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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2491-2500
Expression of the Epstein-Barr Virus Protein LMP1 Mediates Tumor
Regression In Vivo
By
Barry W. Cherney,
Cecilia Sgadari,
Chiharu Kanegane,
Frederick Wang, and
Giovanna Tosato
4From the Division of Hematologic Products, Center for Biologics
Evaluation and Research, Food and Drug Administration, Bethesda, MD;
and the Infectious Disease Division, Brigham and Women's Hospital,
Harvard Medical School, Boston, MA.
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ABSTRACT |
By stimulating the expression of murine IP-10 and Mig, CXC
chemokines that inhibit neovascularization and cause damage to established tumor vasculature, human B cells immortalized with Epstein-Barr virus (EBV) can promote an effective antitumor response in
athymic mice. In the present study, we examined the potential role of
EBV in the induction of this antitumor response. Using a panel of
EBV+ and EBV Burkitt lymphoma (BL) cell
lines, a significant correlation was detected between the expression of
the EBV latency gene LMP1 and the occurrence of spontaneous tumor
regression in athymic mice. Inoculation of LMP1+ and
LMP1 BL cells in the same subcutaneous site resulted in
tumors that completely regressed in a manner indistinguishable from
that induced by EBV-immortalized B cells. EBV-converted BL30 and BL41
sublines infected with B95-8 virus expressed LMP1, generated tumors
that frequently regressed spontaneously, and promoted an effective antitumor response against progressively growing tumors. In contrast, the EBV BL30 and BL41 cell lines and the EBV-converted
BL30 and BL41 infected with P3HR-1 virus did not express LMP1 protein,
and generated progressively growing tumors in nude mice. When
transfected with the LMP1 gene, BL41 cells produced tumors that
regressed spontaneously in most cases, and could induce the regression
of tumors derived from BL41 cells transfected with vector alone. Tumors
induced by LMP1-expressing cells expressed murine IP-10 and Mig and
displayed histological evidence of extensive tumor tissue necrosis and
vascular damage. We conclude that the EBV protein LMP1 is likely
responsible for the antitumor response elicited by EBV-immortalized
cells in athymic mice.
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INTRODUCTION |
WHEN INFECTED WITH Epstein-Barr virus
(EBV), B cells can acquire unlimited growth potential, and can thus
represent a continuous challenge to the EBV-infected individual. T-cell
immunity plays a critical role in the defense against EBV infection,
particularly through a potent cytotoxic T-cell response directed at B
cells latently infected with the virus. A subset of latent cycle EBV antigens is preferentially targeted by EBV-immune T cells, including EBNA2A, 3B and 3C, and LMP2.1 T-cell-independent host
responses also contribute to the defense against EBV disease and
EBV-induced lymphoproliferative diseases are rare even in the context
of severe T-cell immunodeficiency.2
Recently, we have reported on an experimental athymic mouse model that
detects an array of non-T-cell-mediated host responses elicited by
EBV-immortalized cells resulting in a potent antitumor response.3-6 When injected subcutaneously into irradiated
athymic mice, human B cells immortalized with EBV are either
nontumorigenic or produce small tumors that soon regress through tissue
necrosis.3,4,7,8 When injected subcutaneously in
conjunction with a variety of human tumor-derived cell lines that are
malignant in this nude mouse model, the EBV-immortalized cells cause
the tumors to undergo a characteristic regression process through
necrosis and scarring.4 Even established subcutaneous human
Burkitt tumors that reproducibly kill athymic mice can be effectively
treated by local inoculation with EBV-immortalized cells.3
Studies aimed at clarifying the mechanisms of tumor regression induced
by EBV-immortalized cells in athymic mice have showed that inhibition
of tumor neovascularization in conjunction with damage to established
tumor vasculature play a central role in this process,3 and
that the CXC chemokines IP-10 (interferon-inducible protein 10) and Mig
are likely mediators of these effects.5,6 IP-10 and Mig are
expressed at much higher levels in lymphoblastoid-cell line-treated
tumor tissues undergoing regression compared with untreated
progressively growing tumors, and these chemokines can act as
inhibitors of growth factor-induced neovascularization in vivo, cause
damage to established tumor vasculature, and exert antitumor effects in
the nude mouse.5,6,9,10 However, the mechanism by which
EBV-immortalized cells elicit the host response leading to the
rejection of a variety of human tumors established in the nude mouse
has remained elusive.
In the course of investigations designed to study Burkitt lymphoma (BL)
growth in athymic mice, we have observed that certain EBV-carrying BL
cell lines undergo spontaneous tumor regression in a manner similar to
that exhibited by EBV-immortalized cells.11 Based on this
observation, we have now examined in detail the role of EBV as a
potential inducer of the host antitumor response observed in athymic
mice. We report that expression of the latent EBV protein LMP1 can
account for the antitumor effects typically induced by lymphoblastoid
cell treatment of Burkitt tumors.
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MATERIALS AND METHODS |
Cell lines.
BL cell lines were either derived from biopsies at the National Cancer
Institute or obtained from the American Type Culture Collection
(Rockville, MD) or the National Institute of General Medical Sciences
(Camden, NJ). The PA682 cell lines were derived from a patient with
acquired immunodeficiency syndrome-associated BL from different sites
of tumor involvement: peripheral blood (PA682PB), bone marrow
(PA682BM2), and pleural effusion (PA682PE2).12,13 All cell
lines were maintained in RPMI 1640 medium (Biofluids) supplemented with
12% heat-inactivated fetal calf serum (FCS; Reheis, Armour
Pharmaceuticals, Kan Ka Kee, IL), 2 mmol/L L-glutamine (GIBCO-BRL, Grand Island, NY), and 5 µg/mL gentamicin (Sigma Chemical Co, St Louis, MO). LMP1-transfected and control BL41
cells, a gift of Dr E. Kieff (Harvard Medical School, Boston, MA), were maintained in guanosine triphosphate (GTP) selection media as previously described.14 All cell lines were
mycoplasma-free, and were HLA-typed for identification.
Animal studies.
Six- to eight-week-old female BALB/c nu/nu mice (National Cancer
Institute) maintained in pathogen-limited conditions were used
throughout the studies. Twenty-four hours before initiation of the
experiments, the mice received 400-rad total body irradiation. Exponentially growing cell lines with a viability greater than 90%
were injected (107 cells from each of the cell lines, in
0.2 mL RPMI 1640 medium containing 10% FCS) subcutaneously into the
right abdominal quadrant through a 25-gauge needle on plastic 1-mL
syringes. All animals were observed weekly and tumor size estimated in
cm2 as the product of two-dimensional caliper measurements
(longest perpendicular length and width).
Western blot analysis.
Protein extracts from cells were solubilized in sodium dodecyl sulfate
(SDS)-sample buffer, boiled for 10 minutes, subjected to
electrophoresis on a 1% SDS/8.0% polyacrylamide gel
(2 × 105 cell equivalents per lane) and transferred
onto Immobilon-P filters (Millipore Corp, Bedford, MA).
Loading accuracy and efficiency of transfer were evaluated by staining
the gels with Coomassie blue (Sigma) and the membranes with Ponceau S
(Sigma). Membranes were incubated for 1 hour with a mouse
monoclonal antibody (MoAb) to EBNA1 (AB-1 mouse IgG1; Oncogene Science,
Cambridge, MA), EBNA2 (PE2, mouse IgG1), or LMP1 (S12,
mouse IgG2a; or CS1-4, mouse IgG1). Antibody bound to antigen was
detected with a horseradish peroxidase-labeled goat anti-mouse antibody
and a chemiluminescence detection system, following the manufacturer's
recommendations (Amersham Life Science Inc, Arlington Heights,
IL).
RNA preparation and reverse transcriptase-polymerase chain reaction
(RT-PCR) analysis.
Total RNA was extracted from cells by the phenol/guanidinium
thiocyanate method (RNAzol; GIBCO-BRL). The first cDNA strand was
synthesized from 5 µg of total RNA using the SuperScript
preamplification system (GIBCO-BRL). The reaction mixtures containing
oligo (dT)12-18 were incubated for 50 minutes at 42°C.
For PCR analysis of EBV genes primer pairs specific for EBNA1 outer
(GTAACTTAGGAAGCGTTTCT and CAATGCAACTTGGACGTTTT), EBNA1 inner
(AGCTTCCCTGGGATGAGCGT and TCTTCCCCGTCCTCGTCCAT), EBNA2 outer
(ACTTTGAGCCACCCACAGTAACCA and TGGAGTGTCTGACAGTTGTTCCTG), and EBNA2
inner (CATAGAAGAAGAAGAGGATGAAGA and GTAGGGATTCGAGGGAATTACTGA) were
selected.15 PCR amplifications were performed in 50 µL of
reaction mixture containing 5 µL of cDNA template, 20 mmol/L Tris-HCl
(pH 8.4), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.2 mmol/L dNTP
mixture, 0.4 µmol/L for each outer primer pair, and 2.5 U of Taq DNA
polymerase (GIBCO-BRL). For the nested PCR, 2.5 µL of the first
amplified product was used as a template with the inner primers. An
initial denaturation step of 3 minutes at 95°C was followed by 30 cycles of denaturation at 95°C for 1 minute, annealing at 57°C to
62°C (depending on the primers used) for 1 minute, and extension at
72°C for 2 minutes on a RoboCycler (Stratagene, La Jolla, CA).
Amplified product was electrophoresed through a 2% agarose gel
prestained with 1 µg/mL of ethidium bromide and was visualized under
UV light.
For PCR analysis of chemokine expression, 80 ng of cDNA was amplified
using radiolabeled dNTP by a semiquantitative PCR.16 The
number of amplification cycles was selected for each primer pair to
provide a signal within the linear portion of a product versus template
amplification curve.16 Aliquots from each amplification reaction were electrophoresed on 6% acrylamide, Tris-borate EDTA gels
(Long Ranger; AT Biochem, Malvern, PA) followed by
autoradiography and quantitation by phosphorimage analysis. Nucleotide
sequences for murine 5 and 3 primers, respectively (followed in
parentheses by annealing temperature in degrees celsius), were as
follows: IP-10, ACCATGAACCCAAGTGCTGCCGTC and GCTTCACTCCAGTTAAGGAGCCCT
(62); Mig, ACTCAGCTCTGCCATGAAGTCCGC and AAAGGCTGCTCTGCCAGGGAAGGC (60); G3PDH, GCCACCCGAAAGACTGTGGATGGC and CATGTAGGCCATGAGGTCCACCAC (57). The
number of amplification cycles used were 21 for G3PDH, 24 for IP-10,
and 28 for Mig. Primers were designed to discriminate between genomic
and cDNA by spanning at least one intron.
Histology.
Tumors were removed in toto, fixed in 10% neutral buffered formalin
solution (Sigma), embedded in paraffin, sectioned at 5 µm, and
stained with hematoxylin and eosin.
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RESULTS |
Spontaneous regression of EBV+ BL in athymic mice.
To examine potential correlations between the presence of EBV in BL
cells and the spontaneous regression of certain Burkitt tumors, we
injected subcutaneously into athymic mice 17 tumorigenic11 EBV+ and EBV BL cell lines, and looked for
the occurrence of spontaneous tumor regression. Eight of
these BL cell lines are EBV+ and nine are
EBV, as determined by RT-PCR analysis of EBNA-1 mRNA
(Table 1). A dose of 107
exponentially growing viable BL cells was selected for injection because previous experiments had shown that this cell dose generally produces the highest tumor take achievable with BL cells.11 The occurrence of a tumor was defined as the appearance of a
subcutaneous mass at the site of injection, measuring at least 0.25 cm2 in surface area. Tumors were considered as undergoing
regression when three consecutive tumor measurements separated by 1 week showed a size reduction of at least 20%, or when, in the absence of measurable tumor size increase, tumor tissue necrosis and/or fibrosis was found to involve at least 25% of tumor tissue sections through the maximum tumor diameter.5 As shown in Table 1, neither the frequency with which these BL cell lines formed tumors nor
the time to tumor appearance differed significantly among EBV+ and EBV BL cell lines. In contrast, the
frequency of spontaneous tumor regression differed substantially among
EBV+ and EBV cell lines. Although six of
eight EBV+ lines formed tumors that spontaneously regressed
at least 50% of the time, only one of nine EBV cell
lines formed tumors that spontaneously regressed at least 50% of the
time. This difference (6 of 8 versus 1 of 9) of spontaneous regression
among tumors induced by EBV+ and EBV cell
lines was significant (P = .015, Fisher's Exact test), as assessed by chi square analysis of a 2 × 2 contingency table. Two of
the eight EBV+ cell lines, Namalwa and Daudi, produced
tumors that either never or infrequently (1 of 14 tumors) regressed
spontaneously. Seven of the nine EBV cell lines produced
tumors that did not regress spontaneously, and one (Louckes) produced
tumors that regressed infrequently (1 of 6 tumors). Spontaneous
regression occurring in tumors induced by the EBV BL
cell lines (BML 895 and Louckes) occurred later than those noted in
tumors induced by the EBV+ cell lines (94 ± 27
v 44 ± 20 days after tumor appearance), raising the
possibility that different mechanisms of tumor regression might be
operative. Together, these experiments showed a significant correlation
between EBV status of a BL cell line and the line's ability to produce
tumors that spontaneously regress in athymic mice.
Regression of EBV BL tumors induced by
EBV+ BL cells.
When injected subcutaneously into athymic mice, EBV-immortalized B
cells either do not give rise to tumors or give rise to small tumors
that spontaneously regress.3 In addition, we found that
EBV-immortalized cells can promote the regression of a variety of human
tumors, including BL tumors, that would otherwise grow progressively in
the subcutaneous tissue of athymic mice.4 We examined
whether EBV+ BL cells could exert a similar antitumor
effect. To this end, we inoculated athymic mice subcutaneously with the
EBV BL cell line CA46 (that regularly gives rise to
progressively growing tumors in this model) either alone or in
conjunction with one of three EBV+ BL cell lines (PA682
BM2, PA682 PB, and PA682 PE213) that frequently undergo
spontaneous regression in this animal model. As a control,
we coinjected the EBV CA46 cell line with one of two
EBV BL cell lines, EW36 and BL41. EW36 is
nontumorigenic11; BL41 gives rise to progressively growing
subcutaneous tumors in athymic mice (Table 1). As shown in Table
2, when injected alone in nude mice, CA46
formed large progressively growing subcutaneous tumors. In contrast,
when injected in conjunction with one of the EBV+ BL cell
lines (PA682 PB, PA682 PE2, or PA682 BM2), CA46 gave rise to tumors
that spontaneously regressed in 50%, 66%, and 100% of cases,
respectively. The remaining tumors were considered as growing
progressively, despite reduced tumor growth compared with the controls,
and some evidence of tumor tissue necrosis (not shown). It should be
noted that all of the evaluable tumors (three mice were killed during
regression for in vitro studies) considered as spontaneously regressing
disappeared completely, and the animals were cured. The pattern of
tumor regression was indistinguishable morphologically from that of
tumors induced by the inoculation of CA46 cells in conjunction with
EBV-immortalized lymphoblastoid cells. No regression was observed when
CA46 cells were coinjected with the EBV BL cell lines
EW36 or BL41 (Table 2). These results show that certain
EBV+ BL cells can give rise to subcutaneous tumors that
spontaneously regress in athymic mice, and promote the regression of
subcutaneous BL tumors that usually grow progressively in this model.
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Table 2.
Incidence of Tumor Growth and Tumor Regression in
Athymic Mice Injected With CA46 BL Cells Alone or in Conjunction
With EBV+ or EBV BL Cells
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BL cell expression of the EBV-encoded LMP1 protein correlates with
tumor regression.
Two of the EBV+ BL cell lines, Namalwa and Daudi, generally
produced subcutaneous tumors that grew progressively in athymic mice
(Table 1), and thus differed from the other six EBV+ cell
lines that generally produced tumors that spontaneously regressed.
EBV+ BL cell lines are known to differ in the patterns of
EBV gene expression and this determines certain phenotypic differences with respect to the expression of cell activation and adhesion molecules, and growth characteristics of the infected
cells.17 We examined the patterns of EBV gene expression in
the panel of EBV+ BL cell lines. The EBV+
control B95-8 cell line expressed the EBNA1, EBNA2, and LMP1 gene by
immunoblotting with the MoAbs AB-1, PE2, and S12 that specifically
recognize these proteins, respectively, whereas the EBV
control cell line Louckes did not (Fig 1). EBNA1 was detected in all
EBV+ BL cell lines, with the exception of the PA682 BM2,
PA682 PB, and PA682 PE2 cell lines (Fig 1).These cell lines express a mutant EBNA1 protein12 that is
not recognized by the anti-EBNA1 MoAb AB-1 used here. The differences
in the apparent molecular weight of EBNA1 detected in these BL cell
lines (Fig 1) is likely attributable to variations in the internal
repeat region 3 within the EBNA1 coding region.18
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| Fig 1.
Western blot analysis of EBV gene expression in BL cell
lines. Cell extracts from 2 × 105 cell equivalents were
electrophoresed through 8% Tris-glycine gels, transferred onto nylon
membranes, probed with specific MoAb antibodies that recognize EBNA1
(AB-1), EBNA2 (PE2), and LMP1 (S12), and developed.
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EBNA2A or EBNA2B, distinguished by differences in the U2 region
encoding EBNA2,19 were detected by immunoblotting with the PE2 MoAb in five of the eight EBV+ BL cell lines. RT-PCR
analysis failed to detect EBNA2 specific transcripts in PA682 PB, PA682
PE2, and Daudi cell lines, indicating that our failure to detect EBNA2
expression in these BL cell lines was not attributable to low level
expression of EBNA2 or to expression of a mutant EBNA2 protein not
recognized by the antibody used (data not shown). This finding is
consistent with the observation that Daudi cells contain a deletion of
EBNA2 coding sequences.20
Expression of LMP1 protein was detected by Western blotting with the
S12 MoAb in all EBV+ BL lines, with the exception of
Namalwa and Daudi (Fig 1). The observed variations in the relative
molecular weight of LMP1-related bands (Fig 1) has been previously
attributed to variation in the carboxyl terminal domain of
LMP1.18 Expression of the LMP1 protein was also
undetectable in the BL cell lines Namalwa and Daudi by Western blotting
with a pool of MoAbs to LMP1 (CS1-4) that recognize distinct LMP1
epitopes (not shown), indicating that the failure to detect LMP1
expression in Namalwa and Daudi was not attributable to the presence of
LMP1 variants. These results show a direct correlation between the
expression of LMP, but not EBNA1 or EBNA2, and tumor regression
associated with EBV+ BL cell lines.
Effects of EBV infection on BL tumor outcome.
To explore further the relationship between LMP1 expression and the
occurrence of spontaneous BL tumor regression in athymic mice, we
examined whether EBV infection of EBV BL cells might
affect the pattern of progressive tumor growth exhibited by
EBV BL cells. To this end, the EBV BL30
and BL41 cell lines and their EBV-converted sublines BL30-B95-8, BL30-P3HR-1, BL41-B95-8, and BL41-P3HR-1 were tested in nude mice. P3HR-1 virus is a defective EBV that is unable to initiate growth transformation and is deleted for a DNA fragment that includes some of
the IR1 copies, all of U2 and IR2, and a portion of U3 regions coding
for EBNA2 and EBNALP.19 Cells infected with
P3HR-1 generally do not express LMP1 because of a loss of the
transactivating function of EBNA2.21 In contrast, B95-8
virus can induce the immortalization of B cells that express nine EBV
latent viral gene products, EBNA1, EBNA2, EBNA3A, EBNA3B, EBNALP, LMP1,
and LMP2.19 We confirmed that the P3HR-1 and
B95-8-converted BL cell lines BL30 and BL41 are EBV infected because
they expressed EBNA1 by RT-PCR analysis (data not shown). As expected,
the B95-8 but not the P3HR-1-infected BL30 and BL41 cell lines
expressed LMP1 by immunoblotting with S12 MoAb (Fig
2A).
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| Fig 2.
LMP1 expression and tumorigenicity of BL30 and BL41 cell
lines. (A) Western blot analysis of LMP1 expression in the BL30 and BL41 cell lines infected with P3HR-1 or B95-8 EBV. Cell extracts from 2 × 105 cell equivalents were electrophoresed
through 8% Tris-glycine gels, transferred onto nylon membranes, and
probed with a murine MoAb to LMP1 (S12). (B) Growth curves of tumors
induced by subcutaneous inoculation of the EBV and
EBV-converted BL30 and BL41 cell lines. Each data point represents the
mean tumor size of all mice in each group (5 to 10 mice per group).
Mice injected with parental or P3HR1-converted cell lines were killed
when the tumors had reached large sizes and impaired animal mobility.
( ), Parental; ( ), P3HR-1; ( ), B95-8.
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Tumors developed in all mice injected with EBV and
EBV-converted BL30 and BL41 cell lines, except for one mouse injected
with BL41-B95-8 (Fig 2B and Table 3).Tumors induced by the EBV and the P3HR-1-converted BL30
and BL41 cell lines exhibited similar growth patterns, forming large
progressively growing tumors with no signs of tumor regression (Fig
2B). As a group, tumors induced by the B95-8-converted BL30 and BL41
cell lines appeared somewhat later, grew at a diminished rate, and
reached a maximum size after approximately 11 weeks without exhibiting
significant size change over the next 3 to 4 weeks (Table 3, and Fig
2B). All 5 tumors induced by BL30-B95-8 and 7 of 8 tumors induced by
BL41-B95 were considered as undergoing spontaneous regression because
they either exhibited progressive size reductions (at least 20% during
3 weeks observation) or displayed extensive tumor tissue necrosis
and/or fibrosis (at least 25% of tumor sections through the
longest diameter). Among the tumors derived from the BL41-B95-8 cell
line, 4 out of 8 displayed complete tumor regression. Only 1 tumor out
of 13 induced by B95-8-converted BL cell lines grew progressively. Tissue excised from this tumor as well as from other representative tumors induced by B95-8-converted BL cell lines was found to have maintained the expression of LMP1 protein (not shown).
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Table 3.
Incidence of Tumor Growth and Regression in Athymic Mice
Injected With EBV-Converted BL30 and BL41 Cell Lines
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In parallel experiments, we examined whether the B95-8-converted BL30
and BL41 cell lines could induce the regression of autologous EBV cell lines. Coinjection of the cell lines BL30 with
BL30-B95-8, and BL41 with BL41-B95-8 into the same subcutaneous site
produced tumors that underwent spontaneous regression in 4 out of 5 cases and 2 out of 5 cases, respectively (Table 3). Immunoblotting of
tissue extracts from representative tumors derived from coinjection of
EBV and EBV+ BL cell lines, including tumors
that failed to spontaneously regress, detected the presence of LMP1
expression. Thus, infection of EBV BL cells with B95-8
EBV changed the outcome of tumors derived from the cell line.
LMP1-transfected EBV BL cells give rise to tumors
that spontaneously regress.
To examine whether LMP1 expression alone is sufficient to confer
subcutaneous BL tumors an ability to spontaneously regress in nude
mice, we examined the outcome of tumors induced by an LMP1-transfected
BL41 cell line, designated LMP1. A 63-Kd (kilodalton) band, corresponding to LMP1 expressed in B95-8 cells, was
detected in the LMP1 but not the GTP-transfected and untransfected
parental BL41 cells by immunoblotting with the S12 MoAb (Fig
3A). As expected, all tumors induced by
inoculation of the parental EBV BL41 cell line and five
out of six tumors induced by inoculation of the GTP vector control BL41
cell line grew rapidly and gave rise to progressively growing tumors
(Table 4 and Fig 3B, top). In addition,
inoculation of vector-transfected BL41 cells (GTP cell line) in
conjunction with LMP1-transfected BL41 cells (LMP1 cell line) produced
subcutaneous tumors that regressed spontaneously in three out of six
cases (Table 4). These results indicate that expression of LMP1 alone
confers EBV BL cells an ability to generate tumors that
spontaneously regress in athymic mice.
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| Fig 3.
LMP1 expression and tumorigenicity of LMP1-transfected
BL41 cells. (A) Western blot analysis of LMP1 expression in parental, B95-8-EBV-converted, and LMP1-transfected BL41 cells. Cell extracts from 2 × 105 cell equivalents were electrophoresed
through 8% Tris-glycine gels, transferred onto nylon membranes, and
probed with a murine MoAb to LMP1 (S12). (B) Growth curves of tumors
induced by subcutaneous inoculation of LMP1 or vector-transfected BL41
cells. Growth curves of tumors from vector-transfected BL41 cells and
LMP1-transfected BL41 cells are depicted for individual mice.
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Table 4.
Incidence of Tumor Growth and Regression in Athymic Mice
Injected With an LMP1-Transfected BL41 Cell Line
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Histology and chemokine expression in EBV BL
tumors expressing LMP1.
To assess further similarities between Burkitt tumor regression induced
by LMP1-expressing EBV BL cell lines and tumor
regression induced by EBV-immortalized lymphoblastoid cells, we
examined tumor tissue histology and chemokine expression. In earlier
studies, extensive characterization of Burkitt tumor regression induced
by lymphoblastoid cells had showed that tumor tissue necrosis
associated with diffuse vascular damage and expression of the CXC
chemokines IP-10 and Mig are hallmarks of these regressing tumors.
Control progressively growing Burkitt tumors induced by inoculation of
BL41 cells (Fig 4A, B, and C) or EBV-P3HR1-converted BL41 cells not
expressing LMP1 (Fig 4D, E, and F)displayed little tumor necrosis and little evidence of vascular damage.
By contrast, regressing tumors induced by inoculation of
EBV-B95-8-converted BL41 cells expressing LMP1 (Fig 4G, H, and I) and
LMP1-transfected BL41 cells (Fig 4J, K, and L) generally displayed
central and homogeneous central necrosis involving large portions of
the tumor mass and extending from the epidermis to the deeper portions
of the tumor (Fig 4G and H). The tumor vasculature both proximal and
distal to the site of tissue necrosis displayed evidence of
intravascular thrombosis and intimal thickening (Fig 4I and L). The
interface between viable and necrotic tumor tissue was infiltrated with
cells identified morphologically as monocytes (Fig 4H and K).
Inflammatory infiltrates with lymphocytes and neutrophils were
unremarkable. This macroscopic and microscopic morphology is
indistinguishable from that exhibited by regressing tumors induced by
the coinjection of EBV Burkitt cells and lymphoblastoid
cells.3-5
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| Fig 4.
Gross and microscopic morphology of LMP1-expressing and
nonexpressing BL tumors. Female BALB/c nu/nu mice were injected
subcutaneously with 1 × 107 cells from exponentially
growing BL41 EBV cell line (A, B, and C);
EBV-P3HR-1-converted BL41 cell line (D, E, and F);
EBV-B95-8-converted BL41 cell line (G, H, and I); or LMP1-transfected
(J, K, and L) BL41 cell line. Tumors were removed in toto 8 to 14 weeks
after the initial inoculation and processed for histology. (A, D, G,
and J) Gross morphology of Burkitt's tumors removed in toto with
abstract epidermis and dermis showing in (A) and (D) mostly
viable-looking tumor tissue with small areas of central necrosis, and
in (G) and (J) tumors with extensive necrosis and little viable tumor
(no magnification). (B, E, H, and K) Microscopic morphology (original
magnification × 10) of Burkitt's tumors showing in (B) and (E)
viable-looking tumor tissue, and in (H) and (I) the abrupt interface
between necrotic and viable tumor tissue. (C, F, I, and L) Higher power
magnification (original magnification × 40) of viable tumor tissue
with patent capillaries containing red blood cells (C) and (F), and
capillaries occluded with thrombi at various stages of organization (I
and L).
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We looked for IP-10 and Mig gene expression in LMP1-expressing Burkitt
tumors exhibiting macroscopic evidence of tissue necrosis, and compared
it with that of control tumors growing progressively. The results of a
semiquantitative RT-PCR analysis from representative regressing and
progressively growing tumors are shown in Fig
5. We found the RT-PCR products of murine
IP-10 and Mig to be more abundant in regressing tumors induced by
either B95-8-converted or LMP1-transfected BL41 cells than in the
progressively growing tumors induced by either P3HR1-converted or
vector control-transfected BL41 cells. Thus, the pattern of IP-10 and
Mig expression in LMP1-expressing Burkitt tumors is indistinguishable
from that displayed by EBV Burkitt tumors treated with
lymphoblastoid cells.5,6
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| Fig 5.
Chemokine mRNA expression in LMP1-expressing and
LMP1-nonexpressing Burkitt's tumors shown by semiquantitative RT-PCR
analysis. Tumor tissue was obtained from Burkitt's tumors established
in BALB/c nu/nu mice by subcutaneous inoculation of 107
cells from LMP1-nonexpressing (parental BL41, P3HR-1-converted BL41,
or GTP-transfected BL41) or LMP1-expressing BL cells (B95-8-converted BL41, LMP1-transfected BL41, or mixtures of GTP and LMP1-transfected BL41 cells). Tumors were obtained either when tumors were greater than
8 cm2 or when they developed typical tumor necrosis and
scarring lesions that are indicative of tumor regression.
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DISCUSSION |
In this study, we have found evidence supporting a pivotal role for the
EBV latency protein LMP1 as an inducer of T-cell independent antitumor
responses elicited by EBV-immortalized cells. When injected subcutaneously into athymic mice, EBV BL cell lines and
EBV+ BL cell lines that do not express LMP1 generally gave
rise to progressively growing subcutaneous tumors. In contrast,
EBV+ BL cell lines expressing LMP1 produced subcutaneous
tumors that spontaneously regressed. When LMP1-expressing BL cell lines
were injected in conjunction with EBV BL cell lines in
the same subcutaneous site, the resultant tumors often regressed
spontaneously, indicating a bystander effect. The importance of LMP1
expression was confirmed in experiments with the EBV BL
cell lines BL30 and BL41, and the EBV+ converted BL30 and
BL41 cell lines. When the infecting virus was derived from the B95-8
cell line and induced the expression of LMP1 protein, the BL30 and BL41
cell lines produced tumors that spontaneously regressed in most nude
mice. However, when the infecting virus was derived from the P3HR-1
cell line and, as expected, did not induce LMP1 expression, the BL30
and BL41 cell lines always produced progressively growing tumors in
this model. Concordantly, tumors derived from coinjection of autologous EBV and LMP1-expressing BL30 or BL41 cell lines
spontaneously regressed in most cases. Furthermore, the
EBV BL41 cell line transfected with the LMP1 gene alone
gave rise to tumors that spontaneously regressed in many cases.
Histologically, regressing Burkitt tumors induced by the inoculation of
LMP1-expressing EBV Burkitt cell lines were
indistinguishable from the regressing Burkitt tumors induced by the
inoculation of EBV Burkitt cell lines in conjunction
with EBV+ lymphoblastoid cell lines.3 In
addition, Burkitt tumors regressing because of their expression of LMP1
expressed the murine chemokines IP-10 and Mig, indicative of the
induction of a host response similar to that induced by lymphoblastoid
cells.5,6
The effects of LMP1 expression on the tumorigenicity of the
EBV BL41 cell line had previously been examined in
transiently immunosuppressed and SCID mice,14 two murine
models that differ substantially from the nude mouse model used here.
In spite of these experimental differences, these models support the
view that expression of LMP1 reduced the ability of BL cells to grow as
subcutaneous tumors in vivo. The investigators have explained these
earlier results on the basis of LMP1 activities on cell growth, and
this was supported by in vitro results documenting that LMP1 expression
impaired BL cells' growth in suspension and reduced their clonability
in agar.14 Other experiments have confirmed that LMP1 can
be toxic or growth inhibitory to cells when expressed at high
levels.22-24 However, LMP1 was also reported to increase
B-cell survival, possibly through induction of the bcl-2
oncogene.25,26
A direct effect of LMP1 on BL cell growth or survival is unlikely to
explain the frequent occurrence of spontaneous tumor regressions
associated with LMP1 expression. Here, tumor regressions occurred 7 weeks (±3 weeks) after tumor appearance, suggesting that regression
of LMP1-expressing BL cells is a consequence of T-cell independent host
responses elicited by LMP1. This would explain why EBV
tumors of various lineages can also be induced to regress by LMP1-expressing cells through a bystander effect.4 Previous experiments have identified several steps in the murine response to
human BL cells that seem critical to tumor regression, including the
expression of the murine chemokines IP-10 and Mig, mediators that can
inhibit tumor angiogenesis and cause vascular damage.5,6 However, the mechanism by which LMP1 can initiate this host response is
unclear.
LMP1, an integral membrane protein consisting of an amino terminal
cytoplasmic domain 23 amino acids long, six hydrophobic transmembrane
domains, and a carboxy terminal cytoplasmic domain 200 amino acids
long, is emerging as a multifunctional protein that plays a critical
role in EBV transformation, and is responsible for many of the
phenotypic changes characteristic of EBV-immortalized cells. Using EBV
recombinants, the cytoplasmic C-terminal 200 amino acids were found to
be required for efficient B-cell immortalization.27 Within
the first 45 C-terminal amino acids (amino acids 187-231), a domain was
identified that enables LMP1 to associate with tumor necrosis factor
receptor associated factors (TRAFs),28-30 components of a
signal transduction pathway used by members of the tumor necrosis
factor receptor family. Because the transmembrane domains enable LMP1
to aggregate in the plasma membrane, LMP1 may mimic the aggregation of
tumor necrosis factor receptor-ligand complexes that leads to
signaling.31-34 Recently, the LMP1 TRAF binding domain was
found to be critical to transformation of B lymphocytes,35 and to mediate activation of NF- B transcription
factor.36,37 An additional and more potent NF-B
activation domain was identified within the LMP1 C-terminal amino acids
352-386, acting independently of the TRAF binding
domain.30,36,37 Because of NF-B's ability to stimulate
transcription of a variety of genes that contain B
elements,38,39 NF-B might mediate many of the phenotypic changes induced by LMP1.17,36,37,40,41
Based on these structure-function relationships, LMP1 could induce an
effective murine response through activation of NF-B transcription
factor, and secondary secretion of cytokines, such as interleukin-6
(IL-6), tumor necrosis factor- (TNF), and IL-12. In previous
experiments, human IL-6 and TNF failed to reproduce the antitumor
effects exhibited by EBV-immortalized cells in the nude
mouse.3 Human IL-12, produced by EBV-immortalized cells, does not stimulate a chemokine response in the mouse because of its
strict species-specificity. However, other NF-B-induced gene products, alone or in combinations, might serve as inducers of an
effective antitumor response in the mouse. An alternative possibility, also involving NF-B transcription factor activation, is that expression of certain adhesion molecules induced by LMP1, such as LFA1
and ICAM,41,40 might be critical to engagement and activation of the host cells. Previous experiments have identified macrophages and endothelial cells as the murine cells primarily responsible for the secretion of IP-10 and Mig in BL tumors that regress.5,6 These cells constitutively express certain
integrins that could serve as receptors for ligands such as LFA1 and
ICAM1.42 Occupation of integrin receptors by their ligands
can result in signaling and cell activation.43
Among EBV latency genes, LMP1 is essential to B-cell
immortalization,35 and is the only EBV gene that can
transform nonlymphoid cells.44 Expression of LMP1 in a
variety of human malignancies, including nasopharyngeal carcinoma,
Hodgkin's disease, nasal and nasal type T/natural killer (NK) cell
lymphomas and lymphoid granulomatosis, is considered relevant to
tumorigenicity in humans.2 Our observations indicate that
in addition to its role in growth transformation, LMP1 also plays a
role as an inducer of antitumor responses. Biopsy specimens from cases
of nasal or nasal-type T/NK cell lymphomas and lymphoid granulomatosis
usually express LMP1 and display histological evidence of tumor tissue
necrosis and vascular damage that are similar to those of BL tumors
treated with lymphoblastoid cells in the mouse.45 We have
detected high level expression of the chemokines IP-10 and Mig in the
tissue specimens from cases of T/NK cell lymphomas and lymphoid
granulomatosis.46 Thus, chemokine expression, vascular
damage, and tumor tissue necrosis might represent evidence of
LMP1-induced responses.
Elucidation of the mechanisms by which LMP1 mediates antitumor
responses will be important to our understanding of the complexities of
this protein and might lead to novel therapeutic approaches to cancer
treatment.
| |
FOOTNOTES |
Submitted August 20, 1997;
accepted November 17, 1997.
Address reprint requests to Barry W. Cherney, PhD, Center for Biologics
Evaluation and Research, Bldg 29A, Room 2D06, 1401 Rockville Pike
(HFM535), Rockville, MD 20852-1448.
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 |
The authors thank Dr G. Gupta for performing statistical analysis, Dr
J. Teruya-Feldstein for histopathologic analysis, and Dr K. Bhatia for
helpful discussions.
| |
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Abstract
Introduction
Abstract