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REVIEW
SUMMARY
The general problem in cancer treatment centres on nding agents that specically affect cancer cells without
damaging normal cells. The differences between cancer cells and normal cells are usually very subtle but about 15% of
all human cancers involve a virus infection, for example the Epstein-Barr virus associated cancers. In these cancers,
every tumour cell carries the virus in a latent infection but the number of normal cells infected is very low. So a
treatment that could somehow cause the elimination of EBV infected cells would be very specic for the cancer in
such cases. One potential approach could involve nding ways to reactivate the latent virus in cancer cells into the
early part of the lytic cycle, impeding cell proliferation, targeting chemotherapeutic agents to the cancer and causing
the cancer cells to become targets for immune surveillance. This review considers the mechanisms by which EBV
reactivation is controlled and discusses possible therapeutic approaches. Copyright # 2004 John Wiley & Sons, Ltd.
Accepted: 13 September 2004
150
reaction. Different patterns of latent EBV gene
expression are associated with various types of
infected lymphocyte. These can be observed in
cell culture either as a result of infecting primary
resting human B cells (latency III, the growth programme) or in cell lines derived from EBV associated cancers (latency I or II) [1]. The different
latency programmes were discovered in such cell
lines but have now been related to the biology of
EBV infection in vivo.
After entry of the virus into the resting B cell,
nine latent viral proteins and two small RNAs
(the EBER RNAs) constituting the latency III or
growth programme, are expressed [10]: the proteins include EBV nuclear antigens EBNA-1,
EBNA-2, EBNA-3A, 3B, 3C and EBNA-LP as well
as the latent membrane proteins LMP-1, LMP-2A
and LMP-2B (Table 1). As a result, nave B cells
become proliferating blasts. The virus protein
expression later becomes restricted to the latency
II programme [10,11] in which only EBNA-1,
LMP-1, LMP-2A and the EBER RNAs are
expressed. These two latent membrane proteins
are thought to produce the signal for the latently
infected B cell blasts to form germinal centres
[12], which causes the germinal centre B cells to
differentiate into latently infected memory B cells.
The virus enters latent persistence and shuts down
the expression of viral proteins [13], when only
LMP-2A is possibly expressed [14,15]. Latently
infected memory cells circulate between Waldeyers ring and the peripheral blood without
being detected by the immune system [16]. Even
during infectious mononucleosis, where a large
number of B cells carry EBV, no infected proliferating B blasts are found in the blood, only memory
cells [17]. This could be due to EBV actively avoiding immune detection or just efcient killing of
EBV infected B blasts by T cells in the peripheral
blood.
In latently infected memory B cells, the virus
remains silent. Only when memory B cells divide,
a natural process to maintain memory cell numbers, is EBNA-1 expressed [13], allowing the viral
genome to replicate alongside the host chromosomes.
The process of establishing a latent EBV infection by transforming nave B cells into resting
memory cells in germinal centres mimics the natural process of B-cell activation in response to a
foreign antigen [18,19]. In immune-activated B
Copyright # 2004 John Wiley & Sons, Ltd.
Genome maintenance
(binds origin of latent replication)
Transcription factor, binds RBP-J,
activates cellular and viral genes
Like other EBNA3s balances EBNA-2
effect on RBP-J, binds CtBP
Transcriptional regulator
Overcomes cell cycle
checkpoints, binds CtBP
Co-activates EBNA-2 responsive genes,
increases efciency of immortalisation
Activates NF-B, homologue to CD40,
prevents apoptosis
Inhibitor of BCR signalling,
blocks viral lytic cycle,
provides survival signals
Function unclear
Role in tumourigenicity of
Burkitts lymphoma cells
Function unclear, may express
RPMS1 and A73 proteins
()
Immunoblastoid
lymphomas
(Latency III)
Hodgkins
disease, NPC
(Latency II)
NPC, nasopharyngeal carcinoma; EBNA, Epstein-Barr virus nuclear antigen; LMP, latent membrane protein; EBER, Epstein-Barr virus-encoded small
RNA; BARTs, Bam A rightward transcripts.
LMP2B
EBER
RNAs
BARTs
LMP2A
LMP1
EBNA-LP
EBNA-3B
EBNA-3C
EBNA-3A
EBNA-2
EBNA-1
Gene function
Burkitts
lymphoma
(Latency I)
Required for
immortalisation
152
153
trial in patients with nasopharyngeal carcinoma
to test whether it might cause reactivation of the
latent EBV genome and tumour cell death [58].
In fact there was little effect but this was the rst
clinical attempt to apply the strategy of using the
presence of EBV in the tumour cells to target therapy to this type of cancer. Further development of
the strategy will require identication of novel
compounds that can specically cause reactivation
of the EBV lytic cycle in latently infected tumour
cells and these compounds will have to be acceptable for clinical use.
The detailed understanding of the regulation of
Zp and induction of BZLF1 may provide ways to
identify novel compounds that could be used to
induce the early part of the lytic cycle. For example
an Akata cell line containing the Zp luciferase
reporter plasmids has been adapted into a 96well plate format high throughput screening assay
for such compounds [53]. In several of the EBV
associated cancers there is expression of the LMP
proteins, which can prevent the BCR signalling
to Zp [43]. In that case it would be necessary
to identify lytic cycle inducers which acted closer
to the EBV gene regulation itself, downstream
of the signal transduction pathway or even
acted less specically to reactivate the virus (like
butyrate, which non-specically causes histone
deacetylation).
Another approach to induction of the lytic cycle
in EBV-positive tumour cells is transfection with a
BZLF1 expression vector. Introduction of modied
adenovirus vectors expressing EBV early genes led
to the induction of the lytic cycle of EBV and inhibited tumour growth in Burkitts lymphomas and
nasopharyngeal carcinomas grown in nude mice
[59,60]. However, this approach is limited by the
efciency of the gene delivery methods currently
available.
To avoid the problem of inducers of the lytic
cycle causing an EBV viraemia, one could block
EBV DNA replication by giving patients acyclovir.
There has been an ingenious renement of this in
an experimental animal model test of the strategy
that considerably improved the killing of tumour
cells. Investigation of the mechanism by which
the chemotherapy agent cisplatin kills gastric carcinoma cells harbouring EBV in experimental animals revealed that the cisplatin somehow induces
the EBV lytic cycle and this probably plays a role
in the tumour cell death. By also co-administering
Rev. Med. Virol. 2005; 15: 149156.
154
ganciclovir with the cisplatin, improved tumour
cell killing was achieved [61]. The mechanism
probably involves the toxic effects of ganciclovir,
activated by the EBV lytic cycle in some of the
tumour cells, then diffusing and killing bystander
cells in the tumour. A similar result was achieved
in LCLs when combining the chemotherapeutic
drugs gemcitabine or doxorubicin with ganciclovir
[62].
GENERAL APPROACH
Reactivation of the latent viral genome in EBV
associated cancers, causing cancer cell death or
recognition by the immune system is just one
example of the general strategy of targeting therapy based on the selective presence of the virus
in tumour cells. Immunotherapy, by boosting the
immune response to antigens naturally expressed
by the virus in a virus-associated cancer, is being
tested for several viruses, including EBV and
HPV [6365] and adoptive transfer of EBV specic
T cells has also been used to treat some lymphomas successfully. Other possibilities are also being
investigated, for example, the targeted loss of the
viral episome through treatment with low-dose
hydroxyurea [66] or modied adenovirus vectors
containing the EBV oriP origin of replication that
would selectively be toxic or replicate in EBV
infected cells [67,68]. In fact any property of the
virus that is active in the tumour cells could provide the selectivity to target therapywe just
have to be sufciently inventive to think of ways
to take advantage of it!
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