[CANCER RESEARCH 54, 1169-1174, March 1, 1994]

Advances in Brief

WAF1/CIP1 Is Induced in /?53-mediated G! Arrest and Apoptosis1

Wafik S. EI-Deiry, J. Wade Harper, Patrick M. O'Connor, Victor E. Velculescu, Christine E. Canman,
Joany Jackman, Jennifer A. Pietenpol, Marilee Burrell, David E. Hill, Yisong Wang, Klas G. Wiman,
W. Edward Mercer, Michael B. Kastan, Kurt W. Kohn, Stephen J. Elledge, Kenneth W. Kinzler, and Bert Vogelstein
The Oncology Center and Program in Human Genetics and Molecular Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231 W.5. E-D.,
V. E. V., C. E. C., J. A. P., M. B. K., K. W. Ki., B. V.]; The Laboratory of Molecular Pharmacology, Division of Cancer Treatment, Developmental Therapeutics Program, National
Cancer Institute, NIH, Bethesda, Maryland 20892 [P. M. O., J. J., K. W. /Co./; Oncogene Science, Inc., Cambridge, Massachusetts 02142 [M. B., D. E. H,;Department of Tumor
Biology, Karolinska Institutet, Box 60400, S-104 01, Stockholm, Sweden /K. G. W., Y. W.]; Howard Hughes Medical Institute [S. J. E./ and Verna and Marrs McLean Department
of Biochemistry and Institute of Molecular Genetics /J. W. H., S. J. E.,Baylor College of Medicine, Houston, Texas 77030; and Department of Microbiology and Immunology,
Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 W.E. M./

Abstract (4, 5). One pathway can be triggered by exposure of mouse thymo-
cytes to dexamethasone and is independent of p53. The second path
The tumor growth suppressor WAF1/C1Pwas recently shown to be way is dependent upon the induction of p53 and follows exposure to
induced by p53 and to be a potent inhibitor of cyclin-dependent kinases. In
ionizing radiation or other agents associated with DNA damage (11).
the present studies, we sought to determine the relationship between the
Several cellular proteins, including bcl-2 (12), adenoviral E1B (13),
expression of WAFIICIPI and endogenous regulation of p53 function.
and bcr-abl (14), have been identified as inhibitors of apoptosis,
WAF1/CIP1 protein was first localized to the nucleus of cells containing
wild-type p53 and undergoing G| arrest. WAFIICIPI was induced in whereas other proteins, including c-myc (15), adenoviral EIA (16,
wild-type p53-containing cells by exposure to DNA damaging agents, but 17), and bcl-2-associated proteins (18, 19), have been identified as
not in mutant p5.?-containing cells. The induction of WAF1/CIP1 protein inducers of apoptosis. The interactions between these inhibitors and
occurred in cells undergoing either p5.!-associated Garrest or apoptosis the mediators of apoptosis are beginning to reveal a complex regula
but not in cells induced to arrest in G or to undergo apoptosis through tion of cell death (20).
p5J-independent mechanisms. DNA damage led to increased levels of
In the present studies, we sought to determine whether WAF1/CIP1
WAF1/CIP1 in cyclin E-containing complexes and to an associated de
induction occurs in the endogenous DNA damage response pathways
crease in cyclin-dependent kinase activity. These results support the idea
that WAF1/CIP1 is a critical downstream effector in the p5J-specific path
leading to either cell cycle block in G, or apoptosis. We further
analyzed the induction of G, arrest or apoptosis by p5J-independent
way of growth control in mammalian cells.
pathways to determine if WAFIICIPI is a generally induced growth
Introduction inhibitor or a more specific stress/damage-induced mediator of p53
function.
The tumor suppressor p53 is a transcription factor (reviewed in Ref.
1) which has been identified as a participant in the cellular DNA Materials and Methods
damage response resulting in either G! arrest (2, 3) or apoptosis (4, 5).
The mechanism by which p53 induction results in growth suppression Cell Culture Conditions and Treatments. The v-myc retrovirus-induced
murine T-cell lymphoma parental line J3D and the temperature-sensitive mu
is not firmly established, but a potential explanation was provided by
rine Val 135 p53 mutant-transfected cell line M3 have been described previ
the demonstration that p53 transcriptionally activates the production
ously (21). A fourth passage of the WI38 human lung fibroblast cell line was
of a A/r 21,000 protein [WAF1 (6)] which was simultaneously discov obtained from R-W. C. Yen and S. B. Baylin. The murine hematopoietic
ered as a potent inhibitor of cyclin-dependent kinases [CIP1 (7)]. In
(pre-B-cell) cell line BAF3, which undergoes apoptosis when the growth factor
normal diploid fibroblasts, WAF1/CIP1 is associated with every cy- IL32 is withdrawn, was obtained from A. Bedi and R. Jones (22). Cells were
clin-kinase complex examined, including those containing A-, B-, D-, seeded 24 h before drug or radiation treatment and were 50-70% confluent at
and E-type cyclins and Cdc2, Cdk2, Cdk4, and Cdk5 (7, 8). In con the time of such treatment. Radiation treatments of 2-8 Gy were delivered by
trast, cyclin immune complexes from fibroblasts transformed by SV40 a 137Cs y-irradiator at approximately 1 Gy/min. Cells were treated with the
or cells containing germ line p53 mutations contain nearly undetect- chemotherapeutic agent doxorubicin (Adriamycin) at a concentration of 0.2
/ig/ml for 14 or 28 h at 37C.
able levels of WAF1/CIP1 (9). WAF1/CIP1 inhibits growth of both
human tumor cell lines (6) and normal diploid fibroblasts (7) when Immunocytochemistry and Western Blot Analysis. Cells were fixed and
stained with mouse anti-human WAF1 polyclonal antibodies using minor
introduced via transfection, and this effect is largely reversed by
expression of T-antigen in normal fibroblasts (7). Together, these data modifications of previously described methods (23). Cell lysates were har
vested in SDS/PAGE sample-loading buffer and Western blot analysis was
suggest a direct link between p53, G, arrest, and negative regulation performed using pAblSOl (Ab2; Oncogene Science) to detect human p53, Ab3
of the cell cycle kinases required for the GrS transition. (Oncogene Science) to detect murine p53, or mouse anti-human WAF1 poly
Molecular details of the programmed cell death pathway are still clonal sera to detect either human or mouse WAF1/CIP1 protein. The mouse
poorly defined (10), but recent studies indicate that there are at least WAF1 polyclonal sera was obtained following immunization of mice with an
two pathways which mediate apoptosis in hematopoietic cell lineages Escherichia co/i-expressed GST-WAF1 fusion protein (WAF1/CIP1 residues 1
to 164). Mice were immunized by i.p. injection with 7.5 (xg of electroeluted
GST-WAF1 protein at weeks 0, 4, 7, and 15, and serum was obtained from the
Received 12/28/93; accepted 1/20/94. intraorbital plexus on weeks 9 and 16. Serum titers were measured in an
The costs of publication of this article were defrayed in part by the payment of page enzyme-linked immunosorbent assay using soluble, purified GST-WAF1 es
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact. sentially as described (23). Specificity towards WAF1/CIP1 was determined by
' This work was supported by the Preuss Foundation, the Clayton Fund, the National immunoblot using a 1:500 dilution of serum on purified GST-WAF1 and on
Foundation for Cancer Research, the Glaxo Research Institute. Inc. (M. B. K. and lysates of GM cells expressing or not expressing WAF1/CIP1 (6). Rabbit
C. E. C), the Council for Tobacco Research (M. B. K. and C. E. C.), and NIH Grants
GM07309, GM07184, ES05777 (M. B. K.), CA-09071, CA-55541 (W. E. M.). AG-11085
(S. J. E., and J. W. H.), and CA-43460. B. V. is an American Cancer Society research 2 The abbreviations used are: IL3, interleukin 3; SDS, sodium dodecyl sulfate; PAGE,
professor. polyacrylamide gel electrophoresis; FBS, fetal bovine serum.
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 WAFI/CIPI IS INDUCED IN p5.?-MEDIATED G, ARREST AND APOPTOSIS

polyclonal antibodies against cyclin E, used for immunoblotting, were gener

ously provided by J. M. Roberts (24).
Cell Cycle. Thymidine Incorporation, and Northern Blot Analysis. Cell
cycle parameters were assessed by pulsing the cells with bromodeoxyuridine

: ".
and then staining with a fluorescein isothiocyanate-conjugated anti-bromode-
oxyuridinc antibody and propidium iodide (3, 25). Thymidine incorporation in
GM cells, which contain an endogenous mutant p53 and an exogenous dexa-
methasonc-inducible wild-type p53, was carried out as described previously
(26). G, arrest of GM cells (in the absence of wild-type p53 induction) was
carried out using the plant amino acid mimosine as described (27, 28) except
that confluent cells were first serum starved (1% serum) for 3 days and split
into media containing either 10% FBS, 10% FBS plus 0.5 nui mimosine, 10% -U. - f *-
FBS plus 1 /xMdexamethasone, or 10% FBS plus both 0.5 ITM mimosine and
1 fiM dexamethasone. After an 18-h incubation at 37C,cells were pulsed for
2 h with | 'H]thymidine and total RNA was isolated for Northern blot analysis,
performed as described (6).
Cell Viability and DNA Fragmentation Analysis. The viable fraction of
J3D or M3 cells was determined at various time points (up to 37 h) of
incubation at 37Cor 31Cusing trypan blue exclusion as an indicator of cell
i -
viability (29). Total gcnomic DNA was extracted from BAF3 cells, which were
incubated cither in the presence or absence of IL3 and either untreated or
treated with 4 Gy at 5, 8, and 15 h following treatment. The integrity of
genomic DNA was evaluated by agarose gel electrophoresis and cthidium
bromide staining.
In Vitro Cyclin E Kinase Assays. Approximately 10 million cells were
lysed in 0.3 ml containing 50 ITIMTris (pH 8.0); 120 IHMNaCI; 50 ITIMNaF; 0.1
CM UM:
OCX:
QM
-
lOH
+
DEL
_
DEL
+

Fig. 1. WAF1/CIP1 localizes to the nucleus of cells undergoing G, arrest. Anti-WAFl

itiM sodium vanadatc; 2 ITIMEDTA; 10 fig/ml each of chymostalin, leupeptin,
antibodies were used to immunocytochemically stain CiM cells, either uninduced (A ) or
antipain, and pepstatin A (all from Sigma); 2 /xg/ml 4-(2-aminoethyl)benzene-
induced (B) to produce WAF1/C1P1 by dexamethasone (DEX). C. inhibition of DNA
sulfonyl fluoride (Calbiochem); and 0.4% Nonidet P-40. The extracts were synthesis as a result of dexamethasone treatment.
clarified by ccntrifugation at 14,000 rpm for 15 min at 4C.For each condition,
triplicate plates were lysed and assayed independently. Lysates were incubated
for 1.5 h at 4Cwith either a monoclonal antibody against cyclin E (HE111,
Basal WAFI/CIP Expression and Induction following DNA
provided by Drs. Emma Lees and Ed Harlow) or a monoclonal antibody
Damage Correlates with p53 Status. Previous studies have shown
against phage T7 gene 10 (Novagen) as a negative control. Immune complexes
that exogenous wild-type p53 was capable of inducing WAF/CIPI
were collected using 20 /xl of protein G-Scpharose and washed 3 times with 1.0
ml of lysis buffer and once with 1 ml of 20 ITIMTris (pH 7.5)-10 ITIMMgCI2. expression (6). The present studies addressed the question of whether
Misione Hl kinase assays were performed using 0.1 ml (one-tenth of the WAF1ICIP1 expression could be induced by endogenous wild-type
immune complex) as described previously (7). The beads were pelleted and 90 p53 in cells undergoing G, arrest or apoptosis. A survey of WAF1/
CIP1 levels in "normal" and tumor cell lines was performed prior to
(Iof supernatant were removed. The remaining 10 pii of beads were mixed
with 15 fil of a kinasc reaction mix containing 2 (j.g of histonc HI and and following treatment with various DNA-damaging agents. The
[7-32P]ATP. After 30 min, 25 ^il of 2 X SDS-PAGE buffer were added and 20 agents tested included ionizing radiation, UV radiation, treatment with
H\ were analyzed by SDS-PAGE and autoradiography and quantitated on a various chemical agents including etoposide, 5-fluorouracil, Adriamy-
Belagen scanner. cin, genistein, hydrogen peroxide, and methylmethane sulfonate.
These treatments induced cellular p53 protein to varying extents, in
Results both a dose- and time-dependent fashion, as has been observed by
others (2, 3, 11, 30, 31). The chemotherapeutic drug Adriamycin
WAF1/CIP1 Localizes to the Nucleus of Cells Blocked in G,
appeared to be one of the strongest and most reproducible inducers of
following p53 Induction. In orderto determinethe subcellularloca
p53 protein and was therefore used for most of our studies. A variety
tion of WAF1/CIP1 in cells undergoing G, arrest, GM cells (contain of cell lines containing exogenous or endogenous wild-type or mutant
ing an endogenous mutant p53 and an exogenous wild-type p53 which
p53 genes were analyzed (Table 1). All cell lines tested which con
is dexamethasone inducible) were induced with dexamethasone as tained wild-type p53 protein (N = 8) expressed WAF1/CIP1, whereas
reported previously (26). Fig. 1 shows that GM cells treated with cell lines with mutant p53 (N = 7) expressed low or undetectable
dexamethasone produce a nuclear antigen recognized by mouse anti-
levels of WAF1/CIP1 (Figs. 2 and 3 and data not shown). Treatment
human WAF1 polyclonal antibodies (Fig. 15), an antigen not found in of cell lines with Adriamycin induced both pf>3 and WAFI/CIPI
untreated cells (Fig. 1/4). We note that dexamethasone treatment expression only in the cell lines which contained wild-type p53 pro
alone, in the absence of wild-type p53, did not induce WAF1/CIP1 tein, as noted below.
gene expression (Ref. 6 and data not shown). Fig. 1C shows that cells WAFI/CIPI Accumulation Is Associated with Ionizing Radia
were indeed cell cycle arrested as demonstrated by their decreased tion-induced G| Arrest. We investigated the relationshipbetween
incorporation of thymidine compared either to untreated cells or to a WAFI/CIPI levels and G, arrest induced by ionizing radiation in
control cell line (DEL, containing a dexamethasone-inducible mutant lymphoid cell lines in which p53 al-elestatus and G, arrest capacity
p53). No WAF1/CIP1 was detectable in the nucleus or cytoplasm of has been characterized (25). As shown in Fig. 2, WAFI/CIPI was
GM cells not undergoing G, arrest. These results demonstrate that detected in unirradiated cells that contained wild-type p53 (WMN,
WAF1/CIP1 is located in the nucleus of cells undergoing G arrest FWL, and PCI 19) but not mutant p53 (CA46, RAMOS, and ST486).
mediated by p53 and predict a nuclear localization for the various The WMN and PCI 19 cells exhibited higher basal levels of wild-type
cyclin-Cdk-WAFl/CIPl complexes which function to prevent cell p53 and WAFI/CIPI than FWL cells (Fig. 2). In two of the three cell
cycle progression. lines expressing wild-type p53 (WMN and FWL), accumulation of
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 WAfllCIPl IS INDUCED IN p5J-MEDIATED O, ARREST AND APOPTOSIS

WAF1/CIP1 occurred when the cells were induced to arrest in G, by

ionizing radiation (Fig. 2). No induction of WAF1/C1P1 was observed
A
in lymphoid cells containing mutant p53 (CA46, RAMOS, and
ST486), which do not arrest following radiation (25). Of particular
interest were the PCI 19 cells. These cells contain wild-type p53 but
do not arrest in G, following ionizing radiation (25). No induction of
p53 protein (25) or WAF1/CIP1 protein (Fig. 2) was observed fol
lowing irradiation of these cells. These results demonstrate a close 60
correlation between WAF1/CIP1 accumulation and radiation-induced
G! arrest in lymphoid cells. > 40
f
WAF1/CIP1 Induction and Apoptosis. The v-myc retrovirus-in-
duced T-cell lymphoma line J3D expresses no endogenous p53 due to 20

loss of one al-elecoupled with the insertion of a murine leukemia

0
provirus in the second al-ele.The M3 derivative of J3D contains a 0.00 7.40 14.80 2220 29.60 3700

stably integrated murine Val 135 temperature-sensitive p53 gene ex

Time (hours)
pressed at high levels (21). A shift from 37Cto 32Cresults in loss M337 -'
M332 --B-- J3D 37 J3D 32*

of viability and apoptosis induction only in the M3 line. We chose this

system to determine whether WAF1/CIP1 would be induced in the
pathway leading to apoptosis. Fig. 3A shows significant loss of vi
ability at 32Cin the M3 line but not in the J3D parent line. The
results in Fig. 3confirmed the absence of p53 in the parental J3D
line as well as the absence of detectable WAF1/CIP1 (Lanes 1-4). The
M3 line expressed murine p53 under all the experimental conditions,
but WAF1/CIP1 induction was observed only at the temperature re B
sulting in a wild-type p53 conformation and subsequent apoptosis
induction (Lanes 5-8). 123 456 78
The BAF3 murine hematopoietic cell line undergoes apoptosis fol
lowing withdrawal of the growth factor 1L3 (22). Treatment of this
cell line with ionizing radiation results in G, arrest in the presence of ,53
IL3, and a much more rapid apoptosis following irradiation in the

IM

WAF1/CIP1

WAF1/CIP1 RNA

Fig. 3. WAF1ICIP1 is induced in p53-mediated apoptosis. In A, the viability of parental

J3D and daughter M3 (containing an exogenous temperature sensitive Val 135 p53
mutant) T-cell lymphoma cell lines is shown as a function of incubation time at 37Cor
32C.The plotted values represent the mean of triplicate determinations, with a range
within 10% of the mean. B. Western blot analysis of J3D cells (Lanes 1, 2 at 17 h and
Lanes 3, 4 at 37 h) and M3 cells (Lanes 5, 6 at 14 h and Lanes 7, 8 at 31 h). correlating
- + 6.3 GRHV WAFIICIP1 expression with apoptosis induction at 32C(Lanes 2, 4.6,8) or the control
temperature (Lanes 1, 3. 5. 7). Northern blot analysis (bulloni) was performed on lysates
mutant uit/mu iut/uit obtained from M3 cells, demonstrating that WAFHCIPl induction was regulated at Ihe
CR46 RHMOS ST486 PC119 mRNA level.

_+ _+-+ _+-+ -+6.3 GRHV

absence of IL3 (22).3 We sought to determine whether WAFHCIPl
expression was induced in BAF3 cells undergoing either G, arrest or
apoptosis. Fig. 44, lop, shows that BAF3 cells treated with 4 Gy in the
p53 "
presence of 1L3 underwent G, arrest (shown here at 14 h following
irradiation). This was preceded by induction of p53 and WAFHCIPl
expression (shown here at 2 h following irradiation; Fig. 4A, bottom).
WAF1/CIP1 - - After IL3 withdrawal, BAF3 cells underwent apoptosis, with DNA
fragmentation evident 15 h later (Fig. 4,top). lL3-starved BAF3
Fig. 2. Relationship among irradiation-induced GI arrest, p53, and WAF1/CIP1 in
Burkitt's lymphoma cell lines. Top, percentage of the original GI population that remained cells treated with 4 Gy underwent a more rapid DNA fragmentation
in GI 16 h alter addition of the microtubule inhibitor, nocoda/ole (D, 0.4 fig/ml) or associated with induction of both p53 and WAFHCIPl expression
exposure to 6.3 Gy (gamma]-radiation plus nocodo/ole ().Nocodozole was included to (Fig. 4B, bottom).
prevent cells from entering GI of the next cell cycle. The cell cycle distribution was
measured by flow cytometry at 16 h following irradiation. Western blot analysis of p53
and WAF1/CIP1 was performed on cells 4 h after irradiation, wt, wild-type; mu, mutant. 3 C. E. Canman and M. B. Kastan. unpublished observations.

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 WAFIICIPI IS INDUCED IN ^-MEDIATED G, ARREST AND APOPTOS1S

Fig. 4. WAFIICIPI expression is noi induced in

p53-independent apoptosis. A (top), cell cycle
analysis al 14 h following exposure of BAF3 cells
to 4 Gy [gammaj-radiation (IR) in the presence of
IL3. For cell cycle fluorescence-activated cell
sorter analysis, the lower k'ft box, upper left box,
and box lo the right represent G,G^/M and S
phase populations, respectively. The ratio of G|-S-
phase populations is indicated beneath the fluores
cence-activated cell sorter analysis. p53 protein
(middle) and WAFIICIPI mRNA (bottom) levels
were increased 2 h postirradiation. B (top). DNA
fragmentation analysis of BAF3 cells at intervals
(h) following IL3 withdrawal, with or without ex
posure to 4 Gy ionizing radiation at time zero. p53
protein (middle) and WAFIICIPI mRNA (bottom)
levels were increased at 2 h following IL3 with
drawal only in the irradiated BAF3 cells. In the
absence of IL3. unirradiated cells undergoing apop p53i ,53
tosis did not exhibit higher levels of either p53 or
WAFIICIPI expression.

WAF1/CIP1 WAF1/CIP1 *

IR IR

WAFJ/CIPl Expression Is Not Induced in Cells Which Undergo type p53, WAFIICIPI mRNA was expressed, even in the presence of
G Arrest or Apoptosis through p5J-independent Pathways. To mimosine (Fig. 5). Additionally, no WAFIICIPI induction was ob
determine whether WAFIICIPI expression is induced in cells under served in GM cells arrested in G0 by serum starvation in the absence
going/?53-independent G, arrest or apoptosis, two experimental sys of dexamethasone (not shown).
tems were used. In the first, the glioblastoma cell line GM [expressing The BAF3 murine hematopoietic cell line undergoes apoptosis fol
only mutant p53 in the absence of dexamethasone (Table 1)] was lowing withdrawal of the growth factor IL3, but no induction of p53
induced to arrest in G, by treatment with the plant amino acid mi- occurs.3 Under these conditions, there was no associated induction of
mosine (27, 28). Under these conditions, there was no measurable WAFIICIPI gene expression (Fig. 4B; data not shown). When the
increase in WAFIICIPI gene expression (Fig. 5). However, when G! same cells were treated with ionizing radiation both p53 and WAF1/
arrest was caused by dexamethasone induction of exogenous wild- CIP! expression were induced (Fig. 4B).

Table 1 WAFIICIPI induction correlates with p53 status

p53 status WAFI/CII'I induction

Cell line/species Cell type Endo" Exo Adriamycin Radiation Dex Temp
fibroblastPre-B +++
WI38/HBAF3/MRKO/HHCT-116/HWMN/HFWL/HM3/MGM/HSW480/HDEL/HJ3D/MCA46/HRAMOS/HST486/HPC119/HLung
lineColorectal
cell
carcinomaColorectal ++
carcinomaBurkitt's +++ts
lymphomaLymphoblastoidT-cell

lymphomaGlioblastomaColorectal +Dex
Val 135
ind
+_ wt

carcinomaGlioblastomaT-cell _Dex _
ind
mu-----
lymphomaBurkitt's
lymphomaBurkitt's
lymphomaBurkitt's
lymphomaBurkitt's
lymphomawt/wtwt/wtwt/wtwt/wtwt/wtwt/wtmu/-mu/-mu/-mu/-mu/-mu/-mu/-wt/muwt/wt''-1-
**Endo and Exo, endogenous and exogenous p53 status in the cell lines; +, significant expression was induced following the indicated treatment: -, little or no induction of expression
was detected; Dex, treatment of cells with 1 JIMdexamethasone to induce the exogenous p53; Temp, temperature shift to 32C.wt and mu, wild-type and mutant p53 al-eles,respectively.
H and M, human and mouse origin, respectively. WAFJ/CIP1 expression was measured at the protein level, and in some cases also at the mRNA level as indicated in Figs. 3-5.
'' Genotypically wild-type p53 but did not undergo Gj arrest or express higher levels of p53 after radiation (see text).

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 WAFUCIPI IS INDUCED IN p5.9-MEDIATED G, ARREST AND APOPTOSIS

Induction of WAF1/CIP1 Correlates with Inhibition of Cyclin ADRIA:

E-associated Kinase Activity. The results described above led to two
predictions, (a) that there would be an increase in cyclin associated p53
WAFl/CIPl following induction of endogenous p53. (b) that cyclin-
associated complexes would have reduced activity following such
induction. These predictions were tested following induction of en
dogenous wild-type p53 in the wild-type p53 containing RKO and
HCT-116 cell lines. Antibodies to cyclin E were used to immunopre-
i WAF1/CIP1
cipitate cell extracts following Adriamycin treatment, and the immu-
noprecipitates were assessed for WAFl/CIPl association. Cyclin E
associates with Cdk2 during G! (24) and the activity of this complex
is thought to be required for the G, to S transition. WAFl/CIPl was
indeed induced by the drug (Fig. 6A, Lanes l4\ and there was a
-Cyclin E
corresponding increase in the amount of WAFl/CIPl associated with
cyclin E (Fig. 6fl, Lanes 14).No WAFl/CIPl was found in immu-
noprecipitates or total cell extracts of the mutant p53 containing tumor
cell lines SW480 and DEL (Fig. 6, A and B, Lanes 5-8). The immu- B
noprecipitates from RKO and HCT-116 also had significantly reduced
HI kinase activity following Adriamycin treatment (Fig. 6C, Lanes WAF1/CIP1
14).There was little or no decrease in the cyclin E-associated HI
kinase activity of the p53 mutant SW480 and DEL tumor cell lines
(Fig. 6C, Lanes 5-8) when normalized to the amount of cyclin E in the
immunoprecipitates. In the case of SW480, the apparent increase in
HI kinase activity following Adriamycin treatment was likely the
result of an apparent increase in cyclin E in the immunoprecipitate
(Fig. 65, Lanes 5 and 6).
3 4 5 6 7 8
Discussion
H S D
The results presented here support the following model. When Fig. 6. DNA damage-induced WAFl/CIPl associates with and inhibits the kinase
activity of the cyclin E complexes. The tumor cell lines, labelled as R, H, S, or D lo
exposed to agents which cause DNA damage, cells which contain indicate RKO. HCT-116, SW480, or DEL. respectively, were either untreated (-) or
treated ( + ) with Adriamycin and immunoprecipitated as described in "Materials and
Methods." A and B, Western blot analysis of p53 and WAFl/CIPl in total cell lysate or
cyclin E immunoprecipitates, respectively. C, Cyclin E-associatcd HI kinase activity.

endogenous wild-type p53 are stimulated to produce higher levels of

the protein. This induced p53 transcriptionally activates WAFl/CIPl
expression by directly interacting with its regulatory elements. Induc
tion of WAFl/CIPl protein and its transport to the nucleus result in
association and inhibition of the function of cyclin-dependent kinase
complexes. Inhibition of these kinases in turn results in a failure of
-a -
cells to exit G,. This G, arrest following induction of p53 allows
i "normal cells" to check their growth. In tumors, loss of wild-type p53
function prevents the activation of this growth control pathway. This
failure to induce transcriptionally active p53 may play a role in the
unregulated growth of the tumors and also in the failure to respond to
chemotherapeutic agents which normally trigger p53-regulated cell
arrest or death.
The induction of wild-type p53 can occur in cells which are un
Cell Line: GM GM GM GM dergoing either G, arrest or apoptosis. It seems unlikely that p53 itself
DEX: + + is the determinant of whether apoptosis occurs or not. Other factors,
including cell type, developmental stage, additional pathways trig
Mimosine: gered by the p53-inducing signal, growth factors, and the induction of
other proteins which modulate cell death may play important roles in
the cell death decision. Moreover, it is clear that both G, arrest and
apoptosis can occur without activation of either p53 or WAFl/CIPl.
Thus, cells blocked in G0 by serum starvation, blocked in G! by
WF1/CIP1* mimosine, or undergoing apoptosis after IL3 withdrawal did not ex
press higher levels of WAFl/CIPl. These observations suggest a
rationale for the design of anticancer therapy. For the treatment of
Fig. 5. WAFl/CIPl expression is not induced in p53-independent G, arrest. Top. tumors with an intact p53-regulated DNA damage response, it may be
inhibition of thymidine incorporation in GM cells (with mutant p53) treated with mimo advantageous to combine chemotherapeutic agents which induce G]
sine. Northern blot analysis, shown below, revealed that the p53-independent mimosine-
induced cell cycle block does not involve induction of WAFl/CIPl expression. DEA', arrest or apoptosis independently of p53 with agents which activate
dexamethasone. the p53-WAFl/CIPl growth control pathway.
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 WAFl/CIPl IS INDUCED IN pJ3-MEDlATED G, ARREST AND APOPTOSIS

Acknowledgments Bcl-2 proteins. Proc. Nati. Acad. Sci. USA, 89: 7742-7746, 1992.
17. Debbas, M., and White, E. Wild-type p53 mediates apoptosis by EIA, which is
inhibited by E1B. Genes Dev., 7: 546-554, 1993.
The authors thank Bob Fay for animal work.
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WAF1/CIP1 Is Induced in p53-mediated G1 Arrest and Apoptosis
Wafik S. El-Deiry, J. Wade Harper, Patrick M. O'Connor, et al.

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