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E2F activity is essential for survival of Myc-overexpressing human cancer
cells
Eric Santoni-Rugiu*
,1,3
, Dominique Duro
1,5
, Thomas Farkas
1
, Ida S Mathiasen
2
, Marja Ja
¨
a
¨
ttela
¨
2
,
Jiri Bartek*
,1,4
and Jiri Lukas
1
1
Department of Cell Cycle and Cancer, Institute of Cancer Biology, Danish Cancer Society, 2100 Copenhagen E., Denmark;
2
Apoptosis Laboratory, Institute of Cancer Biology, Danish Cancer Society, 2100 Copenhagen E., Denmark
Effective cell cycle completion requires both Myc and
E2F activities. However, whether these two activities
interact to regulate cell survival remains to be tested.
Here we have analysed survival of inducible c-Myc-
overexpressing cell lines derived from U2OS human
osteosarcoma cells, which carry wild-type pRb and p53
and are deficient for p16 and ARF expression. Induced
U2OS-Myc cells neither underwent apoptosis sponta-
neously nor upon reconstitution of the ARF-p53 axis
and/or serum-starvation. However, they died massively
when concomitantly exposed to inhibitors of E2F
activity, including a constitutively active pRb (RbDcdk)
mutant, p16, a stable p27 (p27T187A) mutant, a
dominant-negative (dn) CDK2, or dnDP-1. Similar
apoptotic effect was observed upon down-modulation of
endogenous E2Fs throu gh overexpression of E2F binding
site oligonucleotides in U2OS-Myc cells, upon expres-
sion of RbDcdk or dnDP-1 in the Myc-amplified HL-60
(ARF7; p537) human leukemia cells, and upon co-
transfection of Myc and RbDcdk in SAOS-2 (ARF+;
p537) human osteosarcoma cells but not in human
primary fibroblasts. Consistent with these results, a
dnp53 mutant did not abrogate the Myc-induced
apoptotic phenotype, which instead strictly depended on
caspase-3-like proteases and on Myc transcriptional
activity. Our data indicate that in contrast to normal
cells, Myc-overexpressing human cancer cells need E2F
activity for their survival, regardless of their ARF and
p53 status, a notion that may have important implica-
tions for antineoplastic treatment strategies.
Oncogene (2002) 21, 6498 – 6509. doi:10.1038/sj.onc.
1205828
Keywords: Myc; E2F; human cancer cells; survival
Introduction
The tumor suppressors p16INK4A (p16) and p14/
p19ARF (ARF) are two alternative and structurally
unrelated products encoded by the INK4A/ARF genetic
locus (Duro et al., 1995; Quelle et al., 1995), a frequent
target of inactivation in tumorigenesis (Roussel, 1999).
While p16 inhibits the phosphorylation of the retino-
blastoma protein (pRb), ARF stabilizes p53 and
activates p53-mediated growth-inhibito ry responses
(reviewed in Sherr and Weber, 2000). Deregulated
expression of distinct cell cycle stimulating oncogenes,
such as Myc, E2F-1, E1A, and Ras can upregulate
ARF gene exp ression and result in p53-mediated cell
cycle arrest or ap optosis (Sherr and Weber, 2000). This
appears to be an important fail-safe mechanism to
guard cells that do not carry deletions or mutations of
ARF or p53 from abnormal proliferative signals.
Consistent with that, ARF- or p53-null MEFs are
similarly resistant to Myc-induced a poptosis and when
wild-type or ARF-hemizygote MEFs are immortalized
by Myc, they escape from apoptosis by usually
sustaining mutation of either ARF or p53 but not of
both (Zindy et al., 1998). Moreover, reintroduction of
the missing protein in knockout MEFs resensitizes
them to apoptosis. It has also been shown that B-cell
lymphomas arising in Eu-Myc-transgenic mice are
characterized by ARF deletion or p53 mutation
(Eischen et al., 1999). Crossing Eu-Myc mice with
ARF-null or p53-deficient mice, results in similarly
accelerated malignant phenotype, characterized by
aggressive lymphomas with low apoptotic rates and
resistance to chemotherapeutic drugs (Schmitt et al.,
1999). All these results have lead to the notion that
ARF, as a sensor of oncogenic insults such as Myc
overexpression, regulates a tumor suppressor pathway
operating via p53-dependent apoptosis.
However, it remains unclear how closely this model
is also applicable to human tumor cells. In keeping
with the functional link between ARF and p53, a p53-
mediated cell cycle arrest in G1 and G2/M was elicited
by ectopic ARF expression in ARF-negative human
tumor cells (Stott et al., 1998), but whether ARF-
mediated activation of p53 might also result in
apoptosis was not investigated. Moreover, an inverse
correlation between ARF expression and p53 status
Received 8 April 2002; revised 18 June 2002; accepted 28 June
2002
*Correspondence: E Santoni-Rugiu; E-mail: santoni@dadlnet.dk and
J Bartek; E-mail: bartek@biobase.dk
Current addresses:
3
Department of Pathology, Rigshospitalet,
Frederik d. V’s Vej 11, 2100, Copenhagen Ø., Denmark;
4
Depart-
ment of Cell Cycle and Cancer, Institute of Cancer Biology, Danish
Cancer Society, Strandboulevarden 49, 2100 Copenhagen Ø.,
Denmark;
5
INSERM, Paris, France
Oncogene (2002) 21, 6498 – 6509
ª
2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00
www.nature.com/onc
was observed in several human tumor cell lines (Stott
et al., 1998). More recently, either mutually exclusive
(Fulci et al., 2000; Pinyol et al., 2000) or coexisting
(Gazzeri et al., 1998; Markl and Jones, 1998; Sanchez-
Cespedes et al., 1999) alterations of p53 and ARF have
been reported in human tumors. While the former
studies are consistent with these genes acting along the
same pathway, the latter, together with the fact that
murine ARF-p53 double-null preB cells are more
resistant to Myc-induced apoptosis than cells lacking
p53 or ARF alone (Eischen et al., 1999), support the
idea of additional, p53-independent tumor suppressive
functions of ARF (Weber et al ., 2000). Alternatively,
tumor cells may first undergo p53 mutation and then,
despite little selection against ARF, may sustain
deletions targeting p16 that coincidentally co-delete
ARF (Stott et al., 1998; Sanchez-Cespedes et al., 1999).
To investigate the relation between c-Myc and the
ARF-p53 apoptotic pathway in human tumor cells we
analysed the survival properties of inducible, c-Myc-
overexpressing cell lines derived from osteosarcoma
U2OS cells (Santoni-Rugiu et al., 2000), which carry
wild-type pRb and p53 (Diller et al., 1990) and are
deficient for p16 and ARF expression (Stott et al.,
1998; present study and data not shown). Induction of
Myc alone or after reintroduction of ARF did not
result in death of U2OS cells, even when these cells
were concomitantly serum-starved, suggesting that they
may differ from MEFs and primary murine lympho-
cytes in their response to the activation of the ARF-
p53 pathway in presence of deregulated Myc expres-
sion. Thus, this type of human tumor cells may not
necessarily rely on disruption of this pathway to
counteract Myc-derived apoptotic signals. In an
attempt to identify other potential regulators of
survival in Myc-overexp ressing tumor cells, we tested
whether E2F activity would represent one of these. We
have recently shown that E2F and Myc activities are
concomitantly required to ensure timely and proper
levels of DNA synthesis and orderly completion of cell
cycles, despite these activities being able to promote the
G1/S transition independently of each other (Lukas et
al., 1999a; Santoni-Rugiu et al., 2000). Therefore, we
investigated whether survival of Myc-overexpressing
tumor cells may involve endogenous E2F activity. Our
results indicate that this activity is critical for the
survival of human tumor cells with deregulated Myc
expression, regardless of the intactness of the ARF-p53
pathway, a notion that may have important implica-
tions for antineoplastic therapeutic strategies.
Results
Expression of ARF does not sensitize U2OS cells to
Myc-induced apoptotic signals
It is known from studies in different cell types that
increased Myc expression is able to stimulate either cell
cycle progres sion or apoptosis, if the former is
perturbed by serum starvation or by other types of
cell-stress (reviewed in Prendergast, 1999). In this
respect, flow cytometric analysis of DNA content
showed that stable induction of Myc by culturing
U2OS-Myc cells in tetracycline (TET)-free medium
stimulated progression through all phases of the cell
cycle (Table 1 and Santoni-Rugiu et al., 2000), without
provoking a significant increase in the sub-G1 cell
compartment (Table 1). In agreement with this, the
viability of derepressed U2OS-Myc cells monitored
over 6 days by the trypan blue dye exclusion method
was virtually unaffected in presence of serum and very
modestly perturbed under serum-starvation (Figure
1a). As this could be explained, at least in part, by
the lack of ARF gene expression in these cells (see
Introduction and Figure 1b), we tested whether ectopic
HA-tagged ARF would sensitize them to potential
apoptotic signals prompted by Myc (Juin et al., 1999).
Transfection efficiency was monitored by anti-HA
immunostaining as reported (Santoni-Rugiu et al.,
2000). However, following Myc-induction, no signifi-
cant difference in the amount of apoptotic sub-G1
fraction was observed between U2OS-Myc cells
proficiently transfected with HA-ARF and those
transfected with empty-vector, even in conditions of
serum-starvation (Table 1). Nevertheless, exogenous
ARF was functional, as its expression resulted in
appreciable stabilization of p53 (Figure 1b) and arrest
in G1 and G2/M when cells were kept in serum-
containing medium (Table 1), consistent with previous
observations in U2OS cells (Stott et al., 1998). In
serum-starved U2OS-Myc cells, ARF expression rein-
forced the G1 arrest induced by serum deprivation
(Table 1). Taken together these data imply that human
tumor cells may, at least to some extent, respond
Table 1 Flow cytometric analysis of DNA content in propidium
iodide-stained U20S-Myc cells
a
U20S-Myc cells Apoptotic (%) G0/G1 (%) S (%) G2/M (%)
+Tet 2 56 34 10
+Tet+Vector
b
3582814
+Tet+ARF 2 69 9 22
+Tet7FCS
c
3721810
+Tet+ARF7FCS 2 82 8 10
7Tet 3 34 39 27
7Tet+Vector 2 36 38 26
7Tet+ARF 3 45 19 36
7Tet – FCS 4 63 23 14
7Tet+ARF7FCS 2 71 11 18
a
Each profile was assessed at least three times and in independent
clones with similar results. Untransfected cells were cultured in TET-
containing (+Tet) or -free (7Tet) medium for 4 days and cell cycle
distribution analysed by flow cytometry with CellQuest software and
quantified with ModFit software. Sub-G1 cells were gated and
counted separately by using CellQuest and expressed as percentage of
total number of cells.
b
Cells transfected with 5 mg empty pCMV or
ARF vector and 1 mg pCMV-CD20 were cultured in the same
conditions and CD20-positive cells analysed as above. Empty vector
was added to a total of 25 mg DNA/10-cm-diameter dish.
c
Cultured
in absence of fetal calf serum (7FCS)
Myc-overexpressing cancer cells need E2F for survival
E Santoni-Rugiu et al
6499
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differently than MEFs and primary mouse lymphocytes
(Eischen et al., 1999) to the activation of the ARF-p53
pathway in the presence of deregulated Myc expres-
sion. The possibility that apoptosis in U2OS-Myc cells
could be prevented by ARF-mediated cell cycle arrest
or by impairment of the tumor suppressor apoptotic
pathway at a level downstream of ARF and p53
(Soengas et al., 2001) cannot be ruled out. In any case,
the reconstitution of the ARF-p53 axis does not
sensitize U2OS cells to Myc-dependent apoptotic
signals. We therefore searched for other mechanisms
that could do that.
Constitutively active Rb induces death of U2OS-Myc cells
It is still not known whether the effects of Myc on cell
cycle and survival are regulated independently
(Prendergast, 1999). We have recently shown in a
specifically engineered U2OS-deri ved cell line, U2OS-
RbDcdk/Myc, that M yc can stimulate G1/S transitio n
and persistent DNA replication in cells long-term
deprived of E2F activity (Santoni-Rugiu et al., 2000)
by a constitutively active pRb mutant, pRbDcdk
(Lukas et al., 1997, 1999b). However, both E2F and
Myc activities are required to ensure timely and proper
levels of DNA synthesis and orderly completion of cell
cycles (Lukas et al., 1999a; Santoni-Rugiu et al., 2000).
It remains untested, though, whether these activities
interact in regulating cell survival as well. Therefore, to
assess a possible correlation between E2F function and
viability of tumor cells with deregulated Myc activity,
we have now investigated the survival pr operties of
U2OS-RbDcdk/Myc cells. At day 4 after transgene co-
induction, in addition to the expected accumulation of
cells in S phase (Santoni-Rugiu et al., 2000), almost
50% of the total number of U2OS-RbDcdk/Myc cells
displayed sub-G1 DNA content (Figure 2a). Further-
more at the same time-point, more than 60% of the
cells were unable to exclude the vital dye trypan blue,
indicating that they had lost plasma membrane
integrity (Figure 2b). Comparable results were obtained
in an independent set of experiments where we
transiently transfect ed RbDcdk in U2OS-Myc cells
(see Figure 4) or co-transfected Myc and RbDcdk in
non-clonal parental U2OS cells (data not shown).
Altogether, these data indica te that deregulated
expression of Myc in the presence of constitutively
active Rb induces apoptosis in U2OS cells. It is
noteworthy that decreased survival was not observed
in U2OS-RbDcdk or U2OS-RbDcdk/CycE cell lines,
which express RbDcdk alone or together with another
cell cycle stimulator, cyclin E, respectively (Lukas et
al., 1999b; Santoni-Rugiu et al., 2000 and data not
shown). This implies that the induction of apoptosis is
a specific response to the coexpression of Myc and
RbDcdk.
Figure 1 (a) Viability of U2OS-Myc cells. Trypan blue exclusion
test performed at the indicated time-points after plating and cul-
turing the cells in TET-containing (+Tet) or -free (7Tet) med-
ium. Myc induction caused virtually no cell death in presence
of FCS and minimal toxicity in FCS-deprived cells (7FCS).
The experiment was repeated at least three times with comparable
results and reproduced in independent clones. (b) Ectopic ARF
stabilizes endogenous p53 expression in U2OS-Myc cells. Cells
were transfected with ARF expressing vector (+) or empty vector
(7) and Myc transgene was derepressed by TET removal. Cells
were harvested 2 days later and 50 mg protein lysate/lane were
electrophoretically resolved and immunoblotted with mAb DCS-
240.1 to human ARF and DO-1 to p53. CDK7 expression was
assessed with MO-1 mAb and used as loading control
Figure 2 Coexpression of Myc and constitutively active Rb
causes severe cell death of U2OS cells. (a) Representative flow-
cytometric DNA histograms of PI-stained U2OS-RbDcdk/Myc
cells, grown in medium with (filled area) or without (empty area)
TET for 4 days. Note the massive entry and accumulation of cells
in S phase after transgene induction, as originally described (San-
toni-Rugiu et al., 2000) as well as the massive presence of sub-G1
(apoptotic) cells (M1 gate). The result was reproduced multiple
times in this and other independent U2OS-RbDcdk/Myc clones.
(b) Progressive loss of viability of U2OS-RbDcdk/Myc cells after
induction of the transgenes. Cell integrity was evaluated by trypan
blue exclusion as described in Figure 1a. The experiment was re-
peated at least three times, with comparable results
Myc-overexpressing cancer cells need E2F for survival
E Santoni-Rugiu et al
6500
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The apoptotic phenotype in U2OS-RbDcdk/Myc cells is
dependent on Myc transcriptional activity and is mediated
by caspase-3-like proteases
Although the biological functions of Myc cannot be
entirely explained by the known Myc target genes, the
ability of Myc to induce S phase or cell death has been
linked to its capability of regulating key target genes
associated with proliferation or apoptosis (Prendergast,
1999). Therefore, we assessed the requirement of Myc
transcriptional activity for the apoptotic phenotype
observed in U2OS-RbDcdk/Myc cells. For this purpose
we transfected these cells with MadMyc (MM), a
previously described chimera that antagonizes Myc by
inhibiting its transcriptional regulatory function (Berns
et al., 1997; Santoni-Rugiu et al., 2000). Consistent
with our earlier results (Santoni-Rugiu et al., 2000),
MM abolished the massive entry and accumulation of
the U2OS-RbDcdk/Myc cells in S phase. More
importantly, it prevented apoptosis in these cells when
the transgenes were co-induced by TET-removal, while
U2OS-RbDcdk/Myc cells transfected with empty vector
displayed about the same level of apoptosis as the
untransfected population after TET-removal (Table 2
and Figure 2a). Thus, the transcriptional regulatory
function of Myc is required for the induction of cell
death in U2OS cells with deregulated expression of
Myc and constitutively active Rb.
Next, we further analysed the mechanism of cell
death in U2OS-RbDcdk/Myc cells. Following derepres-
sion of the Myc and RbDcdk transgenes we detected
sustained cleavage of poly(ADP-ribose) polymerase
(PARP) into a specific fragment of 85 kD (Figure
3a), a hallmark of caspase-dependent apoptosis
(Lazebnik et al., 1994). Consistent with this observa-
tion, cell death was prevented, in a dose-dependent
manner, when cells were treated with the pan-caspase
inhibitor ZVAD-fmk or the caspase-3-specific inhibitor
DEVD-CHO (Figure 3b and data not shown).
Furthermore, caspase-3-like activity at day 4 of
induction was almost eightfold higher than the activit y
recorded at day 0 (Figure 3c). In comparison, in
U2OS-Myc cells we observed less than twofold increa se
in caspase-3-like activity at day 4 of induction,
suggesting that Myc alone can induce some activity
but this is insufficient to produce death of U2OS cells
(see Figure 1). Thus, cell death caused by over-
expression of Myc and RbDcdk involves a mechanism
dependent in large part on caspase-3-like proteases, in
line with other models of Myc-dependent apoptosis
(Kagaya et al., 1997; Kangas et al., 1998; Hotti et al.,
2000). Consistent with these results, the anti-apoptotic
protein Bcl2, known to inhibit the mitochondrial
activation of caspase-3-like enzymes and Myc-mediated
apoptosis in other systems (Prendergast, 1999), signifi-
cantly diminished the amount of cell death in U2OS-
RbDcdk/Myc cells (Figure 4).
G1/S inhibitors do not prevent apoptosis in
U2OS-RbDcdk/Myc cells
Inappropriate, sustained expression of the co nstitu-
tively active RbDcdk mutant alone in U2OS cells
results in temporary G1 arrest followed by gradual
entry into S phase due to residual cyclin E-associated
kinase activity stimulated by endogenous Myc activity
(Lukas et al., 1999b; Santoni-Rugiu et al., 2000).
Instead, cells with forced co-expression of Myc and
RbDcdk, rapidly enter S phase and persistently
replicate DNA, owing to the high cyclin E-associate d
kinase activity induced by ectopic Myc, without,
however, being able to productively divide (Santoni-
Rugiu et al., 2000). Therefor e, cell death in U2OS-
RbDcdk/Myc cells could be due to their prominent
entry and accumulation in S phase with active cyclin/
CDK complexes, as cyclin-CDKs have been implicated
in apoptosis of certain cell types (O’Connor et al.,
2000). In order to test this hypothesis, U2OS-RbDcdk/
Myc cells were transfected with different inhibitors of
the G1/S transition, such as the CDK4/6 inhibitor p16,
the stable p27T187A mutant of the CDK2 inhibitor
p27 (Nguyen et al., 1999), and the dominant-negative
CDK2 mutant, dnK2 (van den Heuvel and Harlow,
1993) and were further cultur ed with or without TET.
Although capable of significantly reducing the amount
of cells in S phase, the expression of each of these G1/
S-inhibitors did not decrease the sub-G1 population of
induced U2OS-RbDcdk/Myc cells, but rather increa sed
it (Table 2). This was in contrast to the effect of MM,
which as described above, blocked both S phase entry
and apoptosis in U2OS-RbDcdk/Myc cells. Taken
together, these data suggest that apoptosis caused by
coexpression of Myc and RbDcdk can not be overcome
by the prevention of S phase as such but it is blocked
by the inhibition of Myc transcript ional activity. On
the other hand, the additional reduction in viability
observed in U2OS-RbDcdk/Myc cells after expression
of G1/S inhibitors, supports the idea that E2F activity
may be an important regulatory factor for the survival
of cells with deregulated Myc expression.
Table 2 Effects of G1/S inhibitors on apoptosis in U2OS-RbDcdk/
Myc cells
a
Plasmid Apoptotic (%) G1 (%) S+G2/M (%)
b
CMV 45 35 65
p16 49 65 35
p27T187A 54 79 21
dnCDK2 49 89 11
Apoptotic (%) G1 (%) S (%) G2/M (%)
MM 8 51 39 10
a
CD20-positive cells transfected with the indicated plasmids (CMV,
p16, p27T187A at 5 mg; dnK2 and MM at 15 mg) were assayed for
cell cycle distribution and apoptosis after a 4-day culture in TET-free
medium. Empty vector was added to a total of 25 mg DNA/10-cm-
diameter dish. A representative example of three comparable
experiments is shown.
b
S and G2/M are represented together because
the algorithm was unable to clearly distinguish these two phases in
cell transfected with the indicated plasmids except for MM
Myc-overexpressing cancer cells need E2F for survival
E Santoni-Rugiu et al
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Figure 3 Death of U2OS cells conditionally coexpressing Myc and pRbDcdk occurs via a caspase-3-like dependent mechanism. (a)
PARP cleavage after transgenes’ induction in U2OS-RbDcdk/Myc cells. 50 mg protein lysate/lane were electrophoresed and immu-
noblotted with mAbs to c-Myc (9E10), HA-tagged RbDcdk (HA12CA5) and PARP (C2-10). MCM7 expression, assessed with DCS-
141 mAb, was used as loading control. When expression of the ectopic transgenes is repressed by TET (day 0), PARP is uncleaved.
In contrast, sustained expression of transgenes after TET removal (day 2 and 4) causes increasing PARP cleavage. (b) Caspase-in-
hibitors block apoptosis of activated U2OS-RbDcdk/Myc cells in a dose-dependent manner. Cells (5610
5
) were plated onto 10 cm
dishes in TET-containing (filled area) or -free (empty area) medium and after 12 h the medium was supplemented with the pan-cas-
pase inhibitor ZVAD-fmk or the caspase-3-specific inhibitor DEVD-CHO at concentrations from 1 to 100 m
M, some of which are
omitted in the figure for simplicity. Apoptosis was measured 4 days later by FACS analysis of PI-stained cells. Maximal final con-
centration of DMSO in the medium was 0.01%. Medium with only 0.01% DMSO was used as a control. The percentages of apop-
totic cells in filled areas (transgenes repressed) versus empty areas (transgenes induced) are indicated. (c) Caspase-3-like activity in
U2OS-Myc and U2OS-RbDcdk/Myc cells. DEVDase activity was colorimetrically measured in triplicate on cell lysates prepared at
day 0 and 4 of transgene induction. After quantification, the activity was expressed as relative to that of TET-repressed cells (day 0),
which was set at 1 and previously found in either cell population to be identical to that of parental U2OS cells
Myc-overexpressing cancer cells need E2F for survival
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Other inhibitors of E2F activity can induce cell death in
U2OS-Myc cells
The role of pRb in regulation of apoptosis appears
complex. In recent years several studies have focused
on apoptosis triggered by loss of Rb function and/or
excessive E2F activity (reviewed in Harbour and Dean,
2000; Nevins, 2001). However, pRb has also been
reported to activate cell death through diverse and yet
unclear mechanisms (Bowen et al., 1998; Hsieh et al.,
1999; Knudsen et al., 1999). Thus, we cannot
completely rule out that functions of active Rb
different from the inhibition of the endogenous E2F
activity, could play a causative role in the apoptosis of
U2OS cells co-expressing Myc and RbDcdk. To
elucidate this point U2OS-Myc cells were transfected
with plasmids encoding a dominant-negative DP-1
mutant (dnDP-1), T187Ap27, dnK2, or p16, all
proteins shown to efficiently inhibit E2F activity in
U2OS cells (Lukas et al., 1997, 1999a,b; Santoni-Rugiu
et al., 2000). As shown in Figure 5, after removal of
TET, a significant percentage of U2OS-Myc cells
transfected with these E2F inhibitors, but not with
control vector, became apoptotic. Thus, deregulated
expression of Myc in U2OS cells deprived of E2F
activity by different inhibitors leads to cell death. This
supports the notion that apoptosis of U2OS-RbDcdk/
Myc cells is linked to the inhibition of E2F activity by
RbDcdk, rather than to any other potential function of
this Rb mutant. Instead, transfection of wild-type Rb
did not result in apoptosis of U2OS-Myc cells (Figure
5) most likely because these cells can efficiently
inactivate Rb by phosphorylation, consistent with the
very modest apoptosis seen in U2OS-Myc cells under
conditions of serum-starvation (Figure 1a and Table 1).
Depletion of E2F activity via overexpression of E2F
binding site oligonucleotides resul ts in apoptosis of
U2OS-Myc cells
To strengthen the concept that deprivation of E2F
activity may cause apoptosis of tumor cells with
deregulated expression of Myc, we used another
approach aimed at sequestering and removing endo-
genous E2F from its downstream target promoters.
U2OS-Myc cells were electroporated with oligonucleo-
tides, containing either a wild-type binding site for E2F
transcription factors, E2Fbswt, or a point mutant E2F
binding site, E2FbsMut, unable to bind them (Helin et
al., 1992; Lees et al., 1993). Pilot gene-transfer
experiments coupled with reporter assays showed that
10 m
M was the effective concentration of E2Fbswt
resulting in titration of E2Fs and inhibition of E2F
activity in U2OS cells without unspecific toxic ity while
E2FbsMut had no effe ct on E2F activity at any
concentration tested (not shown). When Myc was
induced by TET removal, the sub-G1 population
remained virtually unchanged in cells electroporated
with E2FbsMut or with an oligonucleotide unrelated to
E2F (SP1RBF). In contrast, a marked increment of the
percentage of U2OS-Myc cells undergoing apoptosis
was observed after electroporation of E2Fbswt (Table
3), in keeping with the idea of E2F activity being a
critical survival factor for these cells. Importantly, the
apoptotic effect of E2Fbswt in induced U2OS-Myc
cells was rescued by coelectroporation of the MM
chimera (Table 3), a result that resembles the rescue by
MM in induced U2OS-RbDcdk/Myc cells (Table 2)
Figure 5 Different inhibitors of E2F activity induce apoptosis of
U2OS-Myc cells. Cells were cotransfected with the indicated plas-
mids (5 mg for each of these plasmids except for dnK2 used at
15 mg), 1 mg CD20 expression vector, and empty vector up to a
total of 25 mg DNA/10-cm-diameter dish. Apoptosis was assayed
by flow cytometry as sub-G1 DNA content of PI-stained, CD-20-
positive cells after a 4-days culture in TET-free medium. Data,
expressed as percentage of total number of cells, are means+s.d.
of three independent experiments
Table 3 Titration of endogenous E2Fs by forced expression of E2F
binding sites causes apoptosis in derepressed U2OS-Myc cells
a
Oligo +Tet
b
(%) 7Tet (%)
E2Fbswt 5 23
E2FbsMut 4 6
E2Fbswt+MM
c
37
SPIRBF 4 5
a
Values are percentage of apoptotic cells measured by flow cytometric
analysis 4 days after electroporation with the indicated oligonucleo-
tides, as described in Materials and methods. Each measurement was
evaluated three times with comparable results.
b
+Tet and 7Tet: cells
cultured in TET-containing and -free medium, respectively.
c
Cells co-
electroporated with MM
Figure 4 Bcl2 but not a dominant negative p53 molecule (p53dd)
inhibits apoptosis in U2OS-RbDcdk/Myc cells. CD-20-positive
cells exposed to 5 mg of the indicated plasmids were cultured
for 4 days in TET-containing (white bars) or -free (black bars)
medium and assayed for sub-G1 DNA content. Values, expressed
as percentage of apoptotic cells, are means+s.d. of three indepen-
dent experiments
Myc-overexpressing cancer cells need E2F for survival
E Santoni-Rugiu et al
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Oncogene
and confirms the requirement for Myc transcriptional
activity in this type of cell death. Thus, deregulated
Myc trans criptional activity combined with inhibition
of E2F activity can lead to cell death of human cancer
cells.
p53 is not involved in cell death induced by co-expression
of Myc and RbDcdk
Previous studies have shown that Myc-mediated
apoptosis can be p53-dependent or -independent
(reviewed in Hoffman and Liebermann, 1998; Prender-
gast, 1999). In this respect, even though cell death in
our U2OS model is ARF-independent, it remains
possible that p53, which is wild-type in U2O S cells,
could contribute to it. In particular, the significant
amount of endoreplication seen in U2OS-RbDcdk/Myc
cells (Santoni-Rugiu et al., 2000) as well as the fact that
deregulated Myc may induce genomic instability in
certain cell types (Felsher and Bishop, 1999; Li and
Dang, 1999; Santoni-Rugiu et al., 2000), raise the
possibility that p53 could be activated in response to
potential DNA damage and be responsible for
apoptosis in U2OS-RbDcdk/Myc cells. To check this
possibility, we transfected these cells with an expression
vector encoding p53dd, a dominant-negative p53
mutant shown to reduce p53-dependent apoptosis
(Bowman et al., 1996). However, even tough p53dd
was functional, in that capable of silencing p53-
responsive reporters such as mdm2-Luc and p21-Luc
(Falck et al., 2001), it did not prevent cell death of
derepressed U2OS-RbDcdk/Myc cells. This result was
consistent with the lack of p53 induction observed in
these cells after transgene activation (ES-R, DD, JL
and JB, unpublished observations). To further rule out
a role for p53 in apoptosis caused by coexpression of
Myc and RbDcdk, p53-deficient human osteosarcoma
SAOS-2 cells were transient ly transfected with Myc
and RbDcdk expression plasmids and 4 days later, the
sub-G1 population was analysed. Overexpression of
either Myc or RbDcdk alone did not show a significant
increase of apoptotic cells compared with transfection
of empty-vector, while this increase occurred when
these transgenes were expressed together (Figure 6a).
Induction of apoptosis by inhibition of E2F activity occurs
in Myc-overexpressing tumor but not normal cells
Contrary to what was observed in U2OS and SAOS-2
cells, the combination of Myc and RbDcdk did not
induce apoptosis in BJ human primary fibroblasts
(Figure 6a), although the latter died effectively when
exposed to Myc and serum-starved (data not shown).
Thus, at least in cells of mesenchymal origin, the loss
of viability due to the activation of Myc in conditions
of E2F inhibition appears to occur specifically in tumor
cells. As this observation may have important implica-
tions for designing strategies of selective tumor cell
killing, HL-60 hum an promyelocytic leukemia cells,
which are characterized by Myc gene amplification
(Nowell et al., 1983; von Hoff et al., 1990; Mangano et
al., 1998), deletion of p53 (Wolf and Rotter, 1985) and
lack of ARF protein expression (Della Valle et al.,
1997), were tested for their response to deprivation of
E2F activity (Figure 6b). No toxic effect was observed
by electroporation of either control vector or wild-type
Rb that is promptly phosphorylated in these cells. In
contrast, forced ex pression of RbDcdk or dnDP-1
provoked considerable levels of apoptosis, which in
turn was strongly reduced by inhibition of Myc activity
via coexpression of MM. These results corroborate the
concept that endogenous E2F activity may be crucia l
for survival of tumor cells with deregulated Myc
activity, independently of ARF and p53.
Discussion
Signals that stimulate or hinder growth and survival
determine whether cells respond to abnormal Myc
expression with proliferation or apoptosis (Prendergast,
Figure 6 Apoptosis caused by inhibition of E2F activity in Myc-
overexpressing tumor cells is p53-independent and tumor specific.
(a) Apoptosis induced by coexpression of Myc and RbDcdk is re-
producible in the p53-deficient, SAOS-2 human osteosarcoma
cells but not in BJ human primary fibroblasts. SAOS-2 and BJ
cells were exposed for 4 days to the indicated plasmids (each
transfected at 5 m g) and assayed for sub-G1 DNA content by flow
cytometry as described in Materials and methods. (b) Inhibition
of endogenous E2F activity in HL-60 cells results in Myc-depen-
dent cell death. Sub-G1 DNA content of E-GFP-positive cells
was analysed 4 days after cell electroporation with 0.3 mgE-
GFP and 3 mg of the indicated plasmids. RbDcdk and dnDP-1
but not wild-type Rb or empty vector increased the level of apop-
tosis, which in turn was inhibited by MM. In (a) empty vector
was added to a total of 25 mg DNA/10-cm-diameter dish, in (b)
to a total of 8.5 mg/dish. Results are means+s.d. of three inde-
pendent experiments
Myc-overexpressing cancer cells need E2F for survival
E Santoni-Rugiu et al
6504
Oncogene
1999). We have previously shown in a U2OS model
that effective cell cycle co mpletion requires both Myc
and E2F activities (Lukas et al., 1999a; Santoni-Rugiu
et al., 2000). However, whether these two activities
interact to regulate cell survival remains to be tested. It
is known that Myc sensitizes cells to apoptosis but
requires additional signals to induce apoptosis (Juin et
al., 1999). Here we show that an important event
triggering apoptosis in human cancer cells overexpres-
sing Myc is the inhibition of E2F activity. Together
with our previous results, this suggests that Myc an d
E2F co-regulate fundamental cell behaviors, like
proliferation and survival of neoplastic cells. Indeed,
it is widely accepted that tumor cells possess high and/
or unscheduled E2F activity, given the constant
activation of mitogenic signaling pathways by onco-
genes and growth factors as well as the variety of
aberrations targeting the p16/cyclin D/CDK 4/Rb/E2F
pathway in virtually all types of human cancer cells
(Bartek et al., 1996; Nevins, 2001). While in normal
cells Rb-E2F may regulate gene expression chiefly by a
mechanism of transrepression and derepression, the
higher levels of free E2F in tumor cells may also enable
and/or accentuate a mechanism of direct transactiva-
tion (Dyson, 1998; Harbour and Dean, 2000; Mu
¨
ller
and Helin, 2000). In addition, many tumor cell types
are characterized by high levels of Myc expression and
activity (Nesbit et al., 1999), thereby facing the risk of
being killed if Myc-mediated apoptotic signals are not
tackled. Therefore, it is legitimate to propose from our
data that human cancer cells overexpressing Myc need
E2F activity not only for coordinated completion of
cell cycles (Lukas et al., 1999a; Santoni-Rugiu et al.,
2000) but also for a process of adaptive survival. This
process possibly occurs via direct transactivation of
E2F target genes implicated in survival, whose
identification has started to emerge through the recent
employment of DNA microarray analysis (Mu
¨
ller et
al., 2001; Ishida et al., 2001; Ren et al., 2002). In
essence according to the model we propose, the
increased E2F activity present in human cancer cells
subverts the normal transcriptional gene regulation,
resulting somehow in the induction of factors that
either directly or indirectly are capable of counteracting
Myc-dependent apoptotic signals. Indeed, very few
serum-starved U2OS-Myc cells die, presumably
because they produce their own survival factors, while,
regardless of the presence of serum, they undergo
apoptosis upon inhibition of E2F activity, indicating
that these factors are at least in part regulated by E2F.
In contrast, in nor mal cells like BJ fibroblasts, the
concomitant inhibition of E2F activity and Myc
expression has no apparent impact on survival, perhaps
because their E2F activity is not high enough to
directly transactivate survival genes and regulate cell
viability or alternatively but not mutually exclusive,
because these cells may lack some factors important for
the pro-apoptotic pathway modulated by Myc and
E2F. However, BJ cells can be killed by deregulated
Myc under condition of serum starvation, consistent
with the notion that survival fact ors/cytokines in the
serum are essential for tolerating upregulated Myc
activity in normal fibroblasts (Harrington et al., 1994).
It is interesting in this context that the DU-145
prostate adenocarcinoma cell line, which in addition
to lacking pRb has mutant p53 (Carroll et al., 1993)
and amplified Myc (Asadi and Sharifi, 1995), can be
rendered sensitive to irradiation- and ceramide-induced
cell death by reintroduction of pRb (Bowen et al.,
1998). Myc amplification also characterizes the Rb-
positive LNCaP prostate cancer cells (Nag and Smith,
1989), which undergo apoptosis upon expression of a
constitutively active form of Rb or upon constitutive
activation of pRb by different signaling pathw ays (Day
et al., 1997, 1999; Zhao et al., 1997), apparently
without involvement of p53 (Day et al., 1999).
Collectively, these results suggest that some types of
human epithelial tumor cells with deregulated Myc
expression may be subject to E2F-dependent and p53-
independent mechanisms regulating survival similar to
those we have identified in osteosarcoma an d leukemia
cells.
It might seem difficult at a first glance to reconcile
our results with the notion that excessive E2F activity
can trigger apoptosi s, as observed in certain cells from
Rb-deficient mice or upon forced expression of E2F
itself in several cell types (reviewed in Harbour and
Dean, 2000; Nevins, 2001). However, our study is not
in contrast with the current knowledge on E2F and
apoptosis but rather it complements it by highlighting
the importance of the endogenous E2F activity for the
homeostasis of human tumor cells with deregulated
Myc function. In this regard, it is worth mentioning
that we could not rescue the apoptotic phenotype of
U2OS-RbDcdk/Myc cells by restoring E2F activity
through overexpression of E2F-1, -2 or -3 in doses able
to saturate RbDcdk, or through expression of an Rb-
binding deficient E2F-1 mutant. Indeed, all our
attempts resulted in generation of E2F activity several
fold higher than the endogenous one and marked
toxicity in either TET-repressed or induced cells (ESR,
DD, unpublished results). This toxic effect after forced
expression of ectopic E2Fs is consistent with that
previously observed in parental U2OS cells (Lukas et
al., 1996) or other cell types (Harbour and Dean, 2000;
Mu
¨
ller and Helin, 2000). Therefore, a delicate balance
must exist in cancer cells between levels of E2F activit y
capable of protecting from apoptotic signals such as
those induced by deregulated Myc an d excessive levels
of E2F resul ting in activation of the apoptotic
machinery and cell toxicity. This balance could, at
least partly, explain why genetic alterations resulting in
either loss or deregulated expression of E2F-1, -2, and -
3, that is, the E2Fs implicated in cell proliferation and
apoptosis, have not been reported in human tumors
(Nevins, 2001). The factors and mechanisms regulating
this balance remain elusive. A candidate could be the
murine double minute 2 (MDM2) oncoprotein and its
human homolog, HDM2. MDM2 can stimulate E2F
activity by interacting with the E2F-1/DP-1 hetero-
dimer and pRb (Martin et al., 1995; Xiao et al., 1995).
Moreover, recent work has shown that exogenous
Myc-overexpressing cancer cells need E2F for survival
E Santoni-Rugiu et al
6505
Oncogene
HDM2 can antagonize E2F-dependent apoptosis,
possibly by down-regulating the levels of E2F an d
DP proteins, and can cooperate with E2F-1/DP-1 in
promoting cell viability and cell cycle progression in a
manner dependent on the DP subunit but independent
of p53 (Loughran and La Thangue, 2000). Based on
these results, a model has been proposed in which an
increased expression of HDM2 might prevent the
potentially toxic effects of excessive E2F activity in
cancer cells and maintain E2F in a continuous state of
growth stimulator rather than apoptosis inducer
(Loughran and La Thangue, 2000). However, this
hypothesis remains to be verified in human cancers that
spontaneously overexpress HDM2. In addition, it
renders a possible implication of HDM2 as major
determinant in the phenomena described in our
manuscript improbable, in that U2OS cells unlike
some other types of osteosarcoma cells, do not bear
HDM2 amplification and express only moderate levels
of HDM2 protein (Bo
¨
ttger et al., 1997). Similarly,
neither SAOS-2 nor HL-60 are characterized by
amplification and/or overexpression of HDM2.
It is also necessary to clarify which are the pro- and
antiapoptotic genes regulated by E2F that may be
orchestrating the net effect of E2F activity on cell
survival. Interestingly, in this respect, recent work has
shown that increased expression of E2Fs induces not
only proapoptotic genes (Moroni et al., 2001; Mu
¨
ller et
al., 2001) but also antiapoptotic genes and survival/
growth factors, such as Bcl2 and TGF-a (Mu
¨
ller et al.,
2001), which can inhibit Myc-induced apoptosis and
cooperate with Myc in tumorigenesis of certain tissues
(Strasser et al., 1990; Bissonnette et al., 1992; Fanidi et
al., 1992; Amundadottir et al., 1996; Santoni-Rugiu et
al., 1998).
Since RbDcdk and the other E2F inhibi tors used in
our study are capable of functionally repressing all
endogenous E2Fs (Lukas et al., 1997, 1999a,b) and given
the above-mentioned toxicity caused by overexpression
of different E2Fs in U2OS-RbDcdk/Myc cells, the
present work is not able to clarify the contribution of
different E2F-family members to the antiap optotic effect
in Myc-overexpressing cancer cells nor whether this
effect is due to a specific E2F or to a cooperat ive effect of
different E2Fs. This deserves future investigations, as
certain laboratories have indicated that in tissue culture
models, E2F-1 to -3 have similar proapoptotic effects
when overexpressed (Mu
¨
ller and Helin, 2000), while
others have implicated only E2F-1 in the induction of
apoptosis (Nevins, 2001) and suggested that Myc
requires distinct E2F activities to induce S phase and
apoptosis in MEFs (Leone et al., 2001).
The type of cell death elicited by concomitant
overexpression of Myc and inhibition of endogenous
E2F activity in cancer cells occurs via a caspase-
mediated apoptotic pathway, as in other models of
Myc-dependent apoptosis (Kangas et al., 1998; Kagaya
et al., 1997; Hotti et al., 2000). Moreover, it appears to
be independent of cell cycle progression, consistent
with previous investigations on Myc-i nduced apop tosis
in other systems (Packh am et al., 1996; Rudolph et al.,
1996). Likewise, cytokine-mediated protection from
Myc-induced apoptosis is not linked to the cytokines’
abilities to promote growth and can occur in cells
whose cell cycle has been arrested (Harrington et al.,
1994) and Bcl2 can inhibit Myc-induced ap optosis but
not proliferation (reviewed in Hoffman and Lieber-
mann, 1998; Prendergast, 1999). Nevertheless, our
results using MM indicate that Myc transcriptional
activity is necessary for the apoptotic effect described
in this report, suggesting a key role for a Myc
transcriptional target in eliciting it. However, unlike
Myc-induced apoptosis in MEFs, it does not require
ARF and/or p53, as it is reproducible in U2OS
(ARF7/7; p53+/+) and SAOS-2 (ARF+/+;
p537/7) cells, in agreement with the fact that Myc-
mediated apoptosis is p53-dependent in certain cell
types but -independent in others (Hoffman and
Liebermann, 1998; Prendergas t, 1999; Blyth et al.,
2000). We also show that reconstitution of the ARF/
p53 axis in osteosarcoma cells deficient for ARF
expression and expressing deregulated Myc, does not
result in cell death, even when the cells are serum-
starved. This may conceivably point toward possible
alterations of the pathway downstream of ARF and/or
p53 (Soengas et al., 2001). Alternatively but not
mutually exclusive, U2OS cells might possess survival
mechanisms interfering and blocking the induction of
apoptosis by this pathway. Regardless of the mechan-
ism, caution should be exercised in designing tumor
gene therapy with ARF or with drugs activating
pathways that induce AR F, in that ARF expression
may not be enough to kill human tumor cells that
overexpress certain classes of oncogenes.
Finally, we show that while human primary fibro-
blasts do not show loss of viability upon concomitant
overexpression of Myc and inhibition of E2F activity,
human HL-60 leukemia cells which have amplified Myc
gene (Nowell et al., 1983; Von Hoff et al., 1990;
Mangano et al., 1998) and deleted p53 (Wolf and
Rotter, 1985) and lack ARF protein expression (Della
Valle et al., 1997), can be killed by blocking their
endogenous E2F activity. Thus, cell death by inhibition
of E2F activity seems to selectively occur in human
neoplastic cells with deregulated Myc and is independent
of their ARF and p53 status. Our observations suggest
that the protection from Myc-elicited apoptotic signals
provided by increased, nontoxic level s of E2F activity in
cancer cells may represent a novel mechanism by which
the disruption of the Rb pathway may cooperate with
deregulated Myc in oncogenesis. They may also have
important implications for therapeutic strategies aimed
at specifically eliminating Myc-overexpressing tumor
cells, in particular those with defects of the tumor
suppressor pathways regulated by ARF and p53.
Materials and methods
Plasmids and chemicals
The hemagglutinin (HA)-tagged pBI-HA-RbDcdk and pECE-
HA-RbDcdk vectors, expressing the phosphorylation-deficient
Myc-overexpressing cancer cells need E2F for survival
E Santoni-Rugiu et al
6506
Oncogene
murine pRb mutant RbDcdk in a tetracycline (TET)-
repressible or constitutive manner, respectively, the PECE-
pRbDB/X expressing wild-type pRb, and the pBI-Myc or
pCMV-Myc vectors for TET-repressed or constitutive c-Myc
expression, as well as pCMV-CD20, pCMV-HA-dnDP-1
(D103-126), and pX-p16, were described before (Lukas et
al., 1997, 1999b; Santoni-Rugiu et al., 2000). The pCMV-
HA-ARF plasmid for expression of human ARF was
generated as previously published (Della Valle et al., 1997).
The vectors pCMV-MadMyc (MM), pCMV-dnCDK2
(dnK2), pCMV-p27T187A, and pCMV-p53dd were as
reported (van den Heuvel and Harlow, 1993; Bowman et
al., 1996; Berns et al., 1997; Nguyen et al., 1999).
Z-Val-Ala-
D,L
-Asp-fluoromethylketone (ZVAD-fmk; Bachem)
was diluted to 10 m
M in methanol and added to the cells at
final concentrations from 1 to 100 m
M. Acetyl-Asp-Glu-Val-
Asp-aldehyde (DEVD-CHO; Neosystems) was diluted to
10 m
M in 10% DMSO and used at final concentrations of 1-
100 m
M.
Cell culture and gene transfer
Human osteosarcoma U2OS cells stably expressing human c-
Myc (U2OS-Myc) or c-Myc and RbDcdk (U2OS-RbDcdk/
Myc) in a tetracycline (TET)-repressible manner were as
described (Santoni-Rugiu et al., 2000). These cells were
cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal calf serum (FCS), penicillin
(10 U/ml), streptomycin (10 U/ml), geneticin (G418; 400 mg/
ml), puromycin (1 mg/ml) and TET (2 mg/ml). Transgene
derepression was performed by removal of TET according to
procedures previously published (Lukas et al., 1999b). SAOS-
2 and BJ cells were cultured in DMEM containing 10% FCS,
penicillin and streptomycin. Calcium-phosphate transfection
of U2OS-derived cell lines and SAOS-2 cells were carried out
as reported previously (Lukas et al., 1996, 1997). Gene
transfer into BJ cells was performed with DOTAP (Sigma)
according to the manufacturer’s instructions. Expression of
the ectopic proteins after transfection was confirmed by
immunocytochemical staining as described (Lukas et al.,
1996, 1999b).
HL-60 cells were maintained in RPMI 1640 supplemented
with 10% FCS, penicillin and streptomycin, keeping cell
density lower than 1 million/ml, and were electroporated as
reported (Chen et al., 2000) with minor modifications.
Briefly, cells were collected, washed once with serum- and
antibiotic-free RPMI 1640 and resuspended in this medium
at working cell density of 2.5610
6
/100 ml. Approximately
4.5610
6
cells/cuvette were electroporated at room tempera-
ture with the indicated DNAs and 0.3 mg of E-GFP DNA
(Clontech), 350 V and 960 mFD by using a Bio-Rad Gene
Pulser. Empty vector was added to a total of 8.5 mg of DNA.
After electroporation the cells were incubated on ice for
15 min and then diluted with 15 ml antibiotic-free RPMI
1640 containing 10% FCS. After 12 h the cells were washed
with fresh medium and then incubated for other 3 days
before being harvested and assayed for viability. Gene
transfer efficiency was verified by immunostaining of
electroporated cells grown on coverslips, as described
(Santoni-Rugiu et al., 2000).
Measurement of cell death
Loss of viability in untransfected U2OS-Myc and U2OS-
RbDcdk/Myc was monitored by flow cytometric analysis of
the sub-G1 content of DNA characteristic of apoptotic cells.
Cells were harvested, fixed in 70% methanol, stained with
propidium iodide and analysed with a Becton-Dickinson
FACScalibur flow cytometer by using the CellQuest software
as previously described (Santoni-Rugiu et al., 2000). In
addition, the analysis of cell integrity via exclusion of the
vital dye, trypan blue, was used to further assess cell viability.
Cells were cultured in 10-cm dishes in TET-containing or
-free DMEM, supplemented or deprived of 10% FCS as
indicated. At the indicated time-points, cells were trypsinized,
washed in DMEM and the number of trypan blue-positive
cells evaluated on a hemocytometer by counting a minimum
of 500 cells. To assess the viability of U2OS or SAOS-2 cells
transfected with the indicated plasmids, the pCMV-CD20
vector encoding the CD-20 cell surface antigen was co-
transfected at 1 mg and the sub-G1 DNA content of CD20-
positive cells was analysed by immunostaining with fluor-
escein isothiocyanate-labeled anti-CD20 monoclonal antibody
(mAb; Becton Dickinson) followed by flow cytometry
analysis according to published procedures (Lukas et al.,
1999b; Santoni-Rugiu et al., 2000). Analogous flow cyto-
metric procedures were used to evaluate the viability of
transfected BJ cells and electroporated HL-60 cells, except
that cells were gated according to their expression of
membrane-bound E-GFP (transferred at 0.5 and 0.3 mg,
respectively) following the indications of commercially
available methods (Clontech).
Immunoblot analysis
Western blot analyses were performed as described previously
(Lukas et al., 1996) using the following primary mAbs: anti-
Myc 9E10 (a kind gift of G Evan) as a hybridoma
supernatant diluted 1 : 3 in a 5% skimmed milk solution in
PBS; anti-HA 12CA5 (Lukas et al., 1997), DCS-24.1 anti-
human ARF (NeoMarkers) and DO-1 for p53 (Vojtesek et
al., 1992), all diluted 1 : 500 and anti-PARP C2-10 at a
dilution of 1 : 15 000 (Lazebnik et al., 1994; Mathiasen et al.,
1999). The MO-1 mAb to detect CDK7 (Tassan et al., 1994)
and DCS-141 for MCM7 (Sørensen et al., 2000) as well as
Ponceau-S staining were used as loading controls. After
incubation with peroxidase-conjugated anti-mouse IgG
(Vector), the immunodetection of proteins was achieved by
using the enhanced chemiluminescence method (Pierce).
Caspase-3-like activity
DEVDase activity was assessed in U2OS-RbDcdk/Myc cell
lysates prepared at day 0 and 4 of transgene induction. Equal
amounts of protein (*5610
5
cells) measured by Bio-Rad
protein assay (Bio-Rad) were used in a colorimetric assay for
the detection of DEVD-specific caspase-3-like activity as
described previously (Mathiasen et al., 1999), using acetyl-
Asp-Glu-Val-Asp-p-nitroaniline (DEVD-pNA) as a probe
(Biomol), DEVD-CHO as reaction inhibitor and a microtiter
plate reader (Molecular Devices).
E2F binding sites oligonucleotides
The DNA oligonucleotides (DNA Technology A/S) repre-
senting a wild-type E2F binding site (E2Fbswt) and a mutant
binding site (E2FbsMut) unable to bind E2Fs (Helin et al.,
1992; Lees et al., 1993) were: 5’-ATTTAAGTTTCGCGC-
CCTTTCTCAA-3’ (E2Fbswt sense); 5’-TTGAGAAAGG-
GCGCGAAACTTAAAT-3’ (E2Fbswt antisense); 5’-ATT-
TAAGTTTCGATCCCTTTCTCAA-3’ (E2FbsMut sense);
5’-TTGAGAAAGGGATCGAAACTTAAAT-3’ (E2FbsMut
antisense). The sequences of the E2F unrelated SP1RBF
oligonucleotides were: 5’-CCTCGCGGACGTGACGCCG-
Myc-overexpressing cancer cells need E2F for survival
E Santoni-Rugiu et al
6507
Oncogene
CGGGCGGAAGT-3’ (sense); 5’-ACTTCCGCCCGCGG-
CGTCACGTCCGCGAGG-3’ (antisense). For transfer into
U2OS-Myc cells, 10 m
M oligonucleotides were heated at 958C
for 5 min followed by incubation at RT for 1 h and then
electroporated into 1.5610
6
cells together with 1 mg E-GFP
and with or without pCMVMadMyc (MM, 5 mg), at 48C,
125 V and 960 mFD. Empty vector was added up to a total
of 8.5 mg plasmid DNA. After electroporation cells were kept
10 min on ice, and then plated onto 10 cm culture dishes in
TET-containing or -free DMEM and 10% FCS. Medium was
changed 12 h later and cells harvested 3 days later. Cell
viability of E-GFP-positive cells was assessed by flow
cytometry analysis as described above.
Acknowledge ments
We thank R Bernards and G Evan for providing important
reagents and K Holm and C Lindeneg for excellent
technical assistance. This work was supported by grants
from the Danish C ancer S ociety, the Human Frontier
Science Programme, and the Danish Medical Research
Council.
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