Ectopic EBP2 expression enhances cyclin E1 expression and induces chromosome instability in HEK293 stable clones.

Abstract

To explore the effects of deregulated expression of the EBNA1 binding protein 2 (EBP2) on cell growth, we generated human HEK293 stable clones constitutively expressing an EBP2-EGFP fusion protein. We found both RNA and protein levels of cyclin E1, a dominant oncoprotein, were elevated in the EBP2- EGFP stable clones. These findings were confirmed by flow cytometry bivariate analysis of cyclin expression versus DNA content. Moreover, the increase in p21 expression and the specific phosphorylation at Ser1981 of ATM and Ser15 of p53 were also observed in these stable clones, and these observations may explain the failure to observe an increase in Cdk2 kinase activity. In addition, after one year of passage culture, the EBP2-EGFP stable clones tended to lose 4 to 5 chromosomes per cell when compared to that of control cells. All of these findings provide a possible link between deregulated expression of EBP2 and tumor development.

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*Corresponding author. Tel: 886-2-2312-3456 ext. 5759;
Fax: 886-2-2394-7927; E-mail: en917210@hotmail.com
Received 14 May 2008, Accepted 23 June 2008
Keywords: Chromosome instability, Cyclin E1, EBP2, p53-p21 sys-
tem, Tumorigenesis
Ectopic EBP2 expression enhances cyclin E1 expression and
induces chromosome instability in HEK293 stable clones
Ming-Cheng Lee1, Chang-Hsun Hsieh1, Shu-Chen Wei3, Shu-Chen Shen2, Chiung-Nien Chen4, Vin-Cent Wu3, Li-Ying
Chuang3, Fon-Jou Hsieh5, C. H. Herbert Wu1 & Jyy-Jih Tsai-Wu2,*
1Institute of Molecular Medicine, College of Medicine, National Taiwan University, Departments of 2Medical Research, 3Internal Medicine,
4Surgery, and 5Obstetrics and Gynecology, National Taiwan University Hospital
To explore the effects of deregulated expression of the EBNA1
binding protein 2 (EBP2) on cell growth, we generated human
HEK293 stable clones constitutively expressing an EBP2-EGFP
fusion protein. We found both RNA and protein levels of cyclin
E1, a dominant oncoprotein, were elevated in the EBP2- EGFP
stable clones. These findings were confirmed by flow cytometry
bivariate analysis of cyclin expression versus DNA content.
Moreover, the increase in p21 expression and the specific phos-
phorylation at Ser1981 of ATM and Ser15 of p53 were also ob-
served in these stable clones, and these observations may ex-
plain the failure to observe an increase in Cdk2 kinase activity.
In addition, after one year of passage culture, the EBP2-EGFP
stable clones tended to lose 4 to 5 chromosomes per cell when
compared to that of control cells. All of these findings provide a
possible link between deregulated expression of EBP2 and tu-
mor development. [BMB reports 2008; 41(10): 716-721]
INTRODUCTION
The EBNA1 binding protein 2 (p40/EBP2/NoBP) is a nucleolar
protein that is expressed at high levels in human neoplasms
(1). The human EBP2 protein exhibits the ability to interact
with the viral EBNA1 protein (2) and human nucleolar FGF3
protein (3), as demonstrated by yeast two-hybrid analysis.
Defects in the yeast EBP2 homologue, p40, inhibited the proc-
essing of 27S-A rRNA into 27S-B rRNA (4,5). Moreover, over-
expression of human EBP2 protein increased mouse NIH3T3
cells growth and counteracted the inhibitory effect of nuclear
FGF3 (3). In addition, suppression of human EBP2 by RNA si-
lencing resulted in cell growth arrest (6). These findings sug-
gest that the EBP2 protein plays a dual role in both rRNA proc-
essing and cell growth regulation.
Development and progression of tumors are often associated
with chromosome abnormalities, including changes in struc-
ture and number (7,8). Centrosome hyperamplification has also
been observed in various cancers and results in an unequal seg-
regation of chromosomes (9). Deregulation of the genes in-
volved in DNA damage repair and cell cycle progression are
implicated in the induction of chromosome instability. Recently,
more attention has been focused on the correlation of cyclin E
deregulation and chromosome instability (10,11).
The cyclin E-Cdk2 complex is an important regulator of en-
try into S phase during the mammalian cell cycle. Previous
studies revealed that cdk2 knockout mice were viable and es-
sentially developed normally (12). However, mice lacking
both cyclins E1 and E2 died during midgestation due to pla-
cental abnormalities (13). These findings raise the possibility
that some functions of cyclin E might be Cdk2-independent. In
addition, ectopic cyclin E expression in established cell lines is
associated with genetic instability and abnormal centrosome
duplication (14,15), indicating that cyclin E also functions as a
dominant oncoprotein.
To characterize the consequences of excess EBP2 during
cell growth, we generated HEK293 stable clones that ectopi-
cally expressed an EBP2-EGFP fusion protein. Here, we dem-
onstrated that ectopic EBP2-EGFP expression enhanced cyclin
E1 expression. Moreover, it is noted that prolonged culture of
the EBP2-EGFP stable clones induced chromosome instability.
These findings provide a possible link between excess EBP2
expression and tumorigenesis.
RESULTS
Generation of EBP2-EGFP stable clones in HEK293 cells
HEK293 cells were transfected with the plasmid pEGFP-
N1-EBP2-EGFP. The expression of the EBP2-EGFP fusion pro-
tein, which localized to nucleoli of interphase cells, was iden-
tified directly by fluorescent microscopy (data not shown). The
EBP2-EGFP transfectants were treated with G418 for 2 weeks
and the stable clones were selected by limiting dilution. The
expression of 67-kD EBP2-EGFP fusion proteins in these stable
clones was confirmed by Western blot analysis using anti-
serum against EBP2 (Fig. 1A).

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Fig. 1. Increased cyclin E1 expression in EBP2-EGFP stable clones.
(A) The expression of the EBP2-EGFP fusion protein in parental cells
(293), EGFP control cells (293G), and EBP2-EGFP stable clones (SC1
and SC3) was identified by Western blot analysis with antiserum
against EBP2. The molecular weight of EBP2-EGFP and endogenous
EBP2 were 67 kD and 40 kD, respectively. (B) Fifty micrograms of
total protein isolated from the asynchronized cells were subjected to
Western blot analysis for cyclin E1. The chemoluminescence in-
tensity of cyclin E1 was normalized against that of β-actin and com-
pared to the amount of wild- type HEK293. The average and the
standard deviation of three independent experiments ± S.D. are
shown (*indicates P ＜ 0.01). (C) Stable clone 1 was transfected
with the plasmid- based EGFP shRNA construct and selected using
puromycin for 1 week. Fifty micrograms of total protein was used
to investigate the expression levels of EBP2-EGFP, cyclin E1, and
p21 using antibodies recognizing EGFP, cyclin E1, and p21,
respectively. (D) Ten micrograms of total RNA was subjected to
Northern blot analysis using DIG-labeled EBP2 RNA probes. The
EtBr-stained image of 28S was used as the loading control.
Fig. 2. Effects of ectopic EBP2-EGFP expression on cyclins D1,
E1, A, and B expression using flow cytometry. The asynchronized
cells were subjected to flow cytometry for analysis of the DNA
content versus cyclin levels. The trapezoidal window indicates the
fluorescence level of the negative control without addition of cy-
clin antibodies. Representative results of three independent experi-
ments are shown.
Ectopic EBP2-EGFP expression increased cyclin E expression
To characterize the effect of EBP2-EGFP expression on cell cy-
cle progression in HEK293 cells, cyclin D1, E1, A, and B were
analyzed by Western blot assay. We found that cyclin E1 ex-
pression was elevated in the asynchronized EBP2-EGFP stable
clones, but not cyclin D1, A, or B (Fig. 1B and data not shown).
Moreover, the elevated cyclin E1 expression was reduced by
suppressing the ectopic EBP2-EGFP expression via the ex-
pression of an EGFP short hairpin RNA (shRNA) (Fig. 1C).
To further investigate whether the increase in the cyclin E1
protein was due to the increase in transcription of cyclin E1 in
the EBP2-EGFP stable clones, the mRNA levels of cyclin E1
were analyzed by Northern blot assay. As shown in Fig. 1D,
we found that the mRNA levels of cyclin E1 were also aug-
mented in the stable clones (SC1 and SC3). These data in-
dicate that ectopic EBP2-EGFP expression in the HEK293 sta-
ble clones enhanced cyclin E1 expression at both the protein
and mRNA levels.
Since the expression of cyclins fluctuate throughout the cell
cycle, we performed flow cytometric bivariate analysis of cy-
clin expression versus DNA content to reveal changes in cy-
clin expression with respect to the cell cycle. We found that
the amount of cyclin E1 was much higher in EBP2-EGFP stable
clones during cell cycle progress relative to that of controls
(Fig. 2). This result was consistent with the finding from
Western blot analysis, as shown in Fig. 1B. The highest level
of cyclin E1 was at the G1 phase, then gradually declined after
entering S, and still further declined through G2/M. Expression
of cyclins D1, A, and B were not apparently different between
control cells (293 and 293G) and EBP2-EGFP stable clones
(SC1 and SC3). These results indicate that cyclin E1 expression
is indeed increased in the EBP2-EGFP stable clones and the ac-
cumulation of cyclin E1 protein is due to the increase in
mRNA levels and not a defect in cyclin E1 degradation.
EBP2-EGFP-induced cyclin E expression does not increase
Cdk2 activity
Previous studies showed that the effects of ectopic cyclin E1
expression on Cdk2 activity and cell cycle progression are dif-
ferent in murine and human cell lines (11,14,16). To assess the
effect of ectopic EBP2-EGFP on Cdk2 activity and cell cycle
progression, the stable clones were examined by flow cy-

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Fig. 3. Excess cyclin E1 expression did not influence Cdk2 kinase
activity by initiating the ATM-p53-p21 system in HEK 293 stable
clones. (A) Cdk2 expression in control cells and stable clones
was determined by Western blot assay. (B) Cdk2 protein was im-
munoprecipitated from total protein and subjected to Cdk2 kinase
activity assay. (C) The expression of p21 in the control cells and
the stable clones was analyzed by Western blot analysis by using
specific antibodies against p21. (D) The expression of p53 was
analyzed by Western blot by using specific antibodies against p53
or serine-15-phosphorylated p53. (E) The expression and phosphor-
ylation levels of ATM and ATR were analyzed by Western blot.
tometry and Cdk2 kinase assays. We did not find a marked al-
teration in the cell cycle between the control and stable clones
(data not shown). In addition, the results of Western blot and
kinase assays of Cdk2 revealed that excess cyclin E expression
did not affect Cdk2 protein expression or the kinase activity of
Cdk2 in the EBP2-EGFP stable clones (Fig. 3A and 3B). These
data suggest that ectopic EBP2-EGFP expression might affect
the Cdk2-independent activity of cyclin E1 but not the Cdk2-
dependent activity in HEK293 cells.
EBP2-EGFP induces the ATM-p53-p21 pathway
In primary human fibroblast cells, excess cyclin E initiates a
homeostatic p53-dependent program that limits Cdk2 activity
via induction of p21 (16). To explore the possibility that Cdk2
kinase activity is not influenced by excess cyclin E1 expression
in EBP2-EGFP stable clones, the expression of p21 was eval-
uated by immunoblot assay. We found that more p21 protein
was detected in EBP2-EGFP stable clones relative to control
cells (Fig. 3C), and this phenomenon was restored by sup-
pression of ectopic EBP2-EGFP via EGFP shRNA (Fig. 1C). To
further investigate whether the elevated p21 expression was
due to the change in p53 expression, p53 expression was ex-
amined in the controls and stable clones. We found that more
p53 was expressed in all of the cell lines and no marked differ-
ence was observed (Fig. 3D). Since cellular stress can induce
p53 phosphorylation, and many distinct phosphorylation sites
affect p53 function in different physiologic contexts (16,17), an
anti-phospho-p53 sampler kit was used to analyze the site-spe-
cific p53 phosphorylation in the EBP2-EGFP stable clones. We
found that an increased phosphorylation level at Ser15 of p53
was detectable in the stable clones (Fig. 3D), while no differ-
ence in phosphorylation at Ser 6, 9, 20, 37, 46, or 392 of p53
was observed (data not shown). Since Ser15 of p53 is one of
the targets of ataxia telangiectasia-mutated kinase (ATM) and
ataxia telangiectasia and Rad3-related kinase (ATR) in response
to DNA damage (16), the expression and phosphorylation lev-
els of ATM and ATR were also examined. We found that high-
er levels of ATM, but not ATR, were observed in the stable
clones. The specific phosphorylation of Ser1981, which is con-
sidered an indicator of ATM kinase activity, was also increased
in the stable clones (Fig. 3E). These findings suggest that the ef-
fect of ectopic EBP2-EGFP expression on the Cdk2-dependent
activity of cyclin E1 may be prevented by the ATM-p53-p21
system in HEK293 cells.
EBP2-EGFP expression induces chromosome instability
Since deregulated cyclin E1 expression may result in chromo-
some instability (15), we further evaluated whether ectopic
EBP2-EGFP expression could alter genomic integrity. After one
year of passage cultures, the EBP2-EGFP stable clones and con-
trol cells were treated with colcemid and subjected to kar-
yotype analysis. There was a significant difference between the
chromosomal content of the EBP2-EGFP stable clones and
control cells. Comparison of the total mean chromosome num-
bers showed that EBP2-EGFP stable clones contained 4.1 to
5.7 fewer chromosomes per cell and EGFP control cells con-
tained 0.7 more chromosomes per cell than that of their paren-
tal control cells (Table 1).
DISCUSSION
In this study, we generated HEK293 stable clones that con-
stitutively expressed the EBP2-EGFP fusion protein. We found
that ectopic expression of EBP2-EGFP enhanced cyclin E1 ex-
pression at both the mRNA and protein levels in the HEK293
stable clones, but did not affect the expression of other cyclins.
These findings were confirmed by flow cytometry bivariate
analysis of cyclins versus DNA content. In addition, the aug-
mented cyclin E1 expression was restored by suppression of
the ectopic EBP2-EGFP expression via EGFP shRNA. The tran-
scription of cyclin E is mainly regulated by the mitogen-cyclin
D-Rb-E2F pathway (18,19) and the neoplasms produced with
aberrant cyclin E1 expression are most frequently a result of
mutations in this pathway (20,21). In this study, we did not de-
tect a marked difference in cell proliferation or cell cycle pro-
gression (data not shown). However, cyclin E1 expression, a
dominant oncoprotein, was indeed augmented in the
EBP2-EGFP stable clones. This result indicates that excess cy-
clin E1 expression caused by excess EBP2 expression may play
a role in tumorigenesis.

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Cell line
Analyzed metaphase
cell number
Mean no. of chromosome
per cell
Chromosome loss/gain
per cell
P value＊
HEK293
HEK293G#
SC1
SC3
57
64
75
63
68.5
69.2
64.4
62.8
＋ 0.7
− 4.1
− 5.7
P = 0.48
P ＜ 0.00001
P ＜ 0.00001
＊The P value was determined using Student T-test.
#HEK293G is a mixed population of EGFP-transfected HEK293 cells.
Table 1. Chromosome number of HEK293 with or without ectopic EBP2-EGFP expression
Overexpression of cyclin E1 can increase Cdk2 kinase activ-
ity in primary REF and MEF cells, but not in primary human fi-
broblasts (11,14,16). This discrepancy might be a result of cell
differences. In cultured normal human cells, cyclin E ex-
pression is tightly controlled, limited to a short period in late
G1 and early S phases. In contrast, cyclin E expression is con-
trolled less strictly, and increased levels of cyclin E often can
be detected during early-mid G1 phase in cultured mouse cells
(10). Minella et al. has reported that cyclin E overexpression
can initiate a p53-dependent response to block excess Cdk2
activity by inducing p21. In addition, phosphorylation at Ser15
of p53 is one of the cell's responses to stresses. DNA damage
could activate the ATM and ATR proteins to induce p53 phos-
phorylation at serine 15 and thus increase p21 expression (16).
These findings indicate that in response to cyclin E dereg-
ulation, p53 and p21 form an inducible barrier to prevent cell
cycle anomalies. In this study, we indeed found that excess cy-
clin E1 expression resulted from ectopic EBP2-EGFP ex-
pression could induce induced p21 expression, and this event
may protect Cdk2 function from deregulation in the presence
of excess cyclin E. This may be the reason that cell pro-
liferation and the cell cycle were not significantly altered in
the EBP2-EGFP stable clones. On the other hand, we also
found that the induction of p21 expression was not due to in-
creased p53 expression, as the total p53 protein levels were
not significantly different between control and EBP2-EGFP sta-
ble clones. This may be the result of increased phosphor-
ylation at Ser15 of p53 via ATM activation (but not ATR) in the
EBP2-EGFP stable clones.
Chromosome instability is believed to play an important
role in carcinogenesis through promoting accumulation of mu-
tations responsible for the malignant phenotypes (9,22). The
association of cyclin E1 with centrosome amplification has
been reported. In mouse embryonic fibroblasts, ectopic cyclin
E1 overexpression can induce centrosome overproduction (23)
and cause chromosome instability (14). In addition, it was
found that centrosome duplication is normal in cdk2 null MEF
cells. This indicates that Cdk2 activity is not required for nor-
mal centrosome duplication. Moreover, a kinase-deficient cy-
clin E1 mutant can still localize to centrosomes and chromatin
during G0 to S phase progression, followed by stimulation of
DNA synthesis (24,25). These finding indicated that the
Cdk-independent functions of cyclin E play a vital role in cell
cycle progression and cell transformation. Whether the chro-
mosome instability in EBP2-EGFP clones is due to cyclin
E1-induced centrosome overproduction will be investigated in
a future study.
In conclusion, our study demonstrated that ectopic EBP2-
EGFP expression elevated the expression of multifunctional cy-
clin E1, a dominant oncoprotein, in stable clones. In addition,
we also noted that prolonged culture of HEK293 cells under
such an intracellular situation leads to chromosome instability.
These findings suggest a possible role for EBP2 deregulation in
tumorigenesis.
MATERIALS AND METHODS
EBP2-EGFP stable clone selection
The human embryonic kidney cell line (HEK293) was cultured
in DMEM (Gibco, Invitrogen Corporation, USA) supplemented
with 10% FBS as well as 100 U/ml of both penicillin and strep-
tomycin (Gibco, Invitrogen Corporation, USA). The human
EBP2 cDNA was cloned into the pEGFP-N1 plasmid (Clontech
Laboratories, USA) to generate the recombinant EBP2-EGFP.
HEK293 cells were transfected using Lipofectamine (Invitrogen
Corporation, USA). For stable clone selection, the transfected
cells were cultured in the presence of 800 μg/ml G418 sulfate
(Calbiochem, EMD Chemicals, Inc., USA). Stably transfected
single cell-derived clones were obtained by limiting dilution.
Knockdown of ectopic EBP2-EGFP by EGFP shRNA
The plasmid-based EGFP shRNA construct was purchased
from Open-Biosystems (Birmingham, AL). The EBP2-EGFP sta-
ble clone (SC1) was transfected with the EGFP shRNA con-
struct using Lipofectamine (Invitrogen Corporation, USA) un-
der the conditions suggested by the manufacturer. A stably ex-
pressing EGFP shRNA clone was generated by adding 2 μg/ml
puromycin (Sigma-Aldrich Corporation, USA) 24 hours post-
transfection. Populations of resistant clones were detected 7
days post-transfection.
Northern blot analysis
Ten micrograms of RNA was fractionated by electrophoresis
and electro-transferred onto a nylon membrane (Roche

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Corporation, Switzerland). The DIG-labeled EBP2 RNA probes
were used for hybridization and detected by anti-DIG anti-
bodies conjugated with alkaline phosphatase (Roche Corpora-
tion, Switzerland). CSPD substrate (Roche Corporation, Switzer-
land) was added and the chemiluminescence signal was re-
corded on x-ray films.
Western blot analysis
Cells were resuspended in lysis buffer (10 mM Tris-HCl, pH 7.5,
200 mM NaCl, 1% Triton X-100, 0.1 mM sodium orthovana-
date, 50 mM sodium fluoride, and 1 mM phenylmethylsulfonyl
fluoride). Fifty micrograms of protein was fractionated via 6 or
10% SDS-PAGE and transferred to a PVDF membrane (Pall
Corporation, USA). Primary antibodies used in this study in-
cluded those against β-actin (Sigma-Aldrich Corporation, USA),
p21, Cdk2, cyclin E (Santa Cruz Biotechnology, Inc., USA),
ATM, ATR, phospho-ATR (Ser428) (Cell Signaling Technology,
Inc. USA), and phospho-ATM (Ser1981) (Rockland, Gilbertsville,
PA). EBP2 antiserum was generated by immunizing rabbits with
synthetic peptide (amino acid residues 253-271: KKKGSKWNT
RESYDDVSSF). An anti-phospho-p53 Sampler Kit (Cell Signaling
Technology, Inc. USA) was used to identify the specific phos-
phorylation sites of p53. Bound primary antibodies were de-
tected with goat anti-mouse or rabbit IgG conjugated with HRP.
The chemiluminescence signal was recorded on x-ray films.
Flow cytometric bivariate analysis
Flow cytometric bivariate analysis was performed according to
Gong et al. (26) with some modifications. Briefly, the cells
were trypsinized and fixed with 80% ethanol. The fixed cells
were blocked in buffer (0.1% Triton X-100 and 10% FBS in
PBS) and hybridized with 1:200 diluted primary antibodies in
washing buffer (0.1% Triton X-100 and 1% FBS in PBS).
Primary antibodies included anti-cyclin D1 (H295), cyclin E1
(HE-12), cyclin A (H432), and cyclin B (GNS1) (Santa Cruz).
Primary antibodies were detected by 1:500 diluted FITC-con-
jugated goat anti-mouse or rabbit IgG (Santa Cruz). Finally,
cells were stained with propidium iodide and subjected to
flow cytometric analysis.
Cdk2 kinase activity assay
Cdk2 proteins were immunoprecipitated using an anti-Cdk2
polyclonal antibody (M2, Santa Cruz) and protein A/G con-
jugated agarose beads (Calbiochem, EMD Chemicals, Inc.,
USA). Beads were washed with lysis buffer, resuspended in
SDS-PAGE loading buffer and subjected to electrophoresis. The
immunoprecipitated proteins were examined by Western blot.
The kinase activity of Cdk2 was assayed according to
Mazumder et al. (27) with some modifications. Briefly, the im-
munoprecipitated beads were washed with kinase buffer (50
mM HEPES, pH 7.3, 10 mM MgCl2, 1 mM DTT, and 5 mM
MnCl2) and suspended in 40 μl of kinase buffer containing 2
μg of histone H1 as the substrate (Sigma), plus 50 μM ATP and
10 μCi of [32P] ATP. After incubation at 37oC for 30 min, the
reactions were stopped with SDS-PAGE loading buffer, boiled
for 5 min, and resolved via 10 % SDS-PAGE. Phosphorylated
histone H1 proteins were visualized by autoradiography.
Karyotype analysis
Sterilized 22 × 22 mm cover slips were placed into 6 well
plates. 5 × 103 cells were seeded onto the cover slips for 24
hours. Cytological preparations were performed following the
standard procedures (28).
Acknowledgments
This investigation was supported by National Science Council
grants to J.-J. Tsai-Wu (NSC94-2320-B002-122) and by National
Taiwan University Hospital grants to V-C Wu (96-000710-
N-013). We would like to thank the staff of the Second Core
Lab, Department of Medical Research, National Taiwan
University Hospital for technical support.
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