Mechanisms of Ageing and Development
103 (1998) 27 – 44

Effect of age and apoptosis on the mouse
homologue of the huWRN gene
Jianguo Wu, Jin He, John D. Mountz *
Uni6ersity of Alabama at Birmingham, Birmingham Veterans Administration Medical Center,
701 South 19th Street, LHRB 473, Birmingham, AL 35294 -0007, USA
Received 25 September 1997; received in revised form 4 January 1998; accepted 8 January 1998

Abstract
Werner’s syndrome (WS) is an inherited disease with clinical symptoms which resemble
premature aging. The Werner’s syndrome gene (WRN), which is located on human chromosome 8p12, encodes a predicted protein of 1432 amino acids and shows significant similarity
to DNA helicases. We have cloned the full-length mouse cDNA homologue of the human WRN
gene encoding a predicted protein of 1320 amino acids and have obtained a full-length 70 kb
genomic clone containing the moWRN gene. This gene has been mapped to chromosome 8A3
in mice. The expression of the moWRN gene was increased during apoptosis after IL-2
deprivation, and decreased in the spleen of aged mice. Lymphoid cells isolated from a patient
with WS exhibited increased apoptosis after incubation with anti-Fas but not after incubation
with the topoisomerase inhibitor VP16. RNase protection reviled dysregulation of the ICE
family of apoptosis molecules in the WS cell line. These results indicate that the WS helicase
is involved in certain pathways of apoptosis, and defective WS gene expression leads to
accumulation of cells that are highly susceptibility to Fas-induced apoptosis. © 1998 Elsevier
Science Ireland Ltd. All rights reserved.
Keywords: WS, Werner’s syndrome; WRN, Werner’s syndrome gene; moWRN, mouse Werner’s
syndrome gene; 7-AAD, 7-aminoactinomycin D; TUNEL, Terminal deoxynucleotide transferase nick end labeling; FACS, Fluorescence activated cell sorter; PCR, Polymerase chain
reaction; RPA, Multi-Probe RNase Protection Assay; LN, lymph node; FISH, Fluorescent
in-situ hybridization; DAPI, 4%,6-diamidino-z phenylindole; ICE, IL-1i converting enzyme

* Corresponding author.
john.mountz@ccc.uab.edu

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0047-6374/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved.
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J. Wu et al. / Mechanisms of Ageing and De6elopment 103 (1998) 27–44

1. Introduction
Apoptosis has been shown to activate several DNA repair enzymes (Cumming et
al., 1996; Huang et al., 1996; McConnell and Dynan, 1996; Wang et al., 1996;
Warbrick, 1996), as well as activate the ice-like enzymes which cleaves the enzyme
PARP involved in DNA repair (Lazebnik et al., 1994a; Casciola-Rosen et al., 1996;
Schlegel et al., 1996). We have recently reported that immunosenescence is associated with decreased apoptosis of T cells that accumulate DNA damage (Zhou et al.,
1995). Lymphoblastoid cell lines from patients with Werner’s syndrome (WS)
exhibit impaired S phase transition (Poot et al., 1992) and altered T cell subsets
(Gupta, 1981). Despite these analyses, the relationship of WS gene induction to
DNA damage, apoptosis and immunosenescence is not clear.
The WRN gene has been mapped to human chromosome 8p12 (Goto et al., 1992;
Thomas et al., 1993; Oshima et al., 1994; Goddard et al., 1996) and has been
recently cloned and identified as being highly homologous to the RecQ and other
genes with helicase domains (Yu et al., 1996, 1997; Yamabe et al., 1997). The
predicted protein size is 1432 amino acids encoded by a 5.2 kb cDNA sequence.
The human WRN gene is highly expressed in the pancreas and in lower amounts in
the placenta, muscle and heart. Four mutations were initially detected in patients
with WS; two mutations being nonsense mutations creating premature stop codons.
A third mutation resulted in a four base pair deletion spanning a splice junction
and the fourth mutation was a 95 base pair deletion from a single exon. All of these
mutations affected the proper coding of the predicted WRN protein in the helicase
homology region VI, or regions down-stream.
Extensive previous analysis of patients with WS or lymphoblastoid (Brown et al.,
1985) and fibroblast (Stecker and Gardner, 1970; Fleischmajer and Nedwich, 1973)
cell lines derived from patients with WS has been carried out. Accelerated senescence has been described by Epstein et al. (1966) or as a ‘segmental’ progeroid
syndrome (Martin, 1979). Patients exhibited certain features of senescence, but also
revealed certain discordances between the phenotype typically observed in aged
humans, including disproportionate severity of osteoporosis of the long bones (as
opposed to vertebral bodies) and apparent resistance to Alzheimer-type pathologies. Cell lines, primarily fibroblast cell lines, reveal substantially decreased replicative life spans compared to age-matched controls (Rubin et al., 1992; Thweatt and
Goldstein, 1993; Lecka-Czernik et al., 1996). Cell lines derived from patients with
WS also exhibit genomic instability (Martin, 1981; Murano et al., 1991; Thweatt et
al., 1992; Oshima et al., 1995; Webb et al., 1996) suggesting that an unusually high
accumulation of chromosomal damage might underlie their accelerated senescence.
Apoptosis genes including c-fos, and phosphorylated RB have been found to be
defective in senescent fibroblasts (Seshadri and Campisi, 1990; Seshadri et al., 1993;
Smith and Pereira-Smith, 1996; Smith et al., 1996). The present paper describes the
cloning of the mouse homologue of the WRN gene and its expression in young and
aged mice and during T cell apoptosis induced by IL-2 depravation and correlation
to apoptosis and apoptosis signaling molecules in WS and control patients.

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2. Materials and methods

2.1. Mice
Young (2 month old) and aged (24 month old) C57BL/6 mice were obtained
from the NIA aged mouse depository. MRL-+ /+mice were obtained from
Jackson Labs (Bar Harbor, ME). These ages of mice were selected since we have
previously showed that T cells from mice of these ages exhibit reproducible
differences in apoptosis (Zhou et al., 1995).

2.2. Cell lines
IL-2-dependent murine cell lines CTLL and HT2 cell lines were obtained from
American Type Culture Collection (ATCC) (Rockville, MD). The cells were
cultured in the presence and absence of IL-2 for different periods of time as
described previously (Seldin et al., 1987). IL-2 was used at a concentration of 50
units/ml (Endogen).

2.3. Analysis of apoptosis of human WS cell lines
Lymphoblastoid cell lines were obtained from the National Institute of Aging
Cell Repository (Coriell Cell Repositories, Camden, NJ). A WS patient cell line
(NIA REP number AG 896A) and age-matched natural sibling (NIA REP number
AG 898) were obtained and characteristics of these lines have been previously
described (Brown et al., 1985). Cells (106 each) derived from a WS patient and an
age-matched control were incubated with anti-Fas or VP-16 and were cultured in
96-well round-bottom plates. After 20 h, cells were collected by centrifugation for
10 min at 1000 rpm at 4°C. The cells were washed twice with FACS buffer (5%
FCS, 0.1% NaN3 in PBS) and incubated on ice for 30 min with 50 ml of 7-AAD
that was diluted to 10 mg/ml in FACS buffer. The stained cells were washed once
with FACS buffer, and then resuspended in 1% paraformaldehyde –PBS supplemented with 50 mg/ml of actinomycin D. 10000 cells were analyzed on a FACS
scanner.

2.4. Selection of homologous probes for PCR localization
The huWRN 5% and 3% probe was selected from the conserved region Ia and
region VI, respectively. These regions are highly homologous between the huWRN
amino acid sequence and the human RecQ amino acid sequence. There was also
100% homology to the moWRN for PCR primers selected for amplification. The
initial probe was obtained by RT-PCR amplification of RNA isolated from the
spleen of a 2 month old C57BL/6 mouse. The 5% primer was 5%-GGAAAGAGTTTGTGCTTCCAG (nucleotides from 1178 to 1198), 3% primer was 5%-AAGTCCATCACGACCAGCTCTACC (nucleotides from 2854 to 2830). PCR was carried out
using the hot start method and then denatured for 15 s at 95°C, annealing 30 s at

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J. Wu et al. / Mechanisms of Ageing and De6elopment 103 (1998) 27–44

68°C, and extension 45 s at 70°C, for 30 cycles. The PCR product was cloned
into a TA2 vector (Invitrogen). Twenty clones were obtained in sequence. Four
identical clones were 95.2% homologous to the conserved region of the huWRN
gene.

2.5. Isolation of full-length cDNA clone for moWRN gene
The putative 850 bp partial moWRN gene was used to screen a C57BL/6
mouse spleen gt11 cDNA phage library (Clontech, Palo Alto, CA). Six positive
clones were obtained from approximately one million plaques. Two identical
clones were subcloned into pGEM 7 plasmid vector and sequenced with an
ABI model automated sequence analyzer.

2.6. Isolation of a mouse genomic clone for fish analysis
The full-length moWRN cDNA was used to screen a EMBL3 SP6/T7 mouse
genomic library (Clontech, Palo Alto, CA). Five overlapping clones, designated
FISH1 to FISH5, were obtained from
3× 106 plaques. The FISH2 bacteriophage clone was selected as a probe for FISH analysis after two sub-screenings
using the 32P-labeled moWRN cDNA fragment corresponding to nt 1803 to
2472. Several exons of genomic clone designated as FISH2 were sequenced and
confirmed to be 100% homologous to the corresponding region of the mouse
cDNA sequence.

2.7. FISH analysis of chromosomes
Mouse chromosomes were prepared according to the published procedure
(Heng et al., 1992; Heng and Tsui, 1993; Fang et al., 1994). Briefly,
lymphocytes were isolated from mouse spleen and stimulated with concanavalin
A and lipopolysaccharide. Chromosome slides were made by conventional methods. The genomic probe was biotinylated with dATP using the BRL BioNick
labeling kit (15°C, 1 h). The procedure for FISH detection was performed according to Heng et al. (1992), Heng and Tsui (1993). Briefly, slides were baked
at 55°C for 1 h. After RNase A treatment, the slides were denatured in 70%
formamide in 2× SSC for 2 m. The slides were then treated at 70°C for 5 m
in a hybridization mix consisting of 50% formamide and 10% dextran sulphate
and mouse DNA and pre-hybridized for 15 m at 37°C. The probe was then
added to the denatured slides. After overnight hybridization, slides were washed
and amplified using published methods (Heng et al., 1992).

2.8. Northern blot analysis
Northern blot analysis of poly-A was carried out as described previously
(Mountz et al., 1985).

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2.9. Mouse WRN gene expression
Total RNA was isolated for the cells using the RNA STAT-60™ kit. 5 mg of total
RNA was subjected to reverse transcription system in a total volume of 20 ml using
the reverse transcription system kit (Promega, Madison, WI). The primers used to
amplify mouse WRN gene were 5% primer 5%-TCTCCCCTTATTTCTCTGATG
(nucleotides from 2081 to 2101) and 3% primer 5%-AATACAGTGAGCCTCATCCACAGC (nucleotides from 2780 to 2756). The primers used to amplify mouse
i-actin were 5% primer (5%-AGACAGCACTGTGTTGGCAT-3%; and 3% primer
(5%-GACCTGACAGACTACCTCAT). The amplification was performed in a 100
ml reaction volume containing 1×reaction buffer (Promega, Madison, WI), 1.5
mM of MgCl2, 200 mM of dNTP, 1 mM of each primer and 2.5 units of Taq DNA
polymerase (Promega). A total of 30 cycles was carried out using a Perkin–Elmer
Gene Amp PCR System 9600. Each cycle consisted of denaturation at 94°C for 12
m, annealing at 54°C for 1 m, and extension at 72°C for 1.5 m. To ensure that
nearly equivalent amounts of template were added initially in each PCR reaction,
concurrent PCRs for amplification of mouse i-actin expression were utilized as a
control. The PCR product was electrophoresed in 1% agarose gel. Gels were blotted
and hybridized to a labeled cDNA for i-actin (control) or Fas ligand to verify
specificity of each product.

2.10. Multi-probe RNase protection assay (RPA)
The human APO-1, APO-2 and APO-3 RPA kits were purchased from PharMingen (San Diego, CA). Total RNA was obtained as previously described (Mountz et
al., 1985). 5 mg of total RNA from WS cell line and control cell line was used as
template for RPA accounting to the instruction of manufacture (PharMingen). The
abundance of protected labeled material was quantitated on a Molecular Dynamics
image scanner. L32 is the ribosomal binding gene and GAPDH is the glyceraldehydephosphate dehydrogenase gene, both are house-keeping genes which were used
as controls for normalization of protected RNA expression.
3. Results

3.1. cDNA cloning of mouse WRN gene
The human WRN gene was aligned with all known helicases, and the two most
conserved regions were chosen for cDNA amplification of normal mouse spleen.
These conserved regions are designated as WS-hm-5% and WS-hm-3% in Fig. 1. The
initial PCR product for the moWRN gene was predicted to be 852 bp long and to
include six conserved domains among the known helicase genes. The PCR product
was cloned into the TA II vector (Invitrogen). Twenty clones were obtained and
sequenced. Four identical clones were 95.2% homologous to the conserved region
of the human WRN gene. There was 100% homologous in the ATPase and in the
DE-HC region (Fig. 1).

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J. Wu et al. / Mechanisms of Ageing and De6elopment 103 (1998) 27–44

Fig. 1. Protein alignment of mouse WRN with human WS and RECQ helicase. The predicted protein
sequence of the mouse WRN gene product. The human WRN amino acid sequence contain two perfect
17 amino acid repeat regions whereas this amino acid repeat is not present in the mouse WS amino acid
sequence (Repeat 1, Repeat 2, as underlined). Helicase domains I – VI were highly conserved, and their
location was adapted from previously described helicase domain alignment. There was 95.2% homology
between the mouse and human WRN gene products.

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The putative 850 bp partial moWRN gene was used to screen a C57BL/6 mouse
spleen gt11 cDNA phage library (Clontech, Palo Alto, CA). Six positive clones
were obtained from approximately one million plaques. Two identical clones were
subcloned into a pGEM 7 plasmid vector and sequenced with an ABI model
automated sequence analyzer. The predicted 1320 AA sequence of the moWRN
gene product exhibited an ATPase domain and 6 helicase domains which were
highly homologous with the human WRN gene and the human RECQ gene (Fig.
1). A unique feature of the human WRN gene that is not found in the mouse amino
acid sequence is the presence of a 27 amino acid repeat sequence located between
150 and 100 amino acids upstream from the ATPase domain.

3.2. Chromosomal localization of moWRN gene to mouse chromosome 8.
The human WRN loci gene is located near the centromere of chromosome 8
between the GSR and FGFR1 genes which flanks the WRN gene. This region is
homologous between mouse and human. To further confirm that the putative clone
represented the moWRN gene, chromosomal mapping was carried out. Fluorescent
in-situ hybridization (FISH) signals are illustrated on several chromosomes (Fig.
2(A); left) and the same mitotic figure was stained with DAPI to identify chromosome 8 (Fig. 2(A); right). Under the conditions used, the hybridization efficiency
was 96% for the probe (among 100 checked mitotic figures, 96 of them showed
signals on one pair of the chromosomes). Since the DAPI banding was used to
identify the specific chromosome, the assignment between signal from probe and
the mouse chromosome 8 was obtained. The moWRN gene was located at chromosome 8, region A3 (Fig. 2(B)).

Fig. 2. Mapping of moWRN gene to chromosome 8, position A3. (A) Chromosomes were prepared from
murine lymphocytes and a 10 Kb genomic probe was used for FISH detection. Slides were also stained
for DAPI staining so that chromosomes could be identified. (B) Location of hybridization probe using
100 mitotic figures. Of the 100 stained probes, 98 were located to chromosome 8, band A3.

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J. Wu et al. / Mechanisms of Ageing and De6elopment 103 (1998) 27–44

Fig. 3. Expression of the moWRN gene in young and aged mice. (A) Total RNA was isolated from
different organs including spleen (S), liver (L), pancreas (P) and heart (H), of young (2 month-old) and
aged (24 month-old) C57BL/6 mice. The expression of the moWRN gene was determined by semi-quantitative PCR analysis and hybridization to the moWRN probe relative to i-actin control (Fig. 3A). (B)
The ratio of moWRN gene expression to i-actin expression is plotted for 22, 26 and 30 PCR cycles. The
upper panels indicate tissues in which expression of the moWRN gene increases with age, and the lower
panel indicates spleen tissue, where expression of the moWRN gene decreased with age.

3.3. Expression of mouse WRN gene in young and old C57BL/6 mice
Total RNA was isolated from different organs including spleen (S), liver (L),
pancreas (P), heart (H) of young (2 month-old) and aged (24 month-old) C57BL/6
mice. The expression of the moWRN gene was determined by semi-quantitative
PCR analysis and hybridization to the moWRN probe relative to i-actin control
(Fig. 3(A)). The ratio of moWRN gene expression to i-actin expression is plotted
for 22, 26, and 30 PCR cycles (Fig. 3(B)). There was increased expression of
moWRN gene in aged heart, pancreas and liver compared to young mice (Fig.
3(B)). There was decreased expression of the moWRN gene in spleen from aged
mice compared to young mice.

3.4. Increased expression of moWRN during apoptosis induced by IL-2 depri6ation
The interleukin-2 (IL-2)-dependent CTLL and HT2 T-cell lines undergo apoptosis leading to cell death within 24–48 h after IL-2 deprivation. CTLL T cells were
analyzed with ( + ) or without ( −) of IL-2 for different times (Fig. 4(A)). The
moWRN gene was strongly induced 18 h after removal of IL-2, indicating this gene

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35

is induced late in the process of apoptosis. Induction of the moWRN gene could
also be seen 18 h after removal of IL-2 in a second cell line, HT2 (Fig. 4(A)).
Greater than 90% of the CTLL cells undergo apoptosis 18 h after IL-2 deprivation
(Fig. 4(B)).

3.5. Lymphoid cells from a WS patient exhibits increased susceptibility to Fas
apoptosis
Apoptosis of a T cell line derived from a WS patient (NIA REP number AG
896A) and of a control patient (NIA REP number AG 898) was analyzed using the
7-aminoactinomycin D (7AAD) staining technique. Apoptosis was induced by
anti-Fas antibody or the topoisomerase inhibitor VP-16. Apoptosis was analyzed
either before treatment (NS), or at 5, 10 and 20 h after treatment (Fig. 5). Cell line
from an age-matched natural sibling exhibited a lower spontaneous rate of apoptosis before treatment compared to a cell line derived from a WS patient. Both cell
lines exhibited a statistically significant increase in anti-Fas mediated apoptosis
after incubation for 10 and 20 h compared to untreated (NS) (pB 0.05). There was
an approximate 2-fold increase in apoptosis induced by anti-Fas in the WS T cell

Fig. 4. Expression of the moWRN gene during apoptosis. (A) The IL-2-dependent T cell line CTLL and
HT2 were cultured with and without IL-2 for different time points as indicated. Equivalent amounts (5
mg) of poly A RNA were blotted and probed with the moWRN cDNA. (B) Apoptosis of CTLL cells in
the absence of IL-2. CTLL cells were incubated for 18 h with (+IL-2) or without ( − IL-2), cytospun
and then analyzed for apoptosis using the TUNEL assay.

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J. Wu et al. / Mechanisms of Ageing and De6elopment 103 (1998) 27–44

Fig. 5. (A) WS cell line have a higher sensibility to anti-Fas-induced apoptosis. A human T cell line (106
each) derived from a WS patient (NIA REP number AG 896A) and of a control patient (NIA REP
number AG 898) were analyzed either before treatment (NS), or at the indicated time points after
incubation with anti-Fas or VP-16. Apoptosis was determined using the 7-AAD method. 100000 cells
were analyzed on a FACS scanner.

line compared to the control T cell line at all time points (pB 0.05). VP-16 induced
high and equivalent levels of apoptosis in both the normal T cell line and the WS
T cell line either before treatment, and at 5, 10 and 20 h after treatment. These
results indicate that the WS T cell line, which exhibits an increase in the accumulation of DNA damage and mutation, also exhibits an increased susceptibility of
Fas-mediated apoptosis. These observations suggest the possibility that increased
sensitivity to anti-Fas antibody-mediated apoptosis in premature aging cell lines
may be an important consequence or pathogenic mechanism associated with
increased DNA damage in premature aging T cells.

3.6. Decreased expression of ice apoptosis pathway genes in WS cells
The increased susceptibility to Fas apoptosis in the WS patient (NIA REP
number AG 896A) compared to the normal control cell line (NIA REP number AG
898) supports our hypothesis that Fas apoptosis is dysregulated in cell lines from
WS patients. To determine the molecular basis for this dysregulation, expression of
several family members of the Fas–TNFR, Bcl-2 and IL-1i converting enzyme
(ICE) were examined using RNase protection assay (PharMingen). The most
prominent difference between expression of these apoptosis-related genes between
WS cell lines and normal cell lines was a marked down regulation of the ICE family

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37

member of cysteine-dependent aspartate-specific proteases (caspases) (Fig. 6). There
was a significant decrease in caspase-8 (FLICE), caspase-2 (ICH-1), caspase-6
(Mch-2), caspase-3 (ICE rel III), caspase-4 (Mch-3) and, caspase-1 (ICE) (Fig.
6(A,D)). There was significantly lower expression of bax which forms a dimer with
bcl-2 and would promote apoptosis. There was also lower expression of bcl-2 which
would inhibit apoptosis (Fig. 6(B,E)). In contrast, there was significantly higher
levels of Fas and Fas associated death domain (FADD) and Fas associated factor
(FAF) in Werner’s Syndrome cell lines compared to control cell lines (Fig. 6(C,F)).
There was no significant difference in the TNF receptor family of apoptosis
molecules including TNF-R1 (P55) and the TNFR associated death domain
molecule TRADD. These results suggest that this regulation of ICE caspases in the
Werner’s Syndrome cell line may inhibit normal apoptosis despite higher level of
Fas and Fas-associated signaling molecules FAP, FADD and FAF. We propose
that increased sensitivity to anti-Fas antibody in-vitro results from increased
accumulation of senescent cells that are otherwise predisposed to undergo apoptosis, but require additional signaling through Fas for this process to occur.

4. Discussion
The human WRN gene, which is predicted to encode a 1432 amino acid protein
with significant homology to DNA helicases, is located on chromosome 8p12. The
present study shows that the mouse equivalent of human WRN is a 1320-amino
acid protein, which is encoded by a gene located on mouse chromosome 8A3. This
newly identified gene is the mouse equivalent of human WRN since it is 95.2%
homologous with the conserved region of the human gene, and maps to the region
on mouse chromosome 8 that is homologous with the human region 8p12. Like the
human WRN gene, the moWRN gene is also homologous to the human DNA
helicase RecQ. RecQ is defective in Bloom’s syndrome which gives rise to variable
immune deficiencies, skin pigmentation, distinct face appearances, erythematosus
skin manifestations and abnormal behavior (Ellis et al., 1995; Korn and
Ramkisson, 1995). RecQ, has been mapped to human chromosome 12p11-p12 on
mouse chromosome 6 (Liston, 1996) and therefore cannot be the moWRN gene
described here.
There is a 27 amino acid repeat in the human WRN amino acid sequence located
between 150 and 100 amino acids upstream from the ATPase domain (Fig. 1). A
gene bank search did not reveal homology between this repeat in and any other
known amino acid consensus sequence. Repeated sequences have been described
both relative to ATPase binding domains (Takemoto, 1995) as well as DNA
binding domain (Laine, 1995; Puranam et al., 1995). We propose that this repeated
amino acid sequence in the human WRN gene might affect the function of the
ATPase domain or the efficiency of the helicase activity in human WRN gene
relative to moWRN gene.
We found that expression of the moWRN in the spleen and brain decreases with
age. In contrast, the expression of the moWRN gene is higher especially in the

J. Wu et al. / Mechanisms of Ageing and De6elopment 103 (1998) 27–44

Fig. 6.

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39

heart, but also in the pancreas, liver, and kidney of aged mice compared to young
mice. This increase may be related to either increased activation of DNA repair genes,
including the helicases, or increased apoptosis. Apoptosis has been reported to be
decreased the in spleen of aged mice (Zhou et al., 1995) but increased in the heart
of aged rats (Kajstura et al., 1996).
The moWRN gene encodes a helicase, which is proposed to be involved in DNA
damage and cell senescence (Martin, 1981; Murano et al., 1991; Thweatt et al., 1992;
Thweatt and Goldstein, 1993; Oshima et al., 1995; Webb et al., 1996). We have shown
previously that one aspect of T-cell senescence is defective apoptosis and removal
of senescent T cells (Zhou et al., 1995). Here, we show the moWRN gene is induced
late during apoptosis by removing an IL-2 growth factor from IL-2-dependent, T-cell
lines. This is consistent with previous reports that apoptosis induces upregulation of
similar genes that are involved in DNA cell cycle proliferation and DNA repair
including the DNA helicases XP-B and XP-D (Ljungman and Zhang, 1996; Wang
et al., 1996). Therefore, we propose that defective moWRN gene expression in the
immune system during aging may play a role in the age-associated defect in apoptosis,
leading to prolonged survival of certain senescent cells including fibroblast and T cells.
An increased number of cells that are overly susceptive to Fas apoptosis in vivo
might result from a defect of normal apoptosis signaling. Down regulation of caspases
that are important for signaling apoptosis were observed in Werner Syndrome cells
compared to control cells. We propose that this down regulation of caspase decreases
the sensitivity of Werner Syndrome cells to other apoptosis signaling pathways in
vivo, since these caspases are common to many apoptosis pathways. This is consistent
with previous results that caspases are activated during DNA damage and apoptosis

Fig. 6. (Left) Expression of apoptosis molecules and apoptosis signaling pathways in Werner’s Syndrome
cell line and control cell line. RNA was prepared from Werner’s Syndrome and control cell line and
analyzed for expression of different apoptosis molecules or apoptosis signaling pathways by RNA
protection assay as described in Section 2. The labeled probe is shown in the first lane (left), the control
and Werner’s Syndrome cell lines are indicated by arrows. The protected fragment is always smaller than
the labeled fragment which contains additional irrelevant sequences. L32 is the ribosomal binding gene
and GAPDH is the glyceraldehydephosphate dehydrogenase gene, both are house-keeping genes which
were used as controls for normalization of protected RNA expression. Each RNA protection assay was
carried out twice (for 6A; the caspase family and 6B; the bcl-2 family), and four times (for 6C; the
Fas-death domain associated molecules) with similar results. Each assay was carried out using 5 mg of
total RNA as described in Section 2. The abundance of protected labeled material was quantitated on
a molecular dynamics image scanner (Palo Alto, CA). The caspases family of molecules is shown in
figures A and D. The bcl-2 family is shown in figures B and E. The Fas and TNFR family is shown in
figures C and F. The abbreviations for the different families are: (a) FLICE = FAAD-like ICE, Granz
B =Granzyme B, Mch = Melanin-concentrating hormone. Rel 3 =ICE related-3. ICH-1 (S/L)=ICE
homologue 1, short and long, Mch-3 = Melanin-concentrating hormone-3, ICE = IL-1i converting
enzyme, Lap6 = ICE-lap6, bclx (L/S)=bclx long and short, bfl1 =bcl-2 family 1, blk = bcl kinase.
bak =bcl kinase, bax = bcl-2 inhibitor, bcl-2 =B cell leukemia 2, Mcl-1 = Mantle cell lymphoma 1,
Flice =FADD-like ICE, FasL = Fas ligand, FADD =Fas associated death domain, DR3 =death
receptor-3, FAP = Fas associated protein, FAF is Fas associated factor, TRAIL = TNF related apoptosis-inducing ligand, TNFR (p55) =TNF receptor p55, TRADD =TNF receptor associated death
domain, RIP =receptor-interacting protein.

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J. Wu et al. / Mechanisms of Ageing and De6elopment 103 (1998) 27–44

utilizing certain members of the caspases family are important for apoptosis after
DNA damage (Subramanian et al., 1995; Dubrez et al., 1996; Marchetti et al.,
1996). Also, these caspases are up regulated under conditions of stress and DNA
damage including UV, and oxidative stress (Subramanian et al., 1995; Dubrez et al.,
1996; Marchetti et al., 1996). These results suggest that the defect of WRN helicase
activity in Werner Syndrome cell lines leads to an increased percentage of cells with
DNA defects. These cells do not undergo apoptosis due to a secondary down
modulation of the ICE family members. These results suggest that up regulation of
ICE family members in vivo may correct this apoptosis defect and lead to removal
of certain cells that undergo accelerated senescence in Werner’s Syndrome.
The present results suggest a model for the relationship between DNA damage
and repair and apoptosis. T cell senescence is associated with telomere shortening
and increased susceptibility to DNA damage (Bodnar et al., 1996; Mei et al., 1996).
Accelerated telomere shortening has been reported in ataxia telangiectasia (Metcalfe et al., 1996). One of the most significant consequence of these impairments are
dysregulation in DNA repair and apoptosis. The first inhibitor of DNA synthesis in
senescent cells was observed in the laboratory of Dr Jim Smith. This gene was
cloned as an inhibitor of DNA synthesis that was over-expressed in terminally
non-dividing senescent human fibroblasts and was designated as senescence-derived
inhibitor-1 (Sdi-1) (Noda et al., 1994). A number of laboratories cloned this same
gene and gave it several names including a p53 transactivated gene (WAF1), and a
cyclin interacting protein (p21, CIP1 and Cap20) (Xiong et al., 1993; El-Deiry et
al., 1994; Johnson et al., 1994). Subsequently, Sdi1 was identified as one of a group
of proteins that inhibit the activity of cyclin–Cdk complexes that control primarily
the transition of cell cycle from G1 to S phase. More interestingly, the induction of
Sdi1 is typically a p53-dependent cellular stress response such as DNA damage
induced by ionizing radiation (Xiong et al., 1993; Dulie et al., 1994; El-Deiry et al.,
´
1994; Johnson et al., 1994; Noda et al., 1994). Therefore, the elevated expression of
cell cycle inhibitors is not unique to senescent cells, but plays a primary function to
induce cell cycle arrest and provide more time for repair of the damaged DNA
before DNA replication and mitosis.
DNA damage results in the induction of DNA repair genes including those
involved in base excision repair, nucleotide excision repair, post-replication repair,
and double strand break repair (Sancar, 1993). It is now clear that DNA damage
and repair pathways and gene products are also very often the same pathways and
gene products involved in the induction of apoptosis. Endoucleases, DNA-dependent protein kinase (DNA-PK) such as Ku, poly(ADP-ribose) polymerase (PARP),
DNA helicase, p53 and proliferating cell nuclear antigen (PCNA) all have been
postulated to have interactions with cell death pathways (Troelstra et al., 1992; de
Murcia, 1994; Li et al., 1994; Finnie et al., 1995; Wang et al., 1995)
The current concept of normal cellular mechanism during stress response suggests that cell cycle arrest following DNA damage can result in DNA repair first
and the removal of the cell cycle block later when the repair is completed. There are
two possible important roles of apoptosis involved in this procedure. First, apoptosis can eliminate cells that have undergone extensive damage and can not be

J. Wu et al. / Mechanisms of Ageing and De6elopment 103 (1998) 27–44

41

repaired. For example, tumor cells with mutant p53 phenotype exhibited defective
apoptosis after ionic-irradiation (Sancar, 1993). The second role of apoptosis during
DNA damage is to suppress or terminate DNA repair. For example, several recent
investigations showed that interleukin 1i-converting enzyme (ICE) family caspases,
the down-stream apoptosis-related proteolysis enzymes, were induced after DNA
damage and these proteases are responsible for the breakdown of DNA repair proteins
such as PARP and DNA-PK (Hayflick, 1965; Lazebnik et al., 1994b; Casciola-Rosen
et al., 1995; Wang et al., 1995; McConnell et al., 1997).
In the senescent T cells, due to their reduced capacity to execute DNA repair, the
frequency and quantity of cell undergoing apoptosis can be increased. Therefore,
unlike the situation in antigen-stimulated cell death or AICD, we postulated there
to be an increased apoptosis during stress response in senescent T cells. However,
as the cells undergo apoptosis, their repairing capacity would be suppressed because
the DNA repair proteins will be cleaved by the down-stream ICE–ICE-like enzymes
(Lazebnik et al., 1994b; Casciola-Rosen et al., 1995; McConnell et al., 1997). The
enhancement of apoptosis may be able to delete some damaged cells; however, it could
also have two possible pathogenic consequences: (i) Only cells with functional
apoptosis capability would be deleted by this process, whereas certain mutant cells
which are not able to undergo apoptosis can escape from this deletion process and
further proliferate into tumor or autoimmune phenotype; and (ii) The increased
apoptosis may be a factor associated with the induction of p21 and the cell cycle block
that contribute to the loss of reproductively, the Hayflick phenomenon that occurs
in senescent cells (Hayflick, 1965).
The discovery that lpr is a fas gene mutation has shed great insights onto the
pathogenic mechanism of autoimmune disease (Zhou et al., 1995). Yet, the majority
of cells appear to have a normal function of Fas. The pathogenic outcome of the
dysregulation of this molecule therefore resides in subtle changes of this gene
throughout the life span. Although our current understandings on the role played
by this molecule in T cell senescence remain incomplete, we propose that defective
Fas apoptosis and aging, error accumulation or genetic program defects. The
alteration of Fas-mediated apoptosis fits in both schemes of the hypotheses.
Future work relating apoptosis to the WS gene and aging should focus on several
questions. (1) There is still a lack of in vivo data demonstrating the role of Fas in
T cell senescence. The in vitro data have completely ignore other endocrine factors,
such as glucocorticoid and estrogen, that can induce dramatic effect on apoptosis.
Therefore, it may not reflect the real life situation in vivo. (2) The role of down stream
signals and co-stimulation signals helping cells to determine to execute cell rescue or
cell death remain to be determined. This is especially important in the field of aging
because alterations of these signals may be critical factors contributing to senescence.
(3) Factors that determine if apoptosis is decreased with aging, allowing the
accumulation of senescent cells, or increased with aging, to remove senescent cells
need to be identified. Much of the data indicates that in vivo, there is a gradual decrease
in apoptosis with aging resulting in accumulation of senescent cells with increased
DNA damage. We believe that identification of these factors will help tremendously
to understand the mechanism of aging and, perhaps, to find ways to prevent aging.

42

J. Wu et al. / Mechanisms of Ageing and De6elopment 103 (1998) 27–44

Acknowledgements
We wish to thank Brenda Bunn for expert secretarial assistance and Fiona
Hunter for review of the manuscript. This work is supported by grants from the
National Institutes of Health R01-AG11653, R01-AR42547 and a Merit Review
Award from the Veterans Administration Medical Center.

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