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Oncogene (2000) 19, 2194 ± 2204
2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00
www.nature.com/onc
c-Myc antagonizes the e€ect of p53 on apoptosis and p21WAF1
transactivation in K562 leukemia cells
Eva Ceballos1, M Dolores Delgado1, Pilar Gutierrez1, Carlos Richard2, Daniel MuÈller3,
Martin Eilers3, Mats Ehinger4, Urban Gullberg4 and Javier LeoÂn*,1
1
Grupo de Biologia Molecular del CaÂncer, Departamento de Biologia Molecular, Unidad Asociada al Centro de Investigaciones
BioloÂgicas, Universidad de Cantabria, Spain; 2Servicio de HematologõÂa, Hospital MarqueÂs de Valdecilla, Santander, Spain;
3
Institute of Molecular Biology and Tumor Research, Phillips University. Marburg, Germany; 4Department of Hematology, Lund
University, Lund, Sweden
c-myc protooncogene positively regulates cell proliferation and overexpression of c-myc is found in many solid
tumors and leukemias. In the present study we used the
K562 human myeloid leukemia cell line as a model to
study the functional interaction between c-Myc and p53.
Using two di€erent methods, we generated K562
transfectant cell lines with conditional expression of
either c-Myc or p53. The cells expressed the p53Vall35
mutant, which adopts a wild-type conformation at 328C,
while c-Myc induction was achieved with a zinc-inducible
expression vector. We found that p53 in wild-type
conformation induces growth arrest and apoptosis of
K562. Expression of c-Myc signi®cantly attenuated
apoptosis and impaired the transcriptional activity of
p53 on p21WAF1, Bax and cytomegalovirus promoters.
The impairment of p21WAF1 transactivation by c-Myc was
con®rmed by transfection of a c-Myc-estrogen receptor
fusion protein and by induction of c-myc by zinc in
transfected cells. Also, p53-mediated up-regulation of
p21WAF1 mRNA protein were signi®cantly reduced by cMyc, while Bax levels were una€ected. Consistently, cMyc increased cyclin-dependent kinase 2 activity in
K562 cells expressing p53 in wild-type conformation.
These results suggest that c-Myc overexpression may
antagonize the pro-apoptotic function of p53, thus
providing a molecular mechanism for the frequently
observed deregulation of c-myc in human cancer.
Oncogene (2000) 19, 2194 ± 2204.
Keywords: myc;
transactivation
p53;
leukemia;
K562;
apoptosis;
Introduction
p53 is a transcription factor which is activated upon
DNA damage and promotes cell growth arrest and
apoptosis (Gottlieb and Oren, 1996; Ko and Prives,
1996; Levine, 1997). Mutations and deletions of the
p53 tumor suppressor gene is a common alteration in
human cancer. In many neoplasias, and particularly
those of hematopoietic origin, loss of normal p53
function occur during tumor progression, and tumors
without wild-type p53 are more aggressive and have
worse prognosis (Wallace-Brodeur and Lowe, 1999;
*Correspondence: J LeoÂn, Dpto. de Biologia Molecular, Facultad de
Medicina, 39011 Santander, Spain
Received 21 June 1999; revised 22 February 2000; accepted 24
February 2000
Kirsch and Kastan, 1999). Under genotoxic stress, p53
binds to speci®c DNA sequences and activates
transcription of di€erent genes. Some p53 target genes
are involved in apoptosis control, e.g. bax, fas, and
IGF-BP3, while others mediate cell cycle arrest, e.g.
cyclin G, gadd45, 14-3-3s and the cyclin-dependent
kinase inhibitor p21WAF1. However, the mechanisms by
which p53 induces apoptosis are not completely
understood (Hansen and Oren, 1997; Amundson et
al., 1998; Bates and Vousden, 1999).
c-Myc is a transcription factor involved in the control
of cell proliferation, di€erentiation and apoptosis.
Mitogenic stimulation induces c-myc expression whereas
growth arrest is usually accompanied by suppression of
c-myc (Henriksson and LuÈscher, 1996; Amati et al.,
1998; Bouchard et al., 1998) c-Myc heterodimerizes with
the protein Max and the dimer binds to regulatory
regions of target genes. These include genes involved in
cell cycle control and DNA and RNA metabolism
(Grandori and Eisenman, 1997; Dang 1999). Consistent
with its positive e€ect on cell growth, deregulated
expression of c-myc is a common ®nding in human
cancer (Garte, 1993; Henriksson and LuÈscher, 1996).
Paradoxically, c-Myc overexpression also mediates
apoptosis in di€erent cell types subjected to sub-optimal
growth conditions, e.g. deprivation of growth factors or
hypoxia (for recent reviews see Ho€man and Lieberman,
1998; Hueber and Evan, 1998; Thompson, 1998). c-Mycmediated apoptosis requires wild-type p53 in ®broblasts
(Hermeking and Eick, 1994; Wagner et al., 1994;
Rupnow et al., 1998), although there are examples
where the apoptosis induced by c-Myc is p53-independent (Selvakumaran et al., 1994; Hsu et al., 1995;
Sakamuro et al., 1995; Trudel et al., 1997; StaÈhler and
Roemer 1998; Hagiyama et al., 1999). Besides, there are
other data revealing a functional interaction between cMyc and p53: (i) wild-type p53 represses c-myc promoter
(Moberg et al., 1992; Ragimov et al., 1993) and mutated
p53 activates c-myc promoter (Frazier et al. 1998); (ii) cMyc induces p53 expression (Hermeking and Eick, 1994;
Reisman et al., 1993; Roy et al., 1994; Kirch et al., 1999)
(iii) c-myc and loss of wild-type of p53 function
cooperate in the development of leukemia and lymphoma (Blyth et al., 1995; Lotem and Sachs, 1995; Eischen
et al., 1999). They also cooperate in the transformation
of mouse ®broblasts (Taylor et al., 1992), the immortalization of hematopoietic precursor cells (Metz et al.,
1995) and the promotion of genomic instability (Yin et
al., 1999).
Chronic myeloid leukemia (CML) is usually diagnosed in a benign chronic phase, characterized by a
Myc antagonizes p53 functions in K562 cells
E Ceballos et al
clonal granulocytosis. The chronic phase lasts 1 ± 3
years and progresses to a fatal blast crisis. The
molecular hallmark of all CMLs is the expression of
the Bcr-Abl kinase, a fusion protein arising from a
translocation (Melo, 1996). The disease evolution
suggests that secondary gene alterations are required
for progression from chronic CML phase to blast
crisis, but there is no evidence, to date, that a single
molecular change is responsible for this progression.
Alterations of p53 are very rare in the chronic phase of
CML, and they occur with frequencies ranging 10 ±
25% of the blast crisis cases (Ahuja et al., 1991;
Prokocimer and Rotter, 1994; Nakai and Misawas,
1995; Stuppia et al., 1997) although this frequency may
be much lower, depending on the population analysed
(Peller et al., 1998). Interestingly, c-myc expression is
higher in cells derived from blast crisis CML than from
the chronic phase (Preisler et al., 1990; Handa et al.,
1997), and recent reports indicate a positive correlation
of c-myc ampli®cation with progression to blast crisis
(Beck et al., 1998; Jennings and Mills, 1998). Moreover, the presence of an extra allele of c-myc
(chromosome 8 trisomy) is the most common karyotypic alteration in CML blast crisis (46% of cases)
(Alimena et al., 1987).
K562 cells derive from CML in blast crisis and are
de®cient in p53, due to the deletion of one allele and a
frameshift mutation in the other (Bi et al., 1993; Law et
al., 1993). We have used the K562 system to study the
role of c-Myc on p53 function. We show here that cMyc counteracts the apoptosis mediated by wild-type
p53 and the up-regulation of p21WAF1 expression.
Results
Wild-type p53 induces apoptosis in K562 cells
Kp53A1 cells are K562 transfected with a p53Vall35
mutant, which were fully characterized previously
(Ehinger et al., 1997). This protein adopts a wild-type
conformation at 328C, whereas it behaves like mutated
p53 at 378C (Michalovitz et al., 1990; Milner and
Medcalf, 1990). As described (Law et al., 1993), no p53
was detected by immunoblot in parental K562 cells,
but signi®cant amounts of p53 protein were detected at
37 and 328C in Kp53A1 and Kp53C1 cells (Figure 1a).
To con®rm the presence of p53 in wild-type conformation at 328C we analysed the expression of bax and
p21WAF1, two well-known transcriptional targets of p53.
Upon transfer to 328C, the mRNA expression of both
genes was clearly induced in Kp53A1, but not in
parental K562 (Figure 1b) or vector-transfected
KCMV cells (not shown). These results demonstrate
that p53 is transcriptionally active at 328C in Kp53A1.
Transfer of cells of 328C resulted in growth arrest,
predominantly in G1 phase (not shown), as well as
progressive decrease in cell viability and appearance of
apoptotic cells. The morphological analysis of cells
revealed a dramatic increase of cells showing typical
features of apoptosis: membrane bebbling, cell shrinkage, chromatin condensation and fragmentation and
formation of apoptotic bodies (Figure 1c). Similar
result was observed for Kp53C1 cells (not shown). The
apoptotic nature of Kp53A1 cell death at 328C was
also con®rmed by the internucleosomal fragmentation
of genomic DNA (Figure 1d). At 328C the cells also
exhibited other markers of apoptosis such as abnormal
chromatin condensation revealed by DAPI staining
and annexin V binding to the cell surface (see below).
Thus, cell death induced by p53Vall35 at 328C in K562
cells had the properties of typical apoptosis. This
apoptosis showed a slow kinetics, as few apoptotic cells
were detected after 24 h at 328C.
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Construction of transfectant cell lines with conditional
expression of c-Myc and p53 in wild-type conformation
To study the e€ect of c-Myc on p53-mediated
apoptosis of K562 we generated double transfectants
with conditional expression of p53 in the wild-type
conformation and c-Myc. Kp53A1 cells were transfected with pHeBO-MT-myc, a zinc-inducible c-Myc
expression vector (Grignani et al., 1990). Individual
clones resistant to G418 (as Kp53A1 cells) and
hygromycin (transfected with c-myc) were isolated.
One cell clone was selected for further experiments and
termed Kp53A1/myc. The construction of this line and
its expected expression pattern is schematized in Figure
2a. The expression of ectopic c-myc mRNA is
markedly induced after 24 h of treatment with 75 mM
ZnSO4 (Figure 2b). The transfected gene lacks the ®rst
non-coding c-myc exon and therefore it produces a
smaller mRNA than the endogenous gene (Grignani et
al., 1990). Kp53A1/myc cells also overexpressed c-Myc
protein after zinc addition, as shown by immunoblot
(Figure 2c). To rule out the possibility that the results
were due to the particular cell clone selected, we
generated another double transfectant cell line by the
inverse pathway to that followed to generate Kp53A1/
myc cells. Thus, we infected KmycB cells (with zincinducible expression of c-myc) (Delgado et al., 1995),
with a retrovirus expressing the p53Vall35 mutant. Cells
resistant to hygromycin (as KmycB cells) and puromycin (infected with the p53 retroviral vector) were
selected and dubbed KmycB/p53 (Figure 2a). The
expression of exogenous c-myc mRNA (Figure 2b) and
protein (Figure 2c) were dramatically induced after
24 h of culture in the presence of 75 mM ZnSO4 in
KmycB/p53 cells. The expression of endogenous c-myc
was reduced at 328C in Kp53A1/myc and KmycB/p53
cells (Figure 2b and data not shown). This result agrees
with the reported induction of c-myc by mutated p53
and repression by wild-type p53 in other systems (see
Introduction).
c-Myc protects K562 cells from p53-mediated apoptosis
We then analysed the e€ect of c-Myc induction on p53mediated apoptosis. As shown in Figure 3a, when cells
were shifted to 328C in the presence of 75 mM ZnSO4,
the loss in cell viability was attenuated. Likewise, the
fraction of cells with apoptotic morphology was
signi®cantly reduced in the presence of 75 mM ZnSO4
(60 versus 20% apoptotic cells after 4 days with zinc).
No signi®cant changes in cell morphology were
observed in those cells not undergoing apoptosis in
the zinc-treated cultures (not shown). The extent of
apoptosis protection was similar for Kp53A1/myc and
KmycB/p53 cell lines (Figure 3a). No changes in
viability were detected among the di€erent cell lines
when the experiments were performed at 378C (not
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Figure 1 Apoptosis mediated by wild-type p53 in K562. (a) Expression of p53 in Kp53A1, Kp53C1 and parental K562 cells. Total
extracts were prepared from cells incubated for 24 h at the indicated temperatures and analysed by immunoblot using the anti-p53
PAb240 antibody as described in Materials and methods. The ®lter was also probed against a-tubulin antibody to verify the protein
loading among samples. (b) Expression of p53, c-myc and p21WAF1 in Kp53A1 and K562 cells. Cells were incubated for 24 h at 37 or
328C. Total RNA was prepared and the expression of p53, p21WAF1 and c-myc mRNA were determined by Northern analysis. The
arrowheads indicate the position of 18S rRNA. (c) Expression of p53 in wild-type conformation induces morphological changes in
Kp53A1 cells that are typical of apoptosis. Parallel cultures of Kp53A1 cells were incubated at 37 or 328C for 48 h and
cytocentrifuge preparations were stained using the May-GruÈnwald-Giemsa method. Three cells with typical apoptotic morphology
are shown in the center of the image of the 328C sample. Arrows indicate apoptotic bodies. (d) Expression of p53 in wild-type
conformation induces DNA laddering typical of apoptosis in K562 cells. K562 and Kp53A1 cells were grown for 48 h at 37 or 328C
and genomic DNA was prepared and analysed by agarose gel electrophoresis
shown). The abrogation of apoptosis mediated by cMyc was also demonstrated by determining the
metabolic rate, using the WST-1 tetrazolium salt
reduction test (not shown). Altogether, the results
con®rmed that c-Myc overexpression protects K562
cells from p53-mediated apoptosis. However, the
induction of c-Myc at 328C (i.e., with p53 in wild-type
conformation) in Kp53A1/myc and KmycB/p53 cells
did not restore full growth ability, and did not change
the cell cycle phase distribution. Cell counts in the
presence of zinc increased about twofold after 24 h at
328C but did not grow further, and no changes were
detected in the extent of G1 arrest with respect to
untreated cells (not shown). The protection from
apoptosis by c-Myc was also determined by the
translocation of phosphatidylserine to the outer layer
of cell membranes, as determined by annexin V
binding. Our results showed that the shift-down to
328C resulted in a dramatic increase in the number of
cells positive for annexin V. However, the presence of
75 mM ZnSO4 resulted in a decrease of about 50% in
Oncogene
the fraction of Kp53A1/myc and KmycB/p53 cells
bound to annexin (Figure 3b).
Next we analysed the e€ect of c-Myc on p53mediated apoptosis by determining the internucleosomal DNA fragmentation. DNA laddering was clearly
reduced in both Kp53A1/myc and KmycB/p53 cell
lines incubated at 328C for 72 h in the presence of
75 mM ZnSO4, with respect to cells incubated without
zinc (Figure 3c). In contrast, extensive DNA fragmentation was observed in Kp53A1 cells either in the
presence or absence of ZnSO4. We also determined the
e€ect of c-Myc expression on apoptosis by analysing
the extent of chromatin condensation by DAPI
staining. KmycB/p53 and Kp53A1/myc cells were
incubated for 72 h at 328C in the presence or absence
of 75 mM ZnSO4, and the fraction of apoptotic cells
was dramatically reduced in zinc-treated cells (Figure
3d). We conclude that c-Myc inhibits the apoptosis
mediated by wild-type p53 in K562. As p53 may be
expressed at di€erent levels among individual cells, it is
formally possible that c-Myc overexpression allows the
Myc antagonizes p53 functions in K562 cells
E Ceballos et al
2197
Figure 2 (a) Scheme of the construction and expression pattern of Kp53A1/myc and KmycB/p53 cells. (b) Induction of ectopic cmyc mRNA by zinc. Total RNA was prepared from samples of the indicated cell lines after 24 h of incubation at 32 or 378C in the
absence or presence of 75 mM ZnSO4 Levels of p53 an c-myc mRNA were determined by Northern analysis. The arrowhead
indicates the position of 18S rRNA. (c) Induction of c-Myc protein expression by zinc. Cell extracts were prepared after incubation
at 328C in the absence or presence of 75 mM ZnSO4, and c-Myc was detected by immunoblot. The ®lter was also probed with antip53 antibody to assess its expression in transfectant cell lines and anti-a-tubulin to assess the protein loading of the samples
growth of those cells with lower p53 expression. Thus,
a population of p53-negative and apoptosis-resistant
cells could be selected during zinc treatment at 328C of
Kp53A1/myc or KmycB/p53 cells. However, the
expression of p53, as assessed by immunoblot, did
not decrease signi®cantly after 96 h at 328C in the
presence of zinc (not shown). Also, p53 is detected by
immuno¯uorescence in the nuclei of Kp53A1/myc cells
incubated at 328C in the presence of zinc (not shown),
ruling out a c-Myc-mediated impairment of p53
translocation to the nucleus.
c-Myc antagonizes the transcriptional activities of p53 in
K562
To investigate possible mechanisms by which c-Myc
could exert its protective e€ect on p53-mediated
apoptosis, we studied the transactivation activity of
p53 on the promoters of bax and p21WAF1, two p53
target genes involved in apoptosis and cell cycle arrest.
We ®rst con®rmed that co-transfection of wild-type
p53 expression vector (pLPC-hp53) into the parental
K562 cells resulted in a dramatic activation of the
p21WAF1 promoter (58-fold, mean of four experiments).
The induction was similar at 328C and 378C (not
shown). The e€ect of c-Myc on p53-dependent
transcription was studied by transfecting into K562,
pLPC-hp53 with and without an expression vector
encoding c-Myc (LMycSN), together with the p53responsive bax, mdmd2 and p21WAF1 promoter-luciferase
reporters. Interestingly, c-Myc reduced the transactiva-
tion of the three promoters by 40 ± 60% depending on
the promoter analysed (Figure 4a). We also tested
whether c-Myc impaired the p53-mediated transcriptional repression of cytomegalovirus (CMV) immediate
early promoter (Subler et al., 1992; Jackson et al.,
1993). The results showed that this repression was
clearly attenuated by co-expression of c-Myc (Figure
4a).
We investigated the c-Myc e€ect in Kp53A1 and
Kp53C1 cells. First, we con®rmed that the shift to
328C of Kp53A1 cells resulted in a notable increase in
the activity of p21WAF1 promoter, in agreement with the
presence of p53 in wild-type conformation (Figure 4b).
This increase was abolished by co-transfection of the
papilloma virus E6 gene expression vector (Figure 4b),
or by deleting the p53-responsive elements of the
p21WAF1 promoter (not shown), demonstrating that p53
was responsible for this transactivation. Next, Kp53A1
and Kp53C1 cells were co-transfected with the di€erent
reporters together with a c-Myc expression vector
(LMycSN) and incubated at 32 and 378C. Transcriptional activation of bax and p21WAF1 promoters was
dramatically induced at 328C (Figure 4c), but it was
reduced by 40 ± 60% upon co-transfection of c-Myc
expression vector. Likewise, p53-mediated repression of
CMV promoter was reduced by c-Myc expression
(Figure 4c). Thus, c-Myc impaired both the transcriptional activation and repression activities of p53 in the
K562 model. In contrast, the p53-independent upregulation of p21WAF1 induced by phorbol esters or
okadaic acid in K562 (Zeng and El-Deiry, 1996) was
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Myc antagonizes p53 functions in K562 cells
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Figure 3 c-Myc overexpression inhibits p53-mediated apoptosis. (a) Cell viability and apoptosis of Kp53A1, Kp53A1/myc and
KmycB/p53. Cells were incubated at 328C in the presence of 75 mM ZnSO4 as indicated. Histograms show percentages of viable cells
as determined with the trypan blue or cells showing apoptotic morphology after Giemsa staining. Bars indicate standard deviations
from three separate experiments. (b) Apoptosis determined by annexin V binding. Cells were incubated for 24 h at 37 or 328C in the
absence (white bars) or presence of 75 mM ZnSO4 (black bars), and the fraction of cells bound to annexin V was determined by ¯ow
cytometry. Data represent the mean of four separate experiments. (c) DNA laddering assay. Kp53A1/myc and KmycB/p53 cells
were grown for 72 h at 37 or 328C in the presence or absence of 75 mM ZnSO4 as indicated. DNA was prepared and electrophoresed
on agarose gels. Molecular mass markers were loaded in M lanes. (d) Apoptotic chromatin condensation of Kp53A1/myc and
KmycB/p53 cells incubated for 72 h at 328C in the presence and absence of 75 mM ZnSO4. Cells were stained with DAPI and
micrographs of representative ®elds were obtained under UV light
only marginally inhibited by c-Myc (5 ± 20%) (not
shown).
As p21WAF1 transactivation correlated to the changes at
protein level (see below), we sought to con®rm the c-Myc
e€ect on p53-induced transactivation of p21WAF1 promoter by three approaches. First, we transfected the
p21WAF1-luciferase construct along with increasing
amounts of c-myc expression vector (6, 10 and 14 mg of
LMycSN) into Kp53A1. The results revealed an
increased blocking e€ect of the p53-induced transactivation of p21WAF1 with higher c-myc doses. With the highest
amount of transfected c-myc expression, the activity of
p21WAF1 promoter fell to 20% of control activity (Figure
5a). Second, we transfected the p21WAF1-luciferase
reporter, along with an expression vector for cMycERTM. This is a fusion protein of c-Myc and a
mutant hormone binding site of estrogen receptor. The cMyc activity of this protein is activated upon addition of
4-hydroxytamoxifen (Littlewood et al., 1995). The
results indicated that p21WAF1 promoter activity was
reduced by 4-hydroxytamoxifen in a concentrationdependent manner, reaching 60% of inhibition in cells
treated with 500 nM 4-hydroxytamoxifen (Figure 5b).
No e€ect of the hormone on promoter activity was
detected in cells transfected with the empty vector.
Finally, we tested p21WAF1 promoter activity in
Kp53A1/myc or KmycB/p53 cells at 328C in the
absence and presence of zinc, i.e., under conditions in
which c-Myc induction was concomitant with apoptoOncogene
sis protection. When the cells were shifted to 328C, the
expression of luciferase was induced by about 10-fold
(Figure 5c, white bars), in accordance with the change
to a wild-type conformation of p53. When the
experiment was conducted in the presence of zinc the
activity of the p21WAF1 promoter was reduced by 40%
compared to the cells without zinc (Figure 5c).
Essentially, the same results were obtained with
KmycB/p53 and Kp53A1/myc cells. The reduction in
p21WAF1 promoter activity upon zinc addition was
reproduced in 10 independent experiments among the
two cell lines. Similar results were observed with 75
and 100 mM ZnSO4 (not shown). Zinc addition also
reduced the activity of Bax promoter in these cell lines
(not shown). As expected, zinc addition did not
decrease p53-mediated p21WAF1 transactivation of
Kp53A1 cells shifted to 328C (Figure 5c) or K562
transfected with a wild-type p53 expression vector (not
shown). To assess that zinc induced active c-Myc, we
used a luciferase reporter carrying a Myc-responsive
promoter (pGL2M4-Luc). The results con®rmed that
c-Myc transcriptional activity is elevated when protecting cells from apoptosis (Figure 5c).
c-Myc antagonizes the up-regulation of p21WAF1 mediated
by p53 in K562
We investigated the levels of mRNA and protein of
Bax, Mdm2 and p21WAF1 in Kp53A1/myc and
Myc antagonizes p53 functions in K562 cells
E Ceballos et al
2199
Figure 4 c-Myc impairs transcriptional activity of p53 in K562. (a) K562 cells were cotransfected with 3 mg of the luciferase
reporter plasmids containing the Bax (`Bax-Luc' in the histogram), p21WAF1 (`p21-Luc') or cytomegalovirus (`CMV-Luc') promoters,
and 9 mg of pLPCX, pLPC-hp53, LXSN or LMycSN. `Vectors' refers to cells transfected with the two empty vectors. Luciferase
activity was determined as described in Materials and methods. (b) Kp53A1 cells were transfected with 3 mg of pGL3-basic vector
(`pGL3'), p21-Luc reporter and p21-Luc with a papillomavirus E6 gene expression vector (CMV16-E6, `E6'). (c) Kp53A1 and
Kp53C1 cells were transfected with the reporter plasmids as indicated above for K562 and co-transfected with 9 mg of LXSN or
LMycSN. Fourteen hours after transfection, aliquots of the culture were incubated at 37 or 328C for 24 h and the luciferase activity
of cell lysates was determined. Several transfections with similar results were performed for each plasmid combination, and
representative results are shown. The maximum luciferase activity value was set at 100% for each reporter and the other data were
normalized to this value
KmycB/p53 cells. As expected from the presence of
p53 in wild-type conformation, the shift to 328C
resulted in the induction of bax and p21WAF1 mRNA
expression (Figure 6a, lanes 4 ± 7). After addition of
75 mM ZnSO4 a dramatic induction of exogenous cmyc mRNA was observed. The endogenous c-myc
expression declined at 328C, in consistency with the
reported e€ect of mutant and wild-type p53. When cMyc expression was induced by zinc addition, no
signi®cant changes in bax mRNA levels (Figure 6a)
and protein (not shown) were observed. In contrast,
levels of p21WAF1 mRNA were clearly decreased in
both cell lines after 24 h of incubation at 328C in the
presence of zinc (Figure 6a, lanes 4 and 7) compared
to parallel cultures without zinc (Figure 6a, lanes 5
and 6). The e€ect of c-Myc on mRNA was also
observed at the level of p21WAF1 protein, as shown by
immunoblot (Figure 6b). The correlation between
p21WAF1 protein down-regulation and inhibition of
cyclin-dependent kinase 2 (CDK2) was studied in
Kp53A1/myc and KmycB/p53 cells in the presence
and absence of c-Myc induction. The result showed
that incubation of the cells at 328C caused a marked
repression of CDK2 activity and that c-Myc counteracted this repression. This result was obtained
assaying both the CDK2- and the cyclin E-associated
kinase activity (Figure 6c). Altogether, our results
show that c-Myc impairs the induction of p21WAF1
expression mediated by wild-type p53 in K562 cells
and that this e€ect occurs at the transcriptional level.
Discussion
The present study provides evidence that c-Myc
antagonizes both the apoptosis and the up-regulation
of p21WAF1 mediated by p53. Using the p53Val135 mutant
we ®rst showed that p53 in wild-type form induces
growth arrest followed by cell death of K562. The cell
death induced by wild-type p53 in K562 transfectants
has the morphological and biochemical features of
typical apoptosis. p53-induced K562 cell death has
been reported (Kremenetskaya et al., 1997; Trepel et
al., 1997). In contrast, other studies indicate that
K562 expressing wild-type p53 are able to grow, albeit
at a slower rate and accompanied by erythroid
di€erentiation (Ehinger et al., 1995; Feinsten et al.,
1992). The most obvious explanation for these
di€erences is that higher levels of p53 in wild-type
conformation are easier to obtain in stable transfectants with a termosensitive mutant, since the transfection with constitutive wild-type p53 will select against
those cell clones overexpressing the pro-apoptotic
protein.
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Myc antagonizes p53 functions in K562 cells
E Ceballos et al
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Figure 5 c-Myc counteracts the transactivation of p21WAF1 by p53. (a) E€ect of increasing doses of c-Myc on p21WAF1 promoter
activity in Kp53A1. Cells were transfected with 2 mg of pGL3-basic and 0, 6, 10 and 14 mg of LMycSN plasmid (from open to solid
histograms). After 14 h at 378C aliquots of the cultures were incubated for 24 h at 32 and 378C before determination of luciferase
activity. Aliquots of transfected cells were incubated for 24 h at 32 and 378C before determination of luciferase activity. (b) E€ect of
c-MycERTM activation by 4-hydroxytamoxifen (TMX). Kp53A1 cells were transfected with 15 mg of a plasmid encoding cMycERTM or the empty vector (pBabePuro) along with the reporter plasmid. After 14 h at 378C, the culture was divided into four
aliquots and incubated for 24 h at 32 or 378C in the presence of 0, 50, 100 and 500 nM 4-hydroxytamoxifen (from open to solid
histograms) before determination of luciferase activity. Bars indicate the standard deviation from the mean of three independent
experiments. (c) E€ect of c-Myc in Kp53A1/myc, KmycB/p53 and Kp53A1. Cells were transfected with 3 mg pGL3-Waf1-Luc (`p21Luc') and 9 mg of pLPCX (as carrier DNA) and incubated at 37 or 328C in the presence or absence of 100 mM ZnSO4 (`Zn'). Right
panel show the transactivation of a Myc-responsive luciferase reporter (pGL2M4-Luc, `M4-Luc') in the presence and absence of
100 mM ZnSO4
Interestingly, ectopic expression of c-Myc counteracts the apoptosis induced by wild-type p53 in K562
cells. This result seems paradoxical as c-Myc induce
apoptosis in di€erent cell types under conditions of
growth factor deprivation. However, c-Myc overexpression induces little apoptosis of K562 cells in
the absence of serum (CanÄelles et al., 1997). This is
consistent with the lack of expression of p53 (Law et
al., 1993), p19ARF (Delgado et al., 2000) and CD95/
FAS (McGahon et al., 1995) in K562, as the three
proteins are required for c-Myc-mediated apoptosis in
®broblasts (Hermeking and Eick, 1994; Hueber et al.,
1997; Wagner et al., 1994; Zindy et al., 1998).
However, c-Myc does not confer an unspeci®c antiapoptotic e€ect on K562 cells, as it does not protect
from other apoptotic stimuli as okadaic acid or
ceramides (CanÄelles et al., 1997; our unpublished
results). The antiapoptotic e€ect of c-Myc has also
been described in lymphoid cells (Sonenshein, 1997;
Felsher and Bishop, 1999) and keratinocytes (Waikel et
al., 1999), but its relationship with p53 function in
these processes is unknown.
How does c-Myc impair the p53-mediated apoptosis in K562 cells? One obvious possibility is that cMyc abrogates the transcriptional activation of p53
target genes. We tested bax and p21WAF1 genes, and
the p53-mediated transactivation of both promoters
Oncogene
was attenuated by c-Myc overexpression. Moreover,
c-Myc attenuates the p53-mediated repression of
CMV immediate early promoter. Thus c-Myc interferes with both the transcriptional activation and
repression mediated by p53. Bax mRNA and protein
levels were unchanged by c-Myc expression, ruling
out the Bax down-regulation as a mechanism for
apoptosis protection. In contrast, the expression of
p21WAF1 mRNA and protein was dramatically reduced.
Inhibition of p53-dependent induction of p21WAF1 is a
newly described activity of c-Myc. In NIH3T3
®broblasts the ectopic expression of c-Myc abrogates
the p21WAF1 inhibitory activity on cyclin dependent
kinases but it does not modify its expression
(Hermeking et al., 1995). During the preparation of
this manuscript, it has been reported that c-Myc
inhibits phorbol ester-induced p21WAF1 up-regulation
in prostate and breast cancer cell lines (Mitchell and
El-Deiry, 1999). Thus, c-Myc-mediated inhibition of
p21WAF1 transactivation described herein for K562
may occur widely among tumor cells. Other recent
studies show that loss of c-Myc in rat Rat-1 cells
results in a reduction of p21WAF1 (Mateyak et al.,
1999). This reinforces the idea that c-Myc does not
directly interact with p21WAF1 or Bax promoters and
that the e€ect on p53 transcriptional activity depends
on the cell type.
Myc antagonizes p53 functions in K562 cells
E Ceballos et al
2201
Figure 6 c-Myc overexpression impairs the induction of p21WAF1 expression by p53. (a) Inhibition of p53-mediated up-regulation
of p21WAF1 mRNA. Cells of the indicated cell lines were incubated at 32 or 378C and parallel cultures were treated with 75 mM
ZnSO4. The cells were incubated for 24 h (lanes 1 ± 11) or 48 h (lanes 12 and 13) at 328C. Total RNA was prepared and the levels of
c-myc, p21WAF1 and bax mRNA determined by Northern analysis. The same ®lter was consecutively hybridized to the indicated
probes. (b) Inhibition of p53-mediated up-regulation of p21WAF1 protein. Cells were incubated at 32 or 378C for 24 h in the presence
or absence of 75 mM ZnSO4 and the levels of p53 were analysed by immunoblot. (c) Increase in CDK2 activity mediated by c-Myc
in K562 expressing wild-type p53. Cells were incubated at 328C for 48 h in the presence or absence of 75 mM ZnSO4 as indicated.
The extracts were immunoprecipitated with anti-CDK2 or anti-cyclin E antibodies as indicated and the kinase activity of the
immune complexes was determined using histone H1 as substrate
Does the c-Myc-mediated down-regulation of
p21WAF1 explain the abrogation of p53-dependent
apoptosis?. Given that the mechanism of p53-induced
apoptosis is not well understood, we can only
speculate on the possible mechanisms that are
responsible for c-Myc-mediated supression of apoptosis. p21WAF1 appears to be necessary for growth
arrest but not for apoptosis induced by wild-type p53
(Bates and Vousden, 1999; Hansen and Oren, 1997).
It is formally possible that p21WAF1 accumulation
eventually results in apoptosis of K562 cells, as has
already been described in other systems as neuroblastoma (Poluha et al., 1996), lung carcinoma
(Adachi et al., 1996), breast carcinoma (Sheikh et
al., 1995), colon carcinoma (Chinery et al., 1997;
Polyak et al., 1996), and lymphoid cells (Wu et al.,
1998). However, it has been reported that expression
of another p53 mutant (p53Val143) in K562 results in
p21WAF1 up-regulation without concomitant apoptosis
(Kobayashi et al., 1995). Also, co-transfection of an
antisense-p21WAF1 expression vector did not alleviate
the apoptosis due to p53 in K562 (our unpublished
results). Thus it seems unlikely that p21WAF1 decline
by itself is responsible for the protective e€ect of cMyc. Our results rather indicate that c-Myc interferes
with the ability of p53 to act as transcription factor
in K562, as it not only antagonizes the activation of
Bax and p21WAF1 promoters but also the repression of
CMV promoter. It must be noted that there might
be many p53-target genes involved in apoptosis, and
therefore it is possible that c-Myc interferes with
p53-dependent induction of other pro-apoptotic
proteins.
Independently of the mechanisms involved, our
results o€er a new clue for the role of c-Myc in the
development of leukemia. On one hand, c-myc overexpression has been implicated in the malignant
progression of human tumors (Garte, 1993; Henriksson
and LuÈscher, 1996; Berns et al., 1992). On the other
hand, apoptosis is a well-documented consequence of
c-Myc ectopic expression in cells subjected to growth
factor deprivation or to hypoxia. Moreover, a
correlation between high c-myc expression and apoptosis has been reported for some solid tumors (Ho€man and Liebermann, 1998; Alarcon et al., 1996;
Donzelli et al., 1999). However, the pro-apoptotic
e€ect of c-Myc is at odds with the frequently found cmyc overexpression in human cancer. This is most
intriguing in those tumors retaining p53 function,
which occurs in a majority of the cases in some
hematological neoplasias, e.g. CML blast crisis (Ahuja
et al., 1991; Prokocimer and Rotter 1994; Nakai and
Misawa, 1995; Stuppia et al., 1997), Burkitt lymphomas (Newcomb, 1995) and mouse plasmacytomas
(Gutierrez et al., 1992). Thus, c-Myc overexpression
must confer some selective advantage which counteracts its apoptotic e€ect. It has been suggested that
tumors produce survival factors able to block the
apoptotic e€ect of c-Myc, leaving the proliferative
pathway unrestrained. Although our data do not
contradict this hypothesis, they indicate that, at least
in some tumors, an additional mechanism may be
Oncogene
Myc antagonizes p53 functions in K562 cells
E Ceballos et al
2202
operating which can explain c-myc overexpression
during tumor progression. Given the fact that p53
plays a pivotal role in mediating drug- or radiationinduced apoptosis, we propose that c-Myc overexpression could protect tumor cells from apoptosis mediated
by p53.
Materials and methods
Cell lines, transfection and retroviral transduction
All cell lines were grown in RPMI 1640 medium (Gibco-Life
Sciences) supplemented with 8% fetal calf serum and
gentamycin (80 mg/ml). Kp53A1 cells (originally termed
K562/ptsp53/A1) are K562 cells expressing a mouse
p53Val 135 mutant (Ehinger et al., 1997). The KCMV cell lines
are K562 transfected with neo-resistant vector (Ehinger et al.,
1997). KmycB are K562 cells transfected with a zincinducible c-myc gene (Delgado et al., 1995). Kp53C1 cells
were generated by infecting K562 cells with retrovirus
containing the vector pBabePuro-p53Val135 (provided by M
Oren, Weizmann Institute, Revohot, Israel). The vector was
electroporated into Phoenix-A cells and the supernatant used
to infect K562 cells in the presence of 6 mg/ml polybrene.
Infected cells were selected with 2 mg/ml puromycin and the
resulting culture of puromycin-resistant cells was termed
Kp53C1. To generate the Kp53A1/myc cell line, Kp53A1
cells were electroporated at 260 v and 1 mFa with pHeBOMT-myc vector which carries a c-myc gene under the control
of metallothionein promoter (Grignani et al., 1990). Cells and
selected with 0.5 mg/ml of G418 and 0.1 mg/ml of hygromycin. Cell clones were isolated by limiting dilution. KmycB/
p53 cells were generated by infecting KmycB cells with
pBabePuro-p53Val135 retroviruses as described above. The p53
transfectant cell lines tended to lose the apoptotic phenotype
after prolonged cell culture, and fresh stocks were used for
most experiments.
Apoptosis determinations
When indicated, exponentially growing cells at a concentration of 26105 cells/ml were transferred to 328C. Cell
morphology was analysed by May-GruÈnwald-Giemsa or
4,6-diamino-2-phenylindole (DAPI) staining of cytocentrifuge
preparations. At least 200 cells were scored for each
determination of apoptotic or metaphasic cells. Cell growth
and viability were assayed with hemocytometer and the
trypan blue exclusion test and by colorimetry based on the
reduction of the tetrazolium salt WST-1 (Roche Molecular
Biochemicals). To determine the presence on internucleosomal fragmentation (DNA laddering), cellular DNA was
prepared and analysed by agarose gel electrophoresis as
described (CanÄelles et al., 1997). Binding of annexin V to cell
surface was carried out by ¯ow cytometry with annexin VFITC kit following the manufacturer's instructions (Genzyme
Diagnostics).
RNA, protein and kinase activity analysis
Total RNA was isolated from cells by the acid guanidine
thiocyanate method and Northern blots were prepared and
hybridized according to standard procedures. Probe for
human c-myc was as described (Delgado et al., 1995); for
p53, a 2.1 kb XhoI ± BamHI fragment from pLTRp53cG
corresponding to mouse cDNA (provided by M Oren); for
p21WAF1 a 0.6 kb NotI fragment from pBSK-p21 corresponding to human cDNA (provided by M Serrano, Centro
Nacional de BiotecnologõÂ a, Madrid); for bax, a 0.5 kb EcoRI
fragment form pSSFV-Bax corresponding to human cDNA
(provided by S Korsmeyer, Washington University Medical
School, St. Louis, USA). For immunoblots, cell lysates were
prepared and immunoblotted as described (CanÄelles et al.,
1997). c-Myc and p21WAF1 were detected with rabbit
polyclonal antibodies, p53 with the monoclonal antibody
PAb240 (Santa Cruz Biotech.), and a-tubulin with a rabbit
polyclonal antibody (provided by N Cowan, New York
University, NY, USA). Immunocomplexes were detected with
peroxidase-conjugated secondary antibodies (Cappel) and
chemiluminescent method (ECL, Amersham). For kinase
assays, cyclin E and CDK2 immunoprecipitations were
performed essentially as described (Mateyak et al., 1999),
using anti-cyclin E (M-20, Santa Cruz) and anti-CDK2 (M2,
Santa Cruz) antibodies. Kinase activity of the immunoprecipitates was assayed using histone H1 as substrate.
Transactivation assays
Cells were transfected by electroporation at 260 v and 1 mFa
in a Bio-Rad electroporator. Cells (4 ± 86106) were cotransfected with the appropriate plasmids and after 14 h of
incubation at 378C, cultures were split into aliquots and
further incubated for 24 h at 32 or 378C in the absence or
presence of 100 mM ZnSO4 or 50 ± 500 nM 4-hydroxytamoxifen. Cells were lysed and the luciferase activity was measured
by a dual-luciferase reporter gene assay system (Promega).
Plasmids used were: pGL3-Waf1-Luc (provided by M Oren)
which contains a 2.3-kb of p21WAF1 promoter upstream of the
®re¯y luciferase cDNA, pGL3-Bax-Luc (provided by M
Oren), CMV-Luc (Bellon et al., 1994), pLPC-hp53 vector
expressing wild-type p53 (provided by M Serrano) and its
corresponding empty vector pLPCX (Serrano et al., 1997);
pGL2M4-Luc (provided by R Eisenman, Fred Hutchinson
Cancer Research Center, Seattle, USA), LMycSN (provided
by R Eisenman) and the empty vector LXSN (Miller et al.,
1993), pCMV16-E6 which carries the human papilloma virus
E6 gene (provided by K Cho, Johns Hopkins University,
Baltimore, USA), and pBpuro-c-mycERTM (provided by T
Littlewood, Imperial Cancer Research Fund, London, UK)
and the vector pBabePuro (Littlewood et al., 1995). In each
transfection experiment, the same amount of total DNA was
transfected for all samples, using the empty vectors as carrier
DNA. One mg of the pRL-TK plasmid encoding for Renilla
luciferase (Promega) was co-transfected in each case.
Promoter activity was de®ned as the ratio between light
units generated by the ®re¯y and Renilla luciferases.
Acknowledgments
We thank Pilar Frade and Rosa Blanco for technical
assistance, and Kathleen Cho, Nicholas Cowan, Riccardo
Dalla Favera, Robert Eisenman, Abelardo LoÂpez-Rivas,
Stanley Korsmeyer, Trevor Littlewood, Moshe Oren and
Manuel Serrano for plasmids and antibodies. We are
gratefully indebted to Dirk Eick and Moshe Oren for
helpful comments on the manuscript. This work has been
supported by grant PM98-0109, Spanish Ministry of
Education and Culture, and Biomed 96-3532 from European Community.
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