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Differential requirement for p19ARF in the
p53-dependent arrest induced by DNA damage,
microtubule disruption, and ribonucleotide depletion
Shireen Hussain Khan*
†
, Janet Moritsugu*, and Geoffrey M. Wahl*
‡
*Gene Expression Laboratory, The Salk Institute, La Jolla, CA 92037; and †Department of Biology, University of California at San Diego, La Jolla, CA 92037
Communicated by Tony Hunter, The Salk Institute for Biological Studies, San Diego, CA, December 21, 1999 (received for review July 9, 1999)
p19ARF has been implicated as a key regulator of p53 stability and
activation. While numerous stresses activate the p53 growth arrest
pathway, those requiring p19ARF remain to be elucidated. We
used p19ARF knockout mouse embryo fibroblasts to show that
DNA damage and microtubule disruption require p19ARF to induce
p53 responses, whereas ribonucleotide depletion and inhibition of
RNA synthesis by low doses of actinomycin D do not. The data
provide evidence that the arrest pathway activated by ribonucle-
otide depletion involves some different signal transducers than
those activated by DNA damage or microtubule disruption. We also
present biochemical analyses that provide insights into the mech-
anism by which p53 and p19ARF cooperate in normal cells to induce
cell cycle arrest.
The tumor suppressors p53 and pRb participate in signal
transduction pathways that respond to various environmen-
tal and intracellular challenges, such as hypoxia, ionizing and
ultraviolet radiation, ribonucleotide depletion, microtubule dis-
ruption, and oncogene activation (1– 8). The responses to these
stresses range from induction of a reversible cell cycle arrest
when DNA damage is not incurred to damage-dependent acti-
vation of pathways that lead to premature senescence or apo-
ptosis. The net result of activation of the pathway, therefore, is
to remove cells exposed to stressing conditions from the repli-
cative cycle. During neoplastic progression, there is a strong
selection for mutations that inactivate the p53–pRb pathways,
thus enabling cells to avoid apoptosis or senescence and to
continue dividing under conditions that can increase genetic
instability (9, 10).
Recent experiments have focused attention on the Ink4a兾ARF
locus, which has the potential to regulate both p53 and pRb
function. The p16Ink4a protein (inhibitor of cdk4) ensures that
pRb remains active by preventing the inhibitory phosphoryla-
tions by cyclin D兾CDK4,6 (11). p19ARF (mouse; p14ARF,
human), partly encoded by exons 2 and 3 of the p16Ink4a gene
in an alternative reading frame, regulates the stability of p53 by
binding to mdm2 (12–15). mdm2 and p53 participate in an
autoregulatory feedback loop in which p53 induces mdm2 ex-
pression (16). mdm2, in turn, mediates p53 degradation and
prevents p53 association with the basal transcriptional machin-
ery (17–19). The binding of mdm2 to p53 is affected by phos-
phorylation of p53 at the N terminus, which compromises mdm2
binding (20, 21). More recently, mdm2 was shown to be regulated
by interaction with p19ARF, which binds to and alters the
subcellular localization of mdm2 so that it has reduced access to
p53 (13, 14, 22, 23).
The available data predict that disruption of the Ink4a兾ARF
locus alone should disable the two tumor suppressors most
commonly mutated in human cancers. Indeed, mice lacking
functional p16Ink4a and兾or p19ARF develop normally but are
highly susceptible to tumor formation (11, 24). However, the
frequency of mutations at the Ink4a and兾or ARF locus is not as
high as the frequency of p53 mutations in human cancers,
suggesting that loss of p53 confers a greater advantage during
tumor progression.
The data discussed above raise the possibility that p19ARF
functions in some, but not all, of the p53-responsive pathways
such that loss of p19ARF may not be functionally equivalent to
loss of p53. It has been suggested that oncogene-mediated
activation of p53 requires p19ARF, and occurs in the absence of
DNA damage (25, 26). By contrast, damage induced by ionizing
radiation (IR) involves p53 activation through N-terminal phos-
phorylations and other modifications, but is reported to be
independent of p19ARF (20, 24, 27). However, E1A-expressing
p19ARF
⫺/⫺
mouse embryo fibroblasts (MEFs) were nearly as
radioresistant after exposure to IR as E1A-expressing p53
⫺/⫺
MEFs (25), and p19ARF deficiency prevents induction of pre-
mature senescence in ATM
⫺/⫺
MEFs (28). One interpretation of
the data is that loss of p53 or p19ARF reduces the E1A-induced
radiosensitivity because of the requirement for these proteins in
arrest responses triggered by E1A. Alternatively, the data raise
the possibility that the observed radioresistance and rescue of
premature senescence is due to the requirement for p53, and
possibly p19ARF, in the arrest induced by DNA damage. To
distinguish between these possibilities, we assessed the role of
p19ARF in the p53-mediated arrest responses induced by IR as
well as other stresses that do not induce DNA damage.
Materials and Methods
Cell Culture. Pools of wild-type, p53
⫹/⫺
, p19ARF
⫺/⫺
, p53
⫺/⫺
, and
p16
⫺/⫺
p19ARF
⫺/⫺
double-knockout MEFs were maintained in
DMEM (Cellgro, Waukesha, WI) containing 10% dialyzed fetal
bovine serum, kanamycin sulfate (GIBCO), L-glutamine
(GIBCO), penicillin兾streptomycin (GIBCO), and nonessential
amino acids (GIBCO). p19ARF
⫺/⫺
MEFs (passage 6) were
generously provided by Charles Sherr and Martine Roussel;
p16
⫺/⫺
p19ARF
⫺/⫺
double-knockout MEFs (passage 3) were
kindly provided by Manuel Serrano. Cells were maintained at
37°C in an atmosphere containing 7% CO
2
.
Cell Cycle Analysis. Cell cultures were split 1:3 or 1:4 into media
containing 0.05
g兾ml nocodazole, 5 nM actinomycin D, or 100
MN-phosphonacetyl-L-aspartate (PALA), or were exposed to
␥
radiation by using a Gammabeam 150-C irradiator with a
60
Co
source at a dose rate of 2 Gy兾min. At the indicated time points,
the fibroblasts were pulse labeled with 10
M BrdUrd for 1 h
before fixation in 70% ethanol. Analysis of BrdUrd-labeled cells
was performed as described previously (3). For DNA content
analysis, cells were stained with propidium iodide only. The
samples were analyzed on a Becton Dickinson FACScan and
Abbreviations: IR, ionizing radiation; MEFs, mouse embryo fibroblasts; PALA, N-
phosphonacetyl-L-aspartate.
‡To whom reprint requests should be addressed at: The Salk Institute, Gene Expression
Laboratory, 10010 North Torrey Pines Road, La Jolla, CA 92037. E-mail: wahl@salk.edu.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073兾pnas.050560997.
Article and publication date are at www.pnas.org兾cgi兾doi兾10.1073兾pnas.050560997
3266–3271
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PNAS
兩
March 28, 2000
兩
vol. 97
兩
no. 7
quantitated by gating events on dot plots by using CELLQUEST
software. All experiments were repeated a minimum of three
times, 10,000 events were analyzed per sample, and data from
representative experiments are shown.
Western Blot Analysis. Treated cells were lysed in RIPA buffer (150
mM NaCl兾1% Nonidet P-40兾0.5% sodium deoxycholate兾0.1%
SDS兾50 mM Tris䡠HCl, pH 8.0) supplemented with fresh 1 mM
PMSF, 1 mM sodium vanadate, 1 mg兾ml leupeptin, 1 mg兾ml
aprotinin, 1 mg兾ml pepstatin, 1 mg兾ml 1,10-phenanthroline䡠HCl,
and 160
g兾ml benzamidine䡠HCl. Lysates were sonicated and
protein concentrations were quantitated with a Bio-Rad protein
assay (modified Lowry assay). Fifty or 100
g of protein was
resolved by electrophoresis on a 10% (p21 and p53) or 12%
(p19ARF) polyacrylamide gel and transferred to a nitrocellulose
membrane (Schleicher and Schuell). p53 was detected by using a
mixture of monoclonal antibodies 421 at 1:50 (Ab-1, Oncogene
Science) and 240 at 1:100 (Ab-3, Oncogene Science). p21 was
detected by using the polyclonal antibody C-19 (Santa Cruz Bio-
technology) diluted 1:100; p19ARF was detected by using a poly-
clonal antibody (200-106; Novus Biologicals, Littleton, CO) diluted
1:200; and protein loading was measured with an anti-actin poly-
clonal antibody (A2066, Sigma) diluted 1:50. Membranes were
incubated with secondar y antibodies conjugated to horseradish
peroxidase, and proteins were detected by using ECL chemilumi-
nescence reagents according to the manufacturer’s protocol (New
England Nuclear). Bands were quantitated by computer scan
densitometry (NIH IMAGE), experiments were repeated at least
three times, and representative blots are shown. In all cases, actin
was used as an internal control to enable quantification of relative
band intensities.
Results
p19ARF
ⴚ/ⴚ
MEFs Become Arrested After Extended Exposure to PALA.
p19ARF has been proposed to mediate p53 activation in response
to oncogene overexpression in a manner that is independent of
DNA damage (25). Depletion of ribonucleotide pools in cells also
activates p53 without producing DNA damage in human and mouse
fibroblasts (3, 29). Therefore, we determined whether p19ARF is
involved in the activation of p53 after exposure to the UMP
synthesis inhibitor PALA (30). As previous reports showed no
significant differences in the results obtained after ribonucleotide
depletion of asynchronous or G
0
兾G
1
synchronized MEFs, the
PALA studies reported here were performed on exponentially
growing populations of MEFs (31).
Fig. 1 shows that PALA induces a cell cycle arrest in MEFs
regardless of their p19ARF status. Approximately 20–25% of
untreated wild-type, p53
⫹/⫺
, p53
⫺/⫺
, or p19ARF
⫺/⫺
MEFs were
in S phase (BrdUrd positive) in exponentially growing popula-
tions (Fig. 1 A–D). The fraction of BrdUrd-positive p53
⫹/⫺
,
p53
⫺/⫺
, and p19ARF
⫺/⫺
MEFs increased after 24 h in PALA,
presumably because of depletion of ribonucleotide and deoxy-
ribonucleotide pools, which slows the progression of replication
forks (32, 33). After 48 h of PALA treatment, the fraction of
S-phase cells decreased in wild-type, p53
⫹/⫺
, and p19ARF
⫺/⫺
populations (Fig. 1 A, B, and D), whereas the percentage of
BrdUrd-positive p53
⫺/⫺
MEFs remained elevated, as observed
previously (Fig. 1C) (31). By 72 h, significantly fewer wild-type
and p19ARF
⫺/⫺
MEFs were in S phase compared with untreated
populations or PALA-treated p53
⫺/⫺
MEFs. Although the per-
centage of p53
⫺/⫺
S-phase cells decreased between 48 and 72 h
of PALA treatment, the fraction of cycling cells was as high as
in the untreated population, and significantly higher than
PALA-treated wild-type, p53
⫹/⫺
, or p19ARF
⫺/⫺
populations.
There appears to be a slight increase in the population of cells
in S phase that are BrdUrd negative (below the diagonal line) in
wild-type, p53
⫹/⫺
, and p19ARF
⫺/⫺
MEFs; however, this popu-
lation accounts for only 5% of the total population. As reported
previously, a majority of the PA LA-treated MEFs that were in
S phase at the beginning of the experiment appear to accumulate
in G
2
兾M, as this population shows a 10% increase relative to the
G
2
兾M population in untreated cells (31). p16
⫺/⫺
p19ARF
⫺/⫺
double-knockout MEFs also became arrested after 72 h of
PALA treatment, suggesting that the loss of pRb regulation does
not abrogate the arrest response (data not shown). These data
clearly show that the p53-mediated arrest induced by PALA is
intact in MEFs lacking p19ARF function.
The p53-dependent cell cycle arrest induced by PALA re-
quires activation of p53 target genes such as p21 (3, 31). An
analysis of the p21 protein levels in p19ARF
⫺/⫺
MEFs revealed
that p21 was up-regulated 48 h after exposure to PALA com-
pared with untreated cells (Fig. 1E). The fold induction of p21
(normalized with actin protein loading) is approximately 2-fold
in p19ARF
⫺/⫺
and p53
⫹/⫺
MEFs after 48 h of PALA treatment.
This finding is consistent with the previous observation that
p19ARF function is not required for the arrest induced by
ribonucleotide depletion. Induction of p21 was not observed in
p53
⫺/⫺
MEFs treated with PALA for 48 h, which supports
previous reports that p21 expression is activated by p53 in
response to ribonucleotide depletion (data not shown) (3).
To assess whether ribonucleotide depletion was unique in
eliciting a p53-dependent arrest that is independent of p19ARF,
we determined whether the arrest induced by the intercalating
agent actinomycin D required p19ARF function. Recent data
indicate that low doses (20 nM) of actinomycin D did not induce
DNA double-strand breaks but were sufficient to inhibit tran-
scription and activate p53 in normal human fibroblasts, whereas
doses of 200 nM induced DNA damage (34, 35). We found that
p53
⫹/⫺
and p19ARF
⫺/⫺
MEFs become arrested after 24 h of
Fig. 1. p19ARF-null MEFs become arrested during ribonucleotide depletion.
Asynchronous populations of wild-type (WT), p53⫹/⫺, p53⫺/⫺, and p19ARF⫺/⫺
MEFs were untreated or treated with 100
M PALA for 24, 48, or 72 h. Cells
were pulse labeled with BrdUrd and analyzed by flow cytometry. Cells that
incorporated BrdUrd are shown above the diagonal line. The percentages of
BrdUrd-positive cells are shown in the upper right corner of each plot. Rep-
resentative dot plots for untreated or PALA-treated MEFs are shown in A(WT),
B(p53⫹/⫺), C(p53⫺/⫺), and D(p19ARF⫺/⫺). (E) Western blot analysis of p21 and
actin after 48-h PALA treatment of p53⫹/⫺and p19ARF⫺/⫺MEFs. Relative band
intensities were quantified in all Western analyses with actin as an internal
control.
Khan et al. PNAS
兩
March 28, 2000
兩
vol. 97
兩
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兩
3267
CELL BIOLOGY
exposure to 5 nM actinomycin D, whereas most of the p53
⫺/⫺
MEFs continue cycling (Table 1). Moreover, 5 nM actinomycin
D treatment induced a reversible arrest in wild-type and
p19ARF
⫺/⫺
MEFs; that is, once the drug was removed, the cells
resumed cycling as efficiently as untreated populations (data not
shown). This result provides further evidence that the arrest
induced by 5 nM actinomycin D is unlikely to result from residual
DNA damage. Therefore, two treatments that reduce RNA
synthesis in the absence of detectable DNA damage activate a
cell cycle arrest requiring p53, but not p19ARF.
p19ARF
ⴚ/ⴚ
MEFs Show an Increase in Polyploidy After Nocodazole
Treatment. We and others showed previously that the microtu-
bule-depolymerizing agents nocodazole and Colcemid provoke
an arrest in a 4NG
1
-like state in cells with an intact p53–pRb
pathway (5, 36–39). By contrast, mutants with defects in this
pathway undergo rereplication during continuous exposure to
these drugs. As with PALA, the arrest induced by these agents
is largely reversible and is not associated with detectable DNA
damage (39). It was shown previously that p16
⫺/⫺
MEFs also
undergo rereplication after exposure to such drugs (39). How-
ever, these MEFs have nonfunctional p16Ink4a and p19ARF
because of targeted disruption of exons shared by the two
proteins (11). The dual inactivation of the proteins in this mutant
raised the important question of whether this damage-
independent stress required p19ARF to induce a cell cycle
arrest, or whether p16Ink4a deficiency alone allowed rereplica-
tion to occur.
We investigated this possibility by determining whether no-
codazole induced rereplication in MEFs solely lacking p19ARF.
Asynchronous populations of p53
⫹/⫺
, p53
⫺/⫺
, p19ARF
⫺/⫺
, and
p16
⫺/⫺
p19ARF
⫺/⫺
MEFs were treated with 0.05
g兾ml nocoda-
zole for 48 h, after which polyploidy was measured by DNA
content analysis (see Materials and Methods). After 48 h in
nocodazole, 8.7% of p53
⫹/⫺
MEFs were polyploid compared
with 60.3% in p53
⫺/⫺
MEFs, 18.3% in p19ARF
⫺/⫺
MEFs, and
37.9% in p16
⫺/⫺
p19ARF
⫺/⫺
double-knockout MEFs (Fig. 2 and
Table 1). In three independent experiments, the percentage of
polyploid cells in nocodazole-treated p19ARF
⫺/⫺
populations
consistently doubled compared with the untreated control (Fig.
2Cand Table 1). Furthermore, the percentage of cells with
greater than 4NDNA content was consistently higher in the
p16
⫺/⫺
p19ARF
⫺/⫺
double-knockout MEFs (Fig. 2C), suggesting
that the loss of both tumor suppressors is additive in this
response. While it remains to be determined whether the loss of
p16Ink4a alone induces polyploidy after microtubule disruption,
we predict this is likely, as pRb deficiency produces substantial
polyploidy (⬇50%) under the same conditions (36).
The polyploid population was significantly higher in p53
⫺/⫺
MEFs compared with either p19ARF
⫺/⫺
or p16
⫺/⫺
p19ARF
⫺/⫺
MEFs, indicating that the loss of p19ARF is not equivalent to
loss of p53 in the nocodazole-induced arrest response. Similarly,
a reduced level of polyploidy was observed in p21
⫺/⫺
MEFs
compared with MEFs lacking p53 (39). This observation may be
due to a p53-dependent activation of additional pathways that
may compensate for the loss of p16, p19ARF, or p21, such that
pRb is eventually restored to the antiproliferative form to
promote cell cycle arrest.
p19ARF
ⴚ/ⴚ
MEFs Exhibit a Defective DNA-Damage Response. The
p53-dependent cell cycle arrest induced by ionizing radiation
requires prolonged induction of p21 (7, 40). Previous reports
indicated that p53 and p21 induction occurs in p19ARF
⫺/⫺
Table 1. ARF dependence of p53-mediated stress responses
MEFs
%S
␥
/% S untreated % BrdUrd-positive Polyploidy
T48 Noc5 Gy 6 Gy 7 Gy 20 Gy Untreated T72 PALA T24 Act-D
p53
⫹/⫺
39.6 ⫾0.5 36.9 ⫾1.8 34.4 ⫾5.2 25.9 ⫾4.5 22.8 ⫾2.4 12.0 ⫾3.1 5.1 ⫾0.1 6.5 ⫾3.4
p53
⫺/⫺
101.0 ⫾1.4 100.8 ⫾3.2 105.2 ⫾8.3 83.5 ⫾0.7 24.4 ⫾3.8 27.7 ⫾3.3 20.8 ⫾5.4 60.1 ⫾2.2
p19ARF
⫺/⫺
75.0 ⫾9.9 85.0 ⫾2.8 81.5 ⫾0.7 64.5 ⫾0.7 19.7 ⫾1.2 6.9 ⫾2.6 4.6 ⫾0.5 26.1 ⫾4.9
Asynchronous populations of p53⫹/⫺, p53⫺/⫺, and p19ARF⫺/⫺MEFs were irradiated with 5, 6, 7, or 20 Gy, pulse-labeled with BrdUrd, and fixed 24 h later. The
values represent the percentage of BrdUrd-positive cells in
␥
-irradiated cultures (% S
␥
) relative to untreated. Cells were also untreated or treated with 100
M
PALA for 72 h or 5 nM actinomycin D for 24 h, and the percentage of BrdUrd-positive cells is shown. The polyploid fraction was assessed by treating cells with
0.05
g/ml nocodazole for 48 h and quantitating the percentage of cells with ⬎4NDNA content. The values represent the mean of at least two independent
experiments and standard deviations are shown.
Fig. 2. Increased polyploidy in p19ARF-null fibroblasts after nocodazole
treatment. Asynchronous cultures of p53⫹/⫺, p53⫺/⫺, p19ARF⫺/⫺, and
p16⫺/⫺p19ARF⫺/⫺MEFs were treated with or without 0.05
g兾ml nocodazole
for 48 h, fixed, and stained with propidium iodide for FACS analysis. Values in
the upper right corner of each plot represent the percentage of cells with ⬎4N
DNA content. Histogram plots of untreated or nocodazole-treated MEFs are
shown in A(p53⫹/⫺), B(p53⫺/⫺), C(p19ARF⫺/⫺), and D(p16⫺/⫺p19ARF⫺/⫺).
3268
兩
www.pnas.org Khan et al.
MEFs 4 h after IR, but protein analyses at later time points were
not carried out and the stringency of the cell cycle arrest was not
shown (24). The increased viability of E1A-expressing
p19ARF
⫺/⫺
MEFs after IR invites the speculation that these
cells are compromised in their ability to undergo cell cycle arrest
or apoptosis in response to IR (25). Hence, we assessed whether
the p19ARF
⫺/⫺
MEFs exhibit an altered cell cycle response
after IR.
The cell cycle responses of asynchronous p53
⫹/⫺
, p53
⫺/⫺
, and
p19ARF
⫺/⫺
MEFs are shown in Fig. 3 and Table 1. In the
experiment shown, the percentage of BrdUrd-positive cells in
irradiated populations relative to untreated cells was approxi-
mately 37% for p53
⫹/⫺
MEFs, 103% in p53
⫺/⫺
MEFs, and 87%
in p19ARF
⫺/⫺
MEFs 24 h after exposure to 6 Gy of
␥
-radiation
(Fig. 3 A, B, and C, respectively). This observation suggests that
loss of p19ARF results in a defective DNA-damage arrest
pathway even though these cells express wild-type p53 and p21.
The experiments were repeated a minimum of five times at
passages 8–10 with various radiation dosages (Table 1). Similar
results were observed in irradiated populations of p19ARF
⫺/⫺
MEFs that were synchronized in G
0
兾G
1
by serum deprivation
(data not shown). We also found that the percentage of irradi-
ated cells in S phase relative to untreated was ⬇70% in
p16
⫺/⫺
p19ARF
⫺/⫺
MEFs after 24 h (data not shown), suggesting
that the loss of pRb regulation does not augment the defective
DNA-damage arrest created by loss of p19ARF alone.
The data show that p19ARF
⫺/⫺
MEFs continue cycling after
DNA damage, although not to the same extent as p53
⫺/⫺
MEFs.
Interestingly, the lack of cell cycle arrest in p19ARF
⫺/⫺
MEFs is
similar to that reported for p21
⫺/⫺
MEFs, in which ⬇80% of the
population continued to cycle 24 h after DNA damage (31). This
observation implies that factors in addition to p19ARF and p21
contribute to the p53-dependent DNA-damage-induced arrest in
wild-type cells.
As the response to ionizing radiation involves rapid p53-
dependent induction of p21, it is conceivable that p53 activation
and p21 induction could be compromised in p19ARF-null cells.
We investigated this possibility by measuring p53 and p21 protein
levels as a function of time after IR. As reported previously, p53
induction is biphasic in wild-type cells, consisting of a rapid
increase, followed by a decrease, and then a second increase to
a level that remained above the uninduced control up to 96 h
after IR (7, 8). The p53 levels in p19ARF
⫺/⫺
MEFs showed a
similar initial increase in abundance 2–10 h after irradiation, but
then diminished to undetectable levels by 24 h and remained
undetectable 48 h after IR (Fig. 4A). In contrast, the p53 levels
were elevated between 1.9- and 3.5-fold at each of the time points
analyzed after IR in wild-type MEFs (Fig. 4A). A possible
explanation for the apparent p53 instability is that the absence
of p19ARF allows a higher proportion of p53 to be bound by
mdm2, which increases p53 turnover.
Consistent with the p53 expression levels, p21 protein levels
started to increase4hafterirradiation in wild-type MEFs and
remained elevated up to 48 h (Fig. 4A). While the p21 levels are
detectable in p19ARF
⫺/⫺
MEFs at all times tested, the induced
levels are lower than observed in wild-type MEFs. Wild-type
MEFs show a 1.9- to 5.0-fold increase in p21 between 2 and 48 h
after exposure to IR, whereas the maximum induction of p21 in
p19ARF
⫺/⫺
MEFs during the same time course is 2-fold.
Furthermore, 24–48 h after IR the levels of p21 are not
increased relative to the untreated control in p19ARF
⫺/⫺
MEFs
(Fig. 4A). p53
⫺/⫺
MEFs showed no detectable levels of p21 at
several time points after exposure to IR; hence the induction of
p21 in this stress response is largely, if not entirely, dependent on
transactivation by p53 (data not shown) (40).
We next determined whether p19ARF is induced by DNA
damage by measuring the p19ARF protein levels at several
intervals after IR. As expected, p19ARF was not present in the
p19ARF-null MEFs, and it is present at high levels in p53
⫺/⫺
MEFs (Fig. 4B) (25, 26). p19ARF levels increased significantly
(2- to 8-fold induction) 2–10 h after
␥
-irradiation in wild-type
MEFs (Fig. 4B). Although the level of p19ARF appears to
Fig. 3. Defective DNA-damage response in p19ARF-null MEFs. Asynchronous
cultures of p53⫹/⫺, p53⫺/⫺, and p19ARF⫺/⫺MEFs were exposed to 6 Gy of
␥
-radiation. After 24 h, cells were pulse labeled with BrdUrd and analyzed by
FACS. Cells above the diagonal line are BrdUrd positive. The values shown in
the upper right represent the percentage of irradiated cells in S phase relative
to untreated (% S irradiated兾% S untreated). Representative plots are shown
for p53⫹/⫺(A), p53⫺/⫺(B), and p19ARF⫺/⫺(C).
Fig. 4. Involvement of p19ARF in the sustained induction of p21 after IR.
Asynchronous populations of wild-type (WT) and p19ARF⫺/⫺MEFs were
treated with 6 Gy of
␥
-radiation and harvested after 2, 4, 6, 8, 10, 24, or 48 h.
For p53 and p21 analysis, 50
g of protein was resolved on an SDS兾10%
polyacrylamide gel. For analysis of p19ARF, 100
g of protein was resolved on
a 12% polyacrylamide gel. (A) p53, p21, and actin protein levels in WT and
p19ARF⫺/⫺MEFs 2 h (T2), 4 h (T4), 6 h (T6), 8 h (T8), 10 h (T10), 24 h (T24), and
48 h (T48) after IR compared with untreated (Unt). (B) p19ARF and actin
protein levels in WT MEFs at the indicated times after IR compared with
untreated (Unt). p53⫺/⫺and p19ARF⫺/⫺MEFs are also shown as positive and
negative controls for p19ARF protein detection.
Khan et al. PNAS
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decrease 24–48 h after IR, the protein levels were still 1.5- to
1.7-fold elevated relative to untreated controls at the latest time
points measured (Fig. 4B). These results show that p19ARF
protein levels increase after exposure to
␥
-radiation.
Discussion
The studies reported here reveal that p19ARF contributes to the
ability of MEFs to undergo p53-dependent cell cycle arrest
induced by IR and by microtubule disruption. The lack of a
damage-induced arrest in p19ARF
⫺/⫺
MEFs correlates with
decreased basal p53 levels and an inability to sustain the
induction of p53. This, in turn, appears to affect the induction of
the CDK inhibitor p21, a key downstream target gene in the
damage response. By contrast, the arrest induced by ribonucle-
otide depletion and actinomycin D treatment is intact in
p19ARF-deficient fibroblasts. The data are consistent with the
model that mdm2 is a key negative regulator of p53 function and
that p19ARF contributes to a subset of p53-dependent arrest
pathways to counteract the inhibitory effects of mdm2.
The requirement for p19ARF in the p53-dependent arrest
induced by DNA damage is consistent with current models that
p19ARF is a regulator of mdm2–p53 interactions (12–15, 22).
p19ARF appears to reduce the ability of mdm2 to interact with
p53 by changing mdm2 subcellular localization (22, 23). Our
observation that p53 levels are reduced in p19ARF-null MEFs
is consistent with the increased p53–mdm2 associations that
should occur in cells lacking p19ARF. It is conceivable that the
reduced p53 levels may contribute to the defective arrest induced
by IR in the p19ARF-null MEFs. However, the arrest induced
by IR appears to be normal in cell lines expressing low amounts
of a transfected wild-type p53 gene and high quantities of
dominant-negative p53 encoded by the mutated endogenous
alleles (e.g., see ref. 41). This observation leads us to favor the
interpretation that it is the altered p53 and p21 induction kinetics
in the p19ARF-null MEFs that prevents them from mounting a
durable arrest response. This view is compatible with the ob-
servation of long-term induction of p53 and p21 in irradiated
fibroblasts that undergo a senescent-like arrest (7, 42). The
difference in the response kinetics at later times suggests that
p19ARF and兾or its induction by DNA damage may contribute
to the sustained activation of p53 and its target genes. While the
mechanism for p19ARF induction remains to be elucidated, it is
tempting to speculate that the transcriptional regulator E2F-1
may be involved, as p19ARF expression can be transactivated by
E2F-1 (43), and recent studies show that IR increases E2F-1
abundance by a posttranslational mechanism (44). We also note
that the rapid induction of p53 and p21 in p19ARF-null MEFs
appears to be equivalent to that of wild-type MEFs. We suggest
that kinases such as ATM, ATR, and others that modify the N
terminus of p53 to limit interactions with mdm2 may be involved
in establishing the early stages of the damage response (20, 21,
45–47).
The involvement of p19ARF in the DNA-damage-inducible
arrest is compatible with studies analyzing the cell cycle re-
sponses and radiosensitivity of MEFs with various genetic
deficiencies in the p53 pathway. p21-null MEFs continue to cycle
to approximately the same extent as p19ARF-null MEFs after
IR, and both are more radioresistant than are wild-type MEFs
(31, 48). By contrast, ATM-deficient MEFs are more radiosen-
sitive than wild-type MEFs, and they senesce prematurely (49,
50). These phenotypes are consistent with the generation and
accumulation of DNA damage when cells cycle in the absence of
the ATM kinase (51). Importantly, premature senescence is
rescued in MEFs doubly deficient in ATM and p19ARF, p53, or
p21 (28, 52, 53). Thus, p19ARF deficiency results in defects
similar to those observed in cells lacking two key members of the
damage-response pathway, p53 and p21. However, it is impor-
tant to note that neither p19ARF, p21, nor p53 deficiency
rescues the radiosensitivity of ATM-deficient mice (28, 53, 54).
This observation is most likely explained by the fact that
intestinal epithelia and other cell types in ATM-deficient mice
appear to be exquisitely sensitive to damage-induced apoptosis
that occurs independent of p53 function (54). Hence, the survival
of ATM-deficient mice after radiation is influenced by factors
outside of the p53 signaling pathway, whereas the isolated cells
of specific tissues appear to be profoundly dependent on p53,
p21, and p19ARF.
Previous studies showed that p19ARF cooperates with p53 to
mediate cell cycle arrest or programmed cell death in response to
activated oncogenes such as E1A, Myc, Ras, and E2F-1 (25, 26, 43,
55). E1A overexpression did not lead to phosphorylation of p53 at
serine-15, which is one residue that is modified after DNA damage
(20, 25, 27). This finding led to the proposal that overexpression of
E1A activates p53 independent of DNA damage. However, phos-
phorylation at serine-15 is just one of many p53 modifications that
occur in response to DNA damage; recent data indicate the
importance of serine-20 phosphorylation in the DNA-damage
response (21, 47). Our data linking p19ARF to the damage-induced
arrest raise the possibility that activated oncogenes may induce
DNA damage, which could then lead to senescence in fibroblasts
with an intact p53 pathway. Indeed, this model would be c ompatible
with numerous reports showing that activated c-myc, ras, or mos can
induce chromosome breakage in cycling cells (refs. 56 –58; O. Vafa
and G.M.W., unpublished results).
Our data demonstrate that stresses such as ribonucleotide de-
pletion or RNA synthesis inhibition clearly function independently
of p19ARF. PALA treatment is associated with increased p53
abundance and activation of p21 transcription, but it remains to be
determined whether induction of mdm2 is involved (3). It is possible
that p19ARF is not required for the arrest induced by stresses that
reduce transcription because such conditions may preferentially
reduce the levels of the unstable mdm2 transcript. Consistent with
this proposal, previous studies showed that low doses of actinomy-
cin D and UV doses that reduce transcription also reduce the
abundance of mdm2 mRNA (44, 59). Similarly, we found reduced
levels of mdm2 protein in PALA-treated cells, but not in those
treated with nocodazole (S.H.K. and G.M.W., unpublished data).
Hence, inhibition of mdm2 expression by agents such as actinomy-
cin D, and possibly PALA, serves as a p19ARF-independent
mechanism to promote the stability of p53.
These and other data also emphasize the complexity of the
biochemical and cell cycle responses to diverse stresses that
activate the p53 pathway. The different responses of cells with
defects in this pathway indicate that additional genes remain to
be elucidated. Thus, as p21
⫺/⫺
and p53
⫺/⫺
MEFs exhibit com-
parable defects in PALA-induced arrest, we surmise that p21 is
a key downstream modulator of this response. By contrast, since
p21
⫺/⫺
and p19ARF
⫺/⫺
MEFs retain a partial ability to undergo
arrest after IR and show less rereplication after nocodazole
treatment than do p53
⫺/⫺
MEFs (Figs. 1, 2, and 3; refs. 31 and
39), we infer that additional upstream effectors and downstream
targets remain to be defined. Additional studies to elucidate the
mechanisms by which p19ARF is activated and the kinetics of
p53, mdm2, and p19ARF associations will enhance our under-
standing of the cellular response to DNA damage and damage-
independent stresses.
We thank Drs. Martine Roussel and Charles Sherr for generously
providing p19ARF
⫺/⫺
MEFs and Dr. Manuel Serrano for kindly pro-
viding p16
⫺/⫺
p19ARF
⫺/⫺
MEFs; Dr. Gretchen Jimenez and Ms. Jayne
Stommel for insightful discussions and critical review of this manuscript;
and Ms. Michelle Beeche for isolating the wild-type and p53
⫹/⫺
MEFs.
These studies were supported by grants to G.M.W. from the National
Institutes of Health (CA61449) and the G. Harold and Leila Y. Mathers
Charitable Foundation and by a National Institutes of Health Predoc-
toral Training Grant to S.H.K. (1T32CA64041).
3270
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