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Antimicrobial Agents and Chemotherapy, July 2001, p. 2082-2091, Vol. 45, No. 7
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.7.2082-2091.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Induction of Epstein-Barr Virus Kinases To
Sensitize Tumor Cells to Nucleoside Analogues
Stacy M.
Moore,1
Jennifer S.
Cannon,1
Yvette C.
Tanhehco,1
Fayez M.
Hamzeh,2 and
Richard
F.
Ambinder1,*
Departments of
Oncology1 and
Medicine,2 Johns Hopkins University
School of Medicine, Baltimore, Maryland 21231
Received 27 January 2000/Returned for modification 7 April
2000/Accepted 25 April 2001
 |
ABSTRACT |
The presence of Epstein-Barr virus (EBV) in the tumor cells of some
EBV-associated malignancies may facilitate selective killing of these
tumor cells. We show that treatment of an EBV+ Burkitt's
lymphoma cell line with 5-azacytidine led to a dose-dependent induction
of EBV lytic antigen expression, including expression of the viral
thymidine kinase (TK) and phosphotransferase (PT). Azacytidine
treatment for 24 h modestly sensitized the cell line to all nucleosides
tested. To better characterize EBV TK with regard to various nucleoside
analogues, we expressed EBV TK in stable cell clones. Two EBV
TK-expressing clones were moderately sensitive to high doses of
acyclovir and penciclovir (PCV) (62.5 to 500 µM) and to lower doses
of ganciclovir (GCV) and bromovinyldeoxyuridine (BVdU) (10 to 100 µM)
compared to a control clone and were shown to phosphorylate GCV.
Similar experiments in a transient overexpression system showed more
killing of cells transfected with the EBV TK expression vector than of
cells transfected with the control mutant vector (50 µM GCV for 4 days). A putative PT was also studied in the transient transfection
system and appeared similar to the TK in phosphorylating GCV and
conferring sensitivity to GCV, but not in BVdU- or PCV-mediated cell
killing. Induction of EBV kinases in combination with agents such as
GCV merits further evaluation as an alternative strategy to gene
therapy for selective killing of EBV-infected cells.
| |
INTRODUCTION |
Epstein-Barr virus (EBV), a human
gammaherpesvirus, is associated with several malignancies, including
AIDS-associated primary central nervous system lymphoma, nasopharyngeal
carcinoma, nasal lymphoma, a subset of Hodgkin's disease,
posttransplant B-cell lymphoproliferative disease, and African
Burkitt's lymphoma (BL) (31, 38, 51, 52, 54, 55). The
presence of viral genomes in malignancies offers unique opportunities
for novel and specific approaches to therapy. The herpesvirus
prodrug-converting enzymes thymidine kinase (TK) and phosphotransferase
(PT) phosphorylate nucleoside analogues, converting these drugs into
intermediates able to inhibit critical cellular processes (13,
14, 25, 34, 46). For example, the nucleoside analogue
ganciclovir (GCV) is very efficiently phosphorylated by the herpes
simplex virus type 1 (HSV-1) TK but is less efficiently phosphorylated by cellular enzymes (10). The phosphorylated compound
inhibits the cellular DNA polymerase, leading to cell death (16,
41). Gene therapy studies illustrate the possible utility of
herpesvirus prodrug-converting enzymes in mediating selective cell
killing. The HSV-1 TK gene has been introduced into brain tumor cells
using retroviral vectors so that these transfected tumor cells might be
targeted by GCV (11). Similarly, allogeneic lymphocytes
used in adoptive immunotherapy programs have been marked with a
retroviral vector encoding HSV-1 TK so that if graft-versus-host
disease develops, the infused cells can be selectively destroyed by
treating with GCV (4).
EBV encodes a TK that shows sequence and functional homology with HSV-1
TK (22, 24, 26, 27, 53). The EBV TK is larger than the
HSV-1 TK and encodes a 243-amino-acid N terminus whose function is
unknown (22, 26). The EBV protein, like its HSV-1 homologue, but unlike the homologues in HSV-2 and varicella-zoster virus, has both TK and thymidylate kinase activity (6,
19). The substrate specificity of the EBV TK with regard to GCV
has been the subject of conflicting reports, although there is general agreement that GCV inhibits EBV lytic replication (19,
24). In addition to EBV TK, EBV also encodes a second kinase.
The open reading frame in BGLF4 encodes a protein that is homologous to other herpesvirus PTs (5, 47). The EBV protein
autophosphorylates and phosphorylates viral protein substrates,
including the EBV early antigen EA-D and a DNA polymerase accessory
factor (8).
In EBV-associated malignancies, there is little expression of lytic
cycle genes, including the TK gene. Studies from several laboratories,
including our own, however, have shown that CpG methylation of the
episome plays an important role in the regulation of EBV gene
expression. Viral genomes are methylated in a variety of EBV-associated
tumors, including BL, Hodgkin's disease, nasopharyngeal carcinoma, and
a subset of posttransplant lymphomas (15, 23, 35, 43, 49).
In vitro, inhibitors of DNA methyltransferase lead to lytic induction
in some BL cell lines (3, 35, 39). We sought to determine
whether azacytidine would activate expression of viral kinases and thus
sensitize EBV+ tumor cells to killing by antiviral
nucleoside analogues such as GCV.
| |
MATERIALS AND METHODS |
Chemicals.
5-Azacytidine,
(E)-5-bromovinyldeoxyuridine (BVdU), and acyclovir (ACV)
were purchased from Sigma (St. Louis, Mo.). S-BVdU was a
gift from Erik De Clerq (Katholieke Universiteit Leuven, Leuven,
Belgium). GCV and penciclovir (PCV) were purchased from Hoffmann-La
Roche Inc. (Nutley, N.J.) and SmithKline Beecham Pharmaceuticals (Philadelphia, Pa.), respectively. Azidodeoxythymidine (AZT) was purchased from Calbiochem (La Jolla, Calif.).
Hypoxanthine-aminopterin-thymidine (HAT) was purchased from Life
Technologies (Gaithersburg, Md.) as a 100× lyophilized supplement and
diluted in water to a 10× working concentration. HAT diluted in water
was added diluted 1:10 to media.
Plasmids.
The plasmid pEBVTK was generated by subcloning the
BamHI fragment of pUCX (from J. R. Arrand, Christie
Hospital and Holt Radium Institute, Manchester, United Kingdom)
(24) into the BamHI site of the mammalian
expression vector pcDNA3 (Invitrogen, Carlsbad, Calif.). In the
recombinant plasmid, the EBV TK is expressed from a cytomegalovirus
promoter. The pcDNA3 parent was used as the vector control.
The plasmid pEBVPT was generated by PCR amplification of the EBV PT
gene using Pfu polymerase (Stratagene, La Jolla, Calif.), followed by cloning into pcDNA3 at the BamHI site. The EBV
PT is encoded by the open reading frame within the BGLF4 fragment of
EBV. Genomic DNA isolated from EBV+ B95.8 cells was used as
template in the PCR amplification. The primers used for amplification
of the 1.3-kb BGLF4 insert were 5',
5'-AGTCAGATCTATGGATGTGAATATGGCTGCGGA-3', and 3',
5'-AATCAGATCTTCCTCGAGCTCATCCACGTCG-3'.
The plasmid pEBVmutTK was generated by site-directed mutagenesis using
the QuickChange site-directed mutagenesis kit (Stratagene)
according to
the manufacturer's instructions, with minor changes.
The plasmid was
amplified using 25 ng of pEBVTK as template and
complementary
oligonucleotides, in which the sequences, with the
exception of a
single point mutation, are identical to that of
the wild-type EBV TK,
as primers. The sequences of the primers
used to generate pEBVmutTK
were 5'-CCATTTGCTGTCGACCTCCGTGGTTTTCCC-3'
and
5'-GGGAAAACCACGGAGGTCGACAGCAAATGG-3'. Input plasmid DNA was
digested with
DpnI at 37°C for 1 h. The remaining
plasmid DNA
containing a point mutation in the EBV TK gene was
transformed
into Epicurian Coli XL1-Blue competent cells
(Stratagene).
The plasmid pHSV1TK was generated by PCR amplification of the HSV-1 TK
gene from plasmid pHSV-106 (from G. Hayward, Johns
Hopkins University)
containing the HSV-1 gene (
tk) inserted at
the
BamHI site of pBR322 (Life Technologies). Amplification was
performed using
Pfu polymerase and the following primers:
5',
5'-TTAGGATCCCGTATGGCTTCGTAC-3', and 3', 5'
ACTGGATCCGTTTCAGTTAGCCTC-3'.
The amplified HSV-1 TK gene was then
cloned into the
BamHI site
of
pcDNA3.
Cloned sequences were confirmed by complete sequencing of both
strands.
Cell culture and transfection.
Rael and CA46 are
EBV+ and EBV
BL cell lines, respectively
(42). These were maintained in 1640 RPMI (Gibco BRL) with
10% fetal bovine serum, 100 U of penicillin/ml, 100 µg of
streptomycin/ml, and 100 mM L-glutamine. The cell line 143b
is derived from a human osteosarcoma and was passaged in 15 µg of
bromodeoxyuridine (Sigma)/ml to maintain the cellular TK
phenotype (33). 293T cells, a cellular TK+
cell line, are derived from human kidney epithelial cells transformed with adenovirus E1A and E1B as well as the simian virus 40 T antigen (40). These cells were obtained from W. Burns (Medical
College of Wisconsin). 143b and 293T cells were maintained in
Dulbecco's modified essential medium supplemented with 10% fetal calf
serum, 100 mM nonessential amino acids, 100 U of penicillin/ml, and 250 µg of streptomycin/ml.
To create a stable cell line expressing the EBV TK, 143b cells were
transfected with pEBVTK. Cells were seeded at 2 × 10
5/ml into six-well plates and allowed to adhere
overnight. After
adherence, cells were transfected using 2 µg of
plasmid DNA and
6 µl of Lipofectin reagent (Life Technologies). For
stable cell
clones, selection was carried out in growth medium
containing
400 µg of G418/ml. Colonies were isolated and expanded.
Colonies
derived from cells transfected with the pEBVTK plasmid were
selected
in 1× HAT for 1 week before experimental use (TK143b.1 and
TK143b.2).
Colonies derived from cells transfected with vector alone
(pcDNA3)
were maintained in G418 and 15 µg of bromodeoxyuridine/ml
(V143b.1).
Transient transfections were performed with 293T cells using the
Lipofectamine-PLUS reagent (Life Technologies) according
to the
manufacturer's protocol. Cells were seeded into the wells
of six-well
plates at a concentration of 4 × 10
5/ml in culture
medium without antibiotics and allowed to grow
until the confluency was
approximately 80%. Cells were transfected
with 2 µg of DNA, 14 µl
of PLUS reagent, and 7 µl of Lipofectamine
per well in the absence of
serum and antibiotics. Twenty-four
hours after transfection, cells were
trypsinized, counted, and
prepared for immunohistochemistry and cell
proliferation and phosphorylation
assays. To assay for gene expression
and transfection efficiency,
cells were cytospun onto microscope slides
for immunohistochemical
analysis. Transfection efficiency was
determined by counting the
number of EBV TK-transfected cells
expressing the EBV TK. The
percentage of antigen-positive cells was
determined by averaging
the number of cells expressing antigen in two
high-power fields
relative to the total number of cells in those
fields.
For analysis of cell proliferation and phosphorylation, the transiently
transfected cells were seeded into 96-well plates
for
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT)
(Sigma) cell proliferation assays and/or were seeded into
six-well
plates for determination of GCV phosphorylation by high-performance
liquid chromatography (HPLC). After adherence overnight, the
transfected
cells were treated with the appropriate concentrations of
cold
nucleoside analogues and, where appropriate,
[
3H]GCV. MTT analysis was performed after incubation with
GCV for
4 days, while cells were extracted for phosphorylation analysis
36 h after incubation with cold GCV and [
3H]GCV.
Analysis of EBV TK and PT expression.
The expression of EBV
TK in TK143b cells was monitored by [3H]thymidine
incorporation and by immunoblotting. For [3H]thymidine
incorporation, cells were first seeded at 2 × 104/well into 96-well plates, pulsed for 48 h with 1 µCi of [3H]thymidine/well, and then harvested onto
glass fiber filters. [3H]thymidine incorporation was
measured by scintillation counting. For immunoblotting, proteins were
separated by sodium dodecyl sulfate-7.5% polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to nitrocellulose filters
(Schleicher & Schuell, Inc., Keene, N.H.). These were blocked overnight
in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10.1 mM
Na2PO4, and 1.8 mM KPO4) with 5%
dry milk-0.1% Tween 20 and then incubated for 1 h with a polyclonal
rabbit antiserum (diluted 1:1,000 in PBS with 5% dry milk-0.1% Tween
20) from a rabbit immunized with a synthetic EBV TK peptide
(GRHESGLDAGYLKSVNDAC). After washing, the filters were incubated with
goat anti-rabbit antibody conjugated to horseradish peroxidase
(Bio-Rad, Hercules, Calif.) (diluted 1:3,000 in PBS-0.1% Tween 20).
The filters were washed and the proteins were detected by ECL
chemiluminescence (Amersham, Arlington Heights, Ill.).
The EBV PT was detected by Northern blot analysis. Rael and CA46 cells
were treated with 1 µM 5-azacytidine overnight. RNA
was extracted
from treated and untreated cells using Trizol (Life
Technologies). Five
micrograms of RNA was electrophoresed through
a 1% agarose-2 M
formaldehyde gel. Following electrophoresis,
the RNA was transferred to
a Hybond-N
+ nylon filter (Amersham Pharmacia Biotech
Limited) in 20× SSC
(1× SSC is 0.15 M NaCl and 0.015 M sodium
citrate). A probe specific
for the EBV PT was generated from the
BamHI fragment of pEBVPT
using [
-
32P]dCTP
and the Ready-To-Go DNA labeling kit (Pharmacia Biotech,
Buckinghamshire, United Kingdom). Prehybridization and hybridization
were carried out at 42°C in a mixture of 50% formamide, 1% bovine
serum albumin, 5% SDS, 0.5 M sodium phosphate, and 100 µg of salmon
sperm DNA/ml. After hybridization, the membrane was washed once
in 3×
SSPE (1× SSPE is 0.18 M NaCl, 1 mM EDTA, 10 mM
NaH
2PO
4)-0.1%
SDS at 65°C, once in 0.3×
SSPE-0.1% SDS at 60°C, and once in 0.1×
SSPE-0.1% SDS at 42°C.
Detection of the EBV PT was performed by
exposing the membrane to Kodak
XAR-5 film at
70°C with
screens.
In vitro viability assays.
Stable cell clones were seeded
into 96-well plates, cultured overnight, and incubated with the
indicated nucleoside analogue for 7 days. Transiently transfected 293T
cells were seeded 24 h after transfection and incubated with GCV
for 4 days. Rael (EBV+) and CA46 (EBV) cells
were seeded into 96-well microtiter plates and treated with 0.5 µM
5-azacytidine for 24 h. After 24 h, the plates were centrifuged to pellet the cells, the medium was removed, and cells were
resuspended in medium containing the indicated nucleoside analogue.
After drug treatment, cell viability was measured using the tetrazolium
salt MTT in a colorimetric 96-well-plate assay (37). MTT
was dissolved in PBS at a concentration of 1 mg/ml. Cells were
incubated with the MTT solution for 4 h at 37°C. The formazan
crystals were solubilized in 0.04 N acid isopropanol. Plates were
assayed at 560 nm using a microplate reader (Cambridge Technologies,
Inc., Watertown, Mass.). Absorbance readings were standardized on
untreated cells. Each experiment was repeated three times, with each
MTT absorbance value representing replicates of six wells. Each bar in
the figures represents the average of three experiments. Error bars
represent the standard errors of the means.
Immunohistochemistry.
Lytic antigen expression was assessed
in Rael cells by immunochemical analysis of cytospin preparations from
treated or untreated cells. Cells (200 µl) diluted to 3 × 105 to 5 × 105 /ml in PBS were cytospun
onto positively charged microscope slides, fixed at 20°C in
acetone-methanol (1:1) for 10 min, and stored dry at 20°C. Before
use, slides were hydrated in distilled H2O, and endogenous
peroxidase activity was blocked. Slides were then incubated with
monoclonal antibodies specific for either EBV Zta (1:200; Dako,
Carpinteria, Calif.) or EBV VCA (1:500; Chemicon International, Inc.,
Temecula, Calif.). In transient transfection analysis, EBV TK
expression was detected using a polyclonal antiserum from a rabbit
immunized with a synthetic EBV TK peptide (described above; 1:200
dilution). Detection was performed by standard indirect detection
methods. The peroxidase-based Dako Duet system (Dako) and 0.5 mg of
diaminobenzidine (Sigma)/ml were used for detection of Zta and VCA. The
alkaline phosphatase-based Vectastain ABC-AP kit (Vector Laboratories,
Burlingame, Calif.) was used for detection of the EBV TK.
Extraction of phosphorylated GCV and HPLC analysis.
TK143b.1, TK143b.2, and V143b.1 cells (2 × 106 to
3 × 106) were seeded into wells of six-well plates
and incubated with [3H]GCV (14.6 Ci/mmol; Moravek
Biochemicals, Brea, Calif.) and 2 to 16 µM GCV (final specific
activity, 0.5 to 4 Ci/mmol) for 60 h at 37°C. Transfected 293T
cells (1.5 × 106 to 2 × 106) were
seeded into wells of six-well plates incubated with
[3H]GCV (14.6 Ci/mmol; Moravek Biochemicals) and 8 µM
GCV for 36 h at 37°C. Following incubation, the cells were
trypsinized, washed three times in PBS, and counted using a
hemacytometer. [3H]GCV was extracted by lysing the cells
in 60% methanol at 80°C for 18 h. Cell lysates were
centrifuged at 13,000 × g for 10 min at 4°C to
remove cell debris and dried in a speed vacuum. Dried extracts were
stored at 80°C until analysis.
Phosphorylated forms of GCV were separated using HPLC with a
strong-anion-exchange column (Whatman Partisil 10-SAX) according
to a
previously described procedure (
14,
45), with minor
modifications.
Cell extracts were reconstituted in 200 µl of
HPLC-grade water
and centrifuged at 13,000 ×
g for 5 min at 4°C, and the supernatant
was injected. Nucleotides were eluted
with a gradient of KH
2PO
4 buffer (pH 3.5) at a
flow rate of 0.5 ml/min (0.02 M KH
2PO
4 [pH
3.5] for 10 min, followed by a linear gradient to 1 M
KH
2PO
4 [pH
3.5] over 45 min and a final 15 min at 1 M KH
2PO
4 [pH 3.5]). Fractions
were
collected every 1 min using an ISCO fraction collector (Lincoln,
Nebr.). Radioactivity was counted using Hydrofluor scintillation
fluid
(National Diagnostics, Atlanta, Ga.) and a Beckman 5000TD
scintillation
counter (Fullerton, Calif.). Picomoles of phosphorylated
and
nonphosphorylated drug were determined by measuring the disintegrations
per minute per picomole of GCV of the extract and of the fractions
after separation. Relative peak retention times for GCV metabolites
were as follows: GCV monophosphate (GCV-MP), 27 to 29; GCV diphosphate
(GCV-DP), 41 to 45; and GCV triphosphate (GCV-TP), 61 to 65. This
procedure had a detection limit of >0.02 pmol of
[
3H]GCV.
| |
RESULTS |
Induction of EBV lytic antigen expression and activity of the viral
TK.
Induction of lytic viral antigen expression was evaluated
after exposure of the EBV+ BL cell line Rael to various
concentrations of 5-azacytidine for 6 h. Expression of EBV Zta,
VCA, and TK was dose dependent. Treatment with 15 µM 5-azacytidine
induced expression of the immediate early antigen Zta in 80% of the
cells and expression of the late lytic antigen VCA in 20% of the cells
at 48 h (Fig. 1A). Immunoreactivity with antiserum to an EBV TK peptide was detected in induced cells as a
70-kDa protein (Fig. 1B), consistent with previous reports of EBV TK
detection by immunoblotting (19, 22, 24). The antiserum
worked well for the immunoblot format but not for immunohistochemical assays, and therefore it was not possible to directly assess the percentage of cells expressing TK in induction experiments.
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FIG. 1.
Induction of lytic antigens by 5-azacytidine. (A) The
EBV+ Burkitt's cell line Rael was incubated with the
indicated concentrations of 5-azacytidine for 6 h, washed three
times, and resuspended in complete medium. Cells were fixed in
acetone-methanol 48 h after incubation with 5-azacytidine. Zta and
VCA were detected by immunohistochemistry. The percentage of
antigen-positive cells was determined by averaging the number of cells
expressing antigen in two high-power fields relative to the total
number of cells in those fields. (B) Detection of EBV TK protein in
5-azacytidine-induced Rael cells by immunoblot. Rael cells were treated
with the indicated concentrations of 5-azacytidine for 6 h and
washed three times, and total cellular protein was isolated 48 h
later. Fifty micrograms of protein per lane was separated by SDS-7.5%
PAGE.
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To determine whether the expression of EBV TK might sensitize cells to
nucleoside analogues, we transfected the TK
143b
osteosarcoma cell line with an EBV TK expression vector
and a control
vector as described in Materials and Methods. An
immunoblot showed
reactivity in the TK-transfected clones (TK143b.1
and TK143b.2) but not
in the control clone (V143b.1) (Fig.
2A).
EBV TK is functional in these cell clones, as evidenced by the
ability
to grow in HAT and by [
3H]thymidine incorporation (Fig.
2B). We measured the sensitivity
of EBV TK-expressing cells and control
cells to several nucleoside
analogues. TK143b cell clones were
moderately more sensitive to
high doses of ACV and PCV (62.5 to 500 µM) and to lower doses
of GCV and BVdU (10 to 100 µM) than was the
control (V143b.1 [TK
]) clone (Fig.
3). Some sensitivity to AZT was found,
but there
was no dose-response relationship. This result is consistent
with
a previous report that EBV TK sensitizes NIH 3T3 cells to BVdU,
but poorly sensitizes them to ACV and GCV (
28).
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FIG. 2.
(A) Immunoblot detection of EBV TK expression in
EBV TK-expressing cell clones (TK143b.1 and TK143b.2). One hundred
micrograms of total cell protein per lane was separated by SDS-7.5%
PAGE. (B) Incorporation of [3H]thymidine in TK-expressing
(TK143b.1 and TK143b.2) and control (V143b.1) cell clones.
Incorporation into V143b.1 cells is similar to background (medium alone
without cells).
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FIG. 3.
Treatment of EBV TK-expressing cells and control cells
with nucleoside analogues in vitro. Cells were seeded into 96-well
microtiter plates at 103 cells/well. After allowing cells
to adhere overnight, cells were incubated with GCV (A), PCV (B), ACV
(C), BVdU (D), S-BVdU (E), or AZT (F) for 7 days. A
colorimetric assay measuring the conversion of MTT to formazan was used
to determine the fraction of cells surviving relative to untreated
controls (100% viable). Each experiment was repeated three times, with
each MTT absorbance value representing replicates of six wells. Each
bar represents the average of three experiments, with error bars
representing the standard errors of the means.
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We carried out further experiments to exclude the possibility that our
results were an artifact of clonal selection. The sensitivity
to GCV
was also assessed by the transient transfection of cellular
TK
+ 293T cells with pEBVTK. Historically, high transfection
efficiencies
can be achieved using this system, and no selection is
employed.
The sensitivity of cellular TK
+ 293T cells
transfected with pEBVTK, but not empty control vector
DNA, to GCV (Fig.
4) supports the hypothesis that the
difference
in sensitivity to nucleoside analogues between EBV
TK-expressing
cells and control cells is due specifically to the
presence of
an overexpressed protein. Previous investigators have
reported
various conflicting results in this regard (
6,
19,
24,
28,
53). We sought to determine whether the sensitivity of
EBV
TK-expressing cells to GCV reflected the specific enzymatic
activity of
EBV TK or reflected a nonspecific cellular toxicity,
possibly
associated with foreign protein overexpression, or cellular
activation
of GCV. To this end, we introduced a point mutation
(A398T) into EBV TK
in the nucleotide-nucleoside binding site
inferred on the basis
of homology with HSV-1 TK (
18). Cells
transfected with
this plasmid (pEBVmutTK) qualitatively expressed
TK, as
assessed by immunohistochemistry (not shown), but were
not sensitized
to GCV (Fig.
4). Cells transfected with HSV-1 TK
(pHSV1TK) showed more
sensitivity to GCV than those transfected
with EBV TK, consistent with
data from other laboratories (
6,
20,
28). Note that at
high levels of GCV (50 µM) both the
EBV and HSV-1 TKs result in the
killing of ~70% of the cells in
the transfection experiment, while
at lower levels of GCV (6.25
µM), EBV TK leads to little or no
killing and HSV-1 TK leads to
the killing of ~60% of the cells,
consistent with a transfection
efficiency of ~70 to 75% (Fig.
4).
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FIG. 4.
Sensitivity of EBV TK-expressing, cellular
TK+ 293T cells to GCV. 293T cells were transfected with
pEBVTK (293-EBV TK), pHSV1TK (293-HSV1 TK), pEBVmutTK (293-EBVmutTK),
or control vector DNA (293-p0 cells). Following transfection, cells
were seeded into a 96-well plate and treated with GCV for 4 days, and
viability was determined by the MTT assay. For each experiment,
concentrations were analyzed in replicates of six wells. Bars represent
the averages of three experiments. Error bars show the standard errors
of the means.
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To directly confirm that EBV TK expression resulted in the
phosphorylation of GCV, we performed HPLC analyses on lysates of
EBV
TK-expressing 143b cells and control cells incubated with
[
3H]GCV. Lysates from EBV TK-expressing cells had 1 to 9 pmol of
GCV-TP/10
6 cells, which was four- to fivefold more
than non-EBV TK-expressing
V143b.1 control cells, when exposed to 2 to
16 µM GCV for 60 h
(Fig.
5A). This
phosphorylation was dose dependent, consistent
with cell killing due to
GCV-TP accumulation at higher concentrations
of GCV. Similarly, in the
transient transfection of 293T cells
with pEBVTK, phosphorylation of
GCV was confirmed by HPLC (Fig.
5B). In further experiments,
transfection with HSV-1 TK was associated
with higher levels of GCV
phosphates (15, 94, and 450 pmol of
GCV/10
6 cells detected
for GCV-MP, -DP, and -TP, respectively) than EBV
TK, while transfection
with EBVmutTK was associated with negligible
levels (data not shown).
Thus, the results of these experiments
parallel the killing
experiments.
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FIG. 5.
(A) Phosphorylation of GCV in TK143b and V143b cells as
determined by HPLC. Cells were incubated with 2 to 16 µM GCV for
60 h. [3H]GCV was added as a tracer, and
phosphorylated products were separated by HPLC. (B) Phosphorylation of
GCV in 293T cells transfected with pEBVTK. Following transfection,
cells were treated with 8 µM GCV using 3[H]GCV as a
tracer, incubated for 36 h at 37°C, and analyzed for GCV
phosphorylation by HPLC. Bars represent the averages of three
experiments. Error bars show the standard errors of the means.
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A second EBV GCV kinase.
Herpesviruses such as
cytomegalovirus, while lacking a TK, are nonetheless able to
phosphorylate GCV (21, 48). The cytomegalovirus GCV kinase
(UL97) has been identified as a PT with homologues throughout the
herpesvirus family (1, 47). Recently we have shown that
human herpesvirus 8 open reading frames with homology to herpesvirus
TKs and PTs both phosphorylate GCV (5). In order to
determine whether the EBV PT homologue, BGLF4, also sensitizes cells to
GCV, we transiently transfected 293T cells with an EBV PT expression
vector as described in Materials and Methods. EBV PT RNA can be induced
by 5-azacytidine in Rael cells (Fig. 6A). Though expression from pEBVPT was not assessed, as shown in Fig. 6
transient transfection of the plasmid pEBVPT into 293T cells was
associated with phosphorylation of GCV and sensitization to GCV-mediated cell killing (25 µM GCV for 4 days, compared with an
empty plasmid control). A comparison with UL97 was not performed. In
contrast to pEBVTK, the putative EBV PT did not sensitize cells to BVdU
or, interestingly, to PCV (data not shown).
View larger version (12K):
[in this window]
[in a new window]
|
FIG. 6.
EBV PT induction and activity. (A) The EBV PT transcript
was detected in Rael cells by Northern blotting after exposure to 1 µM 5-azacytidine overnight. (B) Expression of EBV PT in 293T cells
sensitizes cells to GCV cell killing (25 µM for 4 days), as shown by
MTT analysis. (C) Phosphorylation of GCV as shown by HPLC analysis.
293T cells expressing EBV PT were treated with 8 µM GCV using
[3H]GCV as a tracer as described in Materials and
Methods. Bars represent the averages of three experiments. Error bars
represent the standard errors of the means.
|
|
To determine whether induction of the kinases led to increased
sensitivity to antiviral nucleoside analogues in EBV
+ BL
cell lines, we compared the effects of 5-azacytidine on an
EBV
+ and an EBV
Burkitt's cell line in the
presence and absence of various antiviral
nucleoside analogues.
EBV
+ Rael cells treated with 0.5 µM 5-azacytidine are
induced to express
EBV TK, as demonstrated by an immunoblot (Fig.
7A).
EBV
+ Rael cells were more sensitive than EBV
CA46 cells to the cytotoxic effects of 5-azacytidine, perhaps
as a
result of the induction of lytic viral antigen expression,
reducing
cell numbers by half in the absence of nucleosides and
hampering
evaluation of nucleoside effects. The data suggested
that 5-azacytidine
sensitized an EBV
+, but not an EBV
, BL cell
line to killing by GCV, PCV, BVdU,
S-BVdU, and AZT (Fig.
7B). The effects were not as pronounced as in the transfected
cells
(Fig.
3), and with the exception of AZT, high concentrations
of drug
were necessary to see an effect of 50% or greater. No
nucleoside that
did not sensitize cells was identified.
View larger version (60K):
[in this window]
[in a new window]
|
FIG. 7.
Sensitivity of 5-azacytidine-treated cells to nucleoside
analogues. In separate experiments, Rael (EBV+) and CA46
(EBV) cells were treated with 0.5 µM 5-azacytidine for
24 h. (A) Detection of EBV TK protein in 5-azacytidine-induced
Rael cells by immunoblot analysis. Cells were treated and washed three
times, and total cellular protein was isolated. Five micrograms of
protein per lane was separated by SDS-7.5% PAGE. (B) Cells were
seeded into 96-well microtiter plates at 104 cells/well.
Cells were then treated with 0.5 µM 5-azacytidine for 24 h.
After 24 h, the plates were centrifuged to pellet the cells, the
medium was removed, and cells were resuspended in medium containing GCV
(a), PCV (b), BVdU (c), S-BVdU (d), or AZT (e). Plates were incubated
for an additional 7 days. Viability was determined by the MTT assay.
The values of cells treated with drug are presented as the fractions of
cells surviving relative to untreated controls (100% viable).
Experiments for panels a to d were performed at the same time, while
the experiment for panel e was performed at a later date. Each
experiment was repeated three times, with each MTT absorbance value
representing replicates of six wells. Each bar represents the average
of three experiments, with error bars representing the standard errors
of the means.
|
|
| |
DISCUSSION |
The investigations presented here suggest that pharmacologic
activation of viral kinase gene expression may render cells carrying EBV genomes selectively sensitive to high doses of several nucleoside antiviral agents. EBV TK appeared to sensitize cells to killing by GCV,
PCV, BVdU, and AZT. A putative PT sensitized cells to GCV but not to
BVdU or PCV.
The ability of EBV TK to sensitize cells to killing by GCV has been the
subject of recent conflicting reports (6, 19, 24, 28, 53).
Four previous studies examined the activity (phosphorylation) and
substrate specificity (competition) of purified or partially purified
EBV TK expressed as fusion proteins in bacteria, and all documented TK
function. Three studies further examined GCV: two of the studies showed
no significant thymidine competition by nor phosphorylation of GCV,
whereas one study showed GCV to compete 10-fold more efficiently for
EBV TK than for HSV-1 TK, a result that has not been generally
confirmed. Because it was possible that purified EBV TK lost its
ability to selectively phosphorylate GCV, though not thymidine or
thymidine analogues, when compared with EBV TK protein expressed in
cells, we examined GCV phosphorylation as well as sensitization
to various nucleoside analogues. Our results parallel those of
Loubiere et al., who calculated a selectivity index as the ratio of the
drug concentrations required to reduce thymidine incorporation by 50%
in untransfected parental and EBV TK-transfected NIH 3T3 cells
(28). The selectivity index was 4 for ACV, 12 for GCV, and
1,375 for BVdU. Although the thymidine analogue was >1,000 times more
selective sensitization to purine analogues was suggested. The goal of
this study was to compare two efficient TKs for gene therapy, with EBV
TK serving as a negative control. Since comparisons were made to a
parental line that was neither transfected nor selected with toxic drug and in the absence of an analysis of protein expression or GCV phosphorylation, these results are less conclusive. Gustafson et al.
showed that lysates from an EBV TK+ clone of 143b
TK cells phosphorylated GCV twice as well as a
TK control clone but ~1,000 times less well than an
HSV-1 TK-expressing clone analyzed under the same conditions. However,
they did not see sensitization to GCV or ACV, although they used a
system closely parallel to that used in our studies (19,
20). Some of the discrepancies might be explained by a loss of
viral TK expression, with partial reversion of cells to a
TK+ phenotype, if periodic assessment were not performed.
Such reversion has sometimes been noted in HAT-selected lines (2,
17, 32). To help prevent this problem, our EBV TK-expressing
cell clones were selected in HAT for only 1 week prior to
experimentation. Periodic immunoblotting of total TK143b cell protein
with a polyclonal rabbit antiserum from a rabbit immunized with an EBV
TK peptide confirmed that the EBV TK was being expressed in TK143b cell
clones over time (data not shown). Our sensitization results using two stable (cellular TK) clones were parallel to those of a
transient (cellular TK+) transfection system that
overexpressed EBV TK. However, we were unable to determine transfection
efficiencies, and sensitization was modest (50% at 50 µM GCV for 4 days when EBV TK and EBVmutTK were compared). Our HPLC analysis of a
cell clone that constitutively expressed the EBV TK and of a
transiently transfected cell line overexpressing EBV TK compared with
an empty vector control suggested that the EBV TK does phosphorylate
GCV. Our experiments further raised the possibility that, as suggested
by Gustafson et al. (19), another EBV kinase, the EBV PT,
might phosphorylate and sensitize cells to GCV. Though overall activity
was low, together these experiments suggest that the presence of viral
enzymes could contribute to phosphorylation of and sensitization of
cells to GCV.
Is the use of a combination of a lytio-inducing agent and an antiviral
prodrug appropriate for consideration in the clinical setting in view
of the high doses of antiviral drug that may be required? The
susceptibility of EBV-associated disease in vivo to the combination of
an EBV-inducing agent and GCV has been reported (36; H. Oettle, F. Wilborn, C. A. Schmidt, and W. Siegert, Letter, Blood
82:2257-2258, 1993), supporting the hypothesis that EBV-associated malignancies can be targeted with an inducing agent in
combination with a prodrug. Although the levels of GCV required for
killing in combination with 5-azacytidine in vitro are higher than
those achieved with standard doses of these drugs in vivo, those
standard doses are based on regimens that involve chronic administration. 5-Azacytidine-induced EBV+ cells were
sensitive to GCV when treated with concentrations of GCV at 25 to 50 µM for several days. Peak plasma concentrations of GCV can reach 30 to 40 µM after an intravenous dose of 5 mg of GCV/kg of body weight
(12). The limiting toxicity associated with chronic GCV
administration is myelotoxicity; neutropenia has been associated with
peak plasma levels of GCV exceeding 30 to 50 µM in patients treated
for cytomegalovirus pneumonia (44). GCV administered at
very high doses in a short-term regimen, however, might be associated
with tolerable myelotoxicity when used in combination with myeloid
growth factors. Alternative cancer therapies are consistently
associated with neutropenia. With regard to PCV (active metabolite of
the parent compound famciclovir), no dose-limiting toxicity has been
defined in any regimen and substantially higher plasma levels might be
achieved than are associated with current regimens.
A variety of lytic inducers have been studied in the laboratory. These
include phorbol esters, anti-immunoglobulin antibodies, butyrate, and
DNA methyltransferase inhibitors (3, 30, 50, 56). For the
studies described here, we chose to study a DNA methyltransferase
inhibitor because there is a large amount of clinical experience with
this agent in other settings (7, 9, 29). 5-Azacytidine has
been used clinically in the treatment of leukemia, myelodysplasia, and
hemoglobinopathies for more than two decades at doses that achieve
concentration ranges similar to the concentrations studied. In a recent
clinical trial, 5-azacytidine administered at a dose of 75 mg/m2 per day for 5 to 7 days was associated with
demethylation of EBV genomes in an EBV+ AIDS lymphoma and
in nasopharyngeal carcinoma as assessed by biopsy within 72 h
after treatment (Ambinder, unpublished data). This suggests that
administration of agents that impact on patterns of gene expression in
vitro may also impact on viral gene regulation in vivo.
To implement this therapy in vivo, a large percentage of
EBV+ tumor cells need to be induced. As shown in Fig. 1,
approximately 60% of Rael cells were induced by 5-azacytidine (25 µM) to express the immediate early lytic protein Zta. The ability of
5-azacytidine to induce lytic EBV infection is not limited to one
particular cell line. The Burkitt's-derived Akata cell line and the
nasopharyngeal carcinoma C666 cell line also show lytic activation
following treatment with this agent (unpublished data). Greater levels
of induction are likely to be required in order to target tumors. Other
inducing agents or a combination of inducing agents, such as the
combination of a methyltransferase inhibitor and a histone deacetylase
inhibitor, may be able to induce levels of EBV TK closer to 100%.
Further investigation to optimize induction strategies for specific
EBV-associated tumors is underway.
The principle that EBV-infected cells can be selectively killed by the
combination of an inducer of lytic cycle viral gene expression with an
antiviral nucleoside analogue merits further evaluation. In contrast to
gene therapy approaches for treating EBV+ tumors, the
approach we suggest eliminates the requirement for the introduction of
foreign genes. The demonstration that the EBV TK and PT can be
pharmacologically induced in EBV+ tumor cells and that
these induced cells are sensitive to antiviral nucleoside analogues
suggests a pharmacologic approach which specifically targets
EBV-associated tumor cells.
| |
ACKNOWLEDGMENT |
This work was supported by NIH grant P01 CA81400.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: 1650 Orleans
St., Cancer Research Building, Rm 389, Baltimore, MD 21231. Phone:
(410) 955-5617. Fax: (410) 955-0961. E-mail:
rambind{at}jhmi.edu.
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Antimicrobial Agents and Chemotherapy, July 2001, p. 2082-2091, Vol. 45, No. 7
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.7.2082-2091.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
