From the Division of Molecular Carcinogenesis, and Center for Biomedical Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
Received for publication, January 17, 2003
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ABSTRACT |
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Senescence is generally defined as an
irreversible state of G1 cell cycle arrest in which
cells are refractory to growth factor stimulation. In mouse embryo
fibroblasts (MEFs), induction of senescence requires the presence of
p19ARF and p53, as genetic ablation
of either of these genes allows escape from senescence and leads to
immortalization. We have developed a lentiviral vector that directs the
synthesis of a p53-specific short hairpin transcript, which mediates
stable suppression of p53 expression through RNA interference. We show
that suppression of p53 expression in senescent MEFs leads to rapid
cell cycle re-entry, is associated with loss of expression of
senescence-associated genes, and leads to immortalization. These data
indicate that senescence in MEFs is reversible and demonstrate that
both initiation and maintenance of senescence is
p53-dependent.
Most primary mammalian cells have a limited ability to proliferate
in tissue culture (1, 2). After a variable number of cell divisions,
primary cells will undergo what is believed to be an irreversible form
of growth arrest in the G1 phase of the cell cycle and
become refractory to further growth factor stimulation (3-5). In this
state of growth arrest, referred to as senescence, cells adopt a
typical large and flat morphology and express a number of
senescence-associated markers, including senescence-associated
The triggers for the induction of senescence differ between mouse and
human cells. In cultured rodent fibroblasts senescence is thought to
result from stress signals generated in response to the inadequate
tissue culture environment. This includes supraphysiological oxygen
tension, lack of proper extracellular matrix, and the liberal administration of bovine growth factors (Refs. 6 and 7 and reviewed in
Refs. 5 and 8). Indeed, under more gentle and more defined culture
conditions, primary mouse cells can be convinced to proliferate for
extended periods of time in vitro (9). Tissue culture stress
signals induce expression of a number of anti-proliferative genes,
including p16INK4A and p19ARF (10, 11). The
induction of p19ARF appears more relevant than the
induction of p16INK4A, as mouse embryo fibroblasts (MEFs)
genetically deficient for p19ARF are resistant to induction
of senescence and readily become immortal (12), whereas
p16INK4A-deficient MEFs senesce normally (13, 14).
Likewise, MEFs lacking the downstream effector of p19ARF,
p53, are immortal (15), whereas MEFs lacking the downstream effector of
p16INK4a, pRb, are mortal (16). However, MEFs lacking all
three pRb family members, pRb, p107 and p130 are immortal (17, 18). These data indicate that the Rb family proteins not only act
upstream of the p19ARF-p53 pathway, through regulation of
p19ARF by E2F (19), but also downstream by rendering
cells insensitive to p53 signaling (20).
Expression of oncogenes, such as an activated RAS oncogene, can further
enhance tissue culture stress signals and induce rapid onset of
senescence, referred to as "premature senescence" (21). Oncogenic
RAS stimulates many of the same anti-proliferative genes that are
induced by spontaneous senescence, including p19ARF and
p16INK4A, and again only ablation of the
p19ARF-p53 pathway allows escape from oncogene-induced
premature senescence to cause oncogenic transformation (12, 21). These
observations have led to the suggestion that premature senescence is
part of a fail-safe mechanism that protects cells from oncogenic
transformation (22).
Senescence in human cells differs from senescence in rodent cells in
that most primary human cells lack the catalytic component of
telomerase, hTERT. As a consequence, in vitro propagation of primary human cells is associated with erosion of the chromosome ends,
the telomeres, leading to DNA damage-like anti-proliferative signals
when telomeres become critically short (23-25).
Consequently, most human cells require expression of telomerase to
overcome this barrier to immortality. However, similar to rodent cells, primary human cells (especially those of epithelial origin) also suffer
from "tissue culture stress" and often arrest long before their
telomeres are critically short (8, 26). This tissue culture stress
response of primary human epithelial cells appears to depend on
p16INK4a rather than on p14ARF (4, 27).
However, several diploid human fibroblasts can be immortalized by hTERT
expression, suggesting differential sensitivity of primary human cells
to tissue culture stress.
It has been proposed that the stress-induced replicative arrest induced
by tissue culture stress should be referred to as "stasis" (for
"stimulation and stress induced senescence"), whereas the term
"senescence" should be used for cells that undergo replicative arrest as a result of DNA-damage signals emanating from short telomeres. The different names for both forms of senescence suggest that the mechanisms that underlie both processes are distinct. However,
the signaling pathways involved and the phenotypic consequences of both
forms of replicative arrest are related. For this reason, the study of
rodent cell stasis is likely to be relevant for our understanding of the signaling mechanisms that underlie replicative aging of primary human cells in culture.
We describe here a novel vector system to study the genes that are
required to maintain senescence. We created a virus that can infect
post-mitotic cells and direct the synthesis of short hairpin
transcripts that mediate post-transcriptional gene silencing through
RNA interference. We used this vector system to ask if senescence is a
reversible process.
Plasmid Construction--
The murine p53-specific short hairpin
oligonucleotides were first cloned in pRETRO-SUPER (28).
pRETRO- SUPER vector was digested with BglII and
HindIII, and the annealed oligonucleotides targeting murine
p53,
5'-gatccccGTACATGTGTAATAGCTCCttcaagagaGGAGCTATTACACATGTACtttttggaaa-3' and
5'-agcttttccaaaaGTACATGTGTAATAGCTCCtctcttgaaGGAGCTATTACACATGTACggg-3', were ligated with the vector, yielding pRETRO-SUPER-p53. The 19-mer p53
targeting sequence in the oligonucleotide is indicated in capital
letters. The lentiviral transfer vector HIV-CS-CG (29) was digested
with EcoRI and XhoI to remove the CMV-GFP
sequence. The cassette containing the H1 promoter and the p53 target
sequence was excised from pRETRO-SUPER-mp53 with EcoRI and
XhoI and ligated into HIV-CS to yield pLENTI-SUPER-p53.
Cell Culture, Lentiviral Production, and Infection--
Wild
type Friend virus B-strand (FVB) MEFs,
ST.HdhQ111 mouse striatum cells (30), and 293T
cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum. For production of lentivirus,
293T cells were transfected by the calcium-phosphate method using 10 µg transfer vector HIV-CS-CG or pLENTI-SUPER-p53, 3.5 µg of VSVg
envelope vector pMD.G, 2.5 µg of RSV-Rev, and 6.5 µg of packaging
vector pCMVDR8.2 (29). Lentiviruses were harvested 24 and 48 h
after transfection and filtered through a 0.45-µM filter.
ST.HdhQ111 cells were shifted to 39 °C 14 days prior to lentiviral infection. WT MEFs were cultured to passage
9-10 whereupon cells were counted every 3-4 days 14 days prior to
lentiviral infection. The senescent phenotype was also investigated by
acidic Western Blot Analysis--
Whole cell extracts were separated on
12% SDS-PAGE gels and transferred to polyvinylene diflouride membranes
(Millipore). Visualization was done using enhanced chemiluminescence
(Amersham Biosciences, Inc.) Antibodies used were M-156 (Santa Cruz)
against p16INK4a, ab80-50 (Abcam) against
19ARF, F-5 (Santa Cruz) against p21, Ab-7 (Oncogene)
against p53, and P30620 (Transduction Laboratories) against PAI-1.
Time-lapse Microscopy--
5 × 104 senescent
MEFs were seeded in 3.5-cm dishes and infected with lentivirus.
Time-lapse microscopy was initiated 34 h after infection in a
temperature and CO2-controlled chamber using 10× phase
contrast. Frames were taken every 20 min over a period of 38 h.
We have recently described a vector, pSUPER, which mediates highly
specific and persistent RNA interference through stable expression of
short hairpin RNAs (28). We generated a lentiviral derivative of this
vector by cloning the H1 RNA short hairpin gene expression cassette
targeting murine p53 from pRETRO-SUPER (32) into the self-inactivating
lentiviral vector pHIV-CS (29). We named this vector pLENTI-SUPER-p53
(Fig. 1A). As a control, we
used a lentiviral vector that expresses GFP (HIV-CS-CG (29)).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase, plasminogen activator inhibitor-1 (PAI-1),1 and p21cip1
(5).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase staining at the time of infection (31).
1.8 × 105 senescent WT MEFs in 6 cm dishes were
infected with lentivirus for at least 12 h in the presence of 0.8 µg/ml polybrene and were then allowed to recover for 48 h before
reseeding for colony formation assays and growth curves. 0.5 × 105 or 1 × 105 cells were seeded in 10 cm
dishes for colony formation assays. Cells were fixed and stained with
superstain (50% methanol, 10% acetic acid, 0.1% Coomassie Blue) 16 days after seeding. For growth curves 1.5 × 103 cells
were seeded per 3.5-cm dish, at 3-day intervals cells were fixed with
0.5% formaldehyde, stained with 0.1% crystal violet, followed by
re-solubilization in 10% acetic acid. The OD590 was quantified as a relative measure of cell number.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (23K):
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Fig. 1.
A lentiviral vector that mediates RNA
interference. A, a schematic overview of the lentiviral RNA
interference vector pLENTI-SUPER-p53 (pLS-p53). A DNA
fragment containing the H1 promoter and an oligonucleotide insert
targeting murine p53 were transferred from pRETRO-SUPER (32) to HIV-SC
(29) by digestion of both vectors with EcoRI and
XhoI, followed by ligation of the H1-p53 DNA fragment into
HIV-CS. This vector contains a deletion in the U3 region indicated by a
triangle that silences the enhancer of the LTR, efficiently shutting
off LTR-driven transcription after proviral integration. The predicted
short hairpin RNA targeting murine p53 is shown. B, passage
3 FVB wild type MEFs were infected with either HIV-CS-CG
(CMV-GFP) lentivirus or LENTI-SUPER-p53 virus, respectively.
Fortyeight hours after infection 5 × 104 cells were
seeded in 10-cm dishes for colony formation assays and stained after 14 days. C, 48 h after infection 1.5 × 103 cells were seeded per well in six-well dishes. At
varying time intervals cells were fixed and stained with crystal
violet, which was then solubilized with 10% acetic acid and quantified
at OD590 as a relative measure of cell number. CMV-GFP and
pLS-p53 curves are marked in gray and black,
respectively.
Loss of p53 in primary mouse embryo fibroblasts is associated with acquisition of an immortal phenotype (15). To test whether the lentiviral p53 knockdown vector was capable of inducing a functional inactivation of p53 in MEFs, we infected early-passage primary MEFs with LENTI-SUPER-p53 virus or with control GFP lentivirus and asked whether p53 knockdown caused immortalization. Some 30-40% of control lentivirus-infected cells were GFP-positive, indicating that the primary MEFs were efficiently infected by the lentiviral vectors (data not shown). Fig. 1, B and C, show that infection with LENTI-SUPER-p53, but not with control GFP lentiviral vector, caused efficient immortalization of the infected primary MEFs, indicating that the LENTI-SUPER-p53 virus mediates functional inactivation of p53 expression (see also Fig. 3A).
We next asked whether suppression of p53 expression by lentiviral gene
transfer in senescent cells would allow re-entry into the cell cycle.
We employed two cell systems to address this question. First, we used
conditionally immortalized STHdhQ111 neuronal cells derived
from mouse embryonic striatum. These cells are conditionally
immortalized due to the presence of a temperature-sensitive allele of
SV40 T antigen (30). STHdhQ111 cells proliferate
indefinitely at the permissive temperature (32 °C), but rapidly and
synchronously become post-mitotic and adopt a senescent morphology when
shifted to the non-permissive temperature (39.5 °C) at which T
antigen is inactive (33). We used STHdhQ111 cells that had
been maintained at 39.5 °C for 2 weeks to assure that the entire
population was senescent and then infected the senescent cells with the
LENTI-SUPER-p53 virus or control GFP lentivirus and maintained the
infected cells at 39.5 °C for 2 weeks. Fig.
2A shows that knockdown of p53
led to re-entry into the cell cycle and allowed continued
proliferation, indicating that the senescence-like growth arrest of
STHdhQ111 cells at the non-permissive temperature can be
reversed by suppression of p53.
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Next we asked whether p53 knockdown would allow cell cycle re-entry in
senescent primary MEFs. We cultured primary MEFs of FVB genotype until
the cells no longer proliferated (Fig. 2D) and expressed
high levels of the senescence-associated markers acidic
-galactosidase, PAI-1, p21cip1, p19ARF and
p16INK4a (Figs. 2E and
3A). All cells in the culture
showed a flat senescent morphology and stained intensely for acidic
-galactosidase (Fig. 2E), indicating that these cells
were quantitatively senescent. This notion is also supported by the
growth curves of these late-passage MEFs, which showed a constant
decline in cell number over time (Fig. 2D), indicative of
the absence of spontaneously immortalized cells in the culture. Fig. 2,
B and C, show that lentiviral knockdown of p53 in
these senescent primary MEF cultures triggered a marked degree of
proliferation. Importantly, cell cycle re-entry was associated with
loss of expression of several of the senescence-associated markers,
including PAI-1, p21cip1, and acidic
-galactosidase (Fig. 3,
A and B) and senescence-reverted cells continued
to proliferate for several weeks without any signs of senescence,
suggesting that they had become immortal (Fig. 2B and data
not shown).
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In principle, the observed proliferation following lentiviral knockdown
of p53 could originate from cells that were not truly senescent in the
culture. It was therefore important to follow the cultures of senescent
MEFs in time after lentiviral infection. Fig.
4 shows a series of time-lapse
photomicrographs of senescent MEFs after lentiviral knockdown of p53,
which together indicate that cells with a completely flat and senescent
morphology round up and divide within 48 h after infection with
the p53 knockdown virus (Fig. 4, cells marked by black
arrows). However, not all cell divisions are productive as many
cells divide initially, but die by apoptosis during division or just
after completion of cell division (Fig. 4, cells marked by white
arrows). Assessed by time-lapse photography and colony formation
efficiencies (Fig. 2B), ~0.5-1% of infected cells divide
successfully. A complete movie of the senescent MEFs after infection
with the p53 knockdown vector is provided as supplementary material. No
division or apoptosis could be observed following infection with
control lentivirus encoding GFP (data not shown). We conclude that
cells with all the hallmarks of fully senescent cells rapidly re-enter
the cell cycle after p53 knockdown. We conclude that p53 is not only
required to initiate senescence, but is also required, at least in
MEFs, to maintain senescence.
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DISCUSSION |
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Using a lentiviral vector system that silences gene expression, we
provide evidence that suppression of p53 expression in senescent MEFs
leads to a reversion of the senescent state and causes immortalization.
Several lines of evidence support the notion that the MEFs were fully
senescent at the time of infection with the lentiviral p53 knockdown
vector. First, the cells had stopped proliferating in the presence of
growth factors, indicating that they were senescent and refractory to
growth factor stimulation, rather than quiescent and still responsive
to growth factors (Fig. 1D). Second, they uniformly
manifested a senescent morphology and expressed the
senescence-associated markers acidic -galactosidase, PAI-1,
p21cip1, p19ARF and p16INK4a (Figs.
2E and 3A). When cells emerged from senescence as
a result of p53 knockdown, the cells behaved phenotypically as p53 null MEFs in that they were immortal and had low levels of p21cip1
and high levels of p19ARF (10, 12, 15). Importantly, the
cells that emerged from senescence by p53 knockdown maintained high
levels of p16INK4a (Fig. 3A). As
p16INK4a expression is induced during senescence in a
p53-independent fashion (10), these data indicate that the signaling
pathways that led to the induction of senescence are still operational in senescence-reverted MEFs. This provides further evidence that the
cells that re-entered cell cycle by p53 knockdown were indeed fully
senescent at the time of infection with the p53 knockdown virus.
Our data are in agreement with earlier experiments performed in
senescent human diploid fibroblasts. Thus, ablation of p53 function by
microinjection of p53 antibody in primary human fibroblasts allowed at
least temporary reversal of senescence and re-entry into the cell cycle
(34). However, inactivation of p53 in human fibroblasts delays, but
does not abrogate, replicative senescence, indicating that p53
inactivation alone is not sufficient to mediate stable reversion of
senescence in primary human fibroblasts and requires also induction of
hTERT expression (35, 36). An essential feature of the lentiviral
vector system described here is that suppression of gene expression is
persistent, allowing the study of long term consequences of gene
inactivation in post-mitotic cells. The LENTI-SUPER vector should
therefore be a useful tool to investigate which genes are continuously
required to maintain a post-mitotic state in cells that have exited the
cell cycle.
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ACKNOWLEDGEMENTS |
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We thank Dr. Inder Verma for the self-inactivating lentiviral vector, Lauran Oomen for invaluable assistance with the time-lapse microscopy, Thijn Brummelkamp and Roderick Beijersbergen for discussions, and Katrien Berns for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by a grant from the Center for Biomedical Genetics.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains a movie.
Supported by a long term fellowship of EMBO.
§ To whom correspondence should be addressed. Tel.: 31-20-512-1952; Fax: 31-20-512-1954; E-mail: r.bernards@nki.nl.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.C300023200
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ABBREVIATIONS |
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The abbreviations used are: PAI-1, plasminogen activator inhibitor-1; FVB, Friend virus B-strand; MEF, mouse embryo fibroblast; CMV, cytomegalovirus; GFP, green fluorescent protein; WT, wild type; LTR, long terminal repeat.
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