
abortive DNA replication events that could trigger premature cell-cycle exit
without involving telomeric loss. Thus, an important but unresolved question
is the extent to which the rapid replicative senescence seen in WS fibroblasts
results from an acceleration of normal senescence mechanisms and to what
extent (if any) it results from chromosomal instability and replication-fork
stalling as a result of mutations in WRN.
Although several groups have shown that telomere shortening acts as a
primary driver of senescence in WS fibroblasts
(Ouellette et al., 2000;
Wyllie et al., 2000
;
Choi et al., 2001
), these
studies did not address in detail the signalling pathway downstream of
telomere erosion in WS cells. Replicative senescence is associated, in the
case of dermal fibroblasts from newborns or adults, with activation of p53 as
a transcriptional transactivator (Itahana
et al., 2001
), as shown by the induction of p53-dependent
transcripts such as the cyclin-dependent kinase inhibitor
p21Waf1 or p53-dependent reporter constructs in senescent
fibroblasts (Bond et al., 1994
;
Bond et al., 1995
;
Bond et al., 1996
;
Webley et al., 2000
).
Furthermore, abrogation of p53 function using dominant-negative p53 alleles,
microinjection of anti-p53 antibodies or viral oncoproteins such as human
papilloma virus 16 (HPV16) E6 leads to a bypassing of senescence and extension
of lifespan (Bond et al., 1994
;
Bond et al., 1999
;
Gire and Wynford-Thomas,
1998
).
Accordingly, in the present study, we address whether replicative senescence in WS fibroblasts shows features consistent with an acceleration of the mechanism of senescence that is seen in normal dermal fibroblasts. Two complementary experimental approaches were undertaken: (1) observational studies of key cell-cycle proteins (including those normally involved in the p53 response); and (2) direct experimental abrogation of p53 function using two independent methods.
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Materials and Methods |
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Retroviral gene transfer
Amphotropic retrovirus vectors expressing the HPV16 E6 oncoprotein from a
pLXSN construct, packaged in PA317 cells
(Halbert et al., 1991), were
kindly provided by Denise Galloway (University of Washington, Seattle, WA).
pBABEhTERT (Wyllie et al.,
2000
) is an amphotropic retrovirus expressing hTERT, the catalytic
protein subunit of human telomerase, constructed by cloning the EcoRI
insert of pGRN121 (Nakamura et al.,
1997
) into pBABEpuro
(Morgenstern and Land, 1990
).
For control infections, pBABEneo, or pBABEpuro vectors, packaged in
CRIP
cells, were used (Wyllie et al.,
2000
).
Gene transfer was carried out as described previously
(Bond et al., 1994). Two days
later, fibroblast cultures were passed into medium containing G418 (0.4 mg
ml-1), or puromycin (2.5 µg ml-1), at serial
dilutions of 1:5, 1:10, 1:100 and 1:250, and observed for colony development.
For each gene transfer, the retroviral infections were done twice.
BrdU incorporation assays
Cells were labelled by incubation in 10 µM bromodeoxyuridine (BrdU) for
1 hour, with incorporation detected by immunoperoxidase
(Bond et al., 1996). The
proportion of BrdU-positive cells was assessed in a total count of 500
cells.
Detection of senescence-associated ß-galactosidase activity
Endogenous mammalian senescence-associated ß-galactosidase activity
(SAß-gal) was assessed histochemically (Dimri et al., 1996;
Bond et al., 1999). The
proportion of ß-galactosidase-positive cells was assessed in a total
count of 500 cells.
Apoptosis (TUNEL) assays
Cytospin preparations of trypsinized cultures were prepared, treated and
incubated with terminal deoxynucleotidyl transferase (Promega) and
biotin-16-dUTP (Boehringer Mannheim) as described in
(Bond et al., 1999). Sites of
biotin-16-dUTP localization were visualized by using the mouse-specific
avidin-biotin-peroxidase (ABC) system (Vector Labs)
(Bond et al., 1999
). After
haematoxylin counterstaining, the proportion of apoptotic (brown) cells was
assessed in samples of 500 cells.
Immunocytochemistry
For p21Waf1, cells on coverslips were fixed and
pre-treated as described (Bond et al.,
1999). p21Waf1 was detected by incubation for
1 hour in a 1:500 dilution of an anti-p21Waf1 mouse
monoclonal antibody (6B6; Becton Dickinson), followed by a 1:100 dilution of
biotinylated anti-mouse antibodies using the ABC kit (Vector Labs). The
proportion of p21Waf1-positive cells was assessed in a
total count of 500 cells.
Immunoblotting
Protein samples were prepared, separated on 12%
sodium-dodecylsulphate/polyacrylamide electrophoresis gels (for pRb, a 7.5%
gel was used), electroblotted to Immobilon-P polyvinylidene difluoride
membrane (Millipore) and antibodies applied as described by Bond et al.
(Bond et al., 1999). The
antibodies used were: mouse monoclonal anti-p21Waf1 (6B6;
Becton Dickinson); mouse monoclonal anti-p16Ink4a (DCS50;
Oncogene Research Products); mouse monoclonal anti-p27Kip1
(C20; Transduction Laboratories); mouse monoclonal anti-p53 (DO-1;
Calbiochem); mouse anti-pRb (C3-245; Becton Dickinson). A chemiluminescence
kit (Amersham) was used for visualization using goat anti-mouse secondary
antibodies. After visualization, blots were washed three times for 10 minutes
in 0.1 M glycine (pH 2.5), once in 1 M Tris (pH 8.0) for 5 minutes, and then
in PBS for 30 minutes, before reuse. After use, the filter was stained with
India ink.
Densitometric quantification analysis
Quantification of the specific signal and the amount of protein loaded for
the immunoblot (Fig. 2) was
performed using a Bio-Rad imaging densitometer with Molecular Analyst
software. When normalized based on protein loading, the relative amounts of
p21Waf1 are 1.0 and 1.17 (arbitrary units) for cycling WS
cells and WS cells at M1, respectively. WS cells at M1 have 2.4 times
more total protein per cell than cycling WS cells (not shown), a figure
similar to that seen for IMR90 cells
(Sherwood et al., 1988
). Thus,
taking into account both the higher relative amount of
p21Waf1 determined from
Fig. 2 (1.17 times) and the
increased amount of total protein per cell (2.4 times), WS cells at M1 have
2.7 times as much p21Waf1 per cell than cycling WS
cells.
|
Microinjection of anti-p53 antibodies
Late-passage AG05229 cells were microinjected using an Eppendorf system as
described previously (Bond et al.,
1996). Mouse monoclonal antibody DO-1 (isotype
IgG2a
; Oncogene Research) or control mouse IgG (Sigma) was
injected at a concentration of 2 mg ml-1 in PBS. Rabbit IgG (12 mg
ml-1) (Sigma) was co-injected. Following injection, cells were
incubated in fresh medium for 48 hours and then incubated in 20 µM BrdU for
4 hours. Cells were fixed as described above and BrdU incorporation was
detected by incubation in a 1:100 dilution of anti-BrdU (isotype
IgG1
) (Dako) with DNAase I (25 U ml-1) for 2
hours at 37°C, followed by incubation for 1 hour at room temperature in a
1:50 dilution of fluorescein isothiocyanate (FITC) conjugated goat anti-mouse
IgG1-isotype specific (Southern Biotechnology Associates). The
isotype-specific FITC-IgG1 is used to avoid a cross-reaction with
microinjected DO-1. Microinjected cells were detected using a 1:800 dilution
of goat anti-rabbit-Texas Red conjugate (Southern Biotechnology Associates).
Nuclei were visualized by staining with 0.5 µg ml-1
4'-6-diamidino-2-phenylindole (DAPI) for 5 minutes at room temperature,
and the cells were mounted in 90% glycerol-PBS. All antibody dilutions were in
1% bovine serum albumin in PBS.
Cell-cycle analysis
Cells were washed twice with PBS prior to trypsinization. All cell-culture
media and PBS washes were collected with trypsinized cells and spun down for
10 minutes at 300 g. Cell pellets were adjusted to 1 ml
(3-6x105 cells ml-1). 125 µl of 0.4 mg
ml-1 ethidium bromide and 1% Triton X-100 was added, followed by 50
µl ribonuclease A (10 mg ml-1). Samples were vortexed, incubated
at room temperature for 10 minutes and then immediately analysed by flow
cytometry (Smith et al.,
1999). Direct observation of cell suspensions indicated minimal
doublet formation. Cell samples were analysed using a FACScan flow cytometer
(Becton Dickinson Immunocytometry Systems). Histograms of cell numbers versus
FL2-H (pulse height) provided a measure of the DNA content of the cells.
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Results |
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Most of the AG05229 cells at M1 (>77%) were arrested with a 2N
DNA content as assessed by fluorescence-activated cell sorting (FACS) analysis
(Fig. 1I), which is consistent
with G1 arrest. However, a significant proportion (20%) had
higher levels, reflecting G2 arrest and/or G1 tetraploid
cells. The same overall pattern was seen with control HCA2 cells
(Fig. 1J) and has been reported
for IMR90 cells (Sherwood et al.,
1988
).
Cycling AG05229 cells showed moderate levels of the cyclin-dependent kinase
inhibitor (CdkI) p21Waf1 in 22.5±1.8% of the nuclei
by immunocytochemistry (Fig.
1B). This increased to 91.2±1.3% of the nuclei by the time
the cells reached M1 (Fig. 1F).
Although, when analysed by immunoblot using anti-p21Waf1
antibodies, cycling and M1 AG05229 cells appeared to have similar levels of
p21Waf1 (Fig.
2), once quantified on a per-cell basis (see Materials and
Methods) to take into account the observation that cells at M1 have
approximately twice the protein of cycling cells
(Sherwood et al., 1988), it is
clear that there has been a significant increase in the level of
p21Waf1 as WS cells reach M1. The presence of cycling
cells in the young AG05229 population was indicated by their small size
(Fig. 1A), a BrdU LI of
29.5±2%, low levels of SAß-gal activity (6±1.1%)
(Fig. 1C) and the presence of
low but detectable levels of hyperphosphorylated pRb
(Fig. 2).
We have also examined the levels of two other CdkIs in AG05229 cells. The CdkI p16Ink4a was present at almost undetectable levels in cycling AG05229 cells (Fig. 2) and showed a large increase as these cells reached M1. By contrast, there was no significant difference in the level of the p21Waf1-related CdkI p27Kip1 as the AG05229 cells reached M1 (Fig. 2). The same overall pattern of expression of these CdkIs is seen in HCA2 cells as they proceed to M1 (Fig. 2).
Abrogation of p53 function extends the cellular life span of WS
fibroblasts
Presenescent AG05229 fibroblasts at 20 PDs (BrdU LI of 3.4±0.8%)
were infected with amphotropic retroviral vectors encoding a neomycin
resistance gene (neoR) alone
(Wyllie et al., 1993) or with
HPV16 E6 (Halbert et al.,
1991
). Analysis of G418-resistant colonies (designated 5229.neo
and 5229.E6) began two to three weeks after infection.
As expected, cells expressing neoR alone ceased
proliferating during the first two weeks and entered replicative senescence,
forming clones of up to 5 PDs (32 cells)
(Fig. 3B). The 5229.neo cells
at this stage were essentially identical to uninfected AG05229 cells at M1
(not shown). By contrast, expression of HPV16 E6 resulted in evasion of
senescence and generated rapidly growing colonies as observed visually
(Fig. 3,
Fig. 4A). As the 5229.E6
colonies grew, they consistently went through a brief phase of cell death
coupled with continued colony expansion
(Fig. 3A). Eventually, >80
days post-infection, net growth ceased and the cells entered a state similar
to the second senescence-like state, which we have termed Mint in
HCA2.E6 fibroblasts (Bond et al.,
1999). The presence of cell death makes the absolute determination
of lifespan extension in the 5229.E6 colonies problematical. However, the
Mint-like state was reached with a final cell count equivalent to
15-25 PDs post-infection (10-20 PDs beyond M1;
Fig. 3B). A pooled sample of 30
colonies (5229.E6p30) arrested in a Mint-like state after 21.5 PDs
post-infection (Fig. 3B), but a
crisis-like phase was not obvious, possibly owing to the mixed population of
clones of differing lifespans.
|
|
The young 5229.E6 cells (<13 PDs post-infection) were small (Fig. 4A), showed a high BrdU LI (25.2±1.8%), a low SAß-gal index (3.5±0.8%) (Fig. 4C) and a TUNEL index of <1%, indicating that there was no significant cell death occurring (Fig. 4D).
The WS cells at Mint were very large and highly irregular in
morphology (Fig. 4E), with a
low BrdU LI (1.8±0.6%) and a high SAß-gal index (92.6±1.2%)
(Fig. 4G). FACS analysis showed
a significant proportion (8%) of Mint cells with
>4N DNA content, indicating the presence of polyploid cells (not
shown). An inspection of 5229.E6 cells fixed on coverslips revealed that
9±1.3% of the Mint cells had more than two nuclei
(Fig. 4F). Lower-power
inspection of the culture revealed a low, but significant, level of
phase-bright cells that probably represent mitoses (not shown), and there was
a build up of cell debris in the culture medium, suggesting the occurrence of
cell death. TUNEL analysis showed 5±1.0% positive nuclei
(Fig. 4H), suggesting that some
of this cell death was due to apoptosis. However, overall cell number was
stable (with occasional refeeding) in this state for several weeks.
Expression of cell-cycle proteins in normal and WS fibroblasts
Cycling AG05229 cells showed moderate levels of p21Waf1
in 22.5%±1.8% of the nuclei by immunocytochemistry, which increased to
91.2%±1.3% of the nuclei by the time the cells reached M1. Expression
of HPV16 E6 caused a dramatic decrease in both the intensity and proportion of
nuclei positive for p21Waf1, with only 2.5±0.7% of
nuclei showing detectable immunostaining at 20 days
(Fig. 4B). The cells at this
time had undergone 13 PDs since infection and were growing rapidly. This
was followed by a slow increase in the number of immunopositive nuclei,
reaching 15±1.6% by Mint
(Fig. 4F).
Immunoblot analysis using anti-p21Waf1 antibodies confirmed the immunocytochemistry data (Fig. 2). (The reasons for the apparently similar levels of p21Waf1 protein in cycling and M1 AG05229 cells are outlined above.) In control HCA2 fibroblasts, the level of p21Waf1 was low in cycling cells and high in senescent cells (Fig. 2). When E6 was introduced into the cells, the level of p21Waf1 was reduced to almost undetectable levels, although they rose slightly as the cells entered the Mint, for both 5229.E6 and HCA2.E6 cells (Fig. 2).
p53 was readily detectable in AG05229 and HCA2 cells in both cycling cells and at M1 (Fig. 2). In the E6-infected cells, however, p53 protein was barely detectable, confirming E6-mediated degradation of p53 (Fig. 2). These data, taken together, indicate that p21Waf1 is upregulated at M1 and that abrogation of p53 using E6 results in a low level of expression of p21Waf1 for both AG05229 and HCA2 cells.
The level of the CdkI p16Ink4a is reported to increase
in HCA2 cells when they reach M1 (Bond et
al., 1999). This increases further in E6-infected HCA2 cells as
they reach Mint (Bond et al.,
1999
) (Fig. 2),
suggesting that increased p16Ink4a production might
compensate for the lack of p21Waf1 production in
cell-cycle arrest. An increase in p16Ink4a was seen in WS
cells as they reached M1, but no further increase was found as 5229.E6 cells
reached Mint (Fig.
2). Densitometric analysis indicated that the low level of
p16Ink4a seen at Mint was not due to different
protein loading. In fact, the level of p16Ink4a might
actually have decreased in these cells after E6 production, although the level
increased again as the cells reached Mint without reaching the
level seen at M1.
The CdkI p27Kip1 has been reported to be elevated in
senescent human fibroblasts (Bringold and
Serrano, 2000). Thus we probed the western blot with
anti-p27Kip1 antibodies and found no obvious changes in
the levels of p27Kip1 in any of the WS or HCA2 samples in
this study (Fig. 2).
The presence of cycling cells in some of these populations was indicated by
probing the western blots with an anti-pRb antibody that detects both hyper-
and hypophosphorylated forms of pRb; the hypophosphorylated form of pRb is
growth inhibitory (Stein et al.,
1990). In all the cycling populations, a doublet was detected
(indicated by the arrows), which was not seen in any of the four noncycling
populations (Fig. 2). As noted
above, the upper hyperphosphorylated band was present at a reduced level in
cycling WS cells.
Senescent WS fibroblasts undergo DNA synthesis after injection of a
p53-neutralizing antibody
The lifespan extension of WS cells following production of HPV16 E6 is
consistent with a p53-dependent cell-cycle arrest. However, because of the
many non-p53 targets of E6, we wished to provide an independent method of
abrogating p53 function in WS AG05229 cells. The anti-p53 antibody DO-1
recognizes an epitope within the N terminus of p53 (amino acids 20-25)
(Stephen et al., 1995;
Böttger et al., 1996
) that
includes key residues required for transactivation (amino acids 22 and 23)
(Lin et al., 1995
). Previous
work has shown that microinjection of DO-1 antibodies into senescent HCA2
cells effectively abrogates p53 activity and allows the cells to re-enter the
cell cycle (Gire and Wynford-Thomas,
1998
).
Thus, DO-1 antibodies were introduced by microinjection into senescent AG05229 cells (BrdU LI of 1.8±0.6%). 48 hours after injection, 13/68 (19.1±4%) of the DO-1-injected cells were synthesising DNA as assessed by a 4-hour BrdU pulse, compared with 0/30 (0%) of cells injected with the control antibodies (Fig. 5). Thus, antibody abrogation of p53 function results in these cells re-entering the cell cycle and provides confirmation that M1 arrest in WS cells is a p53-dependent process.
|
Spontaneous escape from Mint in WS fibroblasts
Pooled 5229.E6p30 cells at Mint were maintained with regular
refeeding for several weeks without net gain or loss in cell number
(Fig. 6A). They had a low BrdU
LI (1.8±0.6%) and a high SAß-gal index (>90%) throughout this
period of stationary growth (not shown). Visual inspection revealed a low
level of mitotic cells (Fig.
6B), that most cells had a large, irregular morphology and that
there was a gradual build up of cellular debris.
|
Interestingly, however, 150 days after the onset of the
Mint-like state (257 days post-infection), the culture began to
expand rapidly (Fig. 6A). Most
cells at this time were small with similar morphology to uninfected growing
cells, although there was a significant proportion of Mint-like
cells and many mitoses were present (Fig.
6C). The cells had a high BrdU LI (39.6±2.2%) and a low
SAß-gal index (2.2±0.65%) at a PD level of 26.2 (i.e.
5 PDs
beyond the Mint-like state) (not shown). After more than 280 days
post-infection, the population began to decrease in total cell number,
coincident with a marked increase in the levels of cell death. This behaviour
resembled a crisis state, with the cells having an apoptotic index of
5±1% as assayed by TUNEL analysis (not shown). The cells at this stage
were very irregular in morphology and apoptotic-like cells could be seen
clearly (Fig. 6D). After 410
days post-infection, all the cells of the 5229.E6p30 culture had died.
Mint in WS fibroblasts is a telomere-dependent,
p53-independent event
WS fibroblasts can bypass the M1 senescent state and be immortalized by the
ectopic production of the human telomerase catalytic subunit hTERT
(Ouellette et al., 2000;
Wyllie et al., 2000
),
indicating that M1 is telomere driven. To investigate whether Mint
is also a telomere-driven event, we produced telomerase in post-M1 5229.E6
cells. Late-passage 5229.E6 cells were infected with amphotropic viruses
expressing either the puromycin resistance gene alone or the puromycin
resistance and hTERT genes. The cells used were 5229.E6 clone 6 at a PD level
of 20.5 (post-infection), which are beyond M1 but still distant from
Mint, which occurs at
25 PDs in this clone.
Analysis of puromycin-resistant colonies (designated 5229.E6.puro and
5229.E6.hTERT) was begun 4-5 weeks after infection. The 5229.E6.puro clones
reached a stationary phase resembling Mint after 49 days (BrdU LI
of 3.8±0.85% and SAß-gal index of 91±1.3%) at 9 PDs
post-infection, and no expansion in cell numbers was seen over a further 56
days (Fig. 7A,B). Conversely,
both of the two 5229.E6.hTERT clones continued expanding and have now reached
a total of 33 and 44 PDs post-infection and are still growing
(Fig. 7A,B). These cells were
small (Fig. 7C) and had growth
characteristics similar to young AG05229 cells (BrdU LI of 23.4±1.9%,
p21Waf1 labelling of 1.6±0.6%, SAß-gal index
of 3.4±0.8%, TUNEL level of <1%; data not shown). Thus, it appears
that ectopic expression of hTERT is sufficient for post-M1 E6-infected AG05229
fibroblasts to avoid Mint, suggesting that Mint is a
p53-independent proliferative lifespan barrier that requires telomere
erosion.
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Discussion |
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Expression of HPV16 E6 causes WS cells to bypass M1 and to continue growth
until reaching a second proliferative lifespan barrier, Mint, after
a similar number of PDs to HCA2.E6 cells
(Bond et al., 1999). Most WS.E6
cells at Mint resemble normal Mint fibroblasts in that
they are very large and have a low level of BrdU incorporation and high
SAß-gal activity. In E6-infected WS cells, the level of
p21Waf1 is much reduced and only rises slightly as the
cells reach Mint. Similar changes in p21Waf1
are also seen in HCA2.E6 and IMR90.E6 cells
(Bond et al., 1999
;
Dulic et al., 2000
). This is
correlated with the reduced levels of p53 found in these cells. Together with
data indicating that loss of p21Waf1 production is
sufficient for normal fibroblasts to bypass senescence
(Brown et al., 1997
), it seems
probable that this reduction in p21Waf1 levels is causal
in permitting WS cells to bypass M1.
In contrast to HCA2 cells, however, the level of
p16Ink4a is reduced in E6-infected WS cells and, although
it rises slightly at Mint, it is still at a level below that found
at M1. This is similar to the changes in p16Ink4a found in
IMR90 cells (Dulic et al.,
2000). These differences in Cdk inhibitors at Mint
probably reflect differences between fibroblast strains isolated from
different donors and from different tissues. Similarly, although the
Mint state in WS cells differs from that reported in HCA2 cells
(Bond et al., 1999
) by the
presence of continued cell turnover (with mitotic events being balanced by
cell death), it is similar to that reported in other normal cell strains
(e.g., IMR90 cells) (Dulic et al.,
2000
).
We have observed one example of spontaneous escape from Mint, in
which, 150 days after entry into Mint, a WS.E6 culture began
to expand rapidly. After an extended period, this culture underwent a crisis
during which the cells died, a situation comparable to that previously
reported in normal LF1 cells (Brown et
al., 1997
). This situation has not been reported for HCA2 cells
(Bond et al., 1999
); however,
Filatov et al. (Filatov et al.,
1998
) found that E6-infected F5 neonatal foreskin fibroblasts did
not proceed to a Mint-like state and had few SAß-gal-producing
cells. Instead these cells entered a crisis during which telomerase expression
was induced and, thereafter, population expansion was continuous. Ectopic
production of hTERT in WS.E6 fibroblasts appears to be equally permissive for
continuous expansion.
It remains to speculate about the mechanism of the cell-cycle arrest
observed at Mint in human fibroblasts. In HCA2 cells, the arrest
might be due to the large increase in p16Ink4a, because it
has been shown that p16Ink4a induction in young and
immortalized human fibroblasts can restore growth arrest
(McConnell et al., 1998;
Vogt et al., 1998
). This is
unlikely to be the situation in WS cells, because the level of
p16Ink4a does not increase. Alternatively, the arrest
might be due to the small increase in p21Waf1 levels, as
has been suggested for IMR90 cells (Dulic
et al., 2000
). A final possibility is that there is a mechanism
that links the erosion of telomeres to cell-cycle arrest that is independent
of p53, but whose nature remains obscure
(Bond et al., 1999
).
Cellular senescence in human fibroblasts appears to be triggered by
telomere erosion (Allsop and Harley, 1995;
Bodnar et al., 1998;
Vaziri and Benchimol, 1998
;
Wright and Shay, 2002
). WS
fibroblasts exit the cell cycle at a higher rate than normal fibroblasts and
so senesce more rapidly (Faragher et al.,
1993
), and it appears that their telomeres erode at a greater rate
(Schulz et al., 1996
),
although this is equivocal. Young WS fibroblasts have telomeres of similar
length to young normal cells but they senesce with longer mean telomere
lengths (Schulz et al., 1996
).
This might indicate that WS cells are more sensitive to variations in telomere
length. However, the telomere length measured by Schulz et al.
(Schulz et al., 1996
) is a
mean length and the presence of a subset of much smaller telomeres in the
senescent WS cells is not excluded. More work to resolve this issue is clearly
indicated in the future. The longer mean telomere length at senescence might
explain the observation that WS.E6 cells make a similar number of PDs as
HCA2.E6 cells before the onset of Mint, despite the increased rate
of erosion. The presence of telomere-driven senescence in WS cells
(Ouellette et al., 2000
;
Wyllie et al., 2000
;
Choi et al., 2001
) and the
similarities in the signalling response (this study), would argue for a defect
that manifests itself early in the signalling pathway, perhaps by modulating
the rate of telomere erosion in WS cells. Thus, a role for WRNp in telomere
dynamics is strongly implicated.
The conclusions to be drawn from this work are that WS fibroblasts behave
in a way that is similar to normal fibroblasts and that, despite having a much
reduced in vitro lifespan, the transducing pathways leading to senescence in
WS cells are essentially the same. Thus, the study of premature ageing in WS
patients (presumably resulting from this premature replicative senescence) is
likely also to reflect the effects of cellular ageing in normal individuals,
thus making WS a suitable model for some aspects of the ageing process. This
model, in which the loss of WRNp expression is linked to whole body ageing via
accelerated replicative senescence, does not exclude other aspects of the WS
cellular phenotype contributing to the clinical spectrum of WS; one could
readily postulate a link between chromosome instability and the observed
increase in mesenchymal cancers in WS
(Salk, 1982;
Goto et al., 1996
). WS is a
complex disease and loss of WRNp expression might well contribute to various
aspects in different ways.
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Acknowledgments |
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Allsopp, R. C. and Harley, C. B. (1995). Evidence for a critical telomere length in senescent human fibroblasts. Exp. Cell Res. 219,130 -136.[CrossRef][Medline]
Bodnar, A. G., Ouellette, M., Frolkis, M., Holt, S. E., Chiu, C.
P., Morin, G. B., Harley, C. B., Shay, J. W., Lichtsteiner, S. and Wright, W.
E. (1998). Extension of life-span by introduction of
telomerase into normal human cells. Science
279,349
-352.
Bohr, V. A., Souza Pinto, N., Nyaga, S. G., Dianov, G., Kraemer, K., Seidman, M. M. and Brosh, R. M., Jr (2001). DNA repair and mutagenesis in Werner syndrome. Environ. Mol. Mutagen. 38,227 -234.[CrossRef][Medline]
Bohr, V. A., Brosh, R. M., Jr, von Kobbe, C., Opresko, P. and Karmakar, P. (2002). Pathways defective in the human premature aging disease Werner syndrome. Biogerontology 3,89 -94.[CrossRef][Medline]
Bond, J. A., Wyllie, F. S. and Wynford-Thomas. D. (1994). Escape from senescence in human diploid fibroblasts induced directly by mutant p53. Oncogene 9,1885 -1889.[Medline]
Bond, J. A., Blaydes, J. P., Rowson, J., Haughton, M. F., Smith, J. R., Wynford-Thomas, D. and Wyllie, F. S. (1995). Mutant p53 rescues human diploid cells from senescence without inhibiting the induction of SDI1/WAF1. Cancer Res. 55,2404 -2409.[Abstract]
Bond, J., Haughton, M., Blaydes, J., Gire, V., Wynford-Thomas, D. and Wyllie, F. (1996). Evidence that transcriptional activation by p53 plays a direct role in the induction of cellular senescence. Oncogene 13,2097 -2104.[Medline]
Bond, J. A., Haughton, M. F., Rowson, J. M., Gire, V.,
Wynford-Thomas, D. and Wyllie, F. S. (1999). Control of
replicative life span in human cells: barriers to clonal expansion
intermediate between M1 senescence and M2 crisis. Mol. Cell.
Biol. 19,3103
-3114.
Böttger, V., Böttger, A., Howard, S. F., Picksley, S. M., Chène, P., Garcia-Echeverria, C., Hochkeppel, H.-K. and Lane, D. P. (1996). Identification of novel mdm2 binding peptides by phage display. Oncogene 13,2141 -2147.[Medline]
Bringold, F. and Serrano, M. (2000). Tumor suppressors and oncogenes in cellular senescence. Exp. Gerontol. 35,317 -329.[CrossRef][Medline]
Brown, W. T., Kieras, F. J., Houck, G. E., Jr, Dutkowski, R. and Jenkins, E. C. (1985). A comparison of adult and childhood progerias: Werner syndrome and Hutchinson-Guildford progeria syndrome. Adv. Exp. Med. Biol. 190,229 -244.[Medline]
Brown, J. P., Wenyi, W. and Sedivy, J. M.
(1997). Bypass of senescence after disruption of
p21CIP1/Waf1 gene in normal diploid human fibroblasts.
Science 277,831
-834.
Choi, D., Whittier, P. S., Oshima, J. and Funk, W. D.
(2001). Telomerase expression prevents replicative senescence but
does not fully reset mRNA expression patterns in Werner syndrome cell strains.
FASEB. J. 15,1014
-1020.
Dimri, G. P., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E. E., Linskens, M., Rubelj, I., Pereira-Smith, O. et al. (1995). A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl. Acad. Sci. USA 92,9363 -9367.[Abstract]
Dulic, V., Beney, G.-E., Frebourg, G., Drullinger, L. F. and
Stein, G. H. (2000). Uncoupling between phenotypic senescence
and cell cycle arrest in ageing p21-deficient fibroblasts. Mol.
Cell. Biol. 20,6741
-6754.
Epstein, C. J., Martin, G. M., Schultz, A. L. and Motulsky, A. G. (1996). Werner syndrome: a review of its symptomatology, natural history, pathologic features, genetics and relationship to the natural aging process. Medicine 45,177 -221.
Faragher, R. G. A., Kill, I. R., Hunter, J. A., Pope, F. M., Tannock, C. and Shall, S. (1993). The gene responsible for Werner syndrome may be a cell division `counting' gene. Proc. Natl. Acad. Sci. USA 90,12030 -12034.[Abstract]
Filatov, L., Golubovskaya, V., Hurt, J. C., Byrd, L. L., Phillips, J. M. and Kaufmann, W. K. (1998). Chromosomal instability is correlated with telomere erosion and inactivation of G2 checkpoint function in human fibroblasts expressing human papillomavirus type 16 E6 oncoprotein. Oncogene 16,1825 -1838.[CrossRef][Medline]
Fukuchi, K., Martin, G. M. and Monnat, R. J., Jr (1989). Mutator phenotype of Werner syndrome is characterised by extensive deletions. Proc. Natl. Acad. Sci. USA 86,5893 -5897.[Abstract]
Gire, V. and Wynford-Thomas, D. (1998).
Re-initiation of DNA synthesis and cell division in senescent human
fibroblasts by microinjection of anti-p53 antibodies. Mol. Cell.
Biol. 18,1611
-1621.
Goto, M., Miller, R. W., Ishikawa, Y. and Sugano, H. (1996). Excess of rare cancers in Werner syndrome (adult progeria). Cancer Epidemiol. Biomarkers Prev. 5, 239-246.[Abstract]
Gray, M. D., Wang, L., Youssoufian, H., Martin, G. M. and Oshima, J. (1998). Werner helicase is localized to transcriptionally active nucleoli of cycling cells. Exp. Cell. Res. 242,487 -494.[CrossRef][Medline]
Halbert, C. L., Demers, G. W. and Galloway, D. A. (1991). The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells. J. Virol. 65,473 -478.[Medline]
Hoehn, H., Bryant, E. M., Au, K., Norwood, T. H., Boman, H. and Martin, G. M. (1975). Cytogenetics of Werner's syndrome cultured skin fibroblasts. Cytogenet. Cell Genet. 15,282 -298.[Medline]
Huang, S., Li, B., Gray, M. D., Oshima, J., Mian, I. S. and
Campisi, J. (1998). The premature ageing syndrome protein,
WRN, is a 3'5' exonuclease. Nat.
Genet. 20,114
-116.[CrossRef][Medline]
Itahana, K., Dimri, G. and Campisi, J. (2001).
Regulation of cellular senescence by p53. Eur. J.
Biochem. 268,2784
-2791.
Lin, J., Teresky, A. K. and Levine, A. J. (1995). Two critical hydrophobic amino acids in the N-terminal domain of the p53 protein are required for the gain of function phenotypes of human p53 mutants. Oncogene 10,2387 -2390.[Medline]
Marciniak, R. A., Lombard, D. B., Johnson, F. B. and Guarente,
L. (1998). Nucleolar localization of the Werner syndrome
protein in human cells. Proc. Natl. Acad. Sci. USA
95,6887
-6892.
Martin, G. M., Sprague, C. C. and Epstein, C. J. (1970). Replicative life span of cultivated human cells. Lab. Invest. 23,86 -92.[Medline]
Martin, G. M., Oshima, J., Gray, M. D. and Poot, M. (1999). What geriatricians should know about the Werner syndrome. J. Am. Geriatr. Soc. 47,1136 -1144.[Medline]
McConnell, B. B., Starborg, M., Brookes, S. and Peters, G. (1998). Inhibitors of cyclin-dependent kinases induce features of replicative senescence in early passage human diploid fibroblasts. Curr. Biol. 12,351 -354.
Morgenstern, J. P. and Land, H. (1990). Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18,3587 -3596.[Abstract]
Nakamura, T. M., Morin, G. B., Chapman, K. B., Weinrich, S. L.,
Andrews, W. H., Lingner, J., Harley, C. B. and Cech, T. R.
(1997). Telomerase catalytic subunit homologs from fission yeast
and human. Science 277,955
-959.
Ouellette, M. M., McDaniel, L. D., Wright, W. E., Shay, J. W.
and Schultz, R. A. (2000). The establishment of
telomerase-immortalised cell lines representing human chromosome instability
syndromes. Hum. Mol. Genet.
9, 403-411.
Salk, D. (1982). Werner's syndrome: a review of recent research with an analysis of connective tissue metabolism, growth control of cultured cells, and chromosomal aberrations. Hum. Genet. 62,1 -5.[Medline]
Salk, D., Au, K., Hoehn, H. and Martin, G. M. (1981). Cytogenetics of Werner's syndrome cultured skin fibroblasts: variegated translocation mosaicism. Cytogenet. Cell Genet. 30,92 -107.[Medline]
Schulz, V. P., Zakian, V. A., Ogburn, C. E., McKay, J., Jarzebowicz, A. A., Edland, S. D. and Martin, G. M. (1996). Accelerated loss of telomere repeats may not explain accelerated replicative decline in Werner syndrome cells. Hum. Genet. 97,750 -754.[CrossRef][Medline]
Shen, J.-C. and Loeb, L. A. (2001). Unwinding the molecular basis of the Werner syndrome. Mech. Ageing Dev. 122,921 -944.[CrossRef][Medline]
Sherwood, S. W., Rush, D., Ellsworth, J. L. and Schimke, R. T. (1988). Defining cellular senescence in IMR-90 cells: a flow cytometric analysis. Proc. Natl. Acad. Sci. USA 85,9086 -9090.[Abstract]
Shiratori, M., Sakamoto, S., Suzuki, N., Tokutake, Y., Kawabe,
Y., Enomoto, T., Sugimoto, M., Goto, M., Matsumoto, T. and Furuichi, Y.
(1999). Detection by epitope-defined monoclonal antibodies of
Werner DNA helicases in the nucleoplasm and their upregulation by cell
transformation and immortalization. J. Cell Biol.
144, 1-9.
Smith, P. J., Wiltshire, M., Chin, S. F., Rabbitts, P. and Souès, S. (1999). Cell cycle checkpoint evasion and protracted cell cycle arrest in X-irradiated small cell lung carcinoma cells. Int. J. Radiat. Biol. 75,1137 -1147.[CrossRef][Medline]
Stein, G. H., Beeson, M. and Gordon, L. (1990). Failure to phosphorylate the retinoblastoma gene product in senescent human fibroblasts. Science 249,666 -669.[Medline]
Stephen, C. W., Helminen, P. and Lane, D. P. (1995). Characterisation of epitopes on human p53 using phage-displayed peptide libraries: insights into antibody-peptide interactions. J. Mol. Biol. 248, 58-78.[CrossRef][Medline]
Vaziri, H. and Benchimol, S. (1998). Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended reproductive life span. Curr. Biol. 8,279 -282.[Medline]
Vogt, M. C., Habbblom, J., Yeargin, T., Christiansen-Weber, T. and Haas, M. (1998). Independent induction of senescence by p16Ink4a and p21Cip1 in spontaneously immortalised human fibroblasts. Cell Growth Differ. 9, 139-146.[Abstract]
Webley, K., Bond, J. A., Jones, C. J., Blaydes, J. P., Craig,
A., Hupp, T. and Wynford-Thomas, D. (2000). Posttranslational
modifications of p53 in replicative senescence overlapping but distinct from
those induced by DNA damage. Mol. Cell. Biol.
20,2803
-2808.
Wright, W. E. and Shay, J. W. (2002). Historical claims and current interpretations of replicative aging. Nat. Biotechnol. 20,682 -688.[CrossRef][Medline]
Wyllie, F., Lemoine, N., Barton, C., Dawson, T., Bond, J. and Wynford-Thomas. D. (1993). Direct growth stimulation of normal human epithelial cells by mutant p53. Mol. Carcinogen. 7,83 -88.[Medline]
Wyllie, F. S., Jones, C. J., Skinner, J. W., Haughton, M. F., Wallis, C., Wynford-Thomas, D., Faragher, R. G. A. and Kipling, D. (2000). Telomerase prevents the accelerated ageing of Werner syndrome fibroblasts. Nat. Genet. 24, 16-17.[CrossRef][Medline]
Yu, C. E., Oshima, J., Fu, Y. H., Wijsman, E. M., Hisama, F., Alisch, R., Matthews, S., Nakura, J., Miki, T., Ouais, S. et al. (1996). Positional cloning of the Werner's syndrome gene. Science 272,258 -262.[Abstract]