Suberoylanilide hydroxamic acid induces viral lytic cycle in
Epstein-Barr virus-positive epithelial malignancies and mediates
enhanced cell death
K.F. Hui and Alan K.S. Chiang
Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Queen Mary Hospital,
Pokfulam, Hong Kong SAR, China
In Epstein-Barr virus (EBV)-associated malignancies, the virus is harbored in every tumor cell and persists in tightly latent
forms expressing a very limited number of viral latent proteins. Induction of EBV lytic cycle leads to expression of a much
larger number of viral proteins, which may serve as potential therapeutic targets. We found that 4 histone deacetylase
inhibitors, trichostatin A (TSA), sodium butyrate (SB), valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA), all
significantly induced EBV lytic cycle in EBV-positive gastric carcinoma cells (AGS/BX1, latency II) but only weakly induced in
Burkitt lymphoma cells (AK2003, latency I) and did not induce in lymphoblastoid cells (LCLs, latency III). Interestingly, SAHA
potently induced viral lytic cycle in AGS/BX1 cells at micromolar concentrations (evidenced by 8-fold increase in viral DNA
replication, strong expression of viral lytic proteins and production of infectious virus particles) and mediated enhanced cell
death of EBV-positive AGS/BX1 cells when compared with that of EBV-negative AGS cells, possibly related to cell cycle arrest
at G2/M phase. Furthermore, SAHA effected strong induction of EBV lytic cycle in nasopharyngeal carcinoma but not in NK
lymphoma cells (both expressing EBV latency II pattern), indicating preferential viral lytic induction in epithelial rather than
lymphoid malignancies. In conclusion, SAHA is found to be a potent EBV lytic cycle inducing agent, which warrants further
investigation into its potential application as a novel virus-targeted drug for treatment of EBV-associated epithelial
malignancies.
Epstein-Barr virus (EBV) is a gamma herpesvirus which
infects more than 90% of human populations all over the
world. It causes infectious mononucleosis and is closely
associated with several lymphomas (e.g., endemic Burkitt
lymphoma, Hodgkin lymphoma, nasal NK/T-cell lymphoma
and post-transplant lymphoproliferative disease) and
epithelial cancers (e.g., nasopharyngeal carcinoma and gastric
carcinoma).1
EBV has a biphasic life cycle, including latent and lytic
cycles. In its latency form, the virus remains silent and exists
as a closed circular plasmid in the host cell nucleus. When
EBV switches from its latent to lytic cycle, a series of lytic
proteins, namely the immediate early, early and late proteins
will be expressed sequentially.2 In EBV-associated malignan-
cies, the virus is harbored in every tumor cell and persists in
tightly latent forms (latency I-III) expressing a restricted and
variable number of viral latent proteins.1,3 Strong induction
of EBV lytic cycle, which expresses a larger number of lytic
viral proteins, may lead to development of novel targeted
therapeutic strategies against EBV-associated malignancies.
Several antiviral drugs, including acyclovir, ganciclovir
and forcarnet, have been tested for their antitumor effects
against EBV-associated cancers.4 Although these drugs alone
are not efficacious due to lack of lytically replicating virus,
the combination of EBV lytic cycle inducing agents and the
antiviral agents has shown significant effect in killing EBV-
associated cancers.5 Acyclovir and ganciclovir are nucleoside
analogues which are converted to their cytotoxic forms only
if they are phosphorylated by viral thymidine kinase or some
late viral proteins. The cytotoxic forms can be incorporated
into both viral and cellular DNA and cause premature DNA
strand termination and apoptosis of EBV-infected cancer
cells.6 The success of this combined approach mainly
Key words: suberoylanilide hydroxamic acid, Epstein-Barr virus,
epithelial cancer, lytic cycle, histone deacetylase inhibitor
Abbreviations: EBV: Epstein-Barr virus; HDACi: histone
deacetylase inhibitor; SAHA: suberoylanilide hydroxamic acid; SB:
sodium butyrate; TPA: 12-O-tetradecanoylphorbol-13-acetate; TSA:
trichostatin A; VPA: valproic acid
Part of this work was presented in 2008 Cold Spring Harbor
Laboratory Meeting on Mechanisms and Models of Cancer and in
the M.Phil Thesis of K.F.H.
Grant sponsor: HKU-CRCG; Grant number: 10207979; Grant
sponsor: EBV Research; Grant number: 20004525; Grant
sponsors: HKU Studentship, Ho Tung Paediatrics Education and
Research Fund
DOI: 10.1002/ijc.24945
History: Received 21 Jun 2009; Accepted 29 Sep 2009; Online 8 Oct
2009
Correspondence to: Alan K.S. Chiang, Department of Paediatrics
and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The
University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong
Kong SAR, China, Fax: 852-28551523, E-mail: chiangak@hkucc.hku.hk
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Int. J. Cancer: 126, 2479–2489 (2010) VC 2009 UICC
International Journal of Cancer
IJC

depends on a strong activation of EBV lytic cycle. A variety
of chemical agents were shown to induce the EBV lytic cycle
in latently infected cell lines. For instance, transforming
growth factor-beta (TGF-B),7 protein kinase C activator
(TPA: 12-O-tetradecanoylphorbol-13-acetate)8 and several
chemotherapeutic agents such as cisplatin, gemcitabine and
doxorubicin9 are capable of inducing EBV lytic cycle in dif-
ferent cell lines. Further examples including DNA demethy-
lating agent such as 5-azacytidine10 and histone deacetylase
inhibitors (HDACi) such as sodium butyrate (SB), trichosta-
tin A (TSA) and valproic acid (VPA) were also shown to
trigger the switch of EBV from latent to lytic cycle.8,9,11
Combined administration of such agents can induce EBV
lytic cycle synergistically. Nutter et al. showed that combina-
tion of TPA and SB could activate EBV lytic cycle in Raji
cells.12 Westphal et al. showed that coadministration of radia-
tion and SB can result in a stronger activation of EBV lytic
cycle in vitro and in vivo.11 Feng and Kenney showed that
VPA could enhance chemotherapeutic agents’ induction of
EBV lytic protein expression in EBV-positive lymphoblastoid
and epithelial cells.9 However, to this date, no lytic inducing
agent is employed to treat EBV-associated malignancies in
clinical practice.
Histone modification is involved in a variety of biological
processes such as cell differentiation, cell cycle arrest and cell
death.13 It also plays an important role in lytic cycle induc-
tion of a number of viruses. Acetylation of histones in chro-
matin around the immediately early promoter of human
cytomegalovirus14 and BRLF1 promoter of EBV15 results in
activation of viral lytic cycle. In view of the importance of
histone acetylation in regulation of viral lytic cycle, we tested
TSA, SB, VPA and suberoylanilide hydroxamic acid (SAHA)
for their abilities to induce EBV lytic cycle in a panel of
EBV-positive malignancies. SAHA is a synthetic compound
approved by the Food and Drug Administration (FDA) for
treatment of cutaneous T-cell lymphoma. Furthermore, it
induces differentiation, cell cycle arrest and apoptosis in a va-
riety of cancer cells such as colon cancer, breast cancer and
lymphoma cells.16–18 Although SAHA has been shown to
induce cell death in Hodgkin lymphoma,19 its ability to
induce EBV lytic cycle remains unknown.
The aims of this study are (i) to test a panel of HDACi,
including TSA, SB, VPA and SAHA, for their abilities to
induce viral lytic cycle in EBV-associated cancer cells of la-
tency I-III and (ii) to characterize their effects on the cancer
cells after EBV lytic cycle activation.
Material and Methods
Cell cultures and drug treatment
AK31 and AK2003 (gifts from Prof. P. Farrell, Imperial Col-
lege, UK) are EBV-negative and EBV-positive Burkitt lym-
phoma lines, respectively.20 AGS and AGS/BX1 (gifts from
Prof. L. Hutt-Fletcher, Louisiana State University, USA and
Dr. H.L.Chen, The University of Hong Kong, respectively)
are EBV-negative and EBV-positive gastric carcinoma cell
lines, respectively.21 HK1-EBV and HONE1-EBV (gifts from
Prof. S.W. Tsao, The University of Hong Kong) are EBV-
positive nasopharyngeal carcinoma cell lines.22 YT and NK-
92 (from DSMZ, the German Resource Center for Biological
Material) are EBV-positive NK cell leukemia/lymphoma
lines.23 LCL329 is a lymphoblastoid cell line (LCL) estab-
lished in our laboratory by immortalization of human peri-
pheral blood mononuclear cells with B95-8 strain of EBV.
AK2003 and LCL329 express EBV latency I and III pattern,
respectively, whereas AGS/BX1, HK1-EBV, HONE1-EBV, YT
and NK92 express EBV latency II pattern. All cell lines were
cultured in RPMI-1640 medium and 10% fetal bovine serum
(FBS, Invitrogen, Carlsbad, CA), except AGS and AGS/BX1
cells, which were cultured in Ham’s F-12 medium, 10% FBS.
AGS/BX1, HK1-EBV and HONE1-EBV cells are infected
with recombinant EBV genomes containing neomycin resist-
ant and green fluorescent protein (GFP) genes, requiring
G418 at 350–500 lg/ml for maintenance. NK-92 was supple-
mented with 10 ng/ml interleukin-2. All cultures were main-
tained at 37
C in 5% CO2. To induce EBV lytic cycle, cells
were treated with either TSA, SB, VPA (Sigma-Aldrich, St.
Louis, MO), or SAHA (Cayman Chemicals, Ann Arbor, MI)
at their individual half maximal inhibitory concentrations
(IC50) which were determined by MTT assay (described
later), or 5 lg/ml anti-human IgG antibody (Sigma-Aldrich,
St. Louis, MO) or combination of 2.5 mM SB and 20 ng/ml
TPA (Calbiochem, San Diego, CA) for 48 hr.
Quantitative PCR assay
Cells at 70% confluence were treated with either TSA, SB,
VPA, or SAHA for 48 hr. DNA from cell cultures was
extracted by Qiagen DNeasyVR Blood & Tissue kit (QIAGEN,
Valencia, CA). The amount of EBV DNA was quantified by
Taqman real-time quantitative polymerase chain reaction
(PCR) assay. EBV DNA polymerase gene (EBV pol) was
used for the measurement of EBV viral genome copies and
human b2 microglobulin (b2m) gene was used as an internal
control for human genome copies in the qPCR assay. The
primers and probe used for EBV pol gene were, forward: 50-
CTTTGGCGCGGATCCTC-30, reverse: 50-AGTCCTTCTTGG
CTAGTCTGTTGAC-30 and FAM-labeled probe: 50-CTTTG
GCGCGGATCCTC-30 (Applied Biosystems, Foster City, CA).
The primers and probe used for human b2m gene were,
forward: 50-GGAATTGATTTGGGAGAGCATC-30, reverse:
50-CAGGTACTGGCTCTACAATTTACTAA-30 and VIC-la-
beled probe: 50-AGTGTGACTGGGCAGATCATCCACCT-
TC-30 (Applied Biosystems, Foster City, CA). A total volume
of 25 ll was used for each PCR reaction. After activation of
uracil-N-glycosylase at 50
C for 2 min and Amplitaq Gold at
95
C for 10 min, the reaction samples were amplified at
95
C for 15 sec and at 60
C for 60 sec for 40 cycles. The flu-
orescent signals were detected with ABI Prism 7700 Sequence
Detection System (Applied Biosystems, Foster City, CA).
Namalwa cells, which contain 2 integrated viral genome
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2480 SAHA and EBV lytic cycle induction
Int. J. Cancer: 126, 2479–2489 (2010) VC 2009 UICC