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Journal of Virology, February 2001, p. 1561-1564, Vol. 75, No. 3
Department of Tumor Virology, Institute for
Genetic Medicine, Hokkaido University, Kita-ku, Sapporo 060-8638, Japan
Received 28 August 2000/Accepted 7 November 2000
We have demonstrated that Epstein-Barr virus (EBV) confers enhanced
growth capability in soft agarose, tumorigenesis in the SCID mouse, and
resistance to apoptosis in the Burkitt's lymphoma cell line Akata.
Subsequently, we have shown that EBV-encoded small RNAs (EBERs) are
responsible for these phenotypes. We constantly observed the
upregulation of bcl-2 oncoprotein expression upon EBV infection and
expression of EBERs. To test whether these phenotypes were due to the
upregulation of bcl-2 expression, we introduced bcl-2 into EBV-negative
Akata cells at various levels encompassing the range at which
EBV-positive cells expressed it. As cells expressed bcl-2 at higher
levels, they became more capable of growing in soft agarose and became
resistant to apoptosis. However, clones expressing bcl-2 at a higher
level than EBV-positive Akata cells were negative in the tumorigenesis
assay in the SCID mouse. On the other hand, introduction of bax into
EBV-positive Akata cells reduced the resistance to apoptosis; however,
it failed to reduce the growth capability in soft agarose. These data
indicate that EBV targets not only bcl-2, but also an unknown
pathway(s) to enhance the oncogenic potential of Akata cells.
Previously we established a system
to test whether any cellular phenotypes of latency I Burkitt's
lymphoma (BL) cells were due to Epstein-Barr virus (EBV), by using a
cell line of BL origin, Akata, which has several unique characteristics
among BL cell lines (24-27). We have demonstrated that
EBV contributes to growth capability in soft agarose, tumorigenesis in
immunodeficient mice, and resistance to apoptosis in Akata cells
(12, 24). We also reported that EBV-determined nuclear
antigen 1 (EBNA1) was not responsible for these phenotypes
(12). Similar results were reported by two independent
groups (4, 23). We further clarified that EBV-encoded RNAs
(EBER-1 and -2) are responsible for these phenotypes (11).
The question that remained to be answered was the mechanism by which
EBV contributes to these phenotypes. We constantly observed the
upregulation of bcl-2 oncoprotein expression upon EBV infection or
expression of EBERs in EBV-negative Akata cell clones (11, 12). A similar finding was also described by Ruf et al.
(23). Distinct from other oncogenes, bcl-2 fosters cell
survival rather than promoting cell proliferation. Since it is well
known for its antiapoptotic function (20), it was assumed
that the resistance to apoptosis was due to upregulation of bcl-2
protein. BL cells are predisposed to c-myc-induced
apoptosis, since BL cells possess immunoglobulin (Ig)/c-myc
translocation, which results in constitutive activation of the
c-myc gene (9). Therefore, we hypothesized that
upregulation of bcl-2 expression by EBV infection would protect cells
from c-myc-induced apoptosis and allow c-myc to exert its oncogenic
functions. To test this idea, we employed two approaches: (i)
introduction of bcl-2 into EBV-negative Akata cells to test whether any phenotypes were restored and (ii) introduction of bax into EBV-positive Akata cells to antagonize the function
of bcl-2 to determine whether any phenotypes were reduced.
Effect of bcl-2 expression on oncogenic potential and resistance to
apoptosis in Akata cells.
First, we introduced bcl-2 expression
vector pBcl-2 into EBV-negative Akata cells. This pcDNA3-based vector
carried human bcl-2
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.3.1561-1564.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Role of bcl-2 in Epstein-Barr Virus-Induced
Malignant Conversion of Burkitt's Lymphoma Cell Line Akata
and
ABSTRACT
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under control of a human
cytomegalovirus promoter. We successfully isolated clones that
expressed low to very high levels of bcl-2 protein (Fig.
1A). The expression of bcl-2 protein was
detected by Western blot analysis with antihuman bcl-2 monoclonal
antibody bcl-2/100 (Pharmingen). Neomycin resistance gene
(neo)-transfected cell clones and EBV-reinfected cell clones
were also isolated for use as negative and positive controls,
respectively. The average relative signal intensity representing the
amount of bcl-2 protein expressed was quantified by densitometric
analysis and dot plotted in Fig. 1A. In this experiment, the level of
bcl-2 expression detected by Western blot analysis appeared to be
within the semiquantitative range. The average level of relative bcl-2
expression of EBV-reinfected cell clones was between those of clones
with low and medium levels of bcl-2 expression. The growth rates among
these cell clones were almost the same under serum-rich and low-serum
conditions, except for clones with high and extra-high levels of bcl-2
expression under low-serum conditions. Using these cell clones, we
carried out a soft agarose cloning assay, apoptosis assay, and
tumorigenesis assay in SCID mice.
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FIG. 1.
Effects of bcl-2 expression on clonability in soft agar
and resistance to apoptosis in Akata cells. (A) Western blot analysis
detecting bcl-2 protein of bcl-2-transfected cells, which
expressed low (L), medium (M), high (H), and extra high (EH) levels.
The data presented here are representative of four clones tested. The
average relative signal intensity representing the amount of bcl-2
protein expressed in these cells was quantified by densitometric
analysis and dot plotted. (B) Clonability in soft agarose. The mean
values of the number of colonies that emerged in soft agar were plotted
against the relative amounts of bcl-2 protein. Each dot represents the
average number of colonies that emerged per 104 cells. (C)
Resistance to apoptosis. The mean values of the survival rates against
apoptotic stimuli were plotted against the relative amounts of bcl-2
protein. 
, bcl-2-transfected cells; 
, EBV-reinfected
cells. Horizontal bars represent the mean values of each group. The
bars show the mean values ± standard deviation of four clones.
(A570 of the
blank)]/[(A570 of the control) (A570 of the blank)]} × 100. The mean values of %SRs against all apoptotic stimuli for each clone were plotted against the relative amounts of bcl-2 protein (Fig. 1C). As a result,
%SRs were also found to be in direct proportion to the relative amount
of bcl-2 protein. It was noted that cells became resistant to hypoxic
stress with a minimal increase of bcl-2 expression. This is consistent
with the previous finding that upon hypoxic stress, the greatest
difference of susceptibility to apoptotic cell death was seen between
EBV-positive and -negative clones (11).
The tumorigenic potential of clones expressing higher levels of bcl-2
than EBV-infected Akata cells was tested. A total of 1.5 × 107 cells were inoculated into the thigh subcutis of
4-week-old male SCID mice as described previously (11).
Those clones failed to develop tumor masses in the SCID mice (Table
1). Interestingly, the malignant
phenotype of bcl-2-expressing Akata cell clones scored differently in
the soft agarose colony assay and tumorigenesis assay in the SCID
mouse. Historically, these results have been thought to reflect the
"tumor cell" phenotype; however, our data suggested that this was
not the case.
|
Effect of bax expression on oncogenic potential and resistance to
apoptosis in Akata cells.
Second, we attempted to antagonize the
function of bcl-2 by using bax, a homologue of bcl-2. bax binds to
bcl-2 and inhibits its antiapoptotic function (21). We
speculated that if the malignant phenotype and resistance to apoptosis
depend on bcl-2 protein, expression of bax in EBV-positive Akata cells
should lead to a loss of these phenotypes. We transfected bax
expression plasmid pBax into both EBV-negative [EBV()] and
-positive [EBV(+)] Akata cells. The expression vector for bax (pBax)
was constructed by inserting the bax- cDNA downstream of
the SR promoter, which drives transcription of a bicistronic mRNA
for bax and neor mediated by an
encephalomyocarditis virus internal ribosomal entry site sequence. We
isolated G418-resistant cells that were designated
EBV()/neor, EBV()/bax,
EBV(+)/neor, and EBV(+)/bax.
Expression of bcl-2 and bax protein in these cells was tested by
Western blot analysis with antihuman bcl-2 monoclonal antibody
bcl-2/100 and a rabbit anti-human bax polyclonal antibody (Pharmingen)
(Fig. 2A). A small amount of bax protein was detected in EBV()/neoR and
EBV(+)/neor cells; in contrast,
EBV()/bax and EBV(+)/bax cells expressed approximately 2.1- and 2.5-fold more bax protein than
EBV()/neor and
EBV(+)/neor, respectively. The levels of bcl-2
protein expression in these cells were almost the same, except for
EBV()/bax cells. They expressed 1.9-fold more bcl-2
protein than the others. Since expression of bax might oversensitize
EBV() cells to apoptosis, cells expressing bcl-2 protein at a higher
level seemed to be selected in the cloning process for
EBV()/bax cells. A slightly reduced growth rate was seen
in bax-transfected cells.
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)/neor and
EBV()/bax cells hardly formed colonies in soft agarose. The number of colonies seen for EBV(+)/neor
cells was significantly higher than that for
EBV()/neoR cells, which is consistent with
previous findings (11, 12). The number of colonies of
EBV(+)/bax cells was not significantly less than that of
EBV(+)/neoR cells. In the apoptosis assay,
EBV(+)/neoR cells were more resistant to
apoptosis than EBV()/neoR cells in response to
all stimuli. A slight reduction of %SRs was seen in
EBV()/bax cells compared with
EBV()/neoR cells. In contrast, a significant
reduction of %SRs was found in EBV(+)/bax cells compared
with EBV(+)/neoR cells. There is a report that
bax protein functions in both bcl-2-dependent and -independent fashions
(10, 29). Therefore, it remains a possibility that the
phenotype seen in EBV(+)/bax cells might be partly due to
the bcl-2-independent function of bax protein.
Using the transfectants derived from an EBV-negative Akata cell clone
expressing various levels of bcl-2 proteins encompassing the range of
EBV-reinfected Akata cell clones, we demonstrated that: (i) bcl-2
expression conferred resistance to apoptosis, (ii) bcl-2 expression
contributed to the growth capability in soft agarose, (iii) the effects
of bcl-2 expression in these assays were dose dependent, and (iv) bcl-2
expression was insufficient to support tumorigenesis in the SCID mouse.
In the bax study, we demonstrated that the bax expression reduced the
resistance to apoptosis, whereas the effect on the growth capability in
soft agarose was modest. Those data strongly support the idea that EBV
targets not only bcl-2, but also an unknown cellular factor(s) to
confer the malignant phenotype and resistance to apoptosis seen in the
EBV-positive Akata cells.
The tumorigenic potential of bcl-2 has been clearly demonstrated in
rodent systems by transfection of the bcl-2 expression plasmid into NIH
3T3 cells in vitro (22), and in a bcl-2
transgenic mouse study in which follicular lymphoproliferations
progressed in the long term to high-grade malignant lymphoma (15,
16). Furthermore, it is widely accepted that bcl-2
synergizes with the c-myc oncogene in tumor progression.
This was suggested by clinical investigations indicating that
activation of both c-myc and bcl-2 may have
conferred an aggressive clinical outcome in lymphoma cases (3, 8,
19). This idea was also demonstrated in a transgenic mouse
study, in which bcl-2/c-myc double transgenic mice displayed accelerated lymphomagenesis (6, 14). In
mammalian cells, deregulated expression of c-myc has been
shown to contribute not only to tumorigenesis (13), but
also to induce apoptosis in various cell lines, including BL cell lines
(1, 5, 17). The mechanism of
bcl-2/c-myc synergy seems to be that bcl-2
protects cells from c-myc-induced apoptosis (2, 28). Like
Akata cells (26), all of the BL cells possess a
chromosomal translocation involving the c-myc locus, which
is believed to result in constitutive activation of the
c-myc gene (9). Therefore, BL cells were thought to be predisposed to c-myc-induced apoptosis. Our data imply
that EBV infection upregulates expression of bcl-2 protein to protect
cells from c-myc-induced apoptosis and to allow c-myc to exert its
oncogenic functions. However, other unknown pathways remain to be
verified to explain the mechanism by which EBV contributes to the
genesis of BL.
The role of bcl-2 in the development of BL has been largely unknown.
Although attempts to detect bcl-2 protein expression in tumor biopsy
samples failed (7, 18), several lines of evidence
supported the hypothesis that BL cell lines with type I latency
expressed bcl-2 protein at a low level (18, 23). Since (i)
the level of bcl-2 expression in type I BL cell lines is relatively low
compared to that in type III BL cell lines and EBV-immortalized
lymphoblastoid cell lines (18) and (ii) there is no ideal
tissue culture system available to demonstrate the role of bcl-2 in the
type I BL cell lines, the significance of bcl-2 expression in the
development of BL remains to be validated.
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ACKNOWLEDGMENTS |
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We thank S. Takahashi, T. Miyashita, and K. Shimotohno for bcl-2, bax, and internal ribosomal entry site plasmids, respectively. We thank K. 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 Fund.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Tumor Virology, Institute for Genetic Medicine, Hokkaido University, 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.
Present address: McArdle Laboratory for Cancer Research, Department
of Oncology, University of Wisconsin
Madison, Madison, WI
53706-1599.
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Journal of Virology, February 2001, p. 1561-1564, Vol. 75, No. 3
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.3.1561-1564.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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