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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1721-1727
Antisense to the Epstein-Barr Virus (EBV)-Encoded Latent Membrane
Protein 1 (LMP-1) Suppresses LMP-1 and Bcl-2 Expression and Promotes
Apoptosis in EBV-Immortalized B Cells
By
Jamie L. Kenney,
Mary E. Guinness,
Tyler Curiel, and
Jill Lacy
From the Department of Internal Medicine, Yale University School of
Medicine, New Haven, CT; and the Department of Internal Medicine,
University of Colorado Health Sciences Center, Denver, CO.
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ABSTRACT |
The Epstein-Barr virus (EBV)-encoded latent membrane protein (LMP-1)
is required for viral transformation and functions to protect cells
from apoptotic cell death, in part, by induction of antiapoptotic
genes, including Bcl-2 and A20. We have used antisense
oligodeoxynucleotides targeted to LMP-1 as a strategy to suppress LMP-1
expression and thereby inhibit its functions. We have shown that levels
of LMP-1 protein in EBV-positive lymphoblastoid cell lines can be
reduced by in vitro treatment with unmodified oligodeoxynucleotides
targeted to the first five codons of the LMP-1 open-reading frame.
Furthermore, suppression of LMP-1 was associated with molecular and
phenotypic effects that included downregulation of the LMP-1-inducible
antiapoptotic genes, Bcl-2 and Mcl-1, inhibition of proliferation,
stimulation of apoptosis, and enhancement of sensitivity to the
chemotherapeutic agent, etoposide. These effects were largely
sequence-specific and observed in EBV-positive, but not EBV-negative
cell lines. These studies suggest that lowering expression of LMP-1 in
EBV-associated malignancy might have therapeutic effects and might
synergize with other antitumor agents.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
EPSTEIN-BARR VIRUS (EBV) is associated
with several human lymphoid malignancies, including endemic Burkitt's
lymphoma, acquired immunodeficiency syndrome (AIDS)-related lymphoma,
and lymphoproliferative disease in transplant recipients, as well as
nasopharyngeal carcinoma.1 The biologic activity of EBV
that causally links it to lymphomagenesis is its capacity to
growth-transform resting B cells to immortalized lymphoblastoid cells
that proliferate indefinitely and harbor the virus in a latent
state.2,3 Latent EBV infection is characterized by
restricted expression of viral gene products, including six nuclear
antigens (Epstein-Barr nuclear antigens [EBNAs]-1, -2, -3a, -3b, -3c,
and -LP) and two transmembrane proteins (latent membrane protein
[LMP]-1 and -2) that function cooperatively to initiate and maintain
growth transformation.4,5 Although the precise role of each
of these latent gene products in transformation is not fully
understood, they mediate their transforming functions, in part, by
constitutively activating cellular genes that are involved in
physiologic B-cell activation, proliferation, and survival.
Although transformation of B cells by EBV requires the expression of at
least five latent viral genes (EBNA-1, EBNA-2, EBNA-3a, EBNA-3c, and
LMP-1) and thus cannot be mediated by a single viral gene,3,6-9 LMP-1 most closely mimics a classical oncogene. Transfection of LMP-1 into immortalized rat fibroblasts induces full
phenotypic transformation, rendering them tumorigenic in vivo,10,11 and, in human epithelial cells, LMP-1 expression inhibits differentiation.12 Expression of LMP-1 in B cells
confers a phenotype resembling activated lymphocytes, including
induction of adhesion molecules, homotypic aggregation, increased cell
size, and entry into cell cycle.13,14 In addition, LMP-1
confers a survival advantage to EBV-infected B cells by protecting
cells from apoptosis.15,16 Gene transfer studies in
EBV-negative Burkitt cells have demonstrated that the antiapoptotic
effects of LMP-1 in B cells are mediated, in part, by induction of the antiapoptotic cellular gene, Bcl-2.15,16 In addition, LMP-1 induces expression of other antiapoptotic genes, including A20 and the
homolog of Bcl-2, Mcl-1.17,18 Thus, LMP-1 is a
multifunctional effector of EBV-mediated transformation, modulating not
only cell-surface phenotype and cell growth, but also cell death.
Because LMP-1 is essential for immortalization by EBV and plays a
critical role in preventing apoptosis, suppression of LMP-1 expression
in EBV-immortalized lymphoblastoid cells should have significant
effects on cell growth and survival. To test this hypothesis, we
examined the effects of antisense oligodeoxynucleotides targeted to
LMP-1 sequence in EBV-immortalized lymphoblastoid cells. We observed
suppression of LMP-1 protein levels by antisense targeted to codons one
to five of the LMP-1 open-reading frame. Furthermore, suppression of
LMP-1 was associated with inhibition of proliferation, downregulation
of Bcl-2 and its homolog Mcl-1, induction of apoptosis, and increased
sensitivity to etoposide-mediated cell death.
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MATERIALS AND METHODS |
Cell lines.
X50-7 and 11-23 are EBV-immortalized lymphoblastoid cell lines derived
by infecting umbilical cord lymphocytes with the B958 and FF41 strains
of EBV, respectively. Cells from these two lines are uniformly positive
for EBV as determined by anticomplement immunofluorescence, and express
EBNA-1, EBNA-2, and LMP-1 by immunoblotting. BJAB and Louckes are
EBV-negative Burkitt B-cell lines. X50-7 was generously provided by
George Miller (Yale University, New Haven, CT); BJAB and Louckes were
provided by W.P. Summers (Yale University). 11-23 was derived in our
laboratory. All cells were maintained in RPMI-1640 plus 7.5% fetal
calf serum.
Oligodeoxynucleotides.
Fifteen-mer unmodified oligodeoxynucleotides were custom-made and
purchased from Macromolecular Resources (Colorado State University,
Fort Collins). The appropriate sequence for LMP-1 antisense
corresponding to the complementary sequence of base pairs +1 to +15 of
LMP-1 was derived from the published sequence of the LMP-1 gene of the
B958 strain of EBV.19 Three oligodeoxynucleotide sequences
were used in the described experiments, as follows: LMP-1 antisense
(5 -AAG GTC GTG TTC CAT-3 corresponding to base pairs +1 to +15 of the
LMP-1 open-reading frame) and LMP-1 scrambled antisense sequences,
designated SS1 and SS2 (5-ACG TCA TGC TAG TGT-3 and GTC AGT ACT GCA
TTG-3, respectively, representing random scrambling of the LMP-1
antisense sequence). The lyophilized oligodeoxynucleotides were
resuspended in water just before use and used at a final concentration
of 5, 10, or 50 µmol/L.
Incubation of cells with oligodeoxynucleotides.
Oligodeoxynucleotides were added directly to cell cultures in log phase
of growth (2 to 4 × 105 cells/mL) in RPMI-1640 plus
7.5% fetal calf serum at a final concentration of 5 or 50 µmol/L; at
24-hour intervals, the culture medium was replaced with fresh medium
and oligodeoxynucleotide. Alternatively, to enhance uptake and reduce
the amount of oligodeoxynucleotide, for some experiments, cells were
cultured in the presence of commercially available liposomes
(LIPOFECTIN reagent; GIBCO-BRL, Gaithersburg, MD) in serum-free
artificial medium (Opti-MEM I; GIBCO-BRL) for the first 24 hours of
exposure to oligodeoxynucleotides. Cells were first washed in
serum-free RPMI-1640 twice and resuspended in Opti-MEM with LIPOFECTIN
reagent (50 µg/mL) and oligodeoxynucleotide (10 or 50 µmol/L). For
untreated controls, cells were cultured with liposomes in serum-free
medium in the absence of oligodeoxynucleotides. After 24 hours of
culture, the Opti-MEM was replaced with fresh medium (RPMI-1640 with
7.5% fetal calf serum, as indicated) containing oligodeoxynucleotide
at a concentration of 10 or 50 µmol/L. Cells were exposed to
oligodeoxynucleotide for 24, 30, 48, or 72 hours, as specified. The
Opti-MEM, LIPOFECTIN reagent, and oligodeoxynucleotide were prepared
according to the manufacturerer's protocol (GIBCO-BRL).
Cellular proliferation assays.
Cells (8 × 104) were plated in triplicate in 200 µL
of medium in microtiter wells and cultured for 48 hours with and
without oligodeoxynucleotide (10 µmol/L) using liposomes to enhance
oligodeoxynucleotide uptake during the first 24 hours of culture, as
described earlier. During the last 16 hours of culture, each well was
pulsed with 1 µCi of [3H]thymidine (73 Ci/mmol). Cells
were harvested with a multiple automated sample harvester (Cambridge
Technology, Cambridge, MA), and incorporation of
[3H]thymidine was measured by standard scintillation
counting and expressed as the mean ± SD of triplicate
assays. The two-tailed unpaired t test was used to determine
significance of differences in [3H]thymidine
incorporation between antisense oligodeoxynucleotide- and scrambled
oligodeoxynucleotide-treated cells.
Immunoblotting.
The preparation of cell extracts, electrophoresis, and transfer were
performed as described previously.20 In brief, cells were
obtained after 24, 48, or 72 hours of culture with and without oligodeoxynucleotides, as described earlier. A quantity of 2 × 105 viable cells was used for each condition; cell
viability was determined by trypan blue exclusion, exactly as
previously described.20 For some experiments, if there were
sufficient numbers of viable cells, samples were aliquoted and run in
duplicate sets on the same gel. Total protein from whole-cell lysates
was resolved by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred electrophoretically to
nitrocellulose paper. The immunoblotting procedure was performed
according to the manufacturer's protocol for the chemiluminescent
detection of proteins using the Phototope-HRP Western Blot Detection
Kit (New England Biolabs, Beverly, MA). The following primary
antibodies were used: for LMP-1 detection, a cocktail of four mouse
monoclonal antibodies to LMP-1 was purchased from DAKO (Glostrup,
Denmark); for Bcl-2 detection, mouse monoclonal antibody was purchased
from Alexis (San Diego, CA); for Mcl-1 detection, affinity-purified
rabbit polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA); for EBV nuclear antigen 1 (EBNA-1) detection, human
serum previously characterized as containing high titer of antibody to
EBNA-1 was generously provided by James Jones (National Jewish Center,
Denver, CO); for poly-(ADP-ribose)-polymerase (PARP), rabbit polyclonal
antibody was purchased from Boehringer Mannheim (Indianapolis, IN).
Relative amounts of protein were quantitated by laser scanning
densitometry analysis of the protein bands (Molecular Dynamics,
Sunnyvale, CA).
Apoptosis assays.
To detect apoptosis, immunoblotting assays were performed as described
earlier to detect PARP, a 113-kD protein substrate for
apoptosis-specific proteases from the interleukin-1 -converting enzyme (ICE) family, and its fragments; apoptosis was detected by the
appearance of an 89-kD PARP fragment.21-23 In addition, a
flow cytometric assay based on quantitating DNA breaks was used to
measure apoptosis.24 This method uses terminal
deoxynucleotide transferase (TdT) and Br-dUTP to label exposed 3OH DNA
ends in fixed cells; BrdU-tagged DNA is then quantitated by flow
cytometry using fluorescein-conjugated anti-BrdU antibody. This assay
was performed according to the manufacturer's protocol using the
APO-BRDU kit (Pharmingen, San Diego, CA). The percentage of cells
induced to undergo apoptosis by antisense treatment was
calculated as follows: (percentage of apoptotic antisense-treated
cells  percentage of apoptotic untreated
cells)/(100 percentage of apoptotic untreated cells) × 100.
Drug treatments and cytotoxicity assays.
Etoposide (Sigma, St Louis, MO) was prepared as a 100-mmol/L stock in
dimethyl sulfoxide (DMSO) and stored at 20°C. Before use,
dilutions of the stock solution were made into 30% DMSO. The drug was
added to cell cultures in log phase of growth (2 × 105
cells/mL) plated in triplicate in RPMI-1640 plus 7.5% fetal calf serum
with or without oligodeoxynucleotide (50 µmol/L) at a final concentration of 2, 20, or 200 µmol/L with a final concentration of
DMSO in all cultures that did not exceed 0.3%; control cultures received equivalent DMSO treatment without etoposide. After 24 hours of
exposure to drug, cell viability was determined by trypan blue
exclusion as previously described.20 The percentage of viable cells at each concentration of drug was determined and expressed
as the mean ± SD of triplicate samples.
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RESULTS |
Antisense oligodeoxynucleotides to LMP-1 suppress LMP-1, Bcl-2, and
Mcl-1 proteins.
To determine whether antisense oligodeoxynucleotides to LMP-1 can
suppress LMP-1 protein expression, EBV-positive lymphoblastoid cell
lines were exposed to three different unmodified 15-mer
oligodeoxynucleotide sequences (+1 to +15, +16 to +30, and +8 to +22 of
the LMP-1 open-reading frame). LMP-1 expression was assessed by
immunoblotting after 48 hours of exposure to oligodeoxynucleotides. The
antisense sequence complementary to the first five codons reproducibly
suppressed LMP-1 expression in two different lymphoblastoid cell lines
and was used in all subsequent experiments (Fig
1). Although we observed some variability
in the degree of LMP-1 suppression from experiment to experiment,
protein levels were consistently diminished by more than 70% relative
to untreated cells (range, 70% to >95%). Suppression of LMP-1 was a
sequence-specific effect, since the antisense oligodeoxynucleotide
consistently inhibited LMP-1 expression by no less than 54% relative
to the control oligodeoxynucleotide sequences (range, 54% to >95%).
The control oligodeoxynucleotides either had no effect on LMP-1
expression (SS1; Fig 1, lane 4) or, in some experiments, a modest
inhibitory effect (<40%) relative to untreated cells in the presence
of cationic liposomes (SS2; Fig 2A, lane
3). Furthermore, suppression of LMP-1 by
antisense was protein-specific as well, since expression of the viral
protein EBNA-1 (Fig 1), as well as other nonspecific proteins detected on long exposure of the autoradiographs (data not shown), was unaffected by antisense treatment. Dose-response studies established that unmodified oligodeoxynucleotide at a concentration of 50 µmol/L
consistently suppressed LMP-1 expression (Fig 1). However, with
the addition of cationic liposomes (LIPOFECTIN) during
the first 24 hours of exposure to antisense, the amount of
oligodeoxynucleotide required for reproducible suppression of LMP-1
could be reduced to 10 µmol/L (Fig 2).
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| Fig 1.
LMP-1 antisense oligodeoxynucleotide treatment suppresses
LMP-1 protein levels. Western blot analysis of LMP-1 and EBNA-1 protein
expression in 2 EBV-positive lymphoblastoid cell lines, 11-23 and
X50-7, was performed after exposure to unmodified antisense
oligodeoxynucleotide targeted to LMP-1 (codons 1 through 5) for 48 hours. Lane 1, untreated control; lane 2, LMP-1 antisense-treated (50 µmol/L); lane 3, LMP-1 antisense-treated (5 µmol/L); lane 4, control oligodeoxynucleotide (SS1)-treated (50 µmol/L). Duplicate
sets of samples for each condition were immunoblotted for LMP-1 or
EBNA-1, as described in the Methods.
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| Fig 2.
LMP-1 antisense oligodeoxynucleotide treatment suppresses
LMP-1-inducible antiapoptotic genes, Bcl-2 and Mcl-1. Western blot
analysis of LMP-1, Bcl-2, and Mcl-1 expression in the EBV-positive
lymphoblastoid cell line, X50-7, was performed after exposure to
oligodeoxynucleotide (10 µmol/L in the presence of liposomes) for 48 (A) or 72 (B) hours. Lanes 1, untreated control cultured in liposomes;
lanes 2, antisense-treated; lanes 3, control oligodeoxynucleotide
(SS2)-treated; lane 4, untreated control cultured without liposomes;
lane 5, untreated EBV-negative B-cell line, Louckes. Blots were
sequentially exposed to anti-LMP-1 antibody, anti-Bcl-2 antibody, and
anti-Mcl-1 antibody. Bottom panel shows a representative nonspecific
high-molecular-weight band seen on prolonged exposure of the blot
using LMP-1 antibody, confirming approximately equivalent amounts of
protein loaded per lane.
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LMP-1 has been implicated in the regulation of several cellular genes
that control cell proliferation and cell death.13-18 In
gene-transfer experiments, LMP-1 has been shown to induce expression of
the antiapoptotic gene, Bcl-2, and its homolog, Mcl-1, and to block
apoptosis in serum-starved EBV-negative Burkitt cell lines.15,16,18 To determine whether antisense-mediated
suppression of LMP-1 caused a reduction in the levels of Bcl-2 or Mcl-1
proteins, LMP-1 antisense-treated cells were assayed for Bcl-2 and
Mcl-1 expression by immunoblotting. Exposure to LMP-1 antisense for 48 and 72 hours was associated with suppression of both Bcl-2 and Mcl-1
compared with untreated and control oligodeoxynucleotide-treated cells
(Fig 2). There was no evidence of global suppression of protein
expression by LMP-1 antisense, since multiple nonspecific protein bands
observed on long exposure of the autoradiograph were unaffected by
oligodeoxynucleotide treatment. There was no effect of LMP-1 antisense
on Bcl-2 or Mcl-1 expression in two different EBV-negative cell lines
examined (data not shown).
Antisense to LMP-1 inhibits proliferation of EBV-positive
lymphoblastoid cell lines.
To determine whether antisense-mediated suppression of LMP-1 affected
cellular proliferation, EBV-immortalized lymphoblastoid cell lines were
exposed to LMP-1 antisense or control oligodeoxynucleotides in the
presence of liposomes (LIPOFECTIN) and assayed for proliferation by
[3H]thymidine incorporation. In two different
EBV-positive lymphoblastoid cell lines, LMP-1 antisense treatment for
48 hours significantly decreased proliferation compared with untreated
cells or cells treated with control scrambled oligodeoxynucleotides, as
shown in one of four representative experiments (Fig
3). In contrast to the antiproliferative
effect of LMP-1 antisense in EBV-positive cells, there was no
significant difference in proliferation between antisense-treated and
control oligodeoxynucleotide-treated cells in two different
EBV-negative cell lines. These results suggested the antiproliferative
effect of LMP-1 antisense oligodeoxynucleotide was sequence-specific
and linked to the presence of EBV.
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| Fig 3.
LMP-1 antisense oligodeoxynucleotide treatment inhibits
proliferation of EBV-positive lymphoblastoid cells. Proliferation was
assayed by [3H]thymidine incorporation in 2 EBV-positive
lymphoblastoid cell lines (11-23 and X50-7) and an EBV-negative B-cell
line (BJAB) after exposure to oligodeoxynucleotide (10 µmol/L in
liposomes) for 48 hours. Values represent the mean ± SD of triplicate
assays. ( ) Antisense-treated; ( ) control oligodeoxynucleotide
(SS1)-treated; ( ) untreated control. P values for the mean
difference between antisense and SS1-treated cells are as follows:
11-23, P < .002; X50-7, P = .0003; BJAB, P > .8. P values for the mean difference between SS1-treated
and untreated cells are as follows: 11-23, P > .1; X50-7,
P > .08; BJAB, P > .06.
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LMP-1 antisense stimulates apoptosis.
LMP-1 has been implicated in the suppression of apoptosis of
EBV-immortalized cells by upregulating several antiapoptotic cellular
genes, including Bcl-2, Mcl-1, and A20.15-18 Thus,
suppression of LMP-1 (and Bcl-2 and Mcl-1) expression in lymphoblastoid
cell lines would be predicted to induce apoptotic cell death under conditions of serum deprivation. The antiproliferative effects of LMP-1
antisense treatment supported the possibility that apoptosis would be
stimulated by antisense mediated suppression of LMP-1. To determine
whether LMP-1 antisense treatment promoted apoptotic cell death,
EBV-positive lymphoblastoid cells were exposed to LMP-1 antisense or
control oligodeoxynucleotides in serum-free medium in the presence of
liposomes and assayed for PARP cleavage by immunoblotting at 24 and 48 hours. After 24 hours of exposure to antisense, there was a minimal
decrease in the ratio of the 113-kD PARP to the 89-kD cleavage product
relative to control oligodeoxynucleotide-treated or untreated cells; at
48 hours, the 113-kD PARP was substantially diminished in association
with a further increase in the level of the 89-kD subfragment (Fig 4). A decrease in the ratio of the 113-kD
PARP to the 89-kD subfragment product was not observed in the control
oligodeoxynucleotide-treated cells at 48 hours. These results suggested
that LMP-1 antisense treatment for 48 hours triggered apoptotic cell
death under conditions of serum deprivation.
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| Fig 4.
LMP-1 antisense oligodeoxynucleotide treatment induces
cleavage of PARP. Western blot analysis of PARP expression in the
EBV-positive lymphoblastoid cell line, X50-7, was performed after
exposure to unmodified oligodeoxynucleotide in serum-free medium with
liposomes for 24 and 48 hours. Lanes 1 and 4, untreated controls; lanes
2 and 5, control oligodeoxynucleotide (SS1)-treated; lanes 3 and 6, LMP-1 antisense-treated. The 113-kD PARP and its 89-kD cleavage product
are indicated (arrows).
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To corroborate the finding that antisense to LMP-1 induced apoptosis, a
flow cytometric assay based on quantitation of DNA fragmentation was
used to quantitate the percentage of cells undergoing apoptosis as a
result of LMP-1 antisense treatment. Lymphoblastoid cells (X50-7) or
Louckes cells in log phase of growth were exposed to antisense or
control oligodeoxynucleotide in the presence of liposomes in serum-free
medium for 24 to 48 hours and assayed for apoptosis. Although we
observed background spontaneous apoptosis in the untreated cells
cultured in serum-free medium with liposomes that appeared to increase
with duration of culture, the percentage of apoptotic cells in
antisense-treated cultures was consistently increased relative to
untreated controls or cells treated with control oligodeoxynucleotide
in multiple experiments (range, 22% to 84%; Table 1 and Fig 5).
Treatment with the control scrambled oligodeoxynucleotide did not
increase apoptosis relative to untreated control cultures (Table 1 and
Fig 5). Furthermore, LMP-1
antisense treatment of an EBV-negative cell line (Louckes) did not
stimulate apoptotic cell death by this assay (Fig 5). These findings
provided further support that antisense directed to LMP-1 protein not
only decreased LMP-1 protein levels, but also stimulated the apoptotic pathway in EBV-immortalized lymphoblastoid cells.
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Table 1.
Percentage of EBV-Positive Lymphoblastoid Cells Induced
to Undergo Apoptosis by Treatment With Antisense Oligodeoxynucleotide
Targeted to LMP-1
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| Fig 5.
Flow cytometric analysis of apoptosis using the APO-BRDU
kit. Cells were incubated with Br-dUTP in the presence of TdT enzyme to
incorporate Br-dUTP into exposed 3-OH ends. Quantitation of Br-dUTP
sites was determined by flow cytometric analysis using
fluorescein-conjugated anti-BrdU monoclonal antibody. Positive
(apoptotic) and negative (nonapoptotic) control HL60 cells, treated
with camptothecin or untreated, were provided by the company. (A)
Negative control ( ); positive control (· · ·). (B)
EBV-positive X50-7 cells treated with LMP-1 antisense
oligodeoxynucleotide (· · ·); treated with control SS1
oligodeoxynucleotide (); or untreated (). (C)
EBV-negative Louckes line treated with LMP-1 antisense
oligodeoxynucleotide (· · ·); treated with control SS1
oligodeoxynucleotide (); or untreated ().
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LMP-1 antisense treatment increases sensitivity to the cytotoxic
drug, etoposide.
Previous gene-transfer studies have shown that Bcl-2 expression can
block apoptosis induced by chemotherapeutic agents in vitro.22,25-28 The demonstration that antisense to LMP-1
suppressed not only LMP-1, but also the antiapoptotic proteins, Bcl-2
and Mcl-1, suggested that antisense treatment may sensitize cells to
the cytotoxic effect of chemotherapeutic agents. To determine whether
LMP-1 antisense treatment enhanced sensitivity to the DNA topoisomerase
II inhibitor, etoposide, oligodeoxynucleotide-treated and untreated
lymphoblastoid cells were exposed to increasing concentrations of
etoposide and assayed for viability after 24 hours of exposure. Due to
excessive toxicity of etoposide in the presence of liposomes and
absence of serum, cells were cultured without cationic liposomes using
oligodeoxynucleotide at a concentration of 50 µmol/L for these
experiments. Under these conditions, in the presence of serum, we
observed no significant effect on cell viability of LMP-1-targeted
antisense oligodeoxynucleotide treatment for 24 hours, in the absence
of etoposide, as determined by trypan blue exclusion. In the presence
of etoposide, cell viability diminished with increasing concentrations
of drug in untreated and oligodeoxynucleotide-treated cells. However,
the concentration of etoposide that reduced viability by 50% was
approximately 10-fold less for antisense-treated cells compared with
either control scrambled oligodeoxynucleotide-treated or untreated
cells, as shown in one of four representative experiments (Fig
6). These results indicated that
suppression of LMP-1 using antisense oligodeoxynucleotides enhanced
susceptibility of lymphoblastoid cells to the cytotoxic effects of
etoposide. This effect was sequence-specific, since etoposide
sensitivity of cells treated with control oligodeoxynucleotides was
similar to untreated cells (Fig 6).
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| Fig 6.
LMP-1 antisense oligodeoxynucleotide treatment enhances
sensitivity to etoposide. X50-7 cells were exposed to etoposide in the
presence or absence of oligodeoxynucleotide (50 µmol/L in the
presence of serum) for 24 hours and assayed for viability by trypan
blue exclusion. Values represent the mean ± SD of triplicate samples.
() LMP-1 antisense-treated; () control oligodeoxynucleotide
(SS1)-treated; () untreated control. P values for the mean
difference between antisense-treated and SS1-treated cells in the
presence of etoposide are as follows: 2 µmol/L, P < .002;
20 µmol/L, P < .001; 20 µmol/L, P = .0002.
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DISCUSSION |
The EBV-encoded latent protein, LMP-1, is expressed in the majority of
EBV-associated neoplasms, including posttransplant lymphoproliferative
disease, AIDS-associated lymphoma, and nasopharyngeal carcinoma.29-31 Although the precise role of LMP-1 in the
genesis of these neoplasms is unclear, expression of LMP-1 is an
absolute requirement for immortalization of B cells by
EBV.8 Furthermore, LMP-1 confers the properties of a
classical oncogene when expressed in rodent fibroblasts or human
epithelial cells.10-12 In addition to the profound
phenotypic properties associated with expression of LMP-1 in lymphoid
and nonlymphoid cells, this viral protein has been directly implicated
in protecting cells from apoptotic cell death.15,16
Gene-transfer experiments in a variety of cell lines have shown that
expression of LMP-1 induces the antiapoptotic cellular genes, Bcl-2,
Mcl-1, and A20.15-18 Furthermore, LMP-1 protects cells from
a variety of apoptotic stimuli, including serum withdrawal and p53
activation.15,16,32 The induction of antiapoptotic genes by
LMP-1 presumably underlies the protective effects of LMP-1 on
sensitivity to apoptosis.
Because LMP-1 is expressed in the majority of EBV-associated neoplasms,
but not in normal cells, and has critical functions in promoting cell
survival, it represents a logical target for modulation by antisense
strategies. Our studies have now demonstrated that short-term, daily
treatment of EBV-positive lymphoblastoid cells in vitro with unmodified
antisense oligodeoxynucleotides targeted to the first five codons of
the LMP-1 open-reading frame results in reproducible suppression of
LMP-1 protein. This effect was sequence-specific, since control
oligodeoxynucleotides containing the same base content as the antisense
sequence did not consistently alter LMP-1 levels. The effect was also
protein-specific, since we did not observe global suppression of
protein levels in antisense-treated cells. LMP-1 protein has a very
short half-life,33 and it was possible to observe
suppression of LMP-1 protein as early as 24 hours of antisense
treatment (data not shown); more dramatic suppression was observed
after 48 and 72 hours of exposure to antisense. Due to the instability
of unmodified oligodeoxynucleotides and their associated transient
effects in tissue culture, we used relatively high concentrations of
antisense (50 µmol/L in the absence of liposomes) and treated cells
at 24-hour intervals. However, using nuclease-resistant,
phosphorothioated antisense oligodeoxynucleotides that are more stable
in vivo, we have elicited similar effects on LMP-1 levels and cellular
proliferation using lower concentrations of oligodeoxynucleotide (5 to
10 µmol/L) added at 36-hour intervals in the absence of liposomes
(data not shown).
Importantly, suppression of LMP-1 with antisense was associated with
molecular and phenotypic effects that can be predicted from the known
functions on LMP-1. These effects included downregulation of the
LMP-1-inducible antiapoptotic genes Bcl-2 and Mcl-1, inhibition of
proliferation, stimulation of the apoptotic pathway of cell death under
conditions of serum deprivation, and enhanced sensitivity to the
chemotherapeutic agent, etoposide. The stimulation of apoptosis and
enhanced sensitivity to etoposide likely derived, in part, from
suppression of Bcl-2, since previous studies have shown that Bcl-2
protects EBV-negative B- and pre-B-lymphoid cells from apoptosis induced by serum deprivation or cytotoxic drug
treatment.15,25 The effects of LMP-1 antisense were not
observed in EBV-negative lymphoid cells, confirming that these events
likely derive from sequence-specific suppression of LMP-1.
Surprisingly, we observed minimal nonspecific toxic effects of control
scrambled oligodeoxynucleotides on proliferation, apoptosis, or
viability after exposure to etoposide. This may be attributed, in part,
to the short duration of exposure and the use of unmodified
oligodeoxynucleotides.
The prognosis of EBV-associated lymphomas that occur in the setting of
AIDS or organ transplantation remains poor, and there is a compelling
need for novel, nontoxic therapeutic approaches. Because these
malignancies are EBV-dependent, there is sound rationale for targeting
critical viral products in the design of alternative therapies.
Previously, we have demonstrated that the latent viral gene product,
EBNA-1, can be suppressed using an antisense strategy.20 However, EBNA-1 is a stable protein, and prolonged exposure to antisense oligodeoxynucleotides was required to elicit significant downregulation of protein levels and biologic effects in vitro. We have
now identified a second critical viral gene product, LMP-1, that is
suppressible by antisense oligodeoxynucleotides. Since LMP-1 is
expressed in the majority of EBV-associated neoplasms, and,
importantly, has a short half-life, it represents an ideal target for
suppression by antisense oligodeoxynucleotides as a potential treatment
strategy. Our studies confirm that a relatively short continuous
exposure to LMP-1 antisense in vitro (<72 hours) elicits biologic
effects. Furthermore, the observation that LMP-1 antisense treatment
modulates susceptibility to etoposide has important potential clinical
implications. Since phosphorothioated oligodeoxynucleotides have proven
to be remarkably nontoxic in animals and humans when administered as
continuous infusions,34-36 it may be possible to use
LMP-1-targeted antisense as a nontoxic agent to enhance the
chemosensitivity of these tumors, thereby increasing treatment
efficacy. Indeed, Bcl-2-targeted antisense oligodeoxynucleotide
treatment has recently been shown to enhance chemosensitivity, not only
in vitro, but also in vivo in a murine model of melanoma.34
Further studies using combinations of LMP-1-targeted antisense in
combination with chemotherapeutic agents both in vitro and in vivo
using the murine SCID model of EBV-associated lymphomas should yield
further information regarding the validity and feasibility of this
approach for clinical applications.
| |
FOOTNOTES |
Submitted December 11, 1997;
accepted April 17, 1998.
Supported by Public Health Services Grant No. CA 67396.
Address reprint requests to Jill Lacy, MD, Department of
Internal Medicine, Yale University School of Medicine, 333 Cedar St,
New Haven, CT 06520.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We gratefully acknowledge James Jones (National Jewish Center, Denver,
CO) for providing EBNA-1-positive human serum.
| |
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Abstract
Introduction
Abstract