bcl-2 Inhibits Wild-Type p53-triggered. Apoptosis but not G1. Cell Cycle Arrest and Transactivation of WAF1 and bax1. Yisong. Wang,2. Ismail Okan,. Laszlo.
Vol.
6,
1071-1075,
September
Cell Growth
1995
bcl-2
Inhibits Wild-Type p53-triggered Cell Cycle Arrest and Transactivation
Yisong George
Wang,2 Klein,
Microbiology Stockholm,
Ismail Okan, Laszlo and KIas G. Wiman
and Tumor Sweden
Biology
Center,
Szekely,
that
Institute,
trigger cell cycle arrest in G1 (4-7) or apoptosis p53 protein levels are low in normal cells under conditions but increase upon exposure to DNA-
damaging agents (i 3-i 5), forcing the cell to arrest in G1 and/or enter apoptosis. Thymocytes from p53 gene knockout mice fail to trigger the apoptotic response upon DNA damage (i 6, i 7). Thus, p53 is an essential Component of the cellular response to DNA damage, causing either a cell cycle blockade that allows DNA repair prior to DNA replication or an elimination of cells with damaged DNA by
Abstrad
We have earlier shown that wild-type expressed from a temperature-sensitive p53) triggers p53-negative
can
normal
5-1 71 77
(wt)
p53
construd (ts retrovirus-induced,
apoptosis in the v-myc T-cell lymphoma line J3D
(Y. Wang
1071
Apoptosis but not G1 of WAF1 and bax1
(8-12).
Karolinska
& Differentiation
et a!.,
apoptosis
(1 8). Loss of p53
therefore,
lead
critical for its ability to antagonize tumor growth and progression (i9). Among the genes transactivated by p53, the WAF1/C!P1 gene that encodes a universal inhibitor of cyclmn-dependent
in G1 at 1 8 h after
indudion
of wt p53
shift to 32#{176}C. At this time,
at
least 80% of the cells remained viable. After 30 h at 32#{176}C, around 50% of the cells had died by apoptosis, while most of the remaining cells were still alive in G1,
indicating
that p53-induced
apoptosis
kinases
gene
is inactivated
by
point
mutation
or gene (1 ), indicating
deletion in a large fraction of human tumors that it plays a central role in eliciting apoptosis in cells driven to proliferate continuously by activated oncogenes. In agreement with this notion, p13 gene knockout mice develop tumors with a high incidence (2). The p53 protein is a sequence-specific
Received t This
3/29/95; work
This may
to be important,
at least
p53-trigof bax (23-25), a gene that promotes apoptosis (26). In addition, p53 inhibits expression of bcl-2 (23, 24). This raises the possibility that p53 elicits apoptosis by shifting the balance between bcl-2 and bax.
p53 also induces
for the
expression
We found previously that expression of exogenous wt3 p53 triggers apoptosis in cells with constitutively activated myc genes, such as in human Burkitt lymphoma cells carrying endogenous mutant p53 (10), or in retroviral v-mycinduced mouse T-lymphoma cells lacking endogenous p53 (ii). We also showed that bcl-2 can inhibit p53-induced
apoptosis
in the mouse T-lymphoma
we demonstrate
that
p53-induced
cells (27). In this study, apoptosis
is preceded
by
cell cycle
arrest in G1 in the mouse T-lymphoma cells. We have found that, whereas bcl-2 prevents wt p53-induced apoptosis, it has no effect on wt p53-induced G1 arrest. Furthermore, p53-mediated transactivation of WAF1/CIP1 and
bax
is unaffected
by bcl-2.
Results
Introdudion p53
is likely
gered G1 arrest (20-22).
occurred
following G1 arrest. The G1 cell cycle arrest at 1 8 h after temperature shift to 32#{176}C was reversible, as shown by the fact that the cells readily resumed exponential growth following temperature shift back to 37#{176}C, although viability dropped from around 80 to 65%. Expression of both WAF1 and bax mRNA was induced by wt p53 in both the ts p53 and ts p53/bc!-2 transfeded cells. The kinetics of G1 cell cycle arrest at 32#{176}C was similar in both the ts p53 and the ts p53/bc!-2 double transfedants. This demonstrates that bc!-2 has no effect on wt p53-induced G1 arrest and does not interfere with wt p53-mediated transadivation of WAF1 and bax during wt p53-induced G1 arrest/apoptosis. These findings are consistent with the possibility that p53-mediated WAF1 indudion is responsible for G1 arrest and that p53-mediated indudion of bax accelerates apoptosis.
The
DNA.
to
were
by temperature
with damaged
facilitate tumor development at any point, from the very inception of the malignant clone to advance stages of tumor progression. This idea is further supported by recent work indicating that the ability of p53 to induce apoptosis is
,
arrested
ofcells
may,
the continued
expression
growth
function
Cell Growth & Differ., 4: 467-473, 1 993). We also found that constitutive bc!-2 expression inhibits wt p53triggered apoptosis in these cells (Y. Wang et a!., Oncogene, 8: 3427-3431 1 993). Here we demonstrate that more than 90% of the ts p53-transfeded J3D cells
was
revised supported
DNA
6/i3/95; by
binding
accepted grants
from
transcription
factor
(3)
6/23/95. the
Swedish
Cancer
Society
(Cancerfonden), Magn. Bergvalls Stiftelse, ke Wibergs Stiftelse, and USPHS Grant CAl 4054 awarded by the National Cancer Institute, Department of Health and Human Services. Y. W. is supported by a fellowship from the Cancer Research Institute, New York. K. G. W. is the recipient of a personal grant from The Swedish Cancer Society (Cancerfonden). 2 To whom requests for reprints should be addressed.
Apoptosis Is Preceded by G1 Arrest. To dewhether induction of wt p53 expression by temshift to 32#{176}C affects the cell cycle distribution of
p53-induced
termine perature J3D-ts
p53
cells,
J3D6 and J3D8,
two
were
clones
grown
of J3D-ts
p53
transfectants,
at 32#{176}C, harvested
at various
time points as shown in Fig. 1 , and analyzed with regard to viability and cell cycle distribution as described in “Materials and Methods.” Eight h after temperature shift from
37#{176}C to 32#{176}C, the peak representing cells with a G1 DNA content had increased from around 30 to 55%, while the fraction of cells in S phase had decreased from 60 to 30% compared to cells grown at 37#{176}C (Fig. 1A). By 18 h after
The abbreviations retinoblastoma. 3
used
are:
wt,
wild
type;
Is, temperature
sensitive;
RB,
1072
p53
and
bcl-2
in G,
Arrest
and
Apoptosis
A
A
0
8
18
30
0
6
12
26h
h
J3D6 0
B
.
E
bcl-2-
K
>
hours
Fig. 2. WI p53-induced G, arrest is reversible. The ts p53-carrying )3D6 cells grown at a density of 2 x 107m1 were arrested (or 1 8 h at 32C and then shifted back to 37’C. Cell cycle distribution A) and viability (B) was deterhours
Fig. 1. A, both J3D-ts p53 and J3D-ts p53/bcl-2 clones undergo C, arrest at 32#{176}C.Logarithmically growing cells were switched from 37#{176}C to 32CC at a density of 2 X 1OVnil. Cells were harvested following incubation for 0 to 30 h as indicated, stained with propidium iodide, and processed for tluorescence-activated cell sorting analysis. The X-axis represents relative fluorescence intensity, which is proportional to DNA content. The positions of 2N (G0-G,( and 4N (G-M( DNA contents are indicated. Data for one of three experiments that yielded the same results are shown. Essentially similar results were obtained from clones J3D8 and bcl-2-A (data not shown). B, viability of J3D-ts p53 and J3D-ts p53/bcl-2 cells at 32#{176}C.Viability was determined at each time point by trypan blue exclusion in parallel with cell cycle analysis as described in “Materials and Methods.” The results represent the viability of triplicate cultures; bars, SD. A, parental J3D cells; ts p53 transfectant J3D6; 0, ts p53 transfectant J3D8; U, ts p53/bcl-2 double transfectant bcl-2-K; EL ts p53/bcl-2 double transfectant bcl-2-A.
#{149},
temperature shift to 32#{176}C, about 90% of the ts p53-transfected cells were arrested in C1 (Fig. 1 A). Fig. 1 B shows that approximately 80% of the cells were still alive at this time. By 72 h after temperature shift, viability had dropped to around 1 0% (Fig. 1 B) with a majority of the living cells remaining in C1 (data not shown). This demonstrates that wt p53-induced G arrest occurred prior to apoptosis. wt p53-induced Growth Arrest Prior to Apoptosis Is Reversible. Clone J3D6 cells were first grown at 32#{176}C for 1 8 h. At this time, more than 90% of cells were arrested at G, while the viability remained high (around 80%; Fig. 1). The cells were then returned to 37#{176}C, and viability and cell cycle distribution were analyzed in parallel every 2 or 3 h. After 1 2 h at 37#{176}C, more than 50% of the G1-arrested cells had entered S phase (Fig. 2A). After another 14 h at 37#{176}C,
mined in parallel at each time point alter temperature indicated. The viability and the histogram 01 cell cycle data from three independent experiments.
shitt to 37CC as analysis represent
the surviving cells continued to proliferate and exhibited a normal cell cycle profile (Fig. 2A). Western blot analysis using PAb421 demonstrated that these cells still expressed ts p53 (Fig. 3). A small population of cells continued to die at 37#{176}C. As indicated in Fig. 2B, the viability dropped from 80 to 65% by 6 h after the temperature shift to 37#{176}C. Agarose gel electrophoresis of DNA from the dead cells showed the characteristic DNA fragmentation ladder (Fig. 4). This shows that the G1 arrest triggered by wt p53 is reversible. IJc!-2 Inhibits wt p53-triggered Apoptosis but not G1 Arrest. We demonstrated previously that overexpression of bcl-2 in the ts p53-transfected J3D cells inhibits wt p53induced apoptosis (27). In order to test whether bcl-2 overexpression has any effect on wt p53-induced G1 arrest, two clones of ts p53/bcl-2 double transfectants (bcl-2-A and bcl-2-K) were analyzed for viability and cell cycle distribution after temperature shift to 32#{176}C. After 8 h at 32#{176}C,the fraction of cells in G had increased to around 60’%, and after 18 h, approximately 90% of the cells were in G1 (Fig. 1A). Thus, there were no major differences in the kinetics of wt p53-induced G1 arrest between the ts p53 transfectants and the ts p53/bcl-2 double transfectants. This demonstrates that p53-induced G1 arrest and p53-induced apoptosis are separate events and that bcl-2 can antagonize wt p53-triggered apoptosis but not cell cycle arrest in the G1 phase.
Cell
Growth
& I)ifferentiation
1073
A
2
1
3
4
3 0 54
5
.
1.018516 2?0
‘-71
P53-
--Lr-__., -U
-
-418
B 1
2
3
4 Fig. 4. DNA fragmentation analysis. DNA was prepared as described 1 1). Each DNA sample d)f 3 pg was separated on a 1 .5/ agarose gel and stained with ethidium bromide. Molecular sizes in bp of marker DNA fragments (BRL, Gaithersburg, MD) are indicated. Lane 1, )3D6 cells that had been kept at 32CC for 1 8 h and subsequently grown at 37CC for 6 Ii; Ltne 2, bcl-2-K cells after 6 clays at 32’C; and Lane 3, parental I3D cells atter 6 clays at 32CC.
-30.6
bcl-2-
.‘.
.-.
-11.8
Fig. 3.
Expression
ECL-immunoblotting
cells.
using
equal
p53 amounts
and
hcl-2 of
proteins
lysates
from
was
measured
)3D6
and
by
bcl-2-K
in A and B were exposed for 1 and 2 mm, respectively. in kiloclaltons (Kaleidoscope prestaineci standards; Bio-Rad) are indicated. A: Lane 1, /)cl-2-K cells grown at 32#{176}C br 22 h; Lane 2, bcl-2-K cells grown at 37CC; Lane 3, parental (3D cells; Lane 4, )3D6 cells grown at 32CC for 22 h; Lane 5, 13Db cells grown at 32CC for 1 8 h and then grown at 37CC for 26 h. B: Lanes 1, 2, and 4, parental 3D, bcl-2-K, and J3D6 cells grown at 37#{176}C,respectively; Lane 3, bcl-2-K cells grown at 32CC for 22 h. Molecular
The
of exogenous
induced in both 32#{176}C (Fig. 5).
blots sizes
After growth at 32#{176}C for 6 days, the viability of the ts p53/bcl-2 double transfectants dropped to around 40%, as determined by trypan blue exclusion (Fig. 1 B). The presence of a characteristic DNA fragmentation ladder on agarose gel electrophoresis confirmed that this loss of viability was due to apoptosis (Fig. 4). Thus, even cells overexpressing bcl-2 are susceptible to wt p53-induced apoptosis after prolonged G1 arrest. Bc!-2 Ovexpresssion Does Not Affect Transactivation of WAF1, MDM2, and bax. The ts p53 and ts p53/bcl-2 transfected J3D cells express very little or no WAF1 mRNA when grown at 37#{176}C (Fig. 5). However, WAF1 mRNA is induced in both the ts p53 and ts p53/bcl-2 transfected J3D cells after temperature shift to 32#{176}C. This result shows that although bcl-2 blocks wt p53-induced apoptosis in these cells, it does not interfere with wt p53-mediated transactivation of WAFJ.
We also examined mRNA levels for two other p53responsive genes, bax and MDM2, in the ts p53 and ts p53/bc/-2-transfected J3D cells at 37#{176}C and 32#{176}C. As shown in Fig. 5, the expression of bax rnRNA is relatively low in both the ts p53 and ts p53/bcl-2-transfected J3D cells at 37#{176}C but is increased after temperature shift to 32#{176}C in both types of transfectants. Likewise, MDM2 mRNA levels are
the ts p53
and ts p53/bcl-2
transfectants
at
Discussion p53 can trigger both G1 cell cycle arrest (4-7) and apoptosis (8-12). An important question is whether p53-induced G arrest and apoptosis are independent pathways or whether p53-induced apoptosis is a consequence of or follows G1 arrest. wt p53-induced cell death in Ml mouse myeloid leukemia cells is not preceded by growth arrest (28), although cells in G1 are preferentially susceptible to an apoptosis-inducing signal delivered by wt p53. In contrast, apoptosis triggered by wt p53 in DP-1 murine erythroleukemia cells occurred following G1 arrest (12). We have shown here that wt p53 induces both G1 arrest and apoptosis in the J3D mouse T-lymphoma cells. Thus, p53-induced apoptosis is at least in some situations preceded by G arrest, but the effect of p53 depends on the cellular context. It is likely that other factors that affect the p53dependent growth arrest pathway will determine whether pS3 will trigger G arrest, apoptosis, or both G1 arrest and apoptosis. For instance, expression ofthe human papilloma virus 16 E7 protein, which targets and inactivates pocket proteins including the RB protein, a potential downstream effector of p53, prevents p53-induced G arrest (29-3 1). Loss of RB, or overexpression of the cyclin Dl protein that can stimulate phosphorylation and thus inactivation of RB, may have the same effect. Moreover, p53-induced apoptosis can be averted by growth factors such as interleukin-6 (8) and interleukin-3 (32) and expression of oncogenic kinases like v-Src and activated c-Raf (32). Our finding that cells arrested in G1 18 h after induction of wt p53 by temperature shift to 32#{176}C can be rescued when shifted back to 37#{176}C indicates that the cells remain for some time in G1 before being irreversibly committed to apoptotic death. This is in agreement with previous experiments demonstrating that p53-induced apoptosis occurs predomi-
1074
p53
and
bcl-2
in G,
1
WAF1’’
Arrest
and
2
Apoptosis
would induce
3456
I
a.
be consistent with the observation that p53 can apoptosis in the absence of RNA or protein synthesis (34). If so, bcl-2 may prevent p53-induced apoptosis, at least in part, through its ability to inhibit p53-mediated transcriptional repression (35). The role of p53-mediated transactivation of genes such as WAF1 and bax in p53-induced cell cycle arrest and/or apoptosis could be addressed by using effective antisense strategies that allow specific inhibition of WAF1, bax, or other p53-responsive genes. Introduction of regulatable WAF1 or bax expression constructs in the J3D cells may also be a fruitful approach.
Materials
and Methods
Cells.
The v-myc retrovirus-induced p53-negative murine T-cell lymphoma line J3D, the ts pS3 transfected clones j3DVaI1 35M6 and J3DVaI1 35M8 (abbreviated as J3D6 and J3D8 in this paper), and the ts p53/bcl-2 double-transfected clone bcl-2A have been described previously (1 1 , 27). Clone bcl-2-K was generated by introducing a human bcl-2a cDNA driven by the Moloney murine leukemia virus long terminal repeat into J3D6 cells as described (27). Logarithmically growing cells were seeded at 2 x 105/ml in Iscove’s medium supplemented with 10% FCS at 37#{176}C and 32#{176}C.
bax-
I... MDM2-1
Analysis of Viability, Fragmentation. Viability
aCtlfl
Fig. 5. Expression of ‘NAF1 , bax, and MDM2 mRNA. Ten pg of total RNA was sequentially blot hybridized with WAF1, bax, and MDM2 probes. The same filter was stripped and rehybridized with an a-actin cDNA probe, as a control forthe amountofapplied RNA. Lanes 1, 3, and 5, bcl-2-K cells; Lanes 2, 4, and 6, J3D6 cells; Lane 7, parental J3D cells; Lanes 1 and 2, cells grown at 32#{176}C for 22 h; Lanes 3 and 4, cells grown at 32#{176}C for 8 h; Lanes 5-7, cells grown at 37#{176}C.
nantly in G1 (12, 28). Alternatively, it may simply reflect a difference between the kinetics of p53-induced C1 arrest and that of p53-induced apoptosis in this system. We have found that whereas bcl-2 prevents wt p53induced apoptosis, it has no effect on p53-induced G1 arrest. Furthermore, our Northern blot analysis showed that bcl-2 does not interfere with wt p53-mediated transactivation of WAF1 in the J3D cells. These findings are consistent with the idea that p53-mediated induction of WAF1 causes
G1 arrest
through
binding
and inhibition
of cyclin/cyclin-
dependent kinase complexes but is not directly involved in p53-induced apoptosis. Instead, the wt p53-dependent transactivation of bax that we have observed may trigger or accelerate apoptosis in our J3D cells. Inhibition of p53induced apoptosis by bcl-2 could be due to neutralization of the apoptosis-promoting effect of bax. It is also possible that p53-induced apoptosis does not require transactivation of specific p53-responsive genes but is due to p53-mediated transcriptional repression (33). This
Cell Cycle
Distribution,
and DNA
and cell cycle distribution were determined in parallel at different time points. Viability was assessed using a hemocytometer and trypan blue exclusion. For cell cycle analysis, cells were centrifuged and resuspended in 1 ml of a solution containing 50 pg of propidium iodide (Sigma Chemical Co.) per ml, 0.6% NP4O, and 0.1 % sodium citrate. The stained cells were analyzed in a fluorescence-activated cell sorter (Becton Dickinson) within 24 h. The percentage of cells in different phases of the cell cycle was determined by using the CelIFit program and Sum-of-Broadened-Rectangles model. The integrity of genomic DNA was evaluated by agarose gel electrophoresis and ethidium bromide staining as described (1 1). RNA Analysis. For preparation of total RNA, cells were washed with PBS, lysed with lysis buffer [1 0/ NP4O, 1 50 mtvt NaCI, 100 mM Tris-HCI (pH 8.0), 5 m EDTA, and 1 mti aurintricarboxylic acid; Ref. 36] and centrifuged to remove the nuclei. An equal volume of SDS/urea buffer [7 M urea, 1 % SDS, 350 mM NaCI, 1 0 mi EDTA, and 1 0 mtvt Tris-HCI (pH 7.5)] was added to the lysis supernatant. Phenol extraction, isopropanol precipitation, and Northern blotting were performed as described (37). The 0.74-kb EcoRl fragment of plasmid pCMW 35 containing the whole coding sequences of mouse WAF1 cDNA was used as WAF1 probe. The 943-bp fragment of the clone 70z7 and the 1 .3-kb fragment of the clone 1 1 B were used as the bax and MDM-2 probes, respectively. The murine a-actin probe is a 0.9-kb Pstl-Pstl cDNA fragment (38). Western Blotting. Approximately 5 x 1 0 cells were lysed with 1 x Laemmli buffer, boiled, separated in 10% SDS polyacrylamide gel, and electroblotted to Immobilon polyvinylidene difluoride membranes (Bio-Rad). After blocking with 5% skim milk in PBS, the membranes were incubated with an anti-human bcl-2 mAb (bcl-2/124; DAKO, Copenhagen, Denmark) or with an anti-p53 mAb (PAb42i ; Oncogene Science, Manhasset, NY) at 1 :500 dilution. The filters were washed and subsequently probed with a horse radish peroxidase-conjugated goat anti-mouse
Cell Growth
immunoglobulmn antibody at i :5000 dilution and developed by ECL (Amersham, Buckinghamshire, United Kingdom) according to the manufacturer’s instructions. The blots were
exposed
for i-2
mm on Hyperfilm-ECL
18. Lane, 1992. 19.
Moshe
Oren
Evan for the bcl-2 Stanley I. Korsmeyer MDM2 plasmid.
for providing
the ts p53 Val135
plasmid,
Gerard I. WAF1 plasmid, L. George for the
plasmid, Bert Vogelstein for the mouse for the mouse bax plasmid, and Donna
Symonds,
guardian
H., Krall,
ofthe
genome.
L., Remington,
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