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Cellular Signalling 16 (2004) 1375–1385

Activation of RB/E2F signaling pathway is required for

the modulation of hepatitis C virus core protein-induced cell growth

in liver and non-liver cells$

Mohamed Hassana,*, Hanan Ghozlanb, Ola Abdel-Kaderc

aFaculty of Medicine, Institute of Pathology, University of Dusseldorf, Mooren Str. 5, 40225 Dusseldorf, GermanybDepartment of Microbiology, Faculty of Science, University of Alexandria, Alexandria, Egypt

cMedical Research Institute, University of Alexandria, Alexandria, Egypt

Received 23 March 2004; received in revised form 20 April 2004; accepted 20 April 2004

Abstract

Hepatitis C virus (HCV) core protein is a multifunctional protein that affects transcription and cell growth in vitro and in vivo. Here, we

confirm the proliferative activities of core protein in liver and non-liver cells and delineate part of the mechanism whereby core protein

promotes cell growth. We show that core protein suppresses the expression of tumor suppressor protein p53 and cyclin-dependent kinase

(CDK) inhibitor p21 and enhances the activation of cyclin-dependent kinase 2 (CDK2), the phosphorylation of retinoblastoma (Rb), the

activation of the transcription factor E2F-1, and the expression of E2F-1 and S phase kinase-interacting protein 2 (SKP2) genes. Pretreatment

of core protein-expressing cells with the inhibitor of CDK2, Butyrolactone I, abolished the phosphorylation of Rb, the activation of E2F-1,

and inhibited the expression of E2F-1 gene and cell growth induced. Consistent with these findings, we define a new signaling pathway

whereby the HCV core protein mediates cell growth in infected cells.

D 2004 Elsevier Inc. All rights reserved.

Keywords: HCV; HCC; Signaling pathways; E2F; Cell proliferation

1. Introduction

Hepatitis C virus (HCV) infection results in different

clinical outcomes, and chronic infections are common and

thought to be strongly associated with the development of

HCC [1,2].

HCV, a member of the Flaviviridae family, possesses an

f 9.5 kb positive stranded RNA genome. The virus genome

encodes a single polyprotein precursor of f 3000 amino

acids [3], which is cleaved by cellular and viral proteases

into four structural (core, E1, E2, and P7) and six nonstruc-

tural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins.

HCV core protein is derived from the first 191 amino acids

of the N-terminal of precursor polyprotein. Besides being a

component of viral nucleocapsids, biologically, HCV core

0898-6568/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.cellsig.2004.04.005

$ Supplementary data associated with this article can be found, in the

online version, at doi:10.1016/j.cellsig.2004.04.005.

* Corresponding author. Tel.: +49-211-811-9077; fax: +49-211-811-

9439.

E-mail address: Hassan@med.uni-duesseldorf.de (M. Hassan).

protein is a multifunctional protein [4]. It has been variously

reported to associate with apolipoprotein A II [5], lympho-

toxin-h receptor [6,7], and tumor necrosis factor (TNF)

receptor [8]. Furthermore, the regulation of different cellular

and unrelated viral promoters, by core protein, has been

widely reported [9–11]. Moreover, the HCV core protein

has been shown to be involved in modulation of cell growth.

Relevant observations to this include transformation of

different cell types [12–14] and induction of HCC, by

HCV core protein, in transgenic mice [15].

Cell cycle progression is driven by sequential activation

of cyclin-dependent kinases (CDKs), which are subject to

regulation by positive (cyclins) and negative (CDK-inhibi-

tory proteins) effectors [16]. The disruption of the check-

points in the cell cycle is one mechanism by which

abnormal tumor cells can proliferate. To assess the func-

tional role of HCV core protein in this mechanism, we

examined the effect of HCV core protein on the RB/EF

signaling pathway that is involved in the regulation of G1/S

cell cycle checkpoint. Here, we demonstrated that HCV core

protein-mediated activation of RB/E2F signaling pathway is

M. Hassan et al. / Cellular Signalling 16 (2004) 1375–13851376

involved in the modulation of promoted cell growth in

infected cells.

2. Materials and methods

2.1. Cell lines

Human hepatoma cells (HepG2) and human cervical

carcinoma cells (HeLa) were obtained from ATCC (Rock-

ville, MD, USA). HeLa Tet-Off cells, which constitutively

express the tetracycline-controlled transactivator and the

RetroPackk pT67 cells, were purchased from Clontech

(California, USA). Cells were grown in Dulbecco’s modi-

fied Eagle’s medium (Sigma, Daisenhofen, Germany) sup-

plemented with 10% fetal bovine serum.

2.2. Extraction of RNA from sera, cDNA synthesis, and

plasmid construction

Patients with high HCV RNA titers (105–108 copies/ml)

were selected in order to obtain sufficient amount of RNA.

All patients had detectable HCV RNA of the genotype 4A

as described previously [17]. The viral RNAs were extracted

from 100 Al of serum using QIAamp viral RNA extraction

kit (Qiagen, Hilden, Germany). The complete HCV cDNA

was synthesized with Genscript (Genecraft, Munster, Ger-

many) as described previously [18]. The HCV ds cDNA (nt

280–8306) was amplified from the complete HCV cDNA

by conventional PCR using the following primers: 5V-GACCGT GCA CCA TGA GCA CG-3V (sense) and 5V-CTGCCT ACC GAG CAG GCA GCA-3V. The PCR product

was modified to generate blunt ends and then cloned into

the EcoRV site of pcDNA3.1(+) (Invitrogen, Groningen,

Netherlands) to generate pcDNA3.1-HCV. The HCV core

protein-encoding region (nt 280–852; aa1–191) was am-

plified from the pcDNA3.1-HCV plasmid comprising the

complete HCV cDNA using the following primers having a

start codon (bold) and a HindIII restriction site (underlined):

5V-CCC AAGCTTGGG GAC CGT GCA CCATGA GCA

CG-3V (sense) and 5V-CCC AAGCTT GGG TCG GCG

AAG CGG GGA CAG TC-3V (anti-sense) were used to

amplify the HCV cDNA comprising the entire core protein-

encoding regions of the genotype 4A. The cDNA sequence

of HCV core cDNA was first sequenced and then inserted

into HindIII site of pRevTRE vector.

2.3. Generation of viruses

The packaging cell line RetroPackk pT67 (Clontech)

was grown in DMEM with 10% FCS, 2 mM L-glutamine

(all from Sigma) at 37 jC and in 5% CO2. The cells were

transfected with the appropriate retroviral construct, e.g.,

pRevTet-Off, pRevTRE-luciferase, or pRevTRE-core by

Nucleafectork according to the established protocol

(Amaxa Biosystems, Cologne). Forty-eight hours post-

transfection, the supernatant was collected, filtered through

a 0.45-Am syringe filter, and spun at 50,000� g for 1.5 h.

Pelleted virus was resuspended in a 0.1 or 0.05 of the

original volume of medium at 4 jC for 4 h.

2.4. Infection of target cells

The day before infection, 3� 105 HepG2 cells were

plated in 10-cmm dish. The next day, virus supernatant

(pRevTet-Off) was added and the cells were placed at 37 jCovernight. The next day, the medium was replaced with

fresh medium containing 500 Ag/ml G418 (Gibco BRL,

UK). G418-resistant clones were screened by transient

infection with recombinant retrovirus expressing luciferase.

In a second step, HepG2-pRevTet-Off and HeLa-Tet-Off

cells were infected with recombinant virus expressing the

pRevTRE-core followed by the selection in culture medium

containing 500 Ag/ml G418 (Gibco) and 300 Ag/ml hygrom-

ycin (Clontech). Tetracycline was added at concentration of

4 Ag/ml (Sigma). G418- and hygromycin-resistant clones,

termed HepG2- and HeLa-core transfectants, were screened

for expression of HCV core protein by RT-PCR. Positive

clones, with high induction efficiency, were expanded and

rescreened by RT-PCR and immunoblotting using anti-HCV

core antibody for the expression of HCV core protein.

2.5. Luciferase assay

The luciferase assay was performed using Luciferase

Assay Systems (Promega, USA). The relative light units

(RLU) were measured in illuminometer (EG&G Berthold,

Berthold).

2.6. Preparation of RNA and Northern blot analysis

The HepG2-core and HepG2-mock transfectants, as well

as HeLa-core and HeLa-mock transfectants (2� 106 each),

were plated into a 10-cmm dish (Nunc) and cultured in

medium with ( + Tc) or without (�Tc) 4 Ag/ml tetracycline.

Forty-eight hours later, the cells were harvested and the total

RNA extraction was carried out using RNeasy Mini Kit

(Qiagen). Northern blot analysis was performed as de-

scribed recently [19].

2.7. Immunoblot

Immunoblot analysis was performed according to the

standard procedures. The following antibodies were used at

the indicated dilution: anti-HCV core protein (HCV-C1,

Research Diagnostic, USA), 1:1500; anti-p53 (Sc-6243),

1:2000; anti-p21 (Sc-471), 1:2000; anti-cyclin-dependent

kinase 2 (anti-CDK2) (Sc-749), 1:2000; anti-CDK4

(Sc-260), 1:2000; anti-CDK6 (Sc-177), 1:2000; anti-actin

(SC-1615), 1:5000 (Santa Cruz Biotechnology, USA); anti-

retinoblastoma (anti-Rb) (Biolabs, UK), 1:1500; anti-phos-

pho-Rb (ser 807–811; Biolabs).

M. Hassan et al. / Cellular Signalling 16 (2004) 1375–1385 1377

2.8. Preparation of total cell extracts

The HepG2-core and HepG2-mock transfectants, as well

as HeLa-core and HeLa-mock transfectants (2� 106 each),

were plated into a 10-cmm dish (Nunc) and cultured in

medium with ( + Tc) or without (�Tc) 4 Ag/ml tetracycline.

Forty-eight hours later, the cells were harvested and the total

cell extracts were prepared using RIPA buffer [50 mM Tris

(pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1

mM NaF, 1.0% NP-40, and 0.25% DOC]. After vortexing,

extracts were centrifuged at 12,000 rpm at 4 jC and the

supernatants were collected.

2.9. Preparation of nuclear extracts

The HepG2-core and HepG2-mock transfectants, as well

as HeLa-core and HeLa-mock transfectants (2� 106 each),

were plated into a 10-cmm dish (Nunc) and cultured in

medium with ( + Tc) or without (�Tc) 4 Ag/ml tetracycline.

Forty-eight hours later, the cells were harvested and the

nuclear extracts were prepared as described recently [19,20].

2.10. Electrophoretic mobility shift assay

Electrophoretic mobility shift assays (EMSAs) have been

performed as described [19,20] with minor modification.

Double-stranded synthetic oligonucleotides carrying a bind-

ing site for E2F-1 or for Oct-1 (Santa Cruz Biotechnology)

were end-labeled with [g-32P] ATP (Hartmann Analytika) in

the presence of T4 polynucleotide kinase (Genecraft). The

specificity of binding was analyzed by competition with an

unlabeled oligonucleotide and with supershift assay. The

competition assay was performed in the same manner, except

that unlabeled probes, containing either E2F-1 or Oct-1

sequences, were incubated with nuclear extracts for 20 min

at room temperature before adding the labeled probes. The

supershift assay was performed by incubating the nuclear

extracts with antibodies to either E2F-1 or to Oct-1 for 30 min

at 37 jC and then subjected to EMSA as described for E2F-1

and Oct-1. Electrophoresis was performed for 3 h at 100 V in

0.5� Tris–borate–EDTA running buffer at room tempera-

ture. The dried gel was visualized by exposure to high-

performance autoradiography film.

2.11. In vitro kinase assays

The cells were lysed in 500 Al of buffer L [20 mM

HEPES (pH 7.9), 10 mM EGTA, 40 mM h-glycerophos-phate, 25 mM MgCl2, 2 mM Na3VO4, 1 mM DTT, 1% NP-

40, 5 Ag apoprotein, 1 mM Leupeptin, 1 Ag/ml, pepstatin,

and 1 mM benzamidine]. Lysates were clarified by centri-

fugation and then incubated with specific antibodies to

CDK2, CDK4, and CDK6 for 1 h at 4 jC. The immune

complexes were bound to A-sepharose (5 mg/ml in lysis

buffer) by rotating overnight at 4 jC. The immunoprecipi-

tated complexes were then washed three times with kinase

reaction buffer [80 mM HEPES (pH 7.9), 80 mM MgCl2,

0.1 mM ATP, 2 mM Na3VO4, and 20 mM NaF]. Kinase

activity was determined by incubation with 2 Ag of Rb

(769–921) protein (Santa Cruz Biotechnology) as substrate

for CDK2, CDK4, or CDK6 and 10 ACi of [g-32P] ATP

(Hartmann Analytika) in 15 Al of kinase reaction buffer and

then incubated for 30 min at 37 jC. Reactions were

terminated by addition of 15 Al of sample buffer and

analyzed by SDS-polyacrylamide gel electrophoresis

(PAGE). The gel was dried and autoradiographed.

2.12. 3H thymidine uptake assays

The thymidine uptake was performed as described pre-

viously [21]. The HepG2-core and HepG2-mock transfec-

tants, as well as HeLa-core and HeLa-mock transfectants

(5� 104/well), were plated into a 6-well plates (Nunc) and

cultured in medium with ( + Tc) or without (�Tc) 4 Ag/ml

tetracycline in the presence of 0.5 ACi [3H] thymidine

(Hartmann Analytika) and 5 AM cold thymidine. Forty-

eight hours later, medium was aspirated and cells were

washed twice in 5% trichloroacetic acid (TCA), followed

by three washes in H2O. The fixed cells were solubilized

with 0.1 M NaOH, then mixed with 4-ml scintillant con-

taining 0.4% TCA and counted on Packard Tricard 4000

series scintillation counter.

2.13. MTT assay

The cell number was determined by MTT assay using

cell proliferation kit (Roche, Mannheim, Germany) as

described previously [22]. The HepG2-core and HepG2-

mock transfectants, as well as HeLa-core and HeLa-mock

transfectants (1�103/well), were plated into a microtiter

plate (Nunc) and cultured in medium with ( + Tc) or without

(�Tc) 4 Ag/ml tetracycline. Forty-eight hours later, the

MTT assays were performed in, at least, three independent

experiments in duplicate.

3. Results

3.1. Establishment of cell lines tightly regulated expression

of HCV core protein

To examine the possible mechanisms whereby the HCV

core protein of the genotype 4A mediates cell growth in

liver (HepG2) and non-liver (HeLa) cells, we established a

HepG2- and HeLa-core cells, which can be induced to

express HCV core protein under the control of tetracycline.

The expression of HCV core protein was detected in

HepG2- and HeLa-core transfectants, 48 h after the removal

of tetracycline from culture media (Fig. 1). The induction

ratio was greater than 1000 when the relative amount of the

expressed HCV core protein with or without tetracycline

was analyzed in HepG2- and HeLa-core transfectants (data

Fig. 1. Detection of HCV core protein expression in HepG2- and HeLa-core

transfectants. Whole cell lysates of 100 Ag, collected from either HepG2-

core transfectants or HeLa-core transfectants cultured in medium with

( + Tc) or without (�Tc) 4 Ag/ml tetracycline, were analyzed by

immunoblotting using anti-HCV core antibody. Arrow indicates expressed

HCV core protein. The same blots were reprobed with an anti-actin

antibody to compare loading and transfer. The units at the bottom reflect

densitometric screening of the expression level of HCV core protein.

M. Hassan et al. / Cellular Signalling 16 (2004) 1375–13851378

not shown). The expression levels of HCV core protein in

HepG2- and HeLa-core transfectants were found to be time-

dependent and could be quantitatively regulated by the

Fig. 2. Inhibition of p53 and p21 by core protein in HepG2 and HeLa cells. (A) W

12% SDS-PAGE and subjected to Western blotting using anti-p53 polyclonal anti

loading and transfer. Similar results were shown repeatedly in three independent e

were analyzed by Northern blotting using cDNAs probe specific for p21. The equal

of whole cell lysates (100 Ag) were resolved by 12% SDS-PAGE and subjected t

reprobed with an anti-actin antibody to compare loading and transfer. Similar res

variation of tetracycline concentration that is present in the

culture medium (data not shown). The intensity of the

signals was quantified by the software Reytest (Reytest,

Straubenhardt, Germany).

3.2. Suppression of p53 in cells expressing core protein

To investigate whether the HCV core protein affects the

p53 gene, the expression of p53 was examined in HepG2-

core and HepG2-mock transfectants as well as in HeLa-core

cultured in medium with ( + Tc) or without (�Tc) 4 Ag/ml

tetracycline for 48 h. Results from Western blot (Fig. 2A)

demonstrated a marked decrease at the expression level in

HepG2- and HeLa-core transfectants induced to express

HCV core protein. These findings confirm earlier results

suggesting a negative effect for HCV core protein on the

expression of p53.

3.3. HCV core protein suppresses the expression of p21waf1

in HepG2 and HeLa cells

To investigate whether the HCV core protein also influ-

ences the expression of p21waf1, the target gene of p53, the

expression of p21 was examined in HepG2-core and HepG2-

mock transfectants as well as from HeLa-core and HeLa-

mock transfectants cultured inmediumwith ( + Tc) or without

(�Tc) 4 Ag/ml tetracycline for 48 h. Results from Northern

blot and Western blot analyses (Fig. 2B and C) showed that

HCV core protein blocks the expression of p21 not only at

estern blot: equal amounts of whole cell lysates (100 Ag) were resolved by

body. The same blots were reprobed with an anti-actin antibody to compare

xperiments. (B) Northern blot: equal amounts of total cellular RNA (20 Ag)loading of mRNAwas judged by GAPDH. (C) Western blot: equal amounts

o Western blotting with anti-p21 polyclonal antibody. The same blots were

ults were shown repeatedly in four independent experiments.

M. Hassan et al. / Cellular Signa

mRNA level, but also at the protein level. These data indicate

that the HCV core protein-induced suppression of p21 in both

HepG2- and HeLa-core transfectants may result from the

HCV core protein-mediated suppression of p53.

3.4. Activation of CDK2 but not CDK4 or CDK6 by HCV

core protein

To examine whether the HCV core protein-mediated

suppression of p53 and p21 influences the expression or

the activation of CDK2, CDK4, and CDK6 in HepG2- or

HeLa-core cells induced to express HCV core protein, we

examined the expression and the activation of CDK2,

CDK4, and CDK6 in HepG2-core and HepG2-mock trans-

fectants as well as in HeLa-core and HeLa-mock trans-

fectants cultured in medium with ( + Tc) or without (�Tc) 4

Ag/ml tetracycline for 48 h. Results from Western blot

(Fig. 3) demonstrated that the expression of HCV core

protein did not alter the expression of CDK2, CDK4, or

CDK6 either in HepG2 or HeLa cells. In contrast, when we

performed in vitro kinase assay (Fig. 3), the expression of

HCV core protein enhanced markedly the basal activity of

CDK2 but not those of CDK4 or CDK6, compared to

control cells. These results suggest that the CDK2-induced

activation, by the expression of HCV core protein, in both

HepG2 and HeLa cells may result from the HCV core

protein-induced suppression of the cyclin-dependent kinase

inhibitor p21 and its upstream regulatory protein p53.

Fig. 3. Effect of HCV core protein on the expression and activation of CDK2, CD

HeLa-mock transfectants were cultured for 48 h in medium with ( + Tc) or withou

CDK4, and CDK6 were immunoprecipitated from extracts by using specific anti-C

measured by using the immune complex in a kinase assay with retinoblastoma fus

was determined by Western blot analysis. In vitro kinase assays and Western blots

results.

3.5. HCV core protein enhances the phosphorylation of Rb

in both HepG2 and HeLa cells

To determine whether HCV core protein-induced activa-

tion of CDK2 triggers the phosphorylation of Rb, we

examined both the phosphorylation and the expression of

Rb in HepG2-core and HepG2-mock transfectants, as well

as in HeLa-core and HeLa-mock transfectants cultured in

medium with ( + Tc) or without (�Tc) 4 Ag/ml tetracycline

for 48 h. Results from Western blots (Fig. 4) demonstrated

that the expression of HCV core protein enhances the

phosphorylation of retinoblastoma (Rb) not only in HepG2

cells, but also in HeLa cells, compared to control cells

(Fig. 4). In contrast, when we probed the same blots using

antibody directed against total Rb, we detected a very weak

signal without any marked changes in response to the

expression of HCV core protein either in HepG2 or HeLa

cells (Fig. 4). This finding suggests that the HCV core

protein-induced phosphorylation of Rb, in HepG2 or in

HeLa cells, is likely to be resulted from HCV core pro-

tein-mediated activation of CDK2.

3.6. Enhancement of the DNA-binding activity of the

transcription factors E2F-1 in both HepG2 and HeLa cells

expressing HCV core protein

To examine whether the HCV core protein-induced

phosphorylation of Rb results in the activation of the

lling 16 (2004) 1375–1385 1379

K4, and CDK6. HepG2-core and HepG2-mock as well as HeLa-core and

t (�Tc) 4 Ag/ml tetracycline. Cells were washed and lysed, and the CDK2,

dk2, Cdk4, or Cdk6, respectively. CDK2, CDK4, or CDK6 activities were

ion protein as substrate. Total CDK2, CDK4, and CDK6 protein expression

are representative of three independent experiments performed with similar

Fig. 4. Effect of HCV core protein on the expression and phosphorylation of retinoblastoma. Phosphorylation of retinoblastoma protein, Rb, in HepG2- and

HeLa-core cells. Whole cell lysates of 100 Ag, collected from either HepG2-core and HepG2-mock transfectants or from HeLa-core and HeLa-mock

transfectants cultured in medium with ( + Tc) or without (�Tc) 4 Ag/ml tetracycline, were resolved by 10% SDS-PAGE and probed for the phosphorylated

form of Rb (p-Rb). Blots were stripped and reprobed either for total amounts of Rb or with actin antibody to compare loading and transfer. Western blots are

representative of three independent experiments performed with identical results.

Fig. 5. Effect of HCV core protein on the activation of the transcription factor E2F-1. (A) Activation of E2F-1 in HepG2 and HeLa cells expressing HCV core

protein. The HepG2-core and HepG2-mock transfectants as well as HeLa-core and HeLa-mock transfectants were cultured in medium with ( + Tc) or without

(�Tc) 4 Ag/ml tetracycline. Forty-eight hours later, the nuclear extracts were prepared and assayed for the E2F-1 DNA-binding activity by EMSA. (B)

Supershift bands were observed by adding either E2F-1 antibody or Oct-1 antibody to the protein–oligonucleotides mixtures. EMSAs are representatives of

three independent experiments performed with identical results.

M. Hassan et al. / Cellular Signalling 16 (2004) 1375–13851380

M. Hassan et al. / Cellular Signa

transcription factor E2F-1, we examined the activity of

E2F-1 in HepG2-core and HepG2-mock transfectants, as

well as in HeLa-core and HeLa-mock transfectants cul-

tured in medium with ( + Tc) or without (�Tc) 4 Ag/ml

tetracycline for 48 h. Results from EMSAs (Fig. 5A)

demonstrated that the expression of HCV core protein

enhances markedly the activity of E2F-1 not only in

HepG2, but also in HeLa cells, compared to the control

cells (Fig. 5A). The specificity of E2F-1 and Oct-1 binding

was analyzed by both competition with unlabeled oligo-

nucleotide (data not shown) and supershift assay (Fig. 5B).

These findings demonstrate that the consequence of the

HCV core protein-induced phosphorylation of Rb may

contribute to the activation of the transcription factor

E2F-1 in cells expressing HCV core protein.

3.7. Induction of E2F-1 gene expression in both HepG2 and

HeLa cells by HCV core protein

To examine the functional properties of the HCV core

protein-induced activation of the transcription factor E2F-1,

we analyzed the expression of the S phase associated gene

E2F-1 in HepG2-core and HepG2-mock transfectants, as

well as in HeLa-core and HeLa-mock transfectants cultured

in medium with ( + Tc) or without (�Tc) 4 Ag/ml tetracy-

cline for 48 h. Northern blot analysis (Fig. 6A) demonstrat-

ed that the HCV core protein induces markedly the mRNA

levels of E2F-1 in both HepG2- and HeLa-core transfec-

tants, compared to control cells (Fig. 6A). These observa-

tions suggest that the core protein may induce the

expression of S phase genes including E2F-1 through the

activation of the transcription factor E2F-1.

Fig. 6. Effect of core protein on the expression of E2F-1 and SKP2 genes in HepG

and HepG2-mock transfectants as well as from HeLa-core and HeLa-mock transfec

for 48 h, was analyzed by Northern blot using specific cDNA probes for E2F-1 (A

probe. Northern blots are representative of three independent experiments perform

3.8. HCV core protein induces the expression of S phase

kinase-interacting protein 2 in both HepG2 and HeLa cells

The S phase kinase-interacting protein 2 (SKP2) is a

member of the F-box protein family that is widely reported

to play an important role in the regulation of the mamma-

lian cell cycle progression via CDK2. Therefore, we

investigated the effect of HCV core protein on the mRNA

level of SKP2 in HepG2 and HeLa cells. Results from

Northern blot (Fig. 6B) demonstrated that the HCV core

protein induces markedly the mRNA levels of SKP2 in

HepG2- and HeLa-core transfectants, compared to control

cells (Fig. 6B). These results suggest that the expression of

HCV core protein induces the mRNA of SKP2 in HepG2

and HeLa cells.

3.9. The CDK inhibitor, Butyrolactone I, prevents the

phosphorylation of Rb and activation of E2F-1, abolishes

E2F-1 gene expression, and inhibits cell growth induced by

HCV core protein

The CDK2 inhibitor, Butyrolactone I, has been widely

reported to inhibit the activity of CDK2 [23]. In order to

determine whether the HCV core protein-induced activation

of CDK2 is directly associated with the cell growth induced,

we sought to block CDK2 by using Butyrolactone I and to

determine the effect on the extent of cell growth induced by

HCV core protein. One hour prior to the cultivation of

HepG2-core and HepG2-mock transfectants, as well as

HeLa-core and HeLa-mock transfectants in medium with

( + Tc) or without (�Tc) 4 Ag/ml tetracycline, the cells were

pretreated with 50 Ag/ml Butyrolactone I and the total RNA,

lling 16 (2004) 1375–1385 1381

2 and HeLa cells. Total cellular RNA of 20 Ag, prepared from HepG2-core

tants cultured in medium with ( + Tc) or without (�Tc) 4 Ag/ml tetracycline

) or for SKP2 (B). Filters were then rehybridized with a GAPDH-specific

ed with identical results.

M. Hassan et al. / Cellular Signalling 16 (2004) 1375–13851382

Fig. 7. Inhibition of HCV core protein-induced Rb phosphorylation, E2F-1 activation, E2F-1 expression, and cell growth by Butyrolactone I. One hour prior to the

cultivation of HepG2-core and HepG2-mock transfectants, as well as HeLa-core and HeLa-mock transfectants in medium with ( + Tc) or without (�Tc) 4 Ag/ml

tetracycline for 48 h, the cells were pretreated with 50 Ag/ml Butyrolactone I. In (A), whole cell lysates were prepared and equal amounts of protein (100 Ag) wereresolved by 10% SDS-PAGE gel followed by Western blotting. Membranes were incubated with phosphospecific Rb antibody (upper panel), subsequently

stripped and reprobed with either anti-Rb antibody (middle panel), or with anti-actin antibody to compare loading and transfer. In (B) and (C), the nuclear extracts

were prepared and equal amounts of nuclear protein (4 Ag) were assayed for the E2F-1 DNA-binding activity in liver cells (right) and non-liver cells (left) by

EMSA. The Oct-1 probe was used to monitor the nuclear extract concentration in the different lanes. In (D), the total RNAs were extracted and the expression of

E2F-1was analyzed byNorthern blot in liver (right) and in non-liver cells (left) as described underMaterials andmethods.Membraneswere then rehybridizedwith

a GAPDH-specific probe. In (E) and (F), measurement of cell growth in HepG2- and HeLa-core transfectants induced to express HCV core protein either by 3H

thymidine uptake assays (E) or by MTT assay (F). Results are representative of three separate experiments.

M. Hassan et al. / Cellular Signalling 16 (2004) 1375–1385 1383

nuclear extracts, and total cell extracts were prepared, and

the functional assays were performed. Results from Western

blot (Fig. 7A), EMSA (Fig. 7B), and Northern blots (Fig.

7C) revealed that the inhibition of HCV core protein-

induced activation of CDK2 prevents the phosphorylation

of Rb (Fig. 7A), as well as the activation of the transcription

factor E2F-1 (Fig. 7B and C), and the expression of E2F-1

gene (Fig. 7D) in HepG2 and HeLa cells. In order to

determine the effect of Butyrolactone I on HCV core

protein-promoted cell growth, 1 h prior to the cultivation

of HepG2-core and HepG2-mock transfectants as well as

HeLa-core and HeLa-mock transfectants in medium with

( + Tc) or without (�Tc) 4 Ag/ml tetracycline, the cells were

either pretreated or not treated with 50 Ag/ml Butyrolactone

I. Forty-eight hours later, the proliferation assays were

performed. Results shown in Fig. 7E and F illustrate that

M. Hassan et al. / Cellular Signalling 16 (2004) 1375–13851384

although Butyrolactone I itself does not promote growth rate

either in HepG2 or in HeLa cells, however, pretreatment

with Butyrolactone I was found to inhibit HCV core protein-

induced growth advantage in HepG2- and HeLa-core trans-

fectants induced to express HCV core protein. These results

suggest that the phosphorylation of Rb and the subsequent

activation of the transcription factor E2F-1 occur as a direct

consequence of CDK2 activation. In addition, the ability of

Butyrolactone I to abrogate the induced growth advantage in

both HepG2 and HeLa cells expressing HCV core protein

indicates that the activation of RB/E2F signaling pathway is

required for the modulation of HCV core protein-promoted

cell growth both in liver and non-liver cells.

4. Discussion

In this study, we present an insight into a mechanism

whereby the HCV core protein mediates cell growth in liver

and non-liver cells and confirmed further the oncogenic

activity of HCV core protein.

In this work, the expression of HCV core protein was

found to affect the intracellular proteins, which are involved

in the regulation of RB/E2F signaling pathway parallel to

the promotion of cell growth. The results that we obtained

by controlled expression of HCV core protein are the first to

demonstrate endogenous regulation of cellular genes.

To determine whether results obtained with HepG2 cells

are applicable to cells of non-hepatic origin, the similar

studies were performed also with HeLa cells, and the results

were comparable.

Cancer is a multistep process that requires a cumulative

effect altering both positive and negative regulators of cell

proliferation and cell survival [24,25]. One of these negative

regulators is the universal CDK inhibitor p21 [26], which

can be transcriptionally regulated by p53 and is thus

considered to participate in execution of p53 effects [27].

Therefore, the suppression of p53 expression and its target

gene p21 may result in the activation of CDKs and thereby

lead to deregulation of restriction (R)-point control in G1

phase that subsequently would promote uncontrolled cell

growth.

The tumor-suppressor protein p53 is an important tran-

scription factor, which is widely reported to play a central

role in the cell cycle regulation mechanisms and cell

proliferation control, and its inactivation is considered a

key event in human carcinogenesis. However, there are

contradicting data regarding the effect of HCV core protein

on p53. Two studies demonstrated the activation of p53 and

its target gene p21 by HCV core protein [28,29], while other

studies demonstrated the suppression of p53 [9] and p21

[10] by HCV core protein.

In agreement with prior studies [9,10], we delineate an

eradication at the expression levels of the endogenous p53

and its target gene p21 by the controlled expression of HCV

core protein in liver and non-liver cells. In addition, the

activation of CDK2, the downstream effector of p53 and its

target gene p21, by the expression of HCV core protein

addresses a potential role for HCV core protein-mediated

suppression of p53 and its target gene p21 in the mechanism

of CDK2 activation. Therefore, our finding that the HCV

core protein enhances the phosphorylation of Rb, as well as

the activation of the transcription factor E2F-1, and subse-

quently its target gene E2F-1 [30,31], suggests that the RB/

E2F signaling pathway may play an important role in the

regulation of HCV core protein-mediated cell growth. Thus

far, the inhibition of CDK2 by Butyrolactone I together with

its inhibitory effect on the extent of the growth advantage

induced confirms the essential role of CDK2 in the regula-

tion of HCV core protein-mediated activation of RB/E2F

signaling pathway. In fact, the ability of Butyrolactone I to

abolish HCV core protein-induced effects on Rb, as well as

on the transcription factor E2F-1 and its target gene E2F-1,

parallel to the inhibition of induced cell growth would

suggest the importance of RB/E2F signaling pathway in

the modulation of HCV core protein-mediated cell growth

in both liver and non-liver cells.

Rb that is known as a cell cycle regulated protein can

be phosphorylated at specific points during the cell cycle

by cyclin D1/cdk4 or D1/cdk6 and E/cdk2 [32]. Rb acts

as a master switch for cell cycle progression, holding cells

in G1 until they are ready to progress into S phase by

binding several key regulators of cell cycle progression,

including members of the transcription factors of E2F

family [33]. When Rb is bound to E2F early in G1, it

actively represses transcription from E2F sites. At the G1/

S phase boundary, Rb becomes phosphorylated and

releases the E2F complexes bound to it, allowing these

family members to transcribe E2F-controlled genes includ-

ing E2F-1 [34,35].

In this study, we demonstrated for the first time the

phosphorylation of Rb and the enhancement of the DNA-

binding activity of the transcription factor E2F-1 following

the expression of HCV core protein in liver and in non-liver

cells and confirmed further the upregulation of the endog-

enous gene E2F-1 that is recently reported in HCV core

protein-expressing cells [36].

The S phase kinase-interacting protein 2 (SKP2) has

been reported to reveal an unexpected oncoprotein con-

nection that may control cell growth in normal and tumor

cells [37–40], and its expression has been demonstrated

in moderately and poorly differentiated HCC in surgically

restricted HCC tissue [41]. In vivo, the induction of SKP2

is elevated in response to liver regeneration [42], whereas

in vitro, its induction and phosphorylation have been

elevated in response to various mitogen stimuli [42,43].

The increase of the expression of SKP2, in liver and non-

liver cells, suggests that the HCV core promotes cellular

proliferation for the maintenance of replication and sur-

vival [44–46]. Physiologically, SKP2 has been shown to

target proteins, such as p27 and free Cdk2-unbound

cyclin E, in addition to other cell cycle proteins and

M. Hassan et al. / Cellular Signalling 16 (2004) 1375–1385 1385

E2F-1 [39], and to induce DNA replication of fibroblast

cultured in low serum [38]. Thus, whether HCV core

protein-induced expression of SKP2 is associated with the

oncogenic activity of HCV core protein remains to be

established. However, further studies with other mamma-

lian cell lines, especially primary hepatocytes, should

further clarify the importance of these results.

Taken together, our findings in agreement with several

reports confirm the oncogenic activity of HCV core protein

in hepatocytes and define a new pathway whereby HCV

core protein mediates cell growth. Therefore, the blockage

of the RB/E2F pathway may become an attractive option for

the treatment of HCV-related HCC.

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