MOLECULAR AND CELLULAR BIOLOGY,
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Mar. 2001, p. 1552–1564Vol. 21, No. 5
Change of the Death Pathway in Senescent Human Fibroblasts in
Response to DNA Damage Is Caused by an
Inability To Stabilize p53
ANDREI SELUANOV,* VERA GORBUNOVA,† AYELLET FALCOVITZ, ALEX SIGAL, MICHAEL MILYAVSKY,
IRIT ZURER, GALIT SHOHAT, NAOMI GOLDFINGER, AND VARDA ROTTER
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, 76100, Israel
Received 25 August 2000/Returned for modification 5 October 2000/Accepted 6 December 2000
The cellular function of p53 is complex. It is well known that p53 plays a key role in cellular response to DNA
damage. Moreover, p53 was implicated in cellular senescence, and it was demonstrated that p53 undergoes
modification in senescent cells. However, it is not known how these modifications affect the ability of senescent
cells to respond to DNA damage. To address this question, we studied the responses of cultured young and old
normal diploid human fibroblasts to a variety of genotoxic stresses. Young fibroblasts were able to undergo
p53-dependent and p53-independent apoptosis. In contrast, senescent fibroblasts were unable to undergo
p53-dependent apoptosis, whereas p53-independent apoptosis was only slightly reduced. Interestingly, instead
of undergoing p53-dependent apoptosis, senescent fibroblasts underwent necrosis. Furthermore, we found that
old cells were unable to stabilize p53 in response to DNA damage. Exogenous expression or stabilization of p53
with proteasome inhibitors in old fibroblasts restored their ability to undergo apoptosis. Our results suggest
that stabilization of p53 in response to DNA damage is impaired in old fibroblasts, resulting in induction of
necrosis. The role of this phenomenon in normal aging and anticancer therapy is discussed.
Normal animal cells, with few exceptions, do not divide in-
definitely. Eventually, cell divisions are arrested and cells enter
cellular or replicative senescence (for a review, see reference
10). Replicative senescence is especially stringent in human
cells, which almost never spontaneously immortalize (41)
Cellular senescence is a genetically controlled process (for a
review, see references 56 and 57). There is strong support for
the theory that telomere shortening limits the longevity of
human cells in culture (9, 26, 27). It has been proposed that
telomere shortening eventually causes chromosome instability,
leading to the activation of the DNA damage response path-
way followed by p53-dependent cell cycle arrest and senes-
cence (59). Furthermore, the important role of p53 in cellular
senescence is supported by the following observations. First,
functional inactivation of p53 rescues cells from senescence-
related growth arrest and instead they enter crisis at a delayed
time point (7, 24, 48, 49, 54). Second, the p21WAF1gene, which
encodes an inhibitor of cyclin-dependent kinases (15, 28), is
transcriptionally regulated by p53 (17, 18) and is overexpressed
in senescent cells (45). Third, exposure of human diploid fi-
broblasts to ?-irradiation leads to a p53-dependent, prolonged
G1arrest and induction of p21WAF1expression that is reminis-
cent of senescence (14). Fourth, the DNA binding and tran-
scriptional activities of p53 increase with cell age, and in most
cases this occurs in the absence of any marked increase in the
level of p53 (1, 4, 36, 47, 60).
p53 is more widely recognized for its role as a tumor sup-
pressor which mediates growth arrest and apoptosis in re-
sponse to DNA damage (25, 37). Apoptosis is generally viewed
as the “cleanest” way a cell can die. Apoptotic cell death does
not have a negative effect on surrounding tissues and is not
accompanied by inflammation. Analysis of p53 knockout mice
showed that in the absence of p53, cells with damaged DNA do
not enter cell cycle arrest and die by an alternative death
pathway, necrosis (43). Necrosis is characterized by the loss of
membrane integrity, cell swelling, and release of the cell con-
tents, which may result in inflammation (20). Not much is
known about the molecular mechanisms underlying the ne-
Little is known of how cell aging affects the ability of the cells
to respond to genotoxic stress. It has been observed that se-
nescent cells are resistant to apoptotic stimuli (22, 62). How-
ever the mechanism by which senescent cells resist apoptotic
death is not well understood. A different set of data indicates
that p53 is modified in senescent cells (60). It has recently been
demonstrated that senescence is associated with specific post-
translational modifications of p53 (63).
Based on these observations, we hypothesized that the in-
ability of senescent cells to undergo apoptosis is caused by
changes in the state of p53. Furthermore, as senescent cells are
resistant to apoptosis, we were interested in understanding the
fate of senescent cells subjected to apoptotic stimuli. For this
purpose, we studied the effects of DNA-damaging agents, in-
cluding actinomycin D, UV irradiation, cisplatin, and etopo-
side, that are known to induce different types of damage on
cells at early and late passages in culture. As a model system,
we chose the WI-38 human primary diploid fibroblasts, the
best-characterized cells with respect to cellular senescence (4,
We found that apoptosis induced by actinomycin D, UV, or
a low dose of cisplatin was p53-dependent in WI-38 fibroblasts,
* Corresponding author. Present address: Huffington Center on Ag-
ing, Baylor College of Medicine, One Baylor Plaza, Houston, TX
77030. Phone: (713) 798-3598. Fax: (713) 798-4161. E-mail: Seluanov
† Present address: Huffington Center on Aging, Baylor College of
Medicine, Houston, TX 77030.
whereas apoptosis induced by etoposide or a high dose of
cisplatin was p53-independent. Senescent fibroblasts were un-
able to induce p53-dependent apoptosis, even though p53-
independent apoptosis was only slightly reduced. Instead of
undergoing p53-dependent apoptosis in response to DNA
damage, senescent fibroblasts underwent necrosis. Artificial
stabilization of p53 in old cells by proteasome inhibitors led to
the induction of apoptosis. Moreover, transient expression of
the wild-type p53 in the old cells fully restored their ability to
undergo p53-dependent apoptosis, indicating that the absence
of p53-dependent apoptosis in old cells was due to their in-
ability to stabilize p53. Our results suggest that stabilization of
p53 in response to DNA damage is impaired in old fibroblasts,
resulting in a change of the death pathway from apoptosis to
MATERIALS AND METHODS
Cell culture and transfection. WI-38 human diploid fibroblasts (American
Type Culture Collection) were cultured in minimal essential medium with all
nonessential amino acids, 10% fetal bovine serum, and 1 mM pyruvate. Twice a
week, 105cells were passaged on a new plate using 0.25% trypsin–EDTA. Care
was taken not to let the cells reach 70% confluency.
Based on previous observation (4, 47), cells were defined as young if they had
completed 60% of their life span (32 to 40 population doublings; passage 21) and
as old if they had completed 90% of their life span (54 to 56 population dou-
blings; passage 29).
Genotoxic stress was induced by the addition of one of the following DNA-
damaging agents to the culture medium of fibroblasts at 40 to 50% confluency:
actinomycin D (25 ng/ml), cisplatin (1 or 10 ?g/ml), or etoposide (30 ?M). UV
irradiation (4 J/m2) was performed by a UV cross-linker (Stratagene).
Transfection experiments were carried out using FuGENE-6 (Roche) accord-
ing to the manufacturer’s instructions. Wild-type p53 was expressed under a
cytomegalovirus (CMV) promoter from the pC53-SN3 plasmid (5) provided by
R. Vogelstein (The Johns Hopkins University School of Medicine). The domi-
nant-negative p53 fragment (DD) was expressed under the CMV promoter from
the pCMV-DD plasmid (53) provided by M. Oren (Weizmann Institute of Sci-
ence). E6 was expressed under the simian virus 40 promoter from the pSG5-E6
plasmid (55) provided by L. Sherman (Tel Aviv University).
Enrichment of transfected cells was performed with the MACSorter Kkkit
from Miltenyi Biotec according to the manufacturer’s instructions. Briefly, the
method is based on cotransfection with a fragment consisting of the extracellular
loop of the Kkprotein, followed by application of anti-Kkantibodies bound to
magnetic beads for the selection of transfected cells.
Following the transfection, the cells were allowed to recover for 24 or 48 h and
then were harvested and incubated with antibodies conjugated to magnetic
beads, transfected cells were selected by passing them through a magnetic col-
umn. The efficiencies of transfection and sorting were estimated by fluorescence-
activated cell sorter (FACS) analysis in control experiments using a plasmid
containing green fluorescent protein (GFP) under the CMV promoter.
Detection of apoptosis by DNA ladder. Detached and adherent fibroblasts
were harvested, and equal numbers of cells (5 ? 105) were suspended in 30 ?l of
sample buffer (10% glycerol, 10 mM Tris[pH 8], 0.1% [wt/vol] bromophenol
blue) mixed at 1:1 ratio with 10-mg/ml RNase A solution. The cells were loaded
on an agarose gel which contained two parts: the lower part (from the comb to
the end of the gel) consisted of 0.8% agarose in Tris-borate-EDTA; the upper
part (from the comb to the beginning of the gel) consisted of 0.8% agarose, 2%
sodium dodecyl sulfate (SDS), and 64 ?g of proteinase K/ml in Tris-borate-
EDTA. The cells were electrophoresed for 10 h at 60 V at room temperature.
The gel was stained with 2 mg of ethidium bromide/ml in water for 1 h and then
destained with water (16).
Western blot analysis. Detached and adherent fibroblasts were harvested, and
equal numbers of cells (106) were lysed in protein sample buffer with 5% ?-mer-
captoethanol and 1 mM phenylmethylsulfonyl fluoride, boiled for 10 min, and
loaded on an SDS-polyacrylamide gel. A 7.5 and a 10% running gel were used for
the detection of poly(ADP-ribose) polymerase (PARP) and p53, respectively.
The proteins were transferred to a nitrocellulose membrane using a semidry
transfer cell (Bio-Rad). The blots were probed with either a human p53-specific
monoclonal antibody (DO-1) or anti-PARP monoclonal antibody (Biomol). The
bands were visualized with the aid of the Super Signal kit (Pierce).
Analysis of apoptosis and necrosis by acridine orange staining. Cells were
fixed in 3 ml of a solution containing 80% ethanol and 20% Hanks balanced salt
solution (HBSS) and stored at ?20°C for a period not exceeding 1 week. On the
day of the assay, the cells were gently remixed and centrifuged at 800 rpm on a
Sorval GLC-3 centrifuge. The pellets were gently resuspended, washed in 1 ml of
HBSS, and centrifuged at 1,000 rpm on a Hettich microcentrifuge. The cells were
resuspended in 1 ml of a 1:30 solution of RNase in HBSS and incubated at 37°C
for 1.5 h. The cells were then centrifuged at 1,200 rpm in an Eppendorf centri-
fuge, gently resuspended in 200 ?l of HBSS, and transferred to FACS tubes, and
0.5 ml of 0.1 M HCl in HBSS was added. After approximately 1 min, the acid
denaturation was quenched by the addition of 2 ml of a solution of 90% citric
acid, 10% Na2HPO4, and 0.06% acridine orange (Molecular Probes). The cells
were analyzed in a FACS SORT flow cytometer (Becton Dickinson). An exci-
tation wavelength of 488 nm was used, 575- and 650-nm emissions were collected,
and the data were analyzed with CellQuest software (Becton Dickinson). Low-
speed centrifugation and gentle handling of the fixed cells were critical to the
success of this assay, as they prevented aggregation and breakage of the cells.
Detection of necrosis by release of DNA. The cellular DNA fragmentation
enzyme-linked immunosorbent assay kit from Boehringer Mannheim was used
for the detection of bromodeoxyuridine (BrdU)-labeled DNA released from ne-
crotic cells into the cell culture medium. Briefly, WI-38 human fibroblast cells were
incubated in culture with the thymidine analogue BrdU, which is incorporated
into the genomic DNA. BrdU was added to the medium 24 or 48 h before treat-
ment. The cells were treated with various drugs, as described above, in the pres-
ence of BrdU, and the supernatants of the cell cultures were collected after
different time intervals. BrdU did not affect the rate of growth, apoptosis, or ne-
crosis of the fibroblasts in the time window used, as determined by FACS analysis
of BrdU-treated and untreated cells. The DNA fragments were captured with an
anti-DNA antibody bound to a Nunc-Immuno flat-bottom plate and were detect-
ed by an anti-BrdU antibody-peroxidase conjugate. The photometric measures
were done at a wavelength of 450 nm with a reference wavelength of 690 nm.
Apoptosis in human primary fibroblasts. To examine wheth-
er cellular aging affects the ability of cells to undergo apoptosis
in response to genotoxic stress, we chose WI-38 primary hu-
man fibroblasts. Since the detection of apoptosis in fibroblasts
is controversial (3, 14, 22, 29, 64), we first analyzed the abilities
of various DNA-damaging agents to induce apoptosis in these
Exponentially growing populations of young human fibro-
blasts were treated with the following DNA-damaging agents:
actinomycin D, UV irradiation, etoposide, and low (0.5- to
2-?g/ml) and high (5- to 20-?g/ml) concentrations of cisplatin.
Apoptosis was determined by the appearance of apoptotic
features, such as DNA fragmentation, chromatin condensa-
tion, and cleavage of PARP.
DNA fragmentation was analyzed by the DNA ladder tech-
nique. It was previously shown that fibroblasts produce mostly
DNA fragments of high molecular weight not followed by the
internucleosomal DNA cleavage typical of hematopoietic cells
(8, 16, 46). Furthermore, it was suggested that the cleavage of
DNA into high-molecular-weight, approximately 50- and 300-
kb fragments is an essential early step in apoptosis for all cell
types, in contrast to the later and nonessential internucleoso-
mal DNA fragmentation (8, 46). Therefore, fibroblasts under-
going apoptosis yield a high-molecular-weight smear instead of
the typical 180-bp DNA ladder. The optimal time and drug
concentration for the induction of apoptosis were defined for
every DNA-damaging agent (data not shown). Upon treatment
of young cells with actinomycin D, UV irradiation, etoposide,
and low and high concentrations of cisplatin, we detected the
appearance of high-molecular-weight smears in a time-depen-
dent manner (Fig. 1A). Different levels of DNA fragmentation
were observed for the different agents.
VOL. 21, 2001CHANGE OF DEATH PATHWAY IN OLD FIBROBLASTS1553
FIG. 1. Apoptosis in young and old normal human fibroblasts induced by DNA-damaging agents. Young and old fibroblasts were treated with various
DNA-damaging agents (actinomycin D, UV, etoposide, and low [1-?g/ml] and high [10-?g/ml] concentrations of cisplatin), and induction of apoptosis
was analyzed by DNA ladder (A), acridine orange staining for chromatin condensation (B), and PARP cleavage (C). (A) After 0, 36, 48, and 72 h of
induction, all detached and adherent fibroblasts were collected, and equal numbers of the cells were directly subjected to gel electrophoresis as described
in Materials and Methods. MW, 1-kb ladder standard. (B) Percent apoptosis was determined by FACS analysis after acridine orange staining of
fibroblasts treated with genotoxic agents. The hatched bars represent young fibroblasts, and the solid bars represent old fibroblasts. All of the experiments
were repeated at least three times, and standard errors are shown. (C) Induction of apoptosis in young human fibroblasts was further confirmed by
Western blotting with anti-PARP antibodies. The full-length (113-kDa) and apoptosis-specific (89-kDa) fragments of PARP are indicated.
1554 SELUANOV ET AL.MOL. CELL. BIOL.
Chromatin condensation was analyzed by acridine orange
DNA staining followed by FACS analysis. Figure 1B shows
similar kinetics of apoptosis for young cells treated with acti-
nomycin D, UV irradiation, and a low concentration of cispla-
tin. A peak of apoptosis (20 to 60%) was reached 48 h after
treatment. The reduction in the percentage of apoptosis mea-
sured 72 h after treatment may be due to the disintegration of
the apoptotic cells. The percentage of spontaneous apoptosis
in untreated cells did not exceed 2%. Cells treated with high
concentrations of cisplatin and etoposide exhibited different
kinetics of apoptosis than cells treated with the other drugs. It
should be noted that low and high concentrations of cisplatin
display different patterns of apoptosis: a low concentration
leads to a peak at 48 h, and a high concentration shows a linear
increase up to 72 h. The apoptotic patterns obtained for the
young cells by DNA condensation analysis measured by acri-
dine orange staining followed by FACS are in good correlation
with those obtained by the DNA fragmentation assay.
Finally, cleavage of PARP, which is indicative of apoptosis-
induced caspase-3 and/or -8 activity, was analyzed by Western
blotting with an anti-PARP antibody (Fig. 1C). A typical apo-
ptotic pattern of the uncleaved form of PARP (113 kDa) with
an increase in the level of the cleaved fragment of PARP (89
kDa) was detected between 24 and 36 h after all treatments.
This occurred despite the differences in the kinetics of the
In conclusion, we have demonstrated by three different tech-
niques that young human fibroblasts are able to undergo apo-
ptosis in response to actinomycin D, UV irradiation, etoposide,
Next, we analyzed the induction of apoptosis in old WI-38
human fibroblasts with the same set of DNA-damaging agents
used for young cells. Apoptosis was determined by the DNA
ladder and acridine orange techniques under the same con-
ditions as for the young fibroblasts. According to the DNA
ladder patterns obtained, it appears that young and old cells
exhibit comparable fragmentation smears in response to high
concentrations of cisplatin and etoposide (Fig. 1A). However,
following treatment with actinomycin D, UV irradiation, or a
low concentration of cisplatin, old cells show a much lower
level of apoptosis than young cells. This difference is more
pronounced in the acridine orange analysis. In this case, no
significant levels of apoptosis are detected in old cells in re-
sponse actinomycin D, UV irradiation, or a low concentration
of cisplatin, whereas high levels of apoptosis are evident in
young cells (Fig. 1B). In contrast, similar patterns of apoptosis
were seen in young and old cells in response to etoposide.
Treatment of old cells with a high concentration of cisplatin
resulted in a low level of apoptosis at 36 h, followed by a
marked increase at 48 h after treatment. By 72 h, the level of
apoptosis in old cells was high and comparable to that of young
To summarize, we found that the treatments with etoposide
and high concentrations of cisplatin induced similar levels of
apoptosis in both young and old cells whereas actinomycin D,
UV irradiation, and a low concentration of cisplatin induced
high levels of apoptosis in young cells and much lower levels of
apoptosis in old cells. This possibly means that cellular re-
sponse pathways induced by etoposide and high concentrations
of cisplatin remain unchanged when cells enter senescence,
while response pathways induced by actinomycin D, UV irra-
diation, and a low concentration of cisplatin are altered in
Human fibroblasts undergo p53-dependent or p53-indepen-
dent apoptosis. We were interested in studying whether there
is a correlation between the differential apoptotic responses of
the young and old cells and the tumor suppressor protein p53.
Apoptosis is known to occur via p53-dependent and p53-inde-
pendent pathways, depending on the treatment applied and
the cell type. We first investigated the involvement of p53 in
the induction of apoptosis by genotoxic stress in young human
fibroblasts. Upon treatment of young fibroblasts with actino-
mycin D, cisplatin, and UV, we detected an increase in p53
levels by Western blotting with DO-1 anti-p53 antibodies (Fig.
2A). However, etoposide failed to induce accumulation of p53
(Fig. 2A), which suggests that etoposide, in contrast to actino-
mycin D, cisplatin, and UV, is unable to stabilize p53. In order
to test whether stabilization of p53 is accompanied by its
activation, we analyzed the induction of the p53 downstream
gene Mdm2 upon genotoxic stress. Induction of Mdm2 was
monitored by Western blotting with the anti-Mdm2 antibody
(data not shown). We observed strong correlation between
accumulation of p53 and induction of the Mdm2 gene in the
cells treated with actinomycin D, UV, and low concentrations
of cisplatin. Even though fibroblasts treated with high concen-
trations of cisplatin exhibit high levels of p53, we did not detect
any induction of the Mdm2 gene. Etoposide-treated cells,
which were found not to accumulate p53 (see above), were
unable to induce Mdm2 in response to stress.
We next examined whether the increase of p53 protein is
associated with the induction of apoptosis in fibroblasts. For
this purpose, we used functional depletion of p53 by a domi-
nant-negative fragment and the viral E6 protein. The minimal
requirement for the dominant-negative function of mutant p53
is its C terminus from amino acids 302 to 390. Transient ex-
pression of this minimal dominant-negative p53 fragment, des-
ignated DD, leads to strong functional inactivation of endog-
enous p53 (53). To exclude the possible gain-of-function effect
of the DD fragment on suppression of apoptosis, we used a
second method of p53 inactivation. To this end, we depleted
p53 by the transient expression of the human papillomavirus
type 16 protein E6, which binds to p53 and promotes its rapid
proteolysis (21, 51).
Young fibroblasts were transiently transfected with plasmids
harboring the dominant-negative DD or E6 under the strong
constitutive CMV and simian virus 40 promoters, respectively.
Empty vectors were used as a control. Inactivation of p53 was
confirmed by cotransfection of DD or E6 with the plasmids
carrying the reporter (luciferase) gene under a p53-inducible
RGC or Bax promoter. In the cells transfected with the plas-
mids harboring DD or E6, the level of luciferase expression
was strongly reduced compared to that in control cells trans-
fected with empty vectors (data not shown). The efficiency of
transfection was ?15%, as determined by cotransfection with
the plasmid containing GFP under the CMV promoter fol-
lowed by FACS analysis. We enriched the population for up to
80% of transfected cells using the magnetic cell sorter (see
Materials and Methods). After recovery from transfection and
cell sorting, the cells were treated with actinomycin D, UV
irradiation, etoposide, and low and high concentrations of cis-
VOL. 21, 2001CHANGE OF DEATH PATHWAY IN OLD FIBROBLASTS 1555
FIG. 2. Role of p53 in the induction of apoptosis in young and old human fibroblasts. (A) Accumulation of p53 in young and old fibroblasts
upon treatment with various DNA-damaging agents. After 0, 5, 12, 24, 36, and 48 h of induction, all detached and adherent fibroblasts were
collected, and equal numbers of cells were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) followed by Western blotting with DO-1
anti-p53 antibodies. The blots were stained with India ink to check the equivalence of protein transfer. One-third of each sample was subjected
to SDS-PAGE and stained with Coomassie blue to demonstrate equal loading of samples (shown below each Western blot). (B) Inactivation of
p53-dependent apoptosis by transient expression of dominant-negative p53 fragment (DD) or human papillomavirus type 16 protein E6. Young
fibroblasts transiently transfected with empty vector (hatched bars), plasmid expressing DD (solid bars), or E6 (dark hatched bars) were treated
with various DNA-damaging agents for 36, 48, and 72 h. The level of apoptosis was determined by acridine orange staining followed by FACS
analysis. The open bars represent the level of apoptosis in the transfected but untreated cells. All the experiments were repeated at least three
times, and standard errors are shown.
1556 SELUANOV ET AL.MOL. CELL. BIOL.
platin, and apoptosis was analyzed by the more quantitative
acridine orange method. The functional depletion of p53 in
fibroblasts markedly decreased their ability to undergo apo-
ptosis in response to actinomycin D, UV irradiation, or a low
concentration of cisplatin (Fig. 2B). However, apoptosis in-
duced by etoposide or a high concentration of cisplatin was not
affected. Functional depletion of p53 by the dominant-negative
DD fragment and E6 gave similar results, indicating that only
inactivation of p53 is responsible for the observed effect rather
than other effects of the DD or E6 protein. These results in-
dicate that young WI-38 human fibroblasts undergo p53-de-
pendent apoptosis in response to treatment with actinomycin
D, UV irradiation, and a low concentration of cisplatin. In con-
trast, etoposide and a high concentration of cisplatin induce
As described above, old WI-38 human fibroblasts predomi-
nantly underwent apoptosis in response to high concentra-
tions of cisplatin and etoposide and to a much lower extent in
response to actinomycin D, UV irradiation, and a low concen-
tration of cisplatin. In light of the observations that actinomy-
cin D, UV, and a low concentration of cisplatin induced p53-
dependent apoptosis in young fibroblasts, it appears that old
fibroblasts are able to undergo p53-independent apoptosis but
not p53-dependent apoptosis.
Old human fibroblasts are unable to stabilize p53 in re-
sponse to genotoxic stress. We have found that, unlike young
cells, old fibroblasts exhibit negligible levels of p53-dependent
apoptosis in response to genotoxic stress. It is well accepted that
p53-dependent apoptosis induced by genotoxic stress requires
p53 protein stabilization. Hence, we analyzed the stabilization
of p53 in old fibroblasts after treatment with actinomycin D,
UV irradiation, etoposide, and low and high concentrations of
cisplatin, using Western blotting with the DO-1 anti-p53 anti-
body (Fig. 2A). We detected very low or no stabilization of the
p53 protein in old cells compared to that in young cells. How-
ever, the basal levels of p53 in young and old fibroblasts were
comparable (Fig. 2A), as shown previously (1, 4). This suggests
that the reduction in p53-dependent apoptosis in old fibro-
blasts may be due to their inability to stabilize p53.
Old human fibroblasts that are unable to enter p53-depen-
dent apoptosis undergo necrosis. The above-mentioned results
showed that old human fibroblasts are unable to undergo p53-
dependent apoptosis in response to DNA damage. A question
arises as to the fate of those cells with damaged DNA. During
preliminary microscopic examination, we observed similar
death rates in the young and old cells treated with all the tested
DNA-damaging agents (data not shown). Hence, we suspected
that necrosis was responsible for the death of DNA-damaged
old cells. Release of the cellular matrix from the cell and the
appearance of “empty” ghost cells, consisting of only cell mem-
branes, are typical necrotic features (20, 31, 34). As mentioned
previously, acridine orange staining followed by FACS analysis
was effective in differentiating between viable and apoptotic
WI-38 fibroblasts. Acridine orange binds to DNA and hence
can detect sub-G1DNA content. Moreover, it is dichromatic
and undergoes a shift from green to red fluorescence when it
binds to the condensed DNA typical of apoptosis. Therefore, it
allows the detection of apoptosis even in cells that do not
FIG. 3. Detection of necrotic and apoptotic cells by acridine orange staining followed by FACS analysis. (A) Density plot of normal cell cycle
distribution of untreated fibroblasts. Cell populations at the G1, S, and G2stages of the cell cycle are indicated. (C) Density plot of empty cells
obtained from normal fibroblasts by DNase and RNase treatment for 1.5 h prior to acridine orange staining. This distinct population of empty cells
(without DNA and RNA) was used to set the parameters for the identification of the population of necrotic cells. (B and D) Typical examples of
young and old fibroblasts undergoing apoptosis or necrosis upon treatment with actinomycin D for 48 h.
VOL. 21, 2001 CHANGE OF DEATH PATHWAY IN OLD FIBROBLASTS1557
exhibit a sub-G1content, like fibroblasts. Figure 3C shows an
example of the fluorescence shift of acridine orange due to
apoptosis induced by actinomycin D treatment.
To examine whether the acridine orange technique can also
detect necrotic cells as a population separate from viable and
apoptotic cells, we treated viable cells with DNase and RNase
to produce the empty WI-38 cells typical of necrosis and as-
sayed them by acridine orange. We obtained a clearly segre-
gated population, with a fluorescence lower and greener than
that of viable cells (Fig. 3B), which is probably the result of
acridine orange staining of membrane-bound glycosaminogly-
cans and proteoglycans, as shown previously (13). We there-
fore designated this population necrotic cells.
Old and young human fibroblasts treated with actinomycin
D, UV irradiation, etoposide, and low and high concentrations
of cisplatin were stained with acridine orange and analyzed by
FACS. The accumulation of necrotic and/or apoptotic cells was
observed following all treatments (Fig. 4). Strikingly, in re-
sponse to actinomycin D, UV irradiation, and a low concen-
tration of cisplatin, old fibroblasts exhibited a population that
corresponded exactly to the population defined above as ne-
crotic (compare Fig. 3B and D). Under the same conditions,
FIG. 4. Induction of apoptosis and necrosis in young and old human fibroblasts by various DNA-damaging agents. After 36, 48, and 72 h of
treatment, the cells were stained with acridine orange and analyzed by FACS (see Materials and Methods), which allowed the measurement of
both apoptosis and necrosis. The open and solid bars represent untreated young and old cells, respectively. The levels of apoptosis and necrosis
in young untreated cells were too low to be seen on the graph. The hatched bars represent the level of apoptosis or necrosis in treated cells, and
light and dark hatched bars represent young and old cells, respectively. All the experiments were repeated at least three times, and standard errors
1558 SELUANOV ET AL.MOL. CELL. BIOL.
young cells underwent apoptosis (Fig. 3C). A time-dependent
increase in necrotic cells can be seen in Fig. 4, following treat-
ment with actinomycin D, UV irradiation, and a low concen-
tration of cisplatin. No significant levels of apoptosis were
detected in the old cells in any of these treatments. Treatment
of young and old cells with a high concentration of cisplatin
and etoposide induced apoptosis to similar extents at most
time points used, with no significant levels of necrosis.
To further confirm that cell death in senescent cells occurred
by necrosis, we used a different method for detection of ne-
crosis which is based on the release of DNA from necrotic cells
into the medium. Following treatment of senescent cells with
actinomycin D, UV irradiation, and a low concentration of
cisplatin, an increase in the DNA level detected in the medium
was observed relative to that of untreated cells (Fig. 5). We
have not detected any significant levels of DNA release for
young cells. The DNA release increased in a time-dependent
manner in all cases. This indicates that old fibroblasts treated
with actinomycin D, UV irradiation, and a low concentration
of cisplatin undergo necrosis in response to the treatments.
The kinetics and relative extent of necrosis detected by DNA
release were similar to those detected by acridine orange.
However, in all the treatments the percentage of necrotic cells
detected by DNA release was twice as small as that with acri-
dine orange. This can be explained by the fact that free DNA
released into the medium undergoes rapid degradation, while
empty cells (membrane vesicles) detected by acridine orange
are much more stable.
These findings suggest that towards senescence, human fi-
broblasts change their death pathway from p53-dependent
apoptosis to necrosis in response to genotoxic stress. DNA-
damaging agents that cause p53-dependent apoptosis in young
cells induce necrosis in old cells.
Stabilization of p53 in old human fibroblasts restores their
ability to undergo apoptosis. To examine whether the reduc-
tion in p53-dependent apoptosis in old fibroblasts is due to
their inability to stabilize p53, we used two approaches. One
was aimed at stabilizing p53 in old cells by the inhibition of its
proteolysis, and the other approach was to exogenously express
the p53 protein by transient transfection of wild-type p53.
(i) Stabilization of p53 by proteasome inhibitors. Degrada-
tion of p53 is mediated by the ubiquitin-proteasome pathway
(40). In order to induce the accumulation of endogenous p53
in old fibroblasts, we used the proteasome inhibitors MG-115,
MG-132, and PSI (proteasome inhibitor I), which were shown
to stabilize p53 (11, 23, 39). Importantly, it was demonstrated
that apoptosis of immortalized cells induced by MG-115 and
PSI is p53 dependent, suggesting that stabilization of p53 plays
a key role in apoptosis induced by proteasome inhibitors (39).
Young and old human fibroblasts were treated with protea-
some inhibitors, and the accumulation of p53 was analyzed up
to 48 h after the treatment. All the tested proteasome inhibi-
tors induced rapid and strong accumulation of p53 in young
and old fibroblasts (Fig. 6A). These results clearly demonstrate
that even though p53 in the old fibroblasts is not stabilized in
response to genotoxic stress, it could be stabilized by protea-
some inhibitors. Furthermore, the kinetics of p53 accumula-
tion confirmed previous observations that the rates of synthesis
of p53 in young and old cells are similar (1, 4).
In order to examine the induction of apoptosis in the cells
treated by proteasome inhibitors, the cells were analyzed by
acridine orange DNA staining. Rapid and strong induction of
apoptosis was observed in both young and old fibroblasts
treated with MG-115, MG-132, and PSI (Fig. 6B). It should be
noted that up to 24 h following this treatment, the level of
apoptosis in old cells was lower than that in young cells. How-
ever, both young and old cells reached ?100% apoptosis 48 h
after treatment. The fact that the proteasome inhibitors that
are associated with p53 stabilization were able to induce apo-
ptosis in old cells suggests that induction of apoptosis under
these conditions was p53 dependent. Apoptosis induced by
proteasome inhibitors was higher than apoptosis induced by
other treatments. This can be explained by the fact that pro-
teasome inhibitors lead to the accumulation of other regula-
tory molecules in addition to p53. However, it has been shown
that modulation of p53 turnover is a key event in apoptosis
induced by proteasome inhibitors (39).
(ii) Exogenous expression of wild-type p53. To confirm that
the observed apoptosis induced by the proteasome inhibitors is
indeed due to an increase in p53 levels rather than stabilization
of other factors, we tested whether overexpression of exoge-
nous p53 would force old cells to enter apoptosis instead of
undergoing necrosis. To this end, old human fibroblasts were
transfected with a plasmid carrying wild-type p53 under the
CMV promoter. The efficiency of transfection was ?15%, as
monitored by cotransfection with a GFP-harboring plasmid.
The population of transfected cells was enriched up to 80% by
magnetic cell sorting (see Materials and Methods). The exog-
enous p53 was highly expressed in transfected old fibroblasts
within 48 h after transfection, as shown by Western blotting
(Fig. 7A). This high level of p53 in transfected cells induced
FIG. 5. Induction of necrosis in old human fibroblasts by actino-
mycin D, UV, and low concentration of cisplatin. Necrosis was ana-
lyzed by the release of DNA from necrotic cells into the medium. Total
DNA of the old fibroblasts was metabolically labeled with BrdU for 24
or 48 h prior to induction. DNA released into the medium upon
induction of necrosis was quantified with a cellular DNA fragmenta-
tion enzyme-linked immunosorbent assay kit (see Materials and Meth-
ods). The percent of released DNA from the total labeled DNA is
shown. Experiments were repeated five times, and standard deviations
VOL. 21, 2001CHANGE OF DEATH PATHWAY IN OLD FIBROBLASTS 1559
massive apoptosis even without genotoxic stress (Fig. 7B). Sim-
ilar massive apoptosis was observed with p53-transfected old
fibroblasts that were treated with actinomycin D, UV irradia-
tion, and a low concentration of cisplatin. This may indicate
that following exogenous expression of p53, the cells reached a
maximum level of p53-dependent apoptosis that could not be
further increased by drug treatment. Importantly, the level of
DNA damage-induced necrosis was significantly reduced by
overexpression of p53 in the old cells in comparison to that
observed for the old cells transfected with the control vector.
Therefore, by overexpression of exogenous p53, we were able
to override senescence-related changes in the old cells and
switch them back from the necrotic to the apoptotic pathway of
Taken together, these results demonstrate that the cellular
milieu of old fibroblasts permits the expression of high levels of
p53 sufficient for apoptosis. Furthermore, the apoptotic ma-
chinery downstream of p53 is fully functional in old cells.
We were the first to analyze the response of senescent cells
to genotoxic stress and the role of p53 in this process. Our
main finding is that old cells do not induce p53-dependent
FIG. 6. Stabilization of p53 and induction of apoptosis in young and old fibroblasts treated with proteasome inhibitors. Young and old
fibroblasts were treated with the following proteasome inhibitors: MG-115 (30 ?M), MG-132 (10 ?M), and PSI (30 ?M). (A) Stabilization of p53
at various time points after treatment was analyzed by Western blotting with DO-1 antibodies. The p53 protein is indicated by arrowheads. The
blots were stained with India ink to check the equivalence of protein loading and transfer, and the blots showing equal loading and transfer within
young and old cells are presented. (B) Induction of apoptosis was analyzed by acridine orange staining followed by FACS analysis. The percent
apoptosis in young (light hatched bars) and old (dark hatched bars) cells treated with proteasome inhibitors is shown. The level of apoptosis in
untreated cells (young and old) was too low to be seen on the graph. Experiments were repeated at least three times; standard errors were less
than 3% of the average and are not shown on the graph.
1560 SELUANOV ET AL.MOL. CELL. BIOL.
apoptosis in response to genotoxic stress, which is likely the
result of an inability to stabilize p53. Upon DNA damage,
senescent cells that are unable to undergo apoptosis die by
necrosis. A summary of our results is shown schematically in
Apoptosis in young human fibroblasts. Based on the analy-
sis of DNA fragmentation, chromatin condensation, and the
cleavage of PARP, we demonstrated that young human fibro-
blasts are able to undergo apoptosis in response to DNA in-
sults mediated by actinomycin D, UV irradiation, etoposide,
and low and high concentrations of cisplatin. Assays of chro-
matin condensation and caspase-dependent cleavage of PARP
in treated fibroblasts showed classical apoptosis. In the DNA
fragmentation assay, high-molecular-weight smears were ob-
tained following all treatments, which is in accordance with
previous studies reporting that fibroblasts are unable to pro-
duce internucleosomal DNA fragmentation during apoptosis
(8, 16, 46). This explains why the methods based on detection
of extensive DNA fragmentation (terminal deoxynucleotidyl-
transferase-mediated dUTP-biotin nick end labeling) or ex-
clusion of the small DNA fragments from the nucleus (pro-
pidium iodide-based detection of a sub-G1population) would
FIG. 7. Transient expression of p53 in old fibroblasts restores their ability to undergo apoptosis and inhibits their ability to undergo necrosis.
Old fibroblasts were transiently transfected with a plasmid harboring the wild-type p53 gene under the CMV promoter or with the control plasmid
containing the CMV promoter alone. The population of transfected cells was enriched using the MACSorter Kkkit. (A) The levels of p53 ex-
pression at 48 and 72 h after transfection were analyzed by Western blotting with DO-1 antibodies. The blots were stained with India ink to check
the equivalence of protein transfer. One-third of each sample was subjected to SDS-polyacrylamide gel electrophoresis and stained with Coomassie
blue to demonstrate equal loading of samples (shown below the Western blot). (B) Immediately following the transfection, cells were subjected to
genotoxic stress (actinomycin D, UV, or a low concentration of cisplatin). The levels of induced apoptosis and necrosis 48 and 72 h after treatment
were analyzed by acridine orange staining followed by FACS. Experiments were repeated at least three times, and standard errors are shown.
VOL. 21, 2001 CHANGE OF DEATH PATHWAY IN OLD FIBROBLASTS1561
not be sensitive enough for the analysis of apoptosis in normal
Using functional depletion of p53, we were able to deter-
mine that young fibroblasts undergo p53-dependent apoptosis
in response to actinomycin D, UV irradiation, and a low con-
centration of cisplatin and undergo p53-independent apoptosis
in response to etoposide and a high concentration of cisplatin.
The choice to undergo either p53-dependent or p53-indepen-
dent apoptosis may be a function of the type of DNA damage,
the level of damage, or the cell cycle phase within the different
cellular milieux (2, 33, 42).
Senescent human fibroblasts are resistant to p53-dependent
apoptosis and instead undergo necrosis. We have observed
that old fibroblasts were unable to undergo p53-dependent
apoptosis, while the ability to undergo p53-independent apo-
ptosis was only slightly affected. It was previously reported that,
unlike young cells, old fibroblasts were unable to undergo
apoptosis in response to stress caused by serum deprivation or
oxygen radicals (22, 62). In order to understand what hap-
pened with the damaged old cells which were unable to induce
p53-dependent apoptosis, we performed detailed analysis of
those cells. We found that senescent fibroblasts were dying via
a different pathway—necrosis. The necrotic death that we ob-
served was characterized by the loss of cellular content and a
lack of chromatin condensation and membrane blebbing. Our
observation that the inhibition of an apoptotic pathway results
in necrotic cell death correlates well with data from recent
studies that used broad-spectrum caspase inhibitors to block
apoptosis (12, 30, 32, 61). Furthermore, data from an assess-
ment of the effect of DNA damage on the limb development of
wild-type and p53 knockout mice (43) showed that the damage
induced apoptosis in the limbs of wild-type mice but not those
of p53?/?mice. However, cell death which exhibited necrotic
features was much higher in the limbs of homozygous p53?/?
mice. Collectively, these observations suggest that apoptosis
and necrosis may be alternative pathways in the process of cell
death. The physiological role of necrosis is only starting to be
understood. Future studies will show the role played by the
change of the death pathway from apoptosis to necrosis in
Inability of senescent fibroblasts to undergo p53-dependent
apoptosis is due to lack of p53 stabilization. Our findings
suggest that old cells are unable to undergo p53-dependent
apoptosis due to an inability to stabilize p53. This was demon-
strated by two independent methods. First, proteasome inhib-
itors forced the accumulation of p53 in old cells, which in turn
induced apoptosis. This indicates that the apoptotic machinery
downstream of p53 is functional in old cells. Second, similar
results were obtained following overexpression of exogenous
p53 in these cells. By the forced accumulation of p53, we were
able to restore the inability of old cells to enter p53-dependent
Lack of p53-dependent apoptosis in senescent cells in re-
sponse to genotoxic stress may be caused by impaired function
of the upstream regulators of p53 involved in recognition of
DNA damage, such as PARP, DNA-PK, or ATM. It has been
reported that DNA-PK and PARP are down regulated in se-
nescent fibroblasts (50). On the other hand, studies showing
activation of p53 in senescent cells suggest that p53 itself is
undergoing modifications (4, 60). It has been shown recently
that the phosphorylation pattern of p53 in senescent cells over-
laps but is distinct from that induced by DNA damage (63). It
is possible that the senescence-specific phosphorylation pat-
tern is also responsible for the lack of p53 stabilization upon
DNA damage in senescent cells. We can speculate that the lack
of apoptotic response to DNA damage in senescent cells has
the following physiological rationale. Apoptosis is a protective
mechanism that prevents cancer by killing potentially danger-
ous cells that have mutated DNA. Senescent cells are entering
irreversible growth arrest and do not pose a threat of malig-
nant transformation. Therefore, apoptotic response to DNA
damage becomes unnecessary in senescent cells. Instead, the
preservation of viable cells might be more important in aging
Implications of the change of death pathway from apoptosis
to necrosis in senescent cells for anticancer therapy. One of
the problems in geriatric medicine is the increased sensitivity
of old patients to stress induced by DNA damage and other
types of cellular damage (19). This problem becomes crucial in
oncology, since cancer is an age-associated disease and anti-
cancer chemotherapy results in severe genotoxic stress to nor-
mal tissues. It was observed that some anticancer drugs have a
greater toxicity in the elderly than in young patients (6, 38).
Our finding of a senescence-related transition of the death
pathway from apoptosis to necrosis in old cells provides a good
explanation for this augmented toxicity. Analysis of studies
concerning the application and toxicity of anticancer drugs in
the elderly revealed good correlation between increased tox-
icity of the drug in the elderly and induction of necrosis in old
FIG. 8. Model of the death pathways taken by young and old hu-
man fibroblasts in response to various genotoxic stresses.
TABLE 1. Toxicity of anticancer drugs to the elderly correlated
with the death pathway they induce in senescent fibroblasts
oncologists for use
Death pathway induced
Young cellsOld cells
Cisplatin (high dose)
Most often used
No increased toxicity
Used with acceptable
Not used due to high
Cisplatin (low dose)Apoptosis Necrosis
Actinomycin D Apoptosis Necrosis
aAll the analyzed anticancer drugs are widely used for treatment of young
1562 SELUANOV ET AL.MOL. CELL. BIOL.
human fibroblasts (Table 1). Thus, etoposide is widely used for
anticancer chemotherapy in old patients (38, 44). Our results
show that etoposide induces p53-independent apoptosis in old
cells. Cisplatin is one of the most effective and widely used
anticancer drugs, and it is considered to have acceptable tox-
icity for old patients (35, 38). No increased toxicity was ob-
served in the elderly when the cisplatin dose was increased (52,
58), and it was suggested that its toxicity was inversely depen-
dent on the dose administered. We found that a low dose of
cisplatin causes necrosis in old cells whereas a high dose in-
duces apoptosis. Actinomycin D is widely used in cancer ther-
apy; however, its severe toxicity in old patients has been re-
ported (52). We found that while in young cells actinomycin D
induces apoptosis, in old cells it results in massive necrosis. As
previously mentioned, necrosis can result in inflammation, and
this can explain the increased toxicity of the necrosis-inducing
drugs observed in the elderly. Thus, elucidation of the death
pathways induced by genotoxic stress in the context of cellular
senescence may contribute to optimization of the current che-
This study was supported by a grant from the Israel Cancer Asso-
ciation and in part by grants from the Israel-USA Binational Science
Foundation, the German Israeli Foundation for Scientific Research
and Development, and the Israel Cancer Research Fund (V.R.). A.S.
was supported by a postdoctoral fellowship from the Weizmann Insti-
tute of Science. V.R. holds the Norman and Helen Asher Professorial
Chair in Cancer Research at the Weizmann Institute.
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