Carcinogenesis vol.34 no.3 pp.627–637, 2013
doi:10.1093/carcin/bgs364
Advance Access publication November 24, 2012
(-)-Epigallocatechin-3-gallate inhibition of Epstein–Barr virus spontaneous lytic
infection involves ERK1/2 and PI3-K/Akt signaling in EBV-positive cells
Sufang Liu1,2, Hongde Li1, Lin Chen1, Lifang Yang1,
Lili Li1, Yongguan Tao1, Wei Li1, Zijian Li1, Haidan Liu1,
Min Tang1, Ann M.Bode3, Zigang Dong3 and Ya Cao1,*
1
Cancer Research Institute, Xiangya School of Medicine, Central South
University, Key Laboratory for Cancer and Invasion of Ministry of Education,
Changsha, Hunan 410078, China, 2Division of Hematology, Institute
of Molecular Hematology, the Second Xiangya Hospital, Central South
University, Changsha, Hunan 410011, China and 3The Hormel Institute,
University of Minnesota, 801 16th Avenue NE, Austin, MN 55912, USA
*To whom correspondence should be addressed. Tel: +86-731-84805448;
Fax: +86-731-84470589;
Email: [email protected]
Correspondence may also be addressed to Zigang Dong. Tel: +507-437-9600;
Fax: +507-437-9606;
Email: [email protected]
Introduction
Epstein–Barr virus (EBV) is a human herpes virus that infects 90% of
the human population. EBV infection, as one of many environmental
factors, has been reported to be strongly associated with the development of several human malignancies, including Burkitt’s lymphoma, Hodgkin’s disease and nasopharyngeal carcinoma (NPC) (1,2).
Importantly, an increasing number of tumors are found to be EBVpositive (3,4). Like all other herpes viruses, EBV can establish a latent
or lytic infection in host cells (5). Intriguingly, evidence indicates that
EBV reactivation into the lytic cycle may play a role in the pathogenesis of malignancies. Elevated antibody titers against EBV lytic antigens and increased viral DNA load in serum/plasma, two parameters
that represent EBV lytic infection in vivo, correspond with advanced
cancer stages, poor prognosis or tumor recurrence after therapy (6,7).
Serological studies further suggest that EBV reactivation into lytic
Abbreviations: AP-1, activator protein; DMSO, dimethylsulfoxide; EBV,
Epstein–Barr virus; EGCG, (-)-epigallocatechin-3-gallate; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; LMP1, latent membrane
protein 1; MAPK, mitogen-activated protein kinase; mRNA, messenger RNA;
MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide; NPC,
nasopharyngeal carcinoma; PBS, phosphate-buffered saline; PI3-K, phosphatidylinositol 3-kinase; qPCR, quantitative PCR.
Materials and methods
Cell lines and cell culture
B95.8, an EBV-positive marmoset B-cell line, and Raji, an EBV-positive
lymphoblast-like cell line established from Burkitt’s lymphoma, were obtained
from the American Type Culture Collection. CNE1-LMP1 is a stable LMP1integrated NPC cell line (20,21). All cells were cultured in RPMI 1640 (Gibco
© The Author 2012. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
627
Downloaded from http://carcin.oxfordjournals.org/ by guest on May 26, 2016
Epstein–Barr virus (EBV) reactivation into the lytic cycle plays
certain roles in the development of EBV-associated diseases,
including nasopharyngeal carcinoma and lymphoma. In this study,
we investigated the effects of the tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) on EBV spontaneous lytic infection and the
mechanism(s) involved in EBV-positive cells. We found that EGCG
could effectively inhibit the constitutive lytic infection of EBV at
the DNA, gene transcription and protein levels by decreasing the
phosphorylation and activation of extracellular signal-regulated
kinase 1/2 (ERK1/2) and Akt. By using cellular signaling pathwayspecific inhibitors, we also explored the signaling mechanisms
underlying the inhibitory effects of EGCG on EBV spontaneous
lytic infection in cell models. Results show that specific inhibitors
of Mitogen-Activated Protein Kinase Kinase (MEK) (PD98059)
and phosphatidylinositol 3-kinase [PI3-K (LY294002)] markedly
downregulated gene transcription and expression of BZLF1 and
BMRF1 indicating that the MEK/ERK1/2 and PI3-K/Akt pathways are involved in the EBV spontaneous lytic cycle cascade.
Therefore, one of the mechanisms by which EGCG inhibits EBV
spontaneous lytic infection appears to involve the suppression of
the activation of MEK/ERK1/2 and PI3-K/Akt signaling.
infection could occur months or years before the clinical diagnosis of
NPC, HD (Hodgkin disease) or Burkitt’s lymphoma, serving as a risk
factor for cancer development (8–10).
Upon induction of the lytic program, the two key EBV immediateearly lytic genes, BZLF1 and BRLF1, are expressed. The lytic genes
encode the transcriptional activator BZFL1 (also called Zta, ZEBRA or
EB1), which activates viral and certain cellular promoters through binding to the Zta-responsive element. Others and we have used a strategy to
induce EBV lytic infection in EBV-positive tumor cells, and results seem
promising (11,12). Evidence indicates that the extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK)
signaling pathway might play a critical role in EBV lytic infection (13).
Immunoblotting using a phospho-MAPK antibody revealed that antiIgG-induced rapid phosphorylation of MAPK in cells. The phosphorylation was inhibited by the MAPK/ERKs-specific inhibitor, PD98059.
The expression of the EBV immediate-early BZLF1 messenger RNA
(mRNA), its protein product and the early antigen was also suppressed
by the inhibitor. These results indicate that the Ras/MEK/MAPK pathway contributes to EBV lytic infection (13). A recent study suggests that
phosphatidylinositol 3-kinase (PI3-K) is a determinant of responsiveness to B-cell antigen receptor-mediated EBV activation (14). BRLF1
activates PI3-K signaling in host cells, and the specific PI3-K inhibitor, LY294002, completely abrogates the ability of a BRLF1 adenovirus
vector to induce the lytic form of EBV infection (15). These studies indicated that the Ras/MEK/MAPK and PI3-K signal transduction pathways
are important in the control of the EBV lytic cycle.
Green tea is chemically characterized by the presence of high
amounts of polyphenolic compounds known as catechins. The most
abundant component is epigallocatechin-3-gallate (EGCG), which
appears to be the primary active ingredient responsible for the biological effects of green tea. EGCG reportedly exhibits antioxidative, antibacterial and antitumor effects (16). Others and we have
shown that EGCG modulates multiple signal transduction pathways
including the MAPK and PI3-K/Akt pathways, thereby imparting
strong cancer chemopreventive as well as therapeutic effects (17,18).
However, the effects of EGCG on the spontaneous reactivation of the
EBV lytic cycle in EBV-positive cell lines have not yet been reported.
Latent membrane protein 1 (LMP1), which is the only viral gene product with oncogenic properties among the EBV-encoded proteins, can be
detected in 90% of NPC patients. Through the cytoplasmic carboxy terminus, LMP1 triggers multiple signal transduction cascades, including
MEK/ERKs, PI3-K/Akt, JNKs and STAT3, to alter cell growth and survival (19). Considering that EGCG can modulate signal pathways induced
by LMP1 (17), we wanted to determine the mechanism of EGCG’s effect
on EBV lytic infection by inhibition of the constitutive activation of lytic
infection-associated signaling pathways. LMP1-positive cell lines with
constitutive activation of the MEK/ERK and PI3-K/Akt pathways provide a model to study the effects and mechanisms of EGCG on EBV
spontaneous lytic infection in EBV-positive cells.
The results in this study indicated that EGCG inhibits EBV spontaneous lytic infection at the DNA, gene transcription and protein levels. Further studies showed that EGCG suppresses EBV lytic infection
through the attenuation of MEK/ERK1/2 and PI3-K/Akt signaling.
Because EBV lytic infection might play a critical role in the development of EBV-associated malignancies, we describe here a potential
chemopreventive activity of EGCG in controlling EBV lytic infection.
S.Liu et al.
Downloaded from http://carcin.oxfordjournals.org/ by guest on May 26, 2016
Fig. 1. Detection of EBV-encoded proteins and EBV DNA in cells. (A) Cells were disrupted and immunoblot analysis was performed to determine the
expression of EBNA1, LMP1, BZLF1 and BMRF1. (B) CNE1-LMP1 cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100.
BZLF1 or BMRF1 was visualized with mouse monoclonal anti-BZLF1 (1:100) or anti-BMRF1 (1:100) and a fluorescein isothiocyanate-conjugated secondary
antibody (1:200). Incubation with PBS but no primary antibody and then with secondary antibody was used as a negative control. DNA was stained with
628
EGCG inhibits EBV lytic infection
BRL, Grand Island, NY), supplemented with 10% fetal bovine serum (FBS;
Invitrogen, Carlsbad, CA), 10 µg/ml streptomycin and 10 IU/ml penicillin, and
incubated in a humidified atmosphere of 5% CO2 at 37°C.
Chemicals and cell treatments
EGCG and LY294002 were purchased from Sigma Chemical (St Louis, MO),
and PD98059 was purchased from Calbiochem (La Jolla, CA). EGCG and all
inhibitors were prepared in autoclaved water and dimethylsulfoxide (DMSO;
Sigma), respectively, as stock solutions for in vitro studies. Subconfluent cells
were treated with the respective compounds at various concentrations for different times as indicated. Detailed treatment procedures are described in the
Figure Legend 4. The final concentration of DMSO in the culture media was
maintained at <0.1%, which had no significant effect on cell growth. Vehicle
controls were included for all treatments.
Preparation of cell lysates and immunoblot analysis
Whole cell lysate preparation and immunoblot analysis were performed according to the method described previously (11). Protein concentration was determined using the BCA Assay Reagent (Pierce, Rockford, IL). The following
antibodies were used for immunodetection with appropriate dilutions: mouse
BZLF1 monoclonal antibody (AZ-69; Argene, Varilhes, France), EBNA1 antibody (ab25653; Abcam, Cambridge, UK), BMRF1 antibody (ab6524; Abcam),
β-actin (Ac-15; Sigma), Akt antibody (9272; Cell Signaling Technology,
Danvers, MA) and p-Akt (Ser473) (9271; Cell Signaling Technology). The
antibodies against ERK1 (sc-93), p-ERK (Tyr-204) (sc-7383), goat anti-rabbit
IgG-horseradish peroxidase (sc-2004) and goat anti-mouse IgG-horseradish
peroxidase (sc-2005) were all from Santa Cruz (Santa Cruz, CA).
Immunofluorescence assay
After being washed with phosphate-buffered saline (PBS), cells were fixed
in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. BZLF1
or BMRF1 was visualized with mouse monoclonal anti-BZLF1 (1:100) or
anti-BMRF1 (1:100) and a fluorescein isothiocyanate-conjugated secondary
antibody (1:200). Incubation with PBS but no primary antibody and then with
secondary antibody was used as a negative control. DNA was stained with
4′,6-diamidino-2-phenylindole at room temperature for 30 s. The staining patterns were observed under a fluorescence microscope (Zeiss, Jena, Germany).
Immunohistochemistry
Thirty-three paraffin-embedded NPC tissues and 24 paraffin-embedded
lymphoma tissues [Hodgkin’s lymphoma (19), Burkitt’s lymphoma (4) and
NHL (1)] were obtained from the Department of Pathology at Hunan Tumor
Hospital and Xiangya Hospital, Changsha, China. Immunohistochemistry was
performed on 4.0 µm sections from formalin-fixed, paraffin-embedded tissues. The sections were deparaffinized and rehydrated. Antigen retrieval was
achieved with diluted ethylenediaminetetraacetic acid (50:1) by microwaving
for 10 min. Endogenous peroxidase activity was quenched with a 3% hydrogen
peroxide block for 15 min at room temperature. The sections were treated with
goat serum for 10 min and incubated at 4°C overnight with the primary antibody (BZLF1 diluted 1:100). The secondary antibody, biotinylated horse antimouse IgG (Vector Laboratories, Burlingame, CA.) diluted 1:200, was applied
for 30 min at 37°C, followed by a 30 min incubation with Vectastain ABC
Elite. The reaction was visualized by using diaminobenzidene chromogen
(Dako) following the manufacturer’s instructions, and the slides were counterstained with Mayer’s hematoxylin. All slides were scored by two observers.
The staining intensity and pattern were evaluated using a 0 to 3+ scale (0,
completely negative; 1+, weak; 2+, intermediate; 3+, strong). A final score of
≥2+or greater was required for the case to be considered positive.
MTT assay
The 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay was used to assess the effects of EGCG on cell viability as described
previously (22). Briefly, cells were cultured in 96 well flat-bottomed plates and
were starved in 0.1% FBS RPMI medium 1640 for 24 h. Then, serial dilutions of
EGCG were added to the cells and incubated for the indicated time. Following
the incubation period, 10 µl MTT (5 mg/ml) per well was added and then incubated for another 4 h at 37°C. After discarding the medium, 150 µl DMSO was
added to each well and mixed thoroughly to dissolve the blue formazan crystals.
The plates were subsequently read on a Bio-Rad 3350 microplate reader at a
wavelength of 570 nm. Null control wells contained medium alone. Cell viability
was calculated as the percentage of MTT inhibition using the following formula:
viability (%) = (OD570 treatment − OD570 blank control)/(OD570 no treatment
control − OD570 blank control) × 100%. The experiments were repeated at least
three times.
←
4′,6-diamidino-2-phenylindole at room temperature for 30 s. The staining patterns were observed under a fluorescence microscope (Zeiss). (C) To detect EBV
DNA copies, the BamHI-W fragment was used as an index using a RT–PCR assay. The estimation of viral genomes in each sample was expressed as the number
of viral genomes copy per 500 ng of purified DNA. The details are described in Materials and methods. Statistical significance:*P < 0.05 versus CNE1-LMP1
cells.
629
Downloaded from http://carcin.oxfordjournals.org/ by guest on May 26, 2016
Fig. 2. Immunohistochemical staining for EBV lytic proteins in specimens from patients with NPC and lymphoma. Paraffin sections were screened for the
expression of the EBV lytic proteins with the specific antibodies to detect BZLF1 and BMRF1. Positive signal implies that BZLF1 or BMRF1 expression occurs
in tumor cells as shown by nuclear localization in different cases. Magnification, ×400.
S.Liu et al.
Transient transfection and luciferase assay
Cells were seeded in 24-well plates before transfection. pZp-Luc (kindly
provided by Ichiro Saito, Tsurumi University School of Dental Medicine,
Yokohama, Japan) (23) or pMp-Luc (kindly provided by Paul J.Farrell,
Imperial College Faculty of Medicine, London, UK) (24) was cotransfected
with an internal control pRL-SV40 using the Lipofectamine 2000 Transfection
Reagent (Invitrogen) following the manufacturer’s instructions. At 4 h after
transfection, cells were treated with chemicals as designated. Cells were
harvested at 24 h after transfection and lysates were analyzed for luciferase
activity using the Dual Luciferase Reporter Assay (Promega, Madison, WI)
according to the manufacturer’s directions. The luciferase reporter plasmids
were cotransfected with pRL-SV40 to correct for variations in transfection
efficiency. The relative luciferase activity was normalized to the value of pRLSV40 activity. The data are represented as means ± SD of three independent
experiments performed in triplicate.
DNA extraction and quantification of EBV copy number
To measure EBV genomic copy number, qPCR targeting the BamHI-W
segment was performed with DNA extracted from cells using the universal
genomic extraction kit (TaKaRa) according to the kit handbook. To detect
EBV DNA copies, the BamHI-W fragment was used as an index with a standard of 10 ng DNA as 50 copies of EBV in one Raji cell (26). The Raji DNA
samples were diluted 10-fold from 107 to 102 copies for a standard curve.
DNA (500 ng) was used in the first round of PCR for EBV copies using the
following primers: forward (5′-TCA AAA CTT TAG AGG CGA ATG G-3′)
and reverse (5′-CTG AGA AGG TGG CCT AGC AAC-3′). The second round
of PCR was performed using another pair of primers: forward (5′-CCA GGA
AGC GGG TCT ATG G-3′), reverse (5′-GGC TTA TTC CTC TTT TCC CCT
CTA-3′) and the special TaqMan probe (FAM-TGG CTG CGC TGC TGC TAT
C-TAMRA). The RT–qPCR assay with a fluorogenic probe was performed
using the TaqMan PCR kit and a Model 7700 Sequence Detector (Applied
Biosystems, Foster City, CA).
Flow cytometer analysis
To detect EBV lytic proteins, cells were washed with PBS, followed by fixing
with 4% paraformaldehyde for 30 min and finally treated for 5 min with PBS
containing 0.1% Triton X-100. Cells were then washed with PBS, treated with
Assay of ERKs and Akt kinase activities
ERKs or Akt kinase activity was estimated using an ERKs kinase assay
kit or an Akt kinase assay kit according to the manufacturer’s instructions
(Cell Signaling Technology). In brief, cells were washed once with ice-cold
PBS and disrupted with lysis buffer. ERK or Akt was immunoprecipitated
using a specific ERKs or Akt-immobilized antibody bead slurry with gentle
rocking overnight at 4°C. The cell lysate/immobilized antibody complex was
centrifuged at 13 000 r.p.m. for 30 s at 4°C and the pellet washed two times with
500 µl of 1× cell lysis buffer, and then twice with 500 µl of 1× kinase buffer.
The kinase reactions were carried out in the presence of 200 µM adenosine
triphosphate at 30°C for 30 min using 2 µg of fusion protein Elk-1 or GSK-3 as
a specific substrate for ERKs or Akt, respectively. The phosphorylated proteins
were detected by immunoblotting using a specific phospho-Elk-1 or phosphoGSK-3α/β (Ser21/9) antibody.
Statistical analysis
All statistical calculations were performed with the statistical software program SPSS (v. 12.0). Differences between various groups were evaluated by
the Student’s t-test, and a P value < 0.05 was considered statistically significant.
Results
Detection of EBV lytic proteins and EBV DNA copies in cell lines and
biopsies
EBV latency-associated gene expression is consistently detected in
NPC and lymphoma cells and is, therefore, regarded as one of the factors in the oncogenesis of these malignancies. However, various lines
of evidence indicate that lytic infection of EBV can also occur in NPC
tumor cells and lymphomas (27,28). B95.8 cell line is a well-known
EBV-positive cell line, which is permissive for virus lytic replication.
In this study, B95.8 cells were used as the cell model and also as a
positive control. To determine the EBV presence and lytic infection
in NPC cell model, we used RT–PCR to quantify EBV DNA load
and immunoblotting and immunofluorescence to analyze the EBVassociated lytic proteins.
First, the EBV latent and lytic proteins were examined using monoclonal antibodies. Immunoblot analysis revealed a constitutive expression of EBV latent proteins, EBNA1 and LMP1, and lytic infection gene
products, BZLF1 and BMRF1 (Figure 1A). The expression level of
EBV proteins, LMP1, EBNA1, BZLF1 and BMRF1, in B95.8 cells was
more notable than in CNE1-LMP1 cells. Immunofluorescence staining
Fig. 3. EGCG reduces the viability of CNE1-LMP1 and B95.8 cells in time- and dose-dependent manners. After starving with 0.1% FBS, cells were treated with
EGCG, and then, cell viability was measured by MTT assay. In each treatment group, cells treated with medium serve as a control and their viability was set as
100%. Values are expressed as means ± SD of three experiments (n = 3).
630
Downloaded from http://carcin.oxfordjournals.org/ by guest on May 26, 2016
RT–PCR and qPCR
Total RNA from cultured cells was isolated using TRIzol following the manufacturer’s instructions (Invitrogen). Complementary DNA was synthesized using
1–5 µg of purified RNA, the RevertAid™ First Strand cDNA Synthesis Kit
(Fermentas, EU) and Oligo(dT)18. Quantitative PCR (qPCR) was performed using
a Rotor-Gene 6000 real-time (RT)–PCR instrument (Corbett Research, Sydney,
Australia) and SYBR Premix Ex Taq™ II (TaKaRa, Dalian, China) and 2 µl of complementary DNA with the following primers: BZLF1, 5′-CATGTTTCAACCG
CTCCGACTGG-3′ (sense) and 5′-GCGCAGCCTGTCATTTTCAGATG-3′
(antisense); BMRF1, 5′-CTAGCCGTCCTGTCCAAGTGC-3′ (sense) and
5′-AGCCAAACGCTCCTTGCCCA-3′ (antisense) (25); and β-actin,
5′-TTCCAGCCTTCCTTCCTGGG-3′ (sense) and 5′-TTGCGCTCAGGA
GGAGCAAT-3′ (antisense). Relative mRNA abundance was calculated using
β-actin as an internal control using the 2ΔΔCT method. For each experiment,
mRNA levels from untreated cells were used as controls and set to 1. The mRNA
changes are represented relative to untreated cells.
1% bovine serum albumin in PBS for 1 h and incubated with 1:200-diluted
monoclonal anti-BZLF1 or monoclonal anti-BMRF1 at 4°C overnight. Next,
the cells were washed with PBS and incubated with 1:200-diluted fluorescein
isothiocyanate-conjugated goat anti-mouse IgG (Santa Cruz) for 1 h at 37°C.
Incubation with PBS, but no primary antibody, and then with secondary antibody was used as a negative control. Finally, cells were resuspended in 1%
paraformaldehyde and analyzed on a FACSCalibur flow cytometer using the
CellQuest software (BD, San Diego, CA).
EGCG inhibits EBV lytic infection
Downloaded from http://carcin.oxfordjournals.org/ by guest on May 26, 2016
631
S.Liu et al.
revealed that the CNE1-LMP1 cells constitutively expressed the BZLF1
and BMRF1 proteins (Figure 1B). For quantification of EBV copy
number, a RT–PCR assay targeting the BamHI-W segment of the EBV
genome was developed. The BamHI-W assay has been reported to be
more sensitive and suitable than EBER for applications in EBV detection, presumably because it targets a reiterated sequence that is present at
approximately 10 copies per EBV genome (29). We used the DNA from
Raji cells that contain 50 copies per cell (30). A 10-fold serial dilution of
the reference was used as a standard of the viral DNA number. The estimation of viral genomes in each sample was expressed as the number of
viral genomes copy per 50 ng of purified DNA. Experiments were performed at least twice to improve reliability of the results. We observed
that EBV DNA was present in both CNE1-LMP1 cells and B95.8 cells,
and the copies of EBV DNA in CNE1-LMP1 cells (1.35 × 103 copies/50 ng DNA) were significantly lower than in B95.8 cells (4.82 × 106
copies/50 ng DNA) (P < 0.01) (Figure 1C).
To determine whether EBV lytic protein expression also existed in
vivo, immunohistochemistry to detect BZLF1 and BMRF1 was performed on formalin-fixed, paraffin-embedded tissue from NPC and
lymphomas. Positive signal implies that BZLF1 or BMRF1 expression occurs in tumor cells as shown by nuclear localization in different cases. We observed BZLF1- and BMRF1-positive cells within
the NPC and lymphoma tissues. We detected BZLF1 expression in 9
(27.3%) of the 33 NPC patients and in 7 (29.17%) of 24 lymphoma
patients and observed BMRF1 expression in 2 (6.06%) of the 33
NPC patients and in 1 (4.16%) of the 24 lymphoma patients studied
(Figure 2).
The inhibitory effect of EGCG on EBV spontaneous lytic infection
The viability of CNE1-LMP1 cells and B95.8 cells after treatment
with EGCG was assessed by MTT assay. EGCG exhibited dose- and
time-dependent inhibitory effects on these cells with an IC50 of 20 µM
(Figure 3). Thus, the concentrations of EGCG (5–20 µM) were the
doses chosen for subsequent experiments.
To characterize the effect of EGCG on EBV spontaneous lytic
infection in cells, a transient transfection assay was performed using
632
a BZLF1 and BMRF1 promoter/luciferase reporter system. Results
showed that EGCG effectively suppressed the transcription activity of
the BZLF1 and BMRF1 genes in both CNE1-LMP1 and B95.8 cells
in a dose-dependent manner (Figure 4A). The effect of EGCG on the
mRNA level of EBV spontaneous lytic genes was measured using
RT–qPCR. In accordance with the results from transfection assay, the
mRNA expression levels of the lytic genes, BZLF1 and BMRF1, were
also strongly reduced after EGCG treatment (Figure 4B).
To confirm the mRNA results, the inhibitory effect of EGCG on
EBV lytic protein expression was assessed by immunoblot analysis, which was performed using monoclonal anti-BZLF1 and antiBMRF1. Upon treatment with different concentrations of EGCG,
expression of both BZLF1 and BMRF1 was substantially decreased
in a dose-dependent manner (Figure 4C). Moreover, flow cytometry
analysis of EGCG-treated cells revealed that following treatment with
EGCG (20 µM), the percentage of the cell population expressing
BZLF1 and BMRF1 decreased from 73.69% and 15.57% to 27.39%
and 2.89%, respectively, in CNE1-LMP1 cells, and from 98.54% and
68.99% to 78.98% and 40.27% in B95.8 cells (Figure 4D).
Further, we examined whether EGCG suppresses lytic replication
of the viral load. EBV DNA copies were quantified using a RT–PCR
method. Results indicated that the copy number of EBV DNA in
treated CNE1-LMP1 cells was significantly reduced by EGCG in a
dose-dependent manner. However, our data showed that EGCG had
no statistically significant effect on the copy number of EBV DNA
in treated B95.8 cells (Figure 4E). These results indicate that EGCG
effectively inhibits the constitutive lytic infection of EBV in CNE1LMP1 and B95.8 cells.
Involvement of the MEK/ERK1/2 and PI3-K/Akt pathways in
EGCG’s inhibition of EBV lytic infection
The MEK/ERK1/2 and PI3-K/Akt signaling pathways were reported
to play certain roles in EBV lytic replication, and EGCG was shown
to suppress the activities of these pathways (18). Thus, we hypothesized that EGCG inhibits EBV spontaneous lytic infection by suppressing these signaling pathways. First, using phospho-specific
Downloaded from http://carcin.oxfordjournals.org/ by guest on May 26, 2016
Fig. 4. Inhibitory effects of EGCG on EBV spontaneous lytic infection in CNE1-LMP1 and B95.8 cells. Cells were starved in 0.1% FBS RPMI medium 1640
for 24 h and then cultured with different concentrations of EGCG for 24–48 h. (A) Effects of EGCG on the transactivation activity of BZLF1 and BMRF1 were
determined using transient transfection and luciferase reporter assays. The relative luciferase activity was normalized to the value of the internal control plasmid
pRL-SV40 activity. (B) RT–PCR quantification of mRNA expression levels for the BZLF1 and BMRF1 genes. The mRNA level in untreated cells was set to
1. The data indicated significant decreases in EGCG-treated EBV lytic gene mRNAs. (C) Cells were disrupted and immunoblot analysis was performed to
determine the expression of BZLF1 and BMRF1. β-Actin was detected as a control to verify equal protein loading. (D) Effects of EGCG on the expression of
BZLF1 and BMRF1 were determined using flow cytometry analysis. Incubation with PBS but no primary antibody and then with the secondary antibody was
used as a negative control. (E) The effect of EGCG on EBV DNA replication was assessed using RT–PCR, with specific primers designed to amplify the EBV W
fragment. For each experiment, BamHI-W fragment levels from untreated cells were used as a control and set to 1. The mRNA changes are represented relative
to untreated cells. Statistical significance:*P < 0.05 versus untreated (EGCG 0 µM).
EGCG inhibits EBV lytic infection
antibodies against ERK1/2 and Akt, we found that the phosphorylation of these kinases was suppressed by EGCG (Figure 5A). ERKs or
Akt kinase activity was estimated using an ERK kinase assay kit or an
Akt kinase assay kit. ERK or Akt was first immunoprecipitated from
cell extracts and then incubated with their specific substrate, Elk-1
or GSK-3 fusion protein, respectively, in the presence of adenosine
triphosphate and kinase buffer. ERKs- or Akt-dependent Elk-1 or
GSK-3 phosphorylation was then measured by immunoblotting using
a phospho-antibody that recognizes Elk-1 or GSK-3 when phosphorylated. The kinase assays further confirmed that the activation of
ERK1/2 and Akt was inhibited by EGCG in a dose-dependent manner
(Figure 5B). These data indicate that EGCG suppresses the activation
of the ERK1/2 and Akt signaling pathways.
To determine if the ability of EGCG to inhibit the ERK1/2 and PI3-K/
Akt pathways plays a role in the attenuation of EBV lytic infection,
a pharmacological approach was adopted using each kinase-specific
inhibitor. PD98059, a specific inhibitor of MEK1 that acts by inhibiting activation of ERK1/2, and LY294002, a specific inhibitor of PI3-K
that acts by inhibiting activation of Akt, were tested for their effects on
EBV lytic infection. Our results showed that treatment of cells with
each of these inhibitors dramatically decreased the phosphorylation
levels of ERK1/2 and Akt. In addition, these inhibitors suppressed
the expression of the EBV lytic proteins in a dose-dependent manner
(Figure 6A and B). A transient transfection assay performed using a
BZLF1 and BMRF1 promoter/luciferase reporter system showed that
the inhibitors each effectively and significantly suppressed the transcription activity of the BZLF1 and BMRF1 genes in our cell models
in a dose-dependent manner (Figure 6C and D, P < 0.05).
Next, to determine the inhibitory effect of the inhibitors on EBV
lytic gene expression due to the suppressive effects on the genes at
the mRNA level, we performed qPCR to estimate BZLF1 and BMRF1
mRNA expression after PD98059 or LY294002 treatment. Consistent
with the data from the transfection assay and immunoblot analysis, the
qPCR results showed that the inhibitors each effectively and significantly suppressed the mRNA expression of the BZLF1 and BMRF1
genes in a dose-dependent manner (Figure 6E and F, P < 0.05).
Overall, these results indicated that the MEK/ERK1/2 and
PI3-K/Akt signaling pathways are involved in EGCG inhibition of
EBV spontaneous lytic infection in both CNE1-LMP1 and B95.8
cells.
Discussion
The life cycle of EBV includes latent and lytic stages. In most asymptomatic carriers of EBV, the cells containing latent viral genomes are
periodically reactivated to the lytic cycle. Previous studies focused on
the effects of EBV latent infection and revealed that viral LMP1 is an
oncoprotein (19). But much of the literature points to the fact that EBV
633
Downloaded from http://carcin.oxfordjournals.org/ by guest on May 26, 2016
Fig. 5. Inhibitory effects of EGCG on activation of ERK1/2 and Akt in CNE1-LMP1 and B95.8 cells. (A) Effect of EGCG on phosphorylation of ERK1/2
and Akt. The cells were starved in 0.1% FBS RPMI medium 1640 for 24 h and then cultured with different concentrations of EGCG for 24 h. Cells were then
disrupted and immunoblot analysis was performed. (B) In vitro kinase assays of ERK1/2 and Akt were used to evaluate the effects of EGCG. Cells were starved
in 0.1% FBS RPMI medium 1640 for 24 h and then cultured with different concentrations of PD98059 and LY294002 for 24 h. Cells were then disrupted and
subjected to immunoblot analysis as described in Materials and methods. To ensure that equal amounts of ERK or Akt were immunoprecipitated, we also
immunoblotted the precipitate for total ERK or Akt.
S.Liu et al.
Downloaded from http://carcin.oxfordjournals.org/ by guest on May 26, 2016
634
EGCG inhibits EBV lytic infection
spontaneous lytic replication takes place in various EBV-associated
tumors (8–10). Our results also indicated that the EBV spontaneous
lytic infection exists in NPC and lymphoma cells (Figure 1).
Although the lytic protein BZLF1 can induce the viral replication
cycle and subsequent cell death, accumulating evidence indicates that
expression of BZLF1 does not always result in virus production and
cell death. The abortive lytic infection occurs not only in NPC but
also in Hodgkin’s disease, non-Hodgkin lymphoma in an immunocompromised host and EBV-associated gastric cancer (28,31–33).
Our results also showed that the basal level of early gene BMRF1
in gene transcription and protein expression was much lower than
the immediate-early gene BZLF1 (Figures 1A and 4). In addition,
the results from immunohistochemistry showed that the percentage
of BZLF1-positive cells in NPC and lymphoma biopsies was much
larger than the percentage of BMRF1-positive cells (Figure 2). The
findings of our study imply that abortive lytic infection may occur in
NPC and lymphoma.
Viral lytic genes, such as BZLF1, BMRF1, or cellular genes induced
by viral lytic proteins could potentially encode paracrine factors that
promote tumor growth. BZLF1 has been reported as having various
malignancy-promoting actions (34). BZLF1 has the potential to create the best cellular environment for EBV replication by suppressing
the cell-mediated immunity of the host (35,36), regulating the expression of the cellular proteins and the activities of signaling pathways
(34,37,38), manipulating the cell cycle (38,39) and interacting with
host proteins physically (40,41).
NPC is known to be closely associated with EBV infection. In
addition to the importance of EBV latent infection in carcinogenesis,
several retrospective and prospective investigations suggest that EBV
lytic infection might also play a substantial role in pathogenesis.
Elevated antibody titers against EBV lytic antigens, representing EBV
lytic infection in vivo, correspond with advanced cancer stages, poor
prognosis or tumor recurrence of NPC (6,7). The serologic marker
of EBV reactivation into lytic infection also serves as a risk factor of
NPC (42). In addition, the presence of lytic viral genome and lytic
gene products, such as BZLF1, further supports the notion that EBV
replications do occur in some cases (43). The presence of lytic viral
genome and lytic gene products, such as BZLF1, also occurs in lymphomas (27). EBV reactivation into lytic EBV gene expression contributes to EBV-associated lymphoproliferative disease, potentially
through induction of paracrine B-cell growth factors. BZLF1-induced
IL-13 production facilitates B-cell proliferation and may contribute
to the pathogenesis of EBV-associated lymphoproliferative disorders, such as post-transplantation lymphoproliferative disease and
Hodgkin’s lymphoma (44). A recent study showed the ability of methotrexate (MTX), which is used in patients with rheumatoid arthritis and
polymyositis, to increase the level of lytic infectious EBV in patients.
This, combined with its potent immunosuppressive effects, may collaborate to induce EBV-positive lymphomas (45). Our data confirm
that EBV lytic infection proteins are present not only in NPC and
lymphoma cell lines but also in patient biopsies, consistent with the
previous studies.
The above evidence has challenged the traditional concept that
EBV only transforms cells in its latent infection and has also broadened our knowledge regarding the mechanism of EBV-associated carcinogenesis. By increasing the horizontal transmission of the virus
635
Downloaded from http://carcin.oxfordjournals.org/ by guest on May 26, 2016
Fig. 6. Inhibitory effects of PD98059 and LY294002 on EBV spontaneous lytic infection in CNE1-LMP1 and B95.8 cells. Cells were starved in 0.1% FBS
RPMI medium 1640 and then cultured with different concentrations of PD98059 or LY294002 for 24 h. (A and B) Cells were then disrupted and immunoblot
analysis was performed to determine the expression of total and phosphorylated kinases and lytic proteins. β-Actin was used to verify equal protein loading.
(C and D) Effects of PD98059 and LY294002 on the transactivation activity of BZLF1 and BMRF1 were determined using transient transfection and luciferase
reporter assays. The relative luciferase activity was normalized to the value of the internal control plasmid pRL-SV40 activity. (E and F) RT–PCR quantification
of mRNA expression levels for the BZLF1 and BMRF1 genes. The mRNA level in vehicle cells was set to 1. The data indicate significant decrease in PD98059or LY294002-treated EBV lytic gene mRNAs. Control treatments were with vehicle (DMSO). Statistical significance:*P < 0.05 versus DMSO.
S.Liu et al.
636
In summary, this study presented novel experimental evidence
describing the mechanisms underlying the inhibition of EBV spontaneous lytic replication by EGCG in both epithelial NPC and B-cell
lymphoma models. Our findings support the future investigation
of EGCG as an anticancer and chemoprevention agent for EBVassociated malignancies. Further study regarding the inhibitory effect
of EGCG on EBV lytic infection in vivo is needed.‍
Funding
National Nature Science Foundation of China (No. 81101474,
81072220 and 81161120410); State Key Basic Research and
Development Plan (973) of the Ministry of Science and Technology
of China (No. 2011CB504300); the Joint Research Fund of National
Nature Science Foundation (No. R37CA081064).
Acknowledgements
We thank Weiwei Wang, Faqing Tang from Xiangya Hospital of Central South
University and Liang Zeng from Hunan Tumor Hospital for technical support;
Ming Li from the Basic Medical School of the Central South University for
helpful comments. We thank Dr Ichiro Saito for the gift of the pZp-Luc plasmid and Dr Paul J.Farrell for the gift of the pMp-Luc plasmid. We also thank
Dr Ann M.Bode from The Hormel Institute of University of Minnesota for
critical reading of this manuscript.
Conflict of Interest Statement: None declared.
References
1.Pathmanathan,R. et al. (1995) Clonal proliferations of cells infected with
Epstein-Barr virus in preinvasive lesions related to nasopharyngeal carcinoma. N. Engl. J. Med., 333, 693–698.
2.Young,L.S. et al. (1992) Epstein-Barr virus, lymphomas and Hodgkin’s
disease. Semin. Cancer Biol., 3, 273–284.
3.Sugawara,Y. et al. (2000) Detection of Epstein-Barr virus DNA in hepatocellular carcinoma tissues from hepatitis C-positive patients. Scand.
J. Gastroenterol., 35, 981–984.
4.Kutok,J.L. et al. (2006) Spectrum of Epstein-Barr virus-associated diseases. Annu. Rev. Pathol., 1, 375–404.
5.Rickinson,A.B. et al. (1996) Epstein-Barr virus. In Fields,B.N., Knipe,
D.M. and Howley,P.M. (eds) Fields Virology. 3rd edn. Lippincott-Raven
Publishers, Philadelphia, PA, pp. 2397–2446.
6.Lei,K.I. et al. (2000) Quantitative analysis of circulating cell-free EpsteinBarr virus (EBV) DNA levels in patients with EBV-associated lymphoid
malignancies. Br. J. Haematol., 111, 239–246.
7.Lo,Y.M. (2001) Quantitative analysis of Epstein-Barr virus DNA in plasma
and serum: applications to tumor detection and monitoring. Ann. N. Y.
Acad. Sci., 945, 68–72.
8.Geser,A. et al. (1982) Final case reporting from the Ugandan prospective study of the relationship between EBV and Burkitt’s lymphoma. Int.
J. Cancer, 29, 397–400.
9.Mueller,N. et al. (1989) Hodgkin’s disease and Epstein-Barr virus. Altered
antibody pattern before diagnosis. N. Engl. J. Med., 320, 689–695.
10. Boos,H. et al. (1987) Enhancement of Epstein-Barr virus membrane protein (LMP) expression by serum, TPA, or n-butyrate in latently infected
Raji cells. Virology, 159, 161–165.
11. Liu,S.F. et al. (2008) Aspirin induces lytic cytotoxicity in Epstein-Barr
virus-positive cells. Eur. J. Pharmacol., 589, 8–13.
12. Israel,B.F. et al. (2003) Virally targeted therapies for EBV-associated
malignancies. Oncogene, 22, 5122–5130.
13. Satoh,T. et al. (1999) The interaction of mitogen-activated protein
kinases to Epstein-Barr virus activation in Akata cells. Virus Genes, 18,
57–64.
14. Iwakiri,D. et al. (2004) Phosphatidylinositol 3-kinase is a determinant of
responsiveness to B cell antigen receptor-mediated Epstein-Barr virus activation. J. Immunol., 172, 1561–1566.
15. Darr,C.D. et al. (2001) Epstein-Barr virus immediate-early protein BRLF1
induces the lytic form of viral replication through a mechanism involving
phosphatidylinositol-3 kinase activation. J. Virol., 75, 6135–6142.
16.
Ramos,S. (2008) Cancer chemoprevention and chemotherapy: dietary polyphenols and signalling pathways. Mol. Nutr. Food Res., 52,
507–526.
Downloaded from http://carcin.oxfordjournals.org/ by guest on May 26, 2016
from cell to cell, EBV lytic infection might increase the total number of latently infected cells. In addition, important implications of
EBV lytic infection might include the idea that the infected host cells
or nearby cells are transformed, or that the biologic characteristics
of these cells could be changed and the degree of malignancy might
increase along with the ease with which metastasis can occur. Thus, if
EBV lytic infection is inhibited, the potential of the malignancy in the
infected cells might be suppressed or reversed.
Cancer chemoprevention by the natural compound, EGCG, has
been studied by many investigators and has received much attention
in recent years. The inhibitory effects of EGCG against carcinogenesis at different organ sites have been demonstrated in many animal
models (16,46). Several studies have shown that EGCG can inhibit
EBV lytic infection, but the mechanism involved in the control of
EBV gene expression by EGCG is not well understood. Choi et al.
(47) found that EBV-induced B-cell outgrowth and B lymphocyte
transformation was dramatically inhibited by treatment with EGCG
(50 µM), supporting the important role of acetylation in cancer initiation and progression. Taniguchi et al. (48) and Chang et al. (49) demonstrated that EGCG blocks EBV lytic cycle induced by chemicals in
Burkitt’ lymphoma cell lines. However, in recent years, considerable
progress has been made in the mechanisms of cancer chemoprevention by EGCG. In our studies, we used the NPC cell line (CNE1LMP1) and lymphoblastoid cell line (B95.8) with EBV spontaneous
lytic replication that mimics the natural state of the infected cells. We
found that EGCG inhibits EBV spontaneous lytic replication without
any inducer and involving the inhibition of the activation of MEK/
ERK1/2 and PI3-K/Akt signaling.
EGCG exerts its anticancer effect by modulating a wide spectrum
of molecular targets, thus inhibiting tumor development and progression. Located at the cell membrane, EGCG suppresses the activation
of multiple receptor tyrosine kinases, including epidermal growth
factor receptor, insulin-like growth factor-I receptor and vascular
endothelial growth factor receptor. It also inhibits downstream cytoplasmic signaling molecules, such as Akt and ERK1/2, ultimately
leading to the abnormities of certain transcription factors, including
nuclear factor-kappaB and activator protein (AP-1) (46,50,51). The
BZLF1 gene promoter contains the ZII element, an important positive regulator of Z transcription (52), which has been shown to bind
with multiple transcription factors, including CREB, ATF1, ATF2
and AP-1. c-Jun phosphorylation, the main component of AP-1, activates Z transcription through the ZII element (37). We also previously
reported that EGCG inhibited the phosphorylation of c-Jun (17,53).
Thus, EGCG may disrupt EBV lytic infection-related signaling pathways by directly or indirectly binding to receptors or other target proteins, such as Akt or ERK1/2, and induce the transcription repression
of BZLF1 by inhibiting the activation of transcription factor AP-1,
thus contributing to EBV lytic infection.
The concept of targeted biological therapy against cancer has
emerged over the past decade. Clinical trials studying the efficacy
and tolerability of various targeted agents have shown that most
tumors depend on more than one signaling pathway for their continued growth and survival. Therefore, investigators pursue different
strategies to inhibit multiple signaling pathways by developing multitargeted agents (54). Large amounts of encouraging data from in vitro
and animal models have emerged suggesting that green tea is nature’s
gift molecule endowed with anticancer effects. Epidemiological and
geographical observations suggest that these laboratory data might be
applicable in the human population. The various effects of green tea
polyphenols and especially EGCG clearly seem to have the ability to
be utilized in the multi-targeted therapy of cancer (55).
During the course of studying the effects of EGCG on regulating
different key proteins in various signaling pathways, the direct interaction of proteins with EGCG was found to be a key step in the process
of EGCG exerting its effects. The identification of proteins interacting
directly with EGCG is important in understanding the molecular mechanisms of the effects of EGCG. Several proteins that can directly bind
with EGCG have been identified and include laminin, GRP-78, insulinlike growth factor-I receptor, G3BP1, ZAP-70 and vimentin (56).
EGCG inhibits EBV lytic infection
37.Adamson,A.L. et al. (2000) Epstein-Barr virus immediate-early proteins BZLF1
and BRLF1 activate the ATF2 transcription factor by increasing the levels of
phosphorylated p38 and c-Jun N-terminal kinases. J. Virol., 74, 1224–1233.
38. Cayrol,C. et al. (1996) The Epstein-Barr virus bZIP transcription factor
Zta causes G0/G1 cell cycle arrest through induction of cyclin-dependent
kinase inhibitors. EMBO J., 15, 2748–2759.
39.Mauser,A. et al. (2002) The Epstein-Barr virus immediate-early protein BZLF1 induces both a G(2) and a mitotic block. J. Virol., 76,
10030–10037.
40. Zhang,Q. et al. (1994) Functional and physical interaction between p53
and BZLF1: implications for Epstein-Barr virus latency. Mol. Cell. Biol.,
14, 1929–1938.
41. Adamson,A.L. et al. (1999) The Epstein-Barr virus BZLF1 protein interacts physically and functionally with the histone acetylase CREB-binding
protein. J. Virol., 73, 6551–6558.
42. Chien,Y.C. et al. (2001) Serologic markers of Epstein-Barr virus infection
and nasopharyngeal carcinoma in Taiwanese men. N. Engl. J. Med., 345,
1877–1882.
43. Cochet,C. et al. (1993) Expression of the Epstein-Barr virus immediate
early gene, BZLF1, in nasopharyngeal carcinoma tumor cells. Virology,
197, 358–365.
44. Tsai,S.C. et al. (2009) EBV Zta protein induces the expression of interleukin-13, promoting the proliferation of EBV-infected B cells and lymphoblastoid cell lines. Blood, 114, 109–118.
45. Feng,W.H. et al. (2004) Reactivation of latent Epstein-Barr virus by methotrexate: a potential contributor to methotrexate-associated lymphomas. J.
Natl. Cancer Inst., 96, 1691–1702.
46. Kim,J.W. et al. (2010) Chemoprevention of head and neck cancer with
green tea polyphenols. Cancer Prev. Res. (Phila)., 3, 900–909.
47. Choi,K.C. et al. (2009) Epigallocatechin-3-gallate, a histone acetyltransferase inhibitor, inhibits EBV-induced B lymphocyte transformation via
suppression of RelA acetylation. Cancer Res., 69, 583–592.
48. Taniguchi,S. et al. (2002) Production of bioactive triterpenes by Eriobotrya
japonica calli. Phytochemistry, 59, 315–323.
49.Chang,L.K. et al. (2003) Inhibition of Epstein-Barr virus lytic cycle by (-)-epigallocatechin gallate. Biochem. Biophys. Res. Commun., 301, 1062–1068.
50. Yang,C.S. et al. (2011) Cancer prevention by tea: evidence from laboratory
studies. Pharmacol Res.
51. Yang,C.S. et al. (2009) Cancer prevention by tea: animal studies, molecular
mechanisms and human relevance. Nat. Rev. Cancer, 9, 429–439.
52. Flemington,E. et al. (1990) Identification of phorbol ester response elements in the promoter of Epstein-Barr virus putative lytic switch gene
BZLF1. J. Virol., 64, 1217–1226.
53. Barthelman,M. et al. (1998) (-)-Epigallocatechin-3-gallate inhibition of
ultraviolet B-induced AP-1 activity. Carcinogenesis, 19, 2201–2204.
54. Petrelli,A. et al. (2008) From single- to multi-target drugs in cancer
therapy: when aspecificity becomes an advantage. Curr. Med. Chem., 15,
422–432.
55. Khan,N. et al. (2008) Multitargeted therapy of cancer by green tea polyphenols. Cancer Lett., 269, 269–280.
56. Bode,A.M. et al. (2009) Epigallocatechin 3-gallate and green tea catechins: united they work, divided they fail. Cancer Prev. Res. (Phila)., 2,
514–517.
Received March 22, 2012; revised October 8, 2012; accepted November 8, 2012
637
Downloaded from http://carcin.oxfordjournals.org/ by guest on May 26, 2016
17. Zhao,Y. et al. (2004) Epigallocatechin-3-gallate interferes with EBVencoding AP-1 signal transduction pathway. Zhonghua Zhong Liu Za Zhi,
26, 393–397.
18. Kanwar,J. et al. (2012) Recent advances on tea polyphenols. Front. Biosci.
(Elite Ed)., 4, 111–131.
19. Zheng,H. et al. (2007) Role of Epstein-Barr virus encoded latent membrane protein 1 in the carcinogenesis of nasopharyngeal carcinoma. Cell.
Mol. Immunol., 4, 185–196.
20. Yang,L. et al. (2009) Effect of DNAzymes targeting Akt1 on cell proliferation and apoptosis in nasopharyngeal carcinoma. Cancer Biol. Ther., 8,
366–371.
21. Ding,L. et al. (2005) Epstein-Barr virus encoded latent membrane protein 1
modulates nuclear translocation of telomerase reverse transcriptase protein
by activating nuclear factor-kappaB p65 in human nasopharyngeal carcinoma cells. Int. J. Biochem. Cell Biol., 37, 1881–1889.
22. Liu,S.F. et al. (2008) NF-kappaB inhibitors induce lytic cytotoxicity in
Epstein-Barr virus-positive nasopharyngeal carcinoma cells. Cell Biol. Int.,
32, 1006–1013.
23. Nagata,Y. et al. (2004) Activation of Epstein-Barr virus by saliva from
Sjogren’s syndrome patients. Immunology, 111, 223–229.
24. Amon,W. et al. (2004) Lytic cycle gene regulation of Epstein-Barr virus. J.
Virol., 78, 13460–13469.
25. Taylor,G.M. et al. (2011) Endoplasmic reticulum stress causes EBV lytic
replication. Blood, 118, 5528–5539.
26. Krumbholz,A. et al. (2006) Comparison of a LightCycler-based real-time
PCR for quantitation of Epstein-Barr viral load in different clinical specimens with semiquantitative PCR. J. Med. Virol., 78, 598–607.
27. Labrecque,L.G. et al. (1999) Expression of Epstein-Barr virus lytically
related genes in African Burkitt’s lymphoma: correlation with patient
response to therapy. Int. J. Cancer, 81, 6–11.
28. Martel-Renoir,D. et al. (1995) Qualitative analysis of the expression of
Epstein-Barr virus lytic genes in nasopharyngeal carcinoma biopsies. J.
Gen. Virol., 76 (Pt 6), 1401–1408.
29. Ryan,J.L. et al. (2004) Epstein-Barr virus quantitation by real-time PCR
targeting multiple gene segments: a novel approach to screen for the virus
in paraffin-embedded tissue and plasma. J. Mol. Diagn., 6, 378–385.
30. Aritaki,K. et al. (2001) A rapid monitoring system of human herpesviruses
reactivation by LightCycler in stem cell transplantation. Bone Marrow
Transplant., 28, 975–980.
31. Niedobitek,G. et al. (1996) Epstein-Barr virus infection and the pathogenesis of nasopharyngeal carcinoma: viral gene expression, tumour cell phenotype, and the role of the lymphoid stroma. Semin. Cancer Biol., 7, 165–174.
32. Gutierrez,M.I. et al. (1993) Replicative viral DNA in Epstein-Barr virus
associated Burkitt’s lymphoma biopsies. Leuk. Res., 17, 285–289.
33. Pallesen,G. et al. (1991) Activation of Epstein-Barr virus replication in
Hodgkin and Reed-Sternberg cells. Blood, 78, 1162–1165.
34. Chen,C. et al. (2009) Regulation of cellular and viral protein expression
by the Epstein-Barr virus transcriptional regulator Zta: implications for
therapy of EBV associated tumors. Cancer Biol. Ther., 8, 987–995.
35. Keating,S. et al. (2002) The lytic cycle of Epstein-Barr virus is associated
with decreased expression of cell surface major histocompatibility complex class I and class II molecules. J. Virol., 76, 8179–8188.
36. Hahn,A.M. et al. (2005) Interferon regulatory factor 7 is negatively regulated by the Epstein-Barr virus immediate-early gene, BZLF-1. J. Virol.,
79, 10040–10052.