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Journal of Virology, November 2000, p. 10223-10228, Vol. 74, No. 21
Program in Viral Oncogenesis and Tumor
Immunology, Department of Virology and Molecular
Biology,1 and Department of
Biochemistry,2 St. Jude Children's Research
Hospital, Memphis, Tennessee 38105, and Department of
Biochemistry3 and Department of
Pathology,4 University of Tennessee Health
Sciences Center, Memphis, Tennessee 38163
Received 7 February 2000/Accepted 14 August 2000
The tumorigenic potential of the Burkitt lymphoma (BL) cell line
Akata is dependent on the restricted latency program of Epstein-Barr virus (EBV) that is characteristically maintained in BL tumors. Within
these cells, EBV-mediated inhibition of apoptosis correlates with an
up-regulation of BCL-2 levels in concert with a down-regulation in
c-MYC expression that occurs under growth-limiting conditions. Here we
addressed whether EBV's effects on apoptosis and tumorigenicity are
mediated by the EBV small RNAs EBER-1 and EBER-2. Stable expression of
the EBERs in EBV-negative Akata BL cells, at levels comparable to those
in EBV-positive cells, significantly enhanced the tumorigenic potential
of EBV-negative BL cells in SCID mice, but did not fully restore
tumorigenicity relative to EBV-positive Akata cells. Furthermore, wild-type or greater levels of EBER expression in EBV-negative Akata
cells did not promote BL cell survival. These data therefore suggest
that EBV can contribute to BL through at least two avenues: an
EBER-dependent mechanism that enhances tumorigenic potential independent of a direct effect on apoptosis, and a second mechanism, mediated by an as-yet-unidentified EBV gene(s), that offsets the proapoptotic consequences of deregulated c-MYC in BL.
Burkitt lymphoma (BL) is a human
B-cell tumor characterized by chromosomal translocations that juxtapose
the c-MYC proto-oncogene and an immunoglobulin gene
enhancer, resulting in the constitutive overexpression of c-MYC
(reviewed in reference 35). Although deregulated
expression of c-MYC is clearly a primary contributing factor in the
development of BL, c-MYC overexpression itself is insufficient for the
transformation of primary cells (30, 31), due to the
activation of apoptotic pathways, particularly under growth-limiting
conditions (2, 12). Recent analyses of B-cell tumors that
arise in Eµ-myc transgenic mice have indicated that the
proapoptotic properties of c-Myc are offset in 80% of these lymphomas
by disruption of the ARF-Mdm2-p53 tumor suppressor pathway (10,
22, 46). Indeed, approximately one-third of actual BL tumors
contain a mutated p53 gene (14), similar to the fraction of
tumors in Eµ-myc mice that exhibit loss of p53 function
(10). Based on these observations, inactivation of the
ARF-Mdm2-p53 pathway is likely to be a key step in BL tumorigenesis,
and one would predict that within BLs containing wild-type p53, other components of the pathway are targeted.
In addition to the genetic anomalies associated with BL, there is a
high incidence of latent (noncytolytic) Epstein-Barr virus (EBV)
infection of tumor cells in cases where BL is endemic, i.e., those that
predominant in the equatorial belt of Africa (11, 35). EBV
infection in endemic BL appears to occur prior to the clonal expansion
of tumor cells, suggesting an important role for EBV in this tumor
(38, 40). In support of this concept, loss of the EBV genome
from cells of the Akata BL cell line is associated with loss of
tumorigenic potential (6, 50), whereas reinfection of these
cells with EBV restores tumorigenicity (27, 44). However,
the EBV oncoprotein LMP-1 and other latency-associated EBV gene
products essential for growth transformation (immortalization) of B
lymphocytes in vitro by EBV are not expressed in BL tumors and many BL
cell lines, including Akata (17, 43, 44, 50). The sole
exception is EBNA-1, a protein required for maintenance of the episomal
EBV genome during latent infection (32, 63). Although EBNA-1
is reported to have oncogenic potential (29, 62), enforced
expression of EBNA-1 in EBV-negative Akata cells is not sufficient to
confer tumorigenic potential to these BL cells (27, 44). The
tumorigenic potential of Akata BL cells is therefore dependent on an
EBV gene product or products other than EBNA-1 alone.
The mechanism or mechanisms by which EBV promotes the tumorigenic
potential of BL cells are unclear. We and others have demonstrated that
EBV-positive Akata BL cells are more resistant to the induction of
apoptosis (e.g., by serum withdrawal) than their EBV-negative counterparts and that this resistance correlates in part with modestly
elevated levels of the antiapoptotic BCL-2 protein in EBV-positive
Akata cells (27, 44). However, the most striking difference
in susceptibility to apoptosis between EBV-positive and -negative Akata
BL cells occurs when cells are permitted to reach the stationary phase
of their growth cycle. Under these conditions, c-MYC levels are
down-regulated in EBV-positive cells (but not EBV-negative cells),
which presumably contributes to BL cell survival in the absence of
adequate growth factors (44). The apparent EBV-mediated
up-regulation of BCL-2, in concert with the down-regulation of c-MYC
under growth-limiting conditions, may therefore offset the proapoptotic
consequences of a deregulated c-MYC gene to promote BL tumorigenicity.
Here we have investigated the contribution of the EBV small noncoding
nuclear RNAs EBER-1 and EBER-2 (166 and 172 nucleotides, respectively)
to cell survival and tumorigenic potential in BL. The EBER genes, which
are transcribed by cellular RNA polymerase III (16, 42),
encode the most abundant EBV transcripts in BL tumors, BL-derived cell
lines, and other latently infected cells (1, 42). To address
the role of the EBERs in cell survival and tumorigenicity, we generated
several lines of EBV-negative Akata BL cells that expressed different
levels of the EBERs. Two approaches were taken to stably express the
EBERs in these BL cells. First, a cassette containing the EBER-1 and
EBER-2 genes and their transcriptional regulatory elements (19,
20) was cloned into a shuttle vector [pBluescript IIKS(+);
Stratagene, La Jolla, Calif.] that was then used to generate
EBV-negative EBER-positive (EBV
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Epstein-Barr Virus Small RNAs Potentiate
Tumorigenicity of Burkitt Lymphoma Cells Independently of an
Effect on Apoptosis
ABSTRACT
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/EBER+) cell
lines. These cell lines typically expressed EBERs at ~10% of the
levels expressed in EBV-positive BL cells (data not shown). Second, to
generate cells that stably expressed higher levels of EBER-1 and
EBER-2, the SacI-to-EcoRI fragment of the EBV
EcoRI-J genomic restriction fragment that encodes both EBERs
was cloned by blunt-end ligation into the SalI site of the
amplicon BSAII adjacent to the EBV oriP element
(61). Since the EBER genes reside immediately adjacent to
oriP in the EBV genome, we reasoned that the high level of
EBER gene expression in latently infected cells (~5 × 106 copies per cell) (59) may be dependent on
their position relative to an active oriP. Therefore, this
construct was transfected by electroporation into EBV-negative 3F2 and
A.2 Akata cells that stably express the EBV EBNA-1 protein
(44), which binds to multiple elements within
oriP to maintain the viral episome in proliferating cells
(41, 63). As noted previously, EBNA-1 expression alone is
not sufficient to confer tumorigenic potential to EBV-negative Akata
cells or to protect them from apoptosis (27, 44). Following transfection, the cells were selected in medium containing 200 µg of
G418 and 200 µg of hygromycin B (Gibco BRL, Rockville, Md.) per ml.
Drug-resistant cells were then expanded and analyzed for EBER
expression by RNA (Northern) blot analysis. As demonstrated in Fig.
1, the cell lines generated by this
approach expressed 41 to 127% of the wild-type level of EBER
expression. Furthermore, analysis of these lines over 18 months of
continuous culture indicated that these levels of EBER expression were
stably maintained and often increased by 10 to 25%. Additionally, when
RNA blots were rehybridized to EBER-specific probes, the ratio of
EBER-1 to EBER-2 expression in these
EBV/EBER+ cells was identical to that in the
EBV-positive Akata cells (data not shown).
View larger version (42K):
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FIG. 1.
Restoration of EBER expression in EBV-negative Akata BL
cells. EBER expression in EBV-positive Akata (A.15) cells and in
EBV /EBER+ cell lines derived from two clonal
EBV-negative Akata lines (A.2 and 3F2) was assessed by Northern blot
analysis with a probe spanning the EBER-1 and EBER-2 genes. Each lane
contained 10 µg of total cellular RNA. The level of EBER expression
is reported as a percentage of EBV-positive Akata cells (A.15) and was
determined by phosphorimage analysis and normalization of values to
GAPDH mRNA expression, which served as a loading control. EBER-1 and
EBER-2 are not distinguishable on the blot due to their similar
lengths; however, when the blot was sequentially stripped and
rehybridized to EBER-specific probes, the ratios of EBER-1 to EBER-2
expression were identical in EBV-positive and
EBV/EBER+ Akata cells (data not shown).
We next addressed whether the EBERs could provide a survival advantage
in vitro, and thus account for the increased resistance of EBV-positive
versus EBV-negative Akata BL cells to apoptosis (27, 44). We
examined whether EBV and
EBV/EBER+ Akata cells differed in their
responses to serum withdrawal by measuring the cleavage of
poly-(ADP-ribose)-polymerase (PARP), an early specific marker of
apoptotic cell death (39), by immunoblotting with a
polyclonal rabbit serum to PARP (Roche Biochemicals, Indianapolis, Ind.). As shown in Fig. 2A, the 89-kDa
cleavage product of PARP continued to accumulate equally in
EBV-negative (EBV/Vector) and
EBV/EBER+ cells when shifted to 0.1% serum,
regardless of whether they had been in the logarithmic or stationary
phase (growth-restrictive conditions) of their growth cycle. In
contrast, EBV-positive cells in the stationary phase exhibited little
or no further increase in PARP cleavage (data not shown). Additionally,
when cells from the stationary phase were shifted to 0.1% serum and
their viability was monitored by trypan blue dye exclusion, we again
observed no effect of EBER expression on cell survival in EBV-negative Akata cells (Fig. 2B). Note that whereas EBV-positive Akata cells maintained viability (>90%) over 4 days in low serum, as we
previously reported (44), the
EBV/EBER+ cells rapidly died at a rate
indistinguishable from that of the EBV-negative vector-control cells.
Thus, the EBERs failed to promote BL cell survival under
growth-restrictive conditions.
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EBV/EBER+ Akata BL cell lines were next
evaluated for their ability to induce tumors in SCID mice. These
included one line (3F2 BSK E) that expressed EBERs at less than 10% of
the levels expressed in EBV-positive BL cells and six lines (see Fig.
1) that expressed 41 to 127% of wild-type levels. Male C.B-17 SCID mice, obtained from the colony maintained by the St. Jude Children's Research Hospital Animal Resource Center, were injected subcutaneously in the hind flank with 2 × 107 cells in
phosphate-buffered saline (200 µl). Animals were monitored for up to
20 weeks for tumor growth and were sacrificed, and their tumors were
excised for analysis of EBER expression when tumors were approximately
1 cm in diameter. As summarized in Table
1, tumors were induced in 25 to 100% of
mice injected with cells expressing EBERs at wild-type levels or
greater. In contrast, only 1 of 24 mice injected with vector-control
cells developed a tumor, and this occurred very late (19 weeks
postinjection). All mice injected with EBV-positive Akata cells (Table
1 [A.15]) developed tumors at 4.5 weeks postinjection, similar to the
rates and frequency previously established for EBV-positive and
reinfected EBV-negative Akata cells (44).
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The mean numbers of weeks required for
EBV/EBER+ BL cells to induce tumors were
10.1, 14.4, and 16.2 for the lines that expressed approximately 100, 50, and less than 10% of wild-type levels of EBERs, respectively.
Thus, the EBERs were clearly capable of enhancing the tumorigenic
potential of EBV-negative Akata BL cells, and there was a direct
correlation between the level of EBER expression and the latency to
tumor development. Interestingly, the observation that the EBV-negative
lines that expressed wild-type or greater levels of EBERs did not
induce tumors in all mice injected and took two to three times as long
as EBV-positive Akata cells to induce tumors suggests that the EBERs
may only partially contribute to EBV-dependent tumorigenicity.
Certainly this is consistent with the failure of the EBERs to inhibit
apoptosis in EBV-negative BL cells, whereas EBV efficiently blocks cell
death (Fig. 2B) (27, 44). However, we cannot currently
exclude the possibility that the apparent inability of the EBERs to
restore tumorigenicity to the level of EBV-positive Akata cells is a
reflection of inherent differences between cellular clones of
EBV-negative and -positive Akata cells, and not the inability of the
EBERs to enhance cell survival. Regardless of whether the EBERs are
able to fully restore tumorigenicity or not, they were clearly able to
potentiate tumorigenic potential independent of a measurable effect on
cell survival.
Finally, all tumors that arose in mice injected with the
EBV/EBER+ cells expressed EBERs (Fig.
3 and data not shown). In some instances, the level of EBERs detected in the tumor samples was lower (following normalization for RNA loading) than that seen within the parental cell
line. This was most likely due to the amount of non-BL-derived cellular
material within some tumor samples rather than loss of EBER expression
in the EBV/EBER+ cells. Note, however, that
the tumor derived from A.2 E8 cells (A.2 E8t), which required 10 weeks
to develop (Table 1), expressed EBERs at 99% of the level of
EBV-positive Akata cells (Fig. 3). Only rarely did we observe an
amplification of EBER expression in tumors, even from the cells that
expressed less than 10% of wild-type levels of EBERs (data not shown).
Therefore, the threshold of EBER expression needed to induce a tumor
was less than 10%. Although there was a direct correlation between the
level of EBER expression and tumorigenic potential, the minimal latency
period of tumors obtained with EBV/EBER+
cells that expressed 100% of the wild-type level was still twice as
long as that obtained with EBV-positive cells, underscoring the concept
that the EBERs are only partly responsible for the tumorigenic
potential of these BL cells.
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Given that neither tumorigenicity nor the ability to mediate increased
resistance to apoptosis is dependent on the EBV EBNA-1 protein
(27, 44), the current studies were initiated to identify which of the relatively few EBV genes expressed in BL are responsible for these phenomena. As demonstrated here and also recently by Komano
et al. (26), expression of even modest levels of the EBERs
markedly increases the tumorigenic potential of EBV-negative Akata BL
cells. However, there are two notable differences between our results
and those of Komano et al. First, the reduced tumorigenic potential of
EBV/EBER+ relative to EBV-positive Akata
cells, also reported here, was previously attributed to the failure to
restore EBER expression to wild-type levels and to the gradual loss of
EBER expression during cell culture (26). Here we achieved
wild-type or greater levels of EBER expression in EBV-negative Akata
cells and did not observe loss of EBER expression. Therefore, the EBERs
are sufficient to promote tumorigenicity of EBV-positive Akata BL cells, but the effects appear to be partial relative to EBV infection, which may require other viral gene products (see below). Second, Komano
et al. also reported that an apparent induction of BCL-2 expression by
the EBERs confers a modest survival advantage to EBV/EBER+ relative to EBV Akata
cells under hypoxic conditions (26). In contrast, we have
evaluated the levels of BCL-2 expressed in several lines each of
vector-control and EBV/EBER+ cells and find
no evidence to support the notion that the EBERs induce expression of
BCL-2 (data not shown). This is consistent with our observed lack of an
effect by the EBERs on cell survival (Fig. 2). We have observed,
however, that enforced expression of BCL-2 in EBV-negative Akata cells
(at levels comparable to those in EBV-positive cells) markedly enhances
cell survival and partially restores tumorigenic potential (I. K. Ruf and J. T. Sample, unpublished observations), suggesting that
the EBV-induced expression of BCL-2 in Akata BL cells does contribute
to tumorigenic potential. Also consistent with the failure of EBERs to
protect BL cells from apoptosis, we found that restored EBER expression in EBV-negative Akata cells failed to down-regulate c-MYC (data not
shown), as previously observed in EBV-positive and reinfected EBV-negative Akata cells.
The functions of the EBERs in tumorigenicity and EBV latency are
unclear. Several cellular proteins that interact with one or both of
the EBERs have been identified, including the autoantigen La (a
component of cellular snRNP complexes) (16, 33), the ribosomal protein L22 (9, 58-60), and two proteins that
mediate the antiviral effects of interferons: the double-stranded
RNA-activated protein kinase PKR (7) and (2'
5')
oligoadenylate synthetase (48). However, the functional
significance of these interactions with respect to EBV biology remains
unresolved, primarily due to the absence of a phenotype associated with
loss of EBER expression in vivo within EBV-immortalized
B-lymphoblastoid cell lines (55, 56).
The formation of ribonucleoprotein complexes containing EBERs and La
has been suggested to reduce pools of free La in the nucleus
(16). This, in turn, might affect the stability and function
of cellular polymerase III transcripts normally bound by La. The
interaction of the EBERs with L22 and resulting relocalization of
~50% of L22 to the nucleoplasm suggests that the EBERs may also
target translation of a specific class of cellular or viral mRNAs
(58). Perhaps the most intriguing (yet controversial) aspect
of potential EBER function is their ability to bind and inhibit PKR
(7, 8, 49), a pivotal mediator of the antiviral effects of
interferons (54). In vitro, EBERs block PKR-mediated inhibition of translation, which involves PKR-dependent phosphorylation of the translation initiation factor eIF-2
(34, 37, 51). A dominant-negative form of PKR is capable of transforming immortal murine fibroblasts (NIH 3T3 cells), suggesting that PKR possesses a
tumor suppressor function (28, 36). Thus, EBERs could
possibly promote tumorigenesis through inhibition of PKR. However, the majority of EBERs are present within the cell nucleus (21), whereas PKR is primarily cytoplasmic (23, 24, 47). Moreover, deletion of the EBER genes from the EBV genome does not impair the
antiviral effects of interferon in EBV-immortalized B-lymphoblastoid cell lines (55). Thus, at this juncture, it is unclear if
the EBERs promote tumorigenesis vis-a-vis effects on PKR function.
The apparent inability of the EBERs to fully restore tumorigenic potential or to effect cell survival implicates the involvement of an additional EBV gene(s). The reported survival function of the EBV LMP-2A gene (4), expression of which is detectable in some BL cell lines and tumor biopsies by reverse transcription-PCR (27, 57), suggests LMP-2A as a likely candidate. However, we have not detected LMP-2A protein within tumors derived from EBV-positive Akata cells, and enforced expression of LMP-2A does not confer tumorigenic potential to EBV-negative Akata cells (I. K. Ruf, R. Longnecker, and J. T. Sample, unpublished observations). The only remaining known latency-associated EBV gene expressed in BL is that which encodes the BamHI-A rightward transcripts (BARTs), a family of alternatively spliced polyadenylated RNAs that contain several short open reading frames (3, 5, 15, 18, 25, 45, 52, 53). The true coding capacity of these transcripts and the functions of the putative proteins encoded by this gene are currently undefined.
In summary, we have demonstrated that the EBV EBER RNAs can contribute to the tumorigenic potential of BL cells independent of an effect upon apoptosis. Thus, EBV likely contributes to c-MYC-induced lymphomagenesis through at least two avenues: an EBER-dependent mechanism that may enhance proliferative potential, and a second mechanism, mediated by an as-yet-unidentified EBV gene(s), that counters the proapoptotic consequences of the inappropriate expression of c-MYC in BL cells. The latter appears to occur through the induction of BCL-2 expression and the down-regulation of c-MYC under growth-limiting conditions (27, 44). Intriguingly, these data also indicate that even in BL cells such as Akata, in which the ARF-Mdm2-p53 tumor suppressor pathway has been inactivated (13), additional inhibition of c-MYC-induced apoptosis is an important facet of BL tumorigenesis.
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ACKNOWLEDGMENTS |
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We thank Fred Wang for the BSAII amplicon, Daniel Henson and Elsie White for excellent technical assistance, and Gerard Zambetti for helpful comments and critical reading of the manuscript.
This work was supported by Public Health Service (PHS) grants CA76379 and DK44158 to J.L.C. and CA73544 and CA56639 to J.T.S., Cancer Center support grant CA21765, and the American Lebanese Syrian Associated Charities (ALSAC). I.K.R. was supported by PHS grant T32-AI07372.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105. Phone: (901) 495-3467. Fax: (901) 523-2622. E-mail: jeff.sample{at}stjude.org.
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Journal of Virology, November 2000, p. 10223-10228, Vol. 74, No. 21
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