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Journal of Virology, December 1999, p. 9827-9831, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Oncogenic Role of Epstein-Barr Virus-Encoded RNAs
in Burkitt's Lymphoma Cell Line Akata
Jun
Komano,
Seiji
Maruo,
Koichi
Kurozumi,
Takanori
Oda, and
Kenzo
Takada*
Department of Virology, Cancer Institute,
Hokkaido University School of Medicine, Kita-ku, Sapporo 060-8638, Japan
Received 15 July 1999/Accepted 1 September 1999
 |
ABSTRACT |
Our previous reports indicated that Epstein-Barr virus (EBV)
contributes to the malignant phenotype and resistance to apoptosis in
Burkitt's lymphoma (BL) cell line Akata (N. Shimizu, A. Tanabe-Tochikura, Y. Kuroiwa, and K. Takada, J. Virol.
68:6069-6073, 1994; J. Komano, M. Sugiura, and K. Takada, J. Virol. 72:9150-9156, 1998). Here we report that the EBV-encoded small
RNAs (EBERs) are responsible for these phenotypes. Transfection of the
EBER genes into EBV-negative Akata clones restored the capacity for
growth in soft agar, tumorigenicity in SCID mice, resistance to
apoptotic inducers, and upregulated expression of bcl-2 oncoprotein
that were originally retained in parental EBV-positive Akata cells and
lost in EBV-negative subclones. This is the first report which provides
evidence that virus-encoded RNAs (EBERs) have oncogenic functions in BL cells.
| |
INTRODUCTION |
Epstein-Barr virus (EBV) was
demonstrated to be present in more than 90% of endemic Burkitt's
lymphoma (BL) cases in equatorial Africa and New Guinea and in about
10% of sporadic BL cases in the western hemisphere (reviewed in
reference 23). The important feature of the BL cell
is the juxtaposition of the c-myc locus to the
immunoglobulin H enhancer through a reciprocal translocation [t(8;14)] and, more rarely, to the Ig
locus [t(2;8)] or the
Ig
locus [t(8;22)]. This feature led to the hypothesis that this translocation resulted in constitutive activation of the
c-myc gene, which seemed to be a key step in the initiation
of BL (12). However, the role of EBV in the genesis of BL is
still an open question. To gain insight into this problem, we have
investigated the pathogenic role of EBV in BL cell line Akata. The
Akata cell line is an EBV-positive BL cell line derived from a Japanese
patient and possessing chromosomal translocation t(8;14)
(29). It retains type I latency even in long-term cultures,
in which EBV nuclear antigen (EBNA) 1, EBV-encoded RNAs (EBERs,
specifically EBER-1 and EBER-2), latent membrane protein (LMP)
2A, and BARF0 are expressed (13). In most BL cell lines,
latency is converted from type I to type III in serial cultures, in
which cells express all the EBNAs (1, 2, 3a, 3b, 3c, and LP), LMPs 1, 2A, and 2B), EBERs, and BARF0 (reviewed in reference
1). EBV-immortalized B-cell lines (lymphoblastoid
cell lines) also have type III latency (24).
Hitherto, it has been impossible to demonstrate that the presence of
EBV with type I latency has any biological effects in BL cells. Akata
cells enabled us to resolve this problem. EBV-negative cell clones can
be isolated from parental Akata cells, since EBV DNA has been
spontaneously lost during cultivation (27). Type I latency
was restored by EBV reinfection in EBV-negative Akata cells. We
successfully established a system to confirm whether any phenotypic
differences in cells were due to EBV. This system compared two pairs of
cell clones: (i) EBV-positive and -negative cell clones obtained from
an EBV-positive Akata cell clone and (ii) neomycin resistance gene
(neoR)-transfected and EBV-reinfected cell clones obtained
from an EBV-negative Akata cell clone by use of recombinant EBV
carrying neoR. Using this system, we verified that, in Akata
cells, EBV was necessary for the malignant phenotype, resistance to
apoptosis, and upregulated expression of bcl-2 oncoprotein (13,
27). These were the first reports that provided direct evidence
that the presence of EBV had an oncogenic role and that type I latency
conferred resistance to apoptosis in vitro. We also demonstrated that
EBNA-1 was not responsible for these phenotypes in Akata cells
(13). Similar results were also reported by Ruf et al.
(25).
In this report, we focused upon the possible involvement of EBERs in
the malignant phenotype and resistance to apoptosis in Akata cells.
EBER-1 and EBER-2 are small nuclear RNAs transcribed by the RNA
polymerase III system; they are, respectively, 166 and 172 nucleotides
long (7). Being abundant, EBER-1 has often been targeted as
a probe for in situ hybridization studies to identify the presence of
EBV (5). With an EBER deletion recombinant virus, it was
demonstrated that EBERs were not necessary for the B-cell-immortalizing
function of EBV (28). To date, the biological functions of
EBERs remain unclear.
Here, we present evidence that EBERs play an oncogenic role in Akata
cells. This is the first report which provides evidence that
virus-encoded RNAs (EBERs) have oncogenic functions in BL cells.
| |
MATERIALS AND METHODS |
Cell culture.
The Akata cell line of BL origin was
maintained in RPMI 1640 medium (Sigma) supplemented with 10% fetal
bovine serum (FBS) (Gibco), 40 U of penicillin per ml, and 50 µg of
streptomycin per ml at 37°C in a 5% CO2 humidified atmosphere.
Plasmid construction.
neoR driven by the simian virus
40 promoter was derived from pcDNA3 (Invitrogen). EBER-1 and EBER-2
open reading frames (ORFs) are located at 6,628 to 6,796 bp and at
6,958 to 7,129 bp, respectively, on the EcoRI K fragment of
Akata EBV DNA, which corresponds to the EcoRI J fragment of
B95-8 EBV DNA. pEK contained the EcoRI K fragment of Akata
EBV DNA and neoR. pEKS10 contained 10 tandem repeats of the
SacI-EcoRI subfragment (6,297 to 7,325 bp) from the EcoRI K fragment of Akata EBV DNA and neoR.
At each end of the SacI-EcoRI subfragment from
the EcoRI K fragment, BglII or BamHI
sites were added and concatenated in a direction-restricted fashion.
Transfection and cloning.
Plasmids were introduced into
Akata cells by the electroporation method as described previously
(13). For cloning cells, 104 cells were
transferred to each well of a flat-bottom 96-well plate (Falcon) with
200 µl of medium containing 700 µg of G418 (Gibco) per ml 2 days
after transfection. Half of the medium was changed every 5 days until
colonies emerged.
EBV infection.
Preparation of the virus solution was
described previously (10). Cells (5 × 106)
were suspended in 2 ml of 1:2-diluted EBV solution (Akata strain with
the neoR gene) for 1 h at room temperature with
continuous mild mixing. Then, they were washed three times and cultured
for 2 days. Selection and cloning procedures are described above.
Northern blot analysis.
Total RNA was extracted from Akata
cells with Trizol reagent (Gibco) according to the manufacturer's
instructions. Samples (1 to 2 µg per lane) were electrophoresed in
2% agarose-formaldehyde gels, transferred to Hybond N+ nylon
membranes (Amersham), fixed with alkali, prehybridized, hybridized with
a probe, and washed under stringent conditions (15 mM NaCl, 1.5 mM
sodium citrate, 0.1% sodium dodecyl sulfate). The probe for EBERs was
an EcoRI K fragment, so that both EBER-1 and EBER-2
transcripts were detected. Fifty nanograms of DNA was radiolabeled with
a Ready-Prime labeling kit (Amersham).
RT-PCR.
Reverse transcriptase PCR (RT-PCR) was performed as
described previously (13). Briefly, total RNA was extracted
from Akata cells with Trizol reagent (Gibco). RNA was solubilized in
distilled water and treated with DNase I (Gibco). The primers used for
EBER-1 were AGG ACC TAC GCT GCC CTA GA (5' sequence) and AAA ACA TGC GGA CCA CCA GC (3' sequence); the product was 166 bp (30).
After reverse transcription, 100 ng of the cDNA was subjected to PCR in
the presence of 5 U of Taq polymerase (Gibco) in a total
volume of 50 ml with a model 2400 Thermal Cycler (Perkin-Elmer) for 12 cycles. The amplified products were electrophoresed in 2% agarose gels
and stained with ethidium bromide.
Soft-agar colony assay.
Wells of a six-well plate (Falcon)
were covered with 0.5% SeaPlaque agarose (FMC, Rockland, Maine)
containing RPMI 1640 medium and 12% FBS. Onto this base,
104 cells in 0.4% SeaPlaque agarose containing RPMI 1640 medium and 12% FBS were embedded. After 2 to 3 weeks of incubation,
colonies that contained more than 100 cells were counted.
EBER-1 in situ hybridization.
EBER-1 was detected with a
digoxigenin-labeled oligonucleotide probe complementary to the EBER-1
sequence (5'-AGA CAC CGT CCT CAC CAC CCG GGA CTT GTA-3')
(5). A sense probe against EBER-1 was used for negative
control studies.
Apoptosis assay.
Cells in the log phase were incubated for
16 h in the presence of 20 µg of cycloheximide (CHX) (Wako,
Osaka, Japan) per ml, for 40 h with 1 mM glucocorticoid (Pharmacia
or Upjohn), or for 65 h in a 100% CO2-saturated
humidified atmosphere (hypoxic stress). The viability of cells was
quantified by an MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay
(Cell Titer 96; Promega).
Western blot analysis.
Western blot analysis was performed
as described previously (27). For immunostaining, blots were
incubated with mouse anti-human bcl-2 monoclonal antibody bcl-2/100 (Pharmingen).
| |
RESULTS |
EBER expression in EK- and EKS10-transfected Akata cells.
We
constructed a plasmid, pEK, that carried a single copy of EBER and
neoR. We isolated G418-resistant clones by transfecting pEK
into an EBV-negative Akata cell clone. Compared with the level of EBERs
in EBV-reinfected cell clones, the average signal intensities for EBERs
in four EK-transfected cell clones were 4.8% by Northern blot analysis
and 6.3% by RT-PCR, even though clones expressing the highest levels
were analyzed (Fig. 1A). To obtain higher
levels of EBER expression in transfected cells, we constructed a
plasmid, pEKS10, that contained 10 tandem repeats of the
SacI-EcoRI subfragment from the EcoRI
K fragment of Akata EBV DNA. We isolated G418-resistant clones by
transfecting pEKS10 into an EBV-negative Akata cell clone. Compared
with the level in EBV-reinfected cell clones, the average levels of
EBER expression in four EKS10-transfected cell clones were 81% by
Northern blot analysis and 66% by RT-PCR (Fig. 1B). It was difficult
to obtain levels of EBER expression in transfected cells equivalent to
those in EBV-reinfected cells. The amount of the EBER-1 transcript was
approximately twofold higher than that of the EBER-2 transcript in
EBV-positive, EBV-reinfected, and EBER-transfected Akata cell clones by
a semiquantitative RT-PCR assay.
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FIG. 1.
Relative EBER expression in EK- and EKS10-transfected
Akata cells compared with EBV-reinfected Akata cells. For each EBER
transfectant derived from different EBV-negative clones, more than 50 cell clones were examined for EBER expression, and 4 to 6 clones with
the highest levels of EBER expression were chosen for further studies.
Representative results are shown. Four EBER-transfected cell clones
from an EBV-negative Akata cell clone were mixed and subjected to
quantitative assays for EBER. (A) Schematic drawing of the
EcoRI K fragment of Akata EBV DNA (left) and relative EBER
expression in EK-transfected cells (right). (B) Schematic drawing of
EKS10 (left) and relative EBER expression in EKS10-transfected cells
(right). avr, average.
|
|
Growth characteristics of EK- and EKS10-transfected Akata
cells.
The growth characteristics of EK- and EKS10-transfected
Akata cell clones were compared with those of EBV-reinfected cell clones and neoR-transfected cell clones. First, we performed
a soft-agar colony assay using cell clones derived from three
independent EBV-negative clones (Fig. 2).
EBV-reinfected cell clones formed colonies, but
neoR-transfected cell clones scarcely did. The absolute number of colonies differed among experiments, presumably due to clonal
variation. EK-transfected cell clones formed a few colonies, whereas
EKS10-transfected cell clones formed significantly more colonies than
neoR-transfected cell clones but formed fewer colonies than
EBV-reinfected cell clones (Fig. 2). Compared with data for EBV-reinfected cell clones, the restoration rates for colony production in soft agar in three independent experiments were 21, 13, and 17%
(average, 17%) for EK-transfected cell clones and 95, 57, and 63%
(average, 72%) for EKS10-transfected cell clones. As the level of EBER
expression went up, more cells formed colonies in soft agar.
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FIG. 2.
Colony production in soft agar for
neoR-transfected, EK-transfected, EKS10-transfected, and
EBV-reinfected cells. The individual panels show independent
experiments with different EBV-negative Akata cell clones. Each dot
represents the number of visible colonies emerging per 104
cells. Horizontal bars represent the mean values for each group. The
differences between neoR-transfected and EKS10-transfected
cell clones were significant (P, <0.01), as determined by
the Mann-Whitney U test.
|
|
Second, we examined whether EKS10-transfected cells were tumorigenic in
SCID mice. In three separate experiments, tumors developed
from
EKS10-transfected cells and from EBV-reinfected cells but
not from
neoR-transfected cells (Table
1). The tumors derived
from
EKS10-transfected Akata cells showed a typical lymphoma pattern
with
intensive blood vessel infiltration (Fig.
3). In an in situ
hybridization study,
almost all the tumor cells were positive
for EBER-1 expression.
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FIG. 3.
Histological examination of tumors derived from
EKS10-transfected Akata cells in SCID mice. Adjacent sections from
paraffin-embedded blocks were stained by hematoxylin-eosin or subjected
to an in situ hybridization study. Typical lymphoma histology was
observed (left panel; magnification, ×400). The EBER-1 in situ
hybridization study revealed that almost all the tumor cells were
positive for EBER-1 expression (middle panel; magnification, ×400).
Dark signals representing the presence of EBER-1 transcripts were
specifically present in the nuclei of tumor cells. A negative control
for the EBER-1 in situ hybridization study is also shown (right panel;
magnification, ×400).
|
|
Resistance to apoptosis of EK- and EKS10-transfected Akata
cells.
To test whether the expression of EBERs restored resistance
to apoptosis, we performed an apoptosis assay using a set of cell clones. Apoptosis was induced by exposing cells to inducers such as
CHX, glucocorticoid, and hypoxic stress, and surviving cells were
measured by an MTT assay (Fig. 4). The
survival rate (%SR) was the highest in EBV-reinfected cells and the
lowest in neoR-transfected cells. The difference in %SR
between neoR-transfected cells and EK-transfected cells was
not significant. In contrast, the %SR of EKS10-transfected cells was
significantly higher than that of neoR-transfected cells for
all stimuli. Compared with data for EBV-reinfected cells, the
restoration rates for EK-transfected cells were 16, 18, and 2.2%
(average, 12%) against CHX, glucocorticoid, and hypoxic stress,
respectively, and those for EKS10-transfected cell were 50, 48, and
70% (average, 56%), respectively. These results illustrated that the
more that EBERs were expressed in cells, the greater the resistance to
apoptosis of the cells.
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FIG. 4.
Resistance to apoptosis of neoR-transfected,
EK-transfected, EKS10-transfected, and EBV-reinfected cells tested with
CHX (A), glucocorticoid (Gluc) (B), and hypoxic stress (C). The bars
show the means ± standard deviations for six clones. The data are
typical results from three independent experiments. As determined by a
t test analysis, the differences between mean values for
neoR- and EKS10-transfected cells were significant at
P values of <0.01 (CHX), <0.05 (Gluc), and <0.001
(hypoxic stress).
|
|
EBERs upregulate the expression of bcl-2 oncoprotein.
We next
examined whether the upregulation of bcl-2 oncoprotein had been
restored in EKS10-transfected cells. The level of bcl-2 protein
expression in EBV-reinfected cells was found to be higher than that in
neoR-transfected cells (Fig.
5). bcl-2 expression in EKS10-transfected
cells was higher than that in neoR-transfected cells. We
could not find significant upregulation of bcl-2 protein in
EK-transfected cells. Finally, the average restoration rate for bcl-2
expression in EKS10-transfected cells was estimated to be 65% by
densitometric analysis in several experiments.
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FIG. 5.
Expression of bcl-2 protein in
neoR-transfected, EKS10-transfected, and EBV-reinfected
(rEBV) cells derived from two independent EBV-negative Akata cell
clones. The data are typical results from several experiments.
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|
| |
DISCUSSION |
In the transfection study, we demonstrated that EBER-expressing
Akata cell clones restored the malignant phenotype, resistance to
apoptosis, and upregulated expression of bcl-2 protein to levels comparable to the restoration rates for EBER expression in
EBV-reinfected cell clones. Thus, it was clear that EBERs were
responsible for the malignant phenotype and resistance to apoptosis in
Akata cells. Many RNAs are known to have catalytic functions; however,
there has been no report describing an oncogenic RNA. This is the first paper that provides evidence that RNA polymerase III-transcribed virus-encoded small RNAs affect the malignant phenotype and resistance to apoptosis.
There was a good correlation between the rate of restoration of EBER
expression and the biological activity of EBV-reinfected cells. This
result clearly illustrates that EBERs contribute to the malignant
phenotype and resistance to apoptosis. All the restoration rates for
biological phenotypes estimated from individual experiments were
somewhat lower than the levels of expression of EBERs in EKS10-transfected cells. The reason for this result is probably that
there was a gradual loss of EBER expression in EKS10-transfected cells
during cultivation. RT-PCR revealed that EBER-1 expression in
EKS10-transfected cells at 14 weeks after transfection was 60% that at
6 weeks (data not shown). The reason for this result is largely
unknown; however, it is often seen in stable transformants.
More interestingly, how could EBERs (RNAs) contribute to malignant
phenotypes and apoptosis resistance? EBERs were reported to bind to
some cellular proteins, La (16), EAP/L22 (31,
32), and PKR (6). Above all, the association of EBERs
with PKR is noteworthy. PKR, previously described as an
interferon-induced protein kinase, is induced by interferons and
activated by double-stranded RNAs to phosphorylate eIF-2
, a
translation initiation factor (18). This phosphorylation
results in the blockage of protein synthesis, through which interferons
are thought to exert their antiviral effects. EBERs were shown to bind
to PKR and block the activation of PKR as well as adenovirus VAI and
VAII RNAs, which might result in the enhancement of protein synthesis
of general mRNAs (26). The dominant negative form of PKR has
been shown to predispose cells to malignant transformation or even
initiate it (14, 19). In addition, GCN4, a yeast homologue
of PKR, is known to regulate specific gene expression via translational control (1). Since the levels of expression of 
-actin,
c-myc, bax, and PKR in EBV-positive Akata cells were equal to those in EBV-negative Akata cells (data not shown), whereas the expression of
bcl-2 protein was specifically upregulated in EBV-positive Akata cells,
it is possible that the inactivation of PKR by EBERs enhances the
translation of bcl-2 mRNA more efficiently than that of other mRNAs to
confer the malignant phenotype and resistance to apoptosis.
However, it remains unclear whether the effects of EBERs are mediated
via upregulation of bcl-2 protein. bcl-2 acts in synergy with the c-myc oncogene in tumor progression, this idea was
suggested by clinical investigations indicating that the activation of
both c-myc and bcl-2 may have conferred an
aggressive clinical outcome in lymphoma cases (4, 11, 21).
This idea was clearly demonstrated in a transgenic mouse study in which
bcl-2 c-myc transgenic mice exhibited accelerated
lymphomagenesis (9, 17). In mammalian cells, deregulated
expression of c-myc has been shown to contribute not only to
tumorigenesis (15) but also to the induction of apoptosis in
various cell lines, including BL cell lines (2, 8, 20). The
mechanism of synergy between bcl-2 and c-myc seems to be that bcl-2 protects cells from c-myc-induced apoptosis (3, 22, 33). Like Akata cells (29), all BL cells
possess a chromosomal translocation involving the c-myc
locus and resulting in constitutive activation of the c-myc
gene (12). Therefore, BL cells were thought to be
predisposed to c-myc-induced apoptosis. Our data suggest that EBV
infection would upregulate the expression of bcl-2 protein to protect
cells from c-myc-induced apoptosis and to allow c-myc to exert its
oncogenic functions. In this way, bcl-2 might cooperate with c-myc in
the development of BL.
| |
ACKNOWLEDGMENTS |
We thank Tsuyoshi Takeda for EBER staining and Kumi Adachi for
technical assistance.
This work was supported by grants-in-aid from the Ministry of
Education, Science, Sports, and Culture, Japan, and from the Princess
Takamatsu Research Fund.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Virology, Cancer Institute, Hokkaido University School of Medicine, N15 W7, Kita-ku, Sapporo 060-8638, Japan. Phone: 81-11-706-5071. Fax: 81-11-717-1128. E-mail: kentaka{at}med.hokudai.ac.jp.
| |
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Journal of Virology, December 1999, p. 9827-9831, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
