Previous Article | Next Article 
J Virol, April 1998, p. 3138-3145, Vol. 72, No. 4
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Herpesvirus Saimiri Transforms Human T-Cell Clones
to Stable Growth without Inducing Resistance to Apoptosis
Michael S.
Kraft,1
Golo
Henning,1
Helmut
Fickenscher,1
Doris
Lengenfelder,1
Jürg
Tschopp,2
Bernhard
Fleckenstein,1 and
Edgar
Meinl1,*
Institut für Klinische und Molekulare
Virologie, University of Erlangen-Nürnberg, D-91054 Erlangen,
Germany,1 and
Institute of Biochemistry,
University of Lausanne, CH-1066 Epalinges,
Switzerland2
Received 6 November 1997/Accepted 19 December 1997
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ABSTRACT |
Herpesvirus saimiri (HVS) transforms human T cells to stable growth
in vitro. Since HVS codes for two different antiapoptotic proteins,
growth transformation by HVS might be expected to confer resistance to
apoptosis. We found that the expression of both viral antiapoptotic
genes was restricted to cultures with viral replication and absent in
growth-transformed human T cells. A comparative examination of
HVS-transformed T-cell clones and their native parental clones revealed
that the expression of Bcl-2, Bcl-XL, Bax, and members of
the tumor necrosis factor receptor (TNF-R) superfamily with a death
domain, namely, TNF-RI, CD95, and TRAMP, were not modulated by HVS.
Expression of CD30 was induced in HVS-transformed T cells, and these
cells also expressed the CD30 ligand. Uninfected and transformed T
cells were sensitive to CD95 ligation but resistant to apoptosis
mediated by TRAIL or soluble TNF-
. CD95 ligand was constitutively
expressed on transformed but not uninfected parental T cells. Both cell
types showed similar sensitivity to cell death induction or inhibition of T-cell activation mediated by irradiation, oxygen radicals, dexamethasone, cyclosporine, and prostaglandin E2.
Altogether, this study strongly suggests that growth transformation by
HVS is based not on resistance to apoptosis but, rather, on utilization of normal cellular activation pathways.
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INTRODUCTION |
Escape from apoptosis is frequently
an essential component of growth transformation and tumor development.
For example, overexpression of the antiapoptotic Bcl-2 as a consequence
of t(14;18) translocations leads to a follicular B-cell lymphoma. Bcl-2
may cooperate with cellular oncogenes like c-myc and viral
oncogenes like E1A (15). In some systems, expression of
Bcl-2 converts a lytic viral infection to a persistent one
(32).
Herpesvirus saimiri (HVS), a lymphotropic and oncogenic herpesvirus,
which is closely related to human herpesvirus 8 (HHV-8), induces
leukemia and lymphoma in susceptible primates. Different strains of
subgroup C of HVS transform in vitro T cells from New World monkeys,
rhesus monkeys, and humans to stable growth (4, 18, 37).
Human T cells transformed by HVS express the surface phenotype of
mature activated T cells and retain a functionally intact T-cell
receptor (reviewed in reference 38). In contrast to
native T cells, they become activated via CD2 upon binding to its
ligand CD58 (39). Human transformed T cells harbor HVS exclusively in an episomal state and do not release infectious virus
(17).
HVS encodes two antiapoptotic proteins. The HVS-encoded FLICE
inhibitory protein (HVS-FLIP) represents a novel class of viral effectors that inhibit apoptosis mediated by death receptors like CD95
(3, 27, 50). The second antiapoptotic protein of this virus
is a member of the Bcl-2 family, HVS-Bcl-2 (40). A Bcl-2 homolog is also encoded by HHV-8 and by Epstein-Barr virus (EBV), while
a viral FLIP is present in HHV-8 but missing in EBV. In the present
study, we analyzed whether the stable growth of the HVS-transformed T
cells is based on a resistance to apoptosis and examined how the
regulation of proliferation and cell cycle progression is modified by
HVS. To address these questions, three approaches were used. First, we
analyzed the expression pattern of the antiapoptotic genes of HVS in
transformed human T cells that lack viral replication, in transformed T
cells from New World monkeys that release infectious virus, and in
infected permissive owl monkey kidney cells. Second, we analyzed
whether the transformation of T cells with HVS modulates the expression
of cellular proteins involved in the regulation of apoptosis. Third, we
examined whether transformed T cells and uninfected T cells have
different sensitivities to apoptosis. Since native T cells are a
heterogeneous population, these comparative examinations were done with
uninfected T-cell clones and their HVS-transformed derivatives. The
expression of cellular regulators of apoptosis belonging to the Bcl-2
family and the tumor necrosis factor receptor (TNF-R) superfamily was analyzed at the protein level. The TNF-R superfamily includes a set of
molecules with a death domain through which apoptosis is mediated. This
subfamily includes TNF-RI and CD95 (Apo-1 and Fas), as well as the
recently identified death receptors TRAMP (DR3, WSL-1, Apo-3, and LARD)
(6, 12, 29, 34, 45) and several receptors for TRAIL
(41, 42, 44, 46). In addition to the members of the TNF-R
superfamily that contain a death receptor, TNF-RII and CD30 can be
involved in the induction of apoptosis (31, 53) and were
included in our study.
Apoptosis can be triggered from outside the cell via engagement of
death receptors of the TNF-R family or from inside the cell after DNA
damage or after disturbance of the intracellular homeostasis, e.g., by
oxygen radicals. Both main pathways to induce apoptosis were included
in this study. Since regulation of apoptosis and proliferation can be
intimately associated, we analyzed cell death development,
proliferation, and cell cycle distribution after different treatments
that may induce cell death in susceptible targets and interfere with
T-cell activation. Cyclosporine (CsA), prostaglandin E2
(PGE2), dexamethasone, irradiation, and menadione were
selected for these analyses. CsA inhibits the serine/threonine phosphatase calmodulin, blocks the expression of certain T-cell activation genes like the interleukin-2 (IL-2) gene, and arrests cell
cycle progression at G1 (47). PGE-2 enhances the
intracellular cyclic AMP level and reduces the proliferation of native
T cells by inducing a G1 arrest (51).
Glucocorticoids, such as dexamethasone, reduce the expression of IL-2
and of the IL-2 receptor of mature T cells (8) and induce
apoptosis in immature thymocytes and T-cell hybridomas (13).
Irradiation may lead to both cell cycle arrest in G1 or
apoptosis depending on the cell type (13). Menadione gives
rise to oxygen radicals that induce apoptosis. The sensitivity to this
type of apoptosis is reduced in some cell lines by overexpression of
Bcl-2 (25). For comparison, the human T-cell leukemia cell line Jurkat was included in the functional assays.
Our analysis of the expression of viral antiapoptotic genes and of the
potential modulation of cellular apoptosis regulators and our
functional comparisons of native uninfected T-cell clones with their
HVS-transformed derivatives led to the same conclusion: growth
transformation of human T cells by HVS is not associated with
resistance to apoptosis but, rather, depends on the utilization of
cellular activation pathways.
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MATERIALS AND METHODS |
Reagents.
CsA (Novartis, Nuremberg, Germany),
PGE2 (Cascade, London, United Kingdom), dexamethasone,
concanavalin A, menadione, phorbol-12-myristate-13-acetate (PMA) (all
from Sigma), and human recombinant TNF- (R&D, Heidelberg, Germany)
were purchased. The monoclonal antibody (MAb) CH-11 (Immunotech, Marseilles, France) to CD95 has the immunoglobulin M (IgM) isotype and
induces apoptosis. Recombinant Flag-tagged TRAIL (6) and an
anti-Flag MAb (Integra, Fernwald, Germany) were used to study TRAIL-mediated cell death.
T-cell lines and proliferation assays.
The native
CD4+ T-cell clones SS-BP8 and ES-BP8, which are specific
for myelin basic protein, were infected with HVS. The transformed derivatives were termed SS-BP8T (52) and ES-BP8T
(36). The HVS-transformed human T-cell line CB-15
(4) and the transformed T-cell lines P-1079 and P-1081 from
Callithrix jacchus (17) have been described
previously. The HVS-transformed T-cell line 93C488 was established from
Saguinus fuscicollis. None of the transformed human T-cell
lines analyzed produced infectious virus. The transformed T-cell line
from tamarin marmosets had stopped producing infectious virus, while
both transformed T-cell lines from common marmosets kept producing
infectious virus, as was seen in coculture experiments with owl monkey
kidney (OMK) cells.
Human native T-cell clones were restimulated every 2 to 3 weeks with
irradiated HLA-DR-compatible blood cells and bovine myelin basic
protein (Sigma, Deisenhofen, Germany) in a medium that consisted of
45% cell growth medium (Vitromex, Selters, Germany), 45% RPMI 1640, and 10% fetal calf serum (Boehringer, Mannheim, Germany) supplemented
with 2 mM glutamine and 50 µg of gentamicin (Gibco, Berlin, Germany)
per ml. After 2 days, recombinant human IL-2 (Chiron, Ratingen,
Germany) was added at 100 U/ml. The HVS-transformed T-cell clones were
continuously kept in IL-2-containing medium. The human T-cell leukemia
cell line Jurkat and the murine T-cell hybridoma cell line DO were kept
in RPMI 1640 with 10% fetal bovine serum, 2 mM glutamine, and 50 µg
of gentamicin per ml.
HVS-transformed T cells were seeded in 96-well flat-bottom plates at a
density of 5 × 10
4 cells per well in 200 µl of
culture medium without IL-2. Native
T-cell clones were seeded at the
same density along with 2 × 10
5 irradiated
HLA-compatible irradiated peripheral blood mononuclear
cells and 10 µg of myelin basic protein per ml. The Jurkat cells
were seeded at
5 × 10
3 cells per well. These different cell
concentrations were selected
to obtain similar proliferation levels
after 3 days and to compensate
for the different growth rates of the
HVS-transformed and native
T cells on the one hand and the Jurkat cells
on the other. Subsequently,
the immunosuppressive substances were
added. All experiments were
done in triplicate. Two days later, 0.2 µCi of [
3H]thymidine (Amersham, Braunschweig, Germany)
was added for another
16 h. The cultures were harvested with the
cell harvester Matrix
TM96 (Packard, Frankfurt, Germany). The filters
were dried, exposed
overnight on a
[
3H]thymidine-sensitive screen, and analyzed with a
BAS2000 imaging
system (Fuji Raytest, Straubenhardt, Germany).
Comparative measurements
of the same filters with the direct
-counter (Packard) and the
BAS2000 system showed that these two
evaluation systems had a
correlation of >0.95 and comparable
sensitivities.
Determination of cell death.
To quantify cell death, cells
were collected, washed once with phosphate-buffered saline (PBS),
incubated for about 30 min in PBS containing 20 µg of propidium
iodide (PI) per ml, and analyzed with a flow cytometer (FACStrak;
Becton Dickinson, Heidelberg, Germany). Viable and dead cells were
distinguished by both forward-scatter analysis and fluorescence caused
by PI uptake. The specific cell death was calculated as 100 × (% experimental cell death 
spontaneous cell death)/(100% % spontaneous cell death). DNA fragmentation was detected essentially as
described previously (24). Briefly, the cells to be analyzed
were washed with PBS and incubated in a mild lysis buffer (1% Nonidet
P-40, 100 mM EDTA, 50 mM Tris-HCl [pH 7.5]) to enrich for DNA from
cells undergoing apoptosis. The supernatant was obtained after
centrifugation at 1,600 × g, and the pellet was
treated with the same lysis buffer again. After centrifugation, the
supernatants were pooled and SDS was added to a final concentration of
1%. The material was then digested with RNase A and proteinase K (both
from Boehringer). The DNA was precipitated, washed with 70% ethanol,
loaded onto an agarose gel, and stained with ethidium bromide.
Flow cytometry.
The MAbs directed to CD30 (Ki-1;
Immunotech), CD28 (C293; kindly provided by R. de Waal Malefyt
[DNAX]), CD95 (DX2; Pharmingen, Hamburg, Germany), TNF-RI (16803.1;
R&D), TNF-RII (1888-01; Genzyme), and CD95 ligand (Nok-2; Pharmingen)
were used to detect the surface expression of these molecules. Binding
was detected with a fluorescein isothiocyanate-labeled goat anti-mouse
IgG F(ab')2 fragment (Dianova, Hamburg, Germany) or a
fluorescein isothiocyanate-labeled goat anti-rat IgG
F(ab')2 fragment (Dianova) for the MAb to TNF-RII. TRAMP
expression was determined with a polyclonal rabbit Ab (6). The intracellular expression of members of the Bcl-2 family was analyzed on cells that were fixed with 2% formalin and permeabilized with saponin buffer (PBS, 0.5% saponin [Sigma], 5% fetal calf serum, 0.02% NaN3). The MAb to Bcl-2 (sc-509; Santa Cruz,
Heidelberg, Germany) and the polyclonal rabbit antibodies to Bax (Santa
Cruz) and Bcl-X (Dianova) were applied at 1 µg/ml. The Ab applied to Bcl-X is suitable for detection of both the Bcl-XL and the
Bcl-XS isoforms of Bcl-X. Binding of these Abs was detected
with phycoerythrin-labeled goat anti-mouse IgG F(ab')2
fragment and a donkey anti-rabbit IgG F(ab')2 fragment
(Dianova), respectively. The stained cells were analyzed in a FACStrak
flow cytometer. To determine the cell cycle distribution, the cells
were fixed with 70% ethanol and resuspended, after being washed, in
PBS containing 20 µg of PI per ml, 2 mg of RNase A per ml, and 0.1%
glucose. The cell cycle distribution was measured with an Epics
cytometer and evaluated with the program Multicycle (Coulter). The
proportion of cells in the S phase was also determined with the
bromodeoxyuridine detection kit (Boehringer Mannheim).
Western blot analysis.
The applied lysis buffer contained 50 mM Tris-HCl (pH 8.0), 1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 5 mM
NaF, and 10 µg each of aprotinin and leupeptin (Sigma) per ml.
Cleared lysates were separated under reducing conditions on an
SDS-15% polyacrylamide gel and electroblotted on Immobilon P membrane
(Millipore, Eschborn, Germany). The blots were blocked with PBS
containing 5% low-fat milk and 0.05% Tween 20 and incubated with 1 µg of the primary Abs per ml and then with a 1:1,000 dilution of
peroxidase-conjugated goat anti-mouse F(ab')2 or a
peroxidase-conjugated donkey anti-rabbit F(ab')2 fragment
(Amersham). They were developed with the ECL Western blotting detection
system (Amersham).
Transcript analysis and DNA sequencing.
OMK cells were
infected with HVS C488 and collected for RNA preparation when a strong
cytopathic effect was visible. T-cell lines were stimulated with 2 ng
of PMA per ml for 6 h. RNA was prepared by the phenol-guanidinium
thiocyanate method, and polyadenylated mRNA was obtained with
oligo(dT)-coated Dynabeads (Dynal, Hamburg, Germany). RNA was separated
in a formaldehyde-agarose gel and transferred to nylon membranes
(Hybond N; Amersham). Purified DNA fragments were radiolabeled with
[32P]dATP by the random-labeling method and used for
hybridization under stringent conditions. The signals were analyzed
with a BAS2000 imaging system. Virion DNA of HVS C488 was digested with
HindIII and XbaI in parallel reactions. The
resulting overlapping fragments were cloned into Bluescript KS+ vector
(Stratagene, Heidelberg, Germany). Sequencing was done by the Dye
Didesoxy Terminator method (ABI, Weiterstadt, Germany).
Nucleotide sequence accession number.
The
HVS-flip sequence of HVS C488 is available from the EMBL
nucleotide sequence database under accession no. Y13660.
| |
RESULTS |
Viral antiapoptotic genes.
The expression of
HVS-flip and of HVS-bcl-2 was analyzed in four
different cellular systems: (i) infected permissive OMK cells, (ii)
growth-transformed T cells from New World monkeys that produce infectious virus, (iii) growth-transformed T cells from New World monkeys that stopped releasing virus, and (iv) growth-transformed human
T cells that showed a strictly episomal persistence of HVS. We found
that the expression of both HVS-flip and
HVS-bcl-2 was restricted to cultures with viral replication.
Activation with PMA does not induce lytic replication of HVS in human
transformed T cells but enhances the expression of some viral genes
(17, 30). HVS-flip and HVS-bcl-2
transcripts were not detected by Northern blot hybridization in human
transformed T cells, neither constitutively nor after activation with
PMA (Fig. 1A). To increase the
sensitivity of detection, we used polyadenylated RNA for analysis. Again, no transcripts of HVS-flip and HVS-bcl-2
were detected in human growth-transformed T cells, while strong
expression was observed in lytically infected OMK cells (Fig. 1B).
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FIG. 1.
Differential expression of HVS-bcl-2 and
HVS-flip. (A) Total RNA of the indicated cell lines was
loaded. OMK cells were infected with HVS C488 or left uninfected. The
transformed T-cell line 93C488 from Saguinus fuscicollis had
stopped releasing infectious virus and remained a nonproducer of
infectious virus even after stimulation with PMA, while the
HVS-transformed T-cell line P1081 from Callithrix jacchus
produced infectious virus. Like all studied human T-cell lines, the
CB-15 cell line did not produce infectious virus, either before or
after activation with PMA. The top panel shows the transcripts of
HVS-bcl-2, the middle panel shows the transcripts of
HVS-flip, and the bottom panel shows the corresponding rRNA
as detected by ethidium bromide staining. Three pieces of the same
Northern blot are shown. (B) Poly(A)+ RNA of the two human
transformed T-cell lines CB-15 and ES-BP8T and RNA of lytically
infected OMK cells were analyzed. The bottom panel shows the
hybridization with a gapdh probe. In the top panel of both A
and B, the bands at 1.1, 1.8, and ca. 5 kb are specifically detected by
the HVS-bcl-2 probe. In the middle panel of A and B, the
bands at 5 and 7.5 kb are specific for HVS-flip.
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The completely sequenced prototype strain A11 of HVS does not transform
human T cells to stable growth. Therefore, we cloned
and sequenced the
gene coding for HVS-
flip of strain C488 that
transforms
human T cells and found that the amino acid sequence
identity of the
FLIPs of strains A11 and C488 was 94%. Likewise,
the sequence identity
of the HVS-Bcl-2 of strains A11 and C488
is 92.5% (
30).
This suggests that strain-specific sequence differences
of the two HVS
antiapoptotic genes do not contribute to the different
transforming
capacities.
Cellular proteins regulating cell death.
Molecules of the
TNF-R superfamily containing a death domain were expressed at different
levels. CD95 and TNF-RI continued to be expressed at high levels after
transformation, while TRAMP was hardly detectable on the native and
transformed T-cell clones but was expressed on freshly activated
peripheral blood mononuclear cells (Fig.
2). TNF-RII was expressed to a similar
extent before and after transformation. Remarkably, CD30 expression,
which was absent on the parental clones, was clearly induced after
transformation with HVS (Fig. 2). We followed this up and found that
both HVS-transformed and native T cells expressed CD30 ligand as
detected by reverse transcriptase PCR with published primer sequences
(20) (data not shown). HVS-transformed T cells
constitutively expressed CD95 ligand on the cell surface (Fig.
3). In contrast, CD95 ligand was not
constitutively expressed by uninfected parental T cells, but additional
activation was required to detect surface expression (Fig. 3).
Constitutive expression of CD95 ligand by HVS-transformed T cells was
confirmed by reverse transcriptase PCR (data not shown).
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FIG. 2.
Expression of members of the TNF-R superfamily. The
transformed T-cell clone ES-BP8T (top), the native T-cell clone ES-BP8
(middle), and peripheral blood mononuclear cells that had been
activated 6 days before with concanavalin A (bottom) were stained for
the expression of TNF-RI, CD95, TRAMP, TNF-RII, and CD30. The open
graph represents the negative control, and the solid graph represents
the specific staining.
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FIG. 3.
Constitutive expression of CD95 ligand by
HVS-transformed T cells. (A) HVS-transformed T cells were analyzed for
expression of CD95 ligand. (B and C) Their nontransformed parental T
cells were analyzed 2 weeks after the last restimulation (B) or 1 day
after activation with PMA plus ionomycin (C). The open graph represents
the negative control, and the solid graph represents the specific
staining of CD95 ligand.
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Bcl-2 family members were detected both by flow cytometry and by
immunoblotting. The expression of Bcl-2, Bcl-X, and Bax was
altogether
unaltered by the transformation as shown with either
readout system
(Fig.
4). The Western blot analysis
showed that
both the transformed and parental clones exclusively
expressed
the Bcl-X
L and not the Bcl-X
S isoform
of Bcl-X (Fig.
4). The costimulatory
molecule CD28, which can mediate
the prevention of apoptosis via
induction of Bcl-X
L
(
7), was not expressed in the parental
or the transformed T
cells (data not shown).
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FIG. 4.
Expression of Bcl-2, Bcl-X, and Bax. The uninfected
parental T-cell clone ES-BP8 (lanes 1) and its HVS-transformed
derivative ES-BP8T (lanes 2) were analysed by Western blotting (A) and
flow cytometry (B). (A) Aliquots containing 20 µg of total protein
were used to detect Bcl-2 or Bcl-X, and 5 µg of total protein was
loaded to detect Bax. The positions of prestained molecular mass
markers are indicated in kilodaltons. (B) Staining of the native clone
ES-BP8 is labeled 1, and staining of the transformed clone is labeled
with 2. The open graph represents the negative control, and the solid
graph represents the specific staining.
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Sensitivity to apoptosis.
Ligation of CD95 induced apoptosis
in HVS-transformed T cells, as seen by DNA fragmentation (Fig.
5). To quantify the effects of CD95
ligation, both specific cell death and proliferation were measured, and
almost identical dose-response curves were obtained. HVS-transformed
cells showed a sensitivity to CD95 ligation similar to that of the
parental clones 1 day after activation (Fig.
6). The native T cells that had been
activated 6 days before the CD95 ligation were slightly more sensitive.
Jurkat cells, however, were far more sensitive to this kind of cell
death induction (Fig. 6). TNF- did not induce cell death at
concentrations up to 300 ng/ml in either HVS-transformed or native
T-cell clones, although both TNF-RI and TNF-RII were expressed. To
analyze the sensitivity to TRAIL-mediated apoptosis, the different cell
types were treated with recombinant Flag-TRAIL that was cross-linked by
an anti-Flag MAb. Such a treatment with recombinant TRAIL did not
induce cell death in HVS-transformed or native T-cell clones up to a
concentration of 1 µg of Flag-TRAIL per ml. By contrast, a specific
cell death of about 50% was induced by 0.1 µg of Flag-TRAIL per ml
in Jurkat cells.
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FIG. 5.
Induction of apoptosis in HVS-transformed T cells by
CD95 ligation. The HVS-transformed T-cell clone SS-BP8T was treated
with 2 µg of MAb CH-11 per ml, and 107 T cells were
collected at each indicated time after the addition of CH-11. The
method used to obtain fragmented DNA includes a mild detergent lysis
that leads to an enrichment of apoptotic DNA. The DNA in the right lane
was obtained from untreated SS-BP8T (neg). The DNA fragmentation after
treatment with MAbs to CD95 was visualized on an agarose gel. The DNA
size marker is on the left; sizes are given in kilobases.
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FIG. 6.
Quantification of the effects of CD95 ligation.
HVS-transformed T-cell clones ES-BP8T and SS-BP8T, their native
parental T-cell clones, and Jurkat cells were compared. The native
T-cell clones have been activated with irradiated HLA-compatible PBMC
and myelin basic protein. One portion of the native T-cell clones
received MAb CH-11 to CD95 1 day after restimulation, and another
portion of the native T cells was treated with this MAb 6 days after
activation. Spontaneous autocrine proliferation was assessed for the
HVS-transformed T cells and the Jurkat cells. About 24 h after
addition of the MAbs, the cultures were labeled with
[3H]thymidine. The results obtained with the two native
T-cell clones treated 1 day after activation (solid circles) or 6 days
after activation (open circles), the two HVS-transformed T-cell clones
(squares), and Jurkat cells (triangles) were combined. The
proliferation in the absence of anti-CD95 was set as 100%, and the
inhibition (± standard error of the mean [SEM]) was calculated.
Three to six independent experiments were performed per cell line.
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HVS-transformed T cells, native T-cell clones, and Jurkat cells showed
a similar sensitivity to irradiation (Fig.
7) or oxygen
radicals (Fig.
8). We noted that about one-third of the
HVS-transformed
T cells were dead in the absence of any cell
death-inducing treatment.
This level of spontaneous cell death was seen
constantly throughout
our study and distinguishes HVS-transformed T
cells from other
transformed lymphocytic cell lines (data not shown).
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FIG. 7.
Sensitivity to CsA (A), PGE2 (B),
dexamethasone (C), and irradiation (D). HVS-transformed T-cell clones
ES-BP8T and SS-BP8T, their native parental clones, and Jurkat cells
were compared. To induce proliferation, native T-cell clones were
activated with irradiated HLA-compatible peripheral blood mononuclear
cells and myelin basic protein. Spontaneous autocrine proliferation was
assessed for HVS-transformed T cells and Jurkat cells. T cells were
irradiated shortly before seeding, and immunosuppressive reagents were
added at the beginning of the culture. Two days later,
[3H]thymidine was added, the cells were harvested and
another 16 h later. The results obtained with the two native
clones (circles), the two transformed T-cell clones (squares), and the
Jurkat cells (triangles) were combined. Proliferation in the absence of
inhibitor was set as 100%, and the inhibition (± SEM) of three to
four independent experiments was calculated.
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FIG. 8.
Sensitivity to menadione-induced cell death. The
HVS-transformed T-cell clones ES-BP8T and SS-BP8T, their native
parental clones, and Jurkat cells were cultured in the presence of
different concentrations of menadione, and after 48 h PI uptake
was measured and the specific cell death was determined. The native
T-cell clones were treated 6 or 20 days after their last restimulation.
Since no difference in the sensitivity to menadione was detected at
these time points after restimulation, the results of the 10 experiments with the native T-cell clones were combined (triangles).
The results of four experiments per cell line with the two transformed
T-cell clones (squares) and the Jurkat cells (circles) were also
combined, and the mean values (± SEM) are shown.
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Regulation of proliferation and cell cycle progression.
Irradiation of HVS-transformed T cells induced both cell death and an
arrest of the cell cycle at G1 (Table
1). We followed this up and studied the
effects of CsA, PGE2, and dexamethasone in addition to
irradiation. The spontaneous proliferation of HVS-transformed T-cell
clones and the activation-induced proliferation of native T-cell clones
were inhibited to similar extents (Fig. 7). By contrast, the autonomous
proliferation of Jurkat cells was not reduced by PGE2 or
CsA. Cell cycle analysis showed that PGE2, CsA, and
dexamethasone shifted the cell cycle distribution toward
G1. Remarkably, PGE2 stopped the proliferation
almost completely without inducing significant cell death, indicating
that proliferation and cell death are regulated independently. The
sensitivity of the HVS-transformed T cells to dexamethasone was similar
to the sensitivity of the native T-cell clones (Fig. 7) but different
from the reactivity of murine T-cell hybridomas. Murine T-cell
hybridoma cells all died in response to 10
7 M of
dexamethasone (data not shown), whereas at the high concentration of
103 M dexamethasone, only 25% of the HVS-transformed T
cells died within 48 h (Table 1).
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DISCUSSION |
T-cell regulation after transformation with HVS.
The
comparative analysis of HVS-transformed T-cell clones and their native
parental T-cell clones revealed two major points. First, the
transformation with HVS was not associated with an altered sensitivity
to apoptosis, as elaborated in three different approaches. Second,
inhibitors of T-cell activation blocked the spontaneous proliferation
of the transformed T cells and the T-cell receptor-mediated
proliferation of native T cells to a similar extent.
Key mediators in the regulation of cell death are members of the TNF-R
superfamily, in particular, those that contain a death
domain, namely,
TNF-RI, CD95, TRAMP, and receptors for TRAIL.
Our analysis revealed
differences in the expression and in the
functional consequences of
ligation of these four death receptors
in HVS-transformed T cells.
TNF-RI continued to be expressed after
transformation, but both the
transformed and the native T-cell
clones were resistant to
TNF-
-mediated cell death. These cell
types were also resistant to
TRAIL-mediated apoptosis but sensitive
to CD95-mediated cell death.
HVS-transformed T cells and native
T-cell clones were equally reactive
to CD95-mediated apoptosis
1 day after restimulation, whereas native
T-cell clones were more
sensitive 6 days after activation. This is
consistent with the
phenotype of HVS-transformed T cells and the
observation that
activation of native T cells reduces their sensitivity
to CD95-mediated
cell death (
43). The recently identified
death receptor TRAMP
was hardly expressed by either native or
transformed T-cell clones
but was found on activated peripheral blood
mononuclear cells.
This suggests that both native and transformed T
cells downregulate
TRAMP during long-term culture. Our studies of these
four death
receptors led to the same conclusion: HVS does not modulate
their
expression. Importantly, the sensitivity to cell death induction
by these death receptors was not altered through transformation.
This
might be surprising at first, since the HVS-encoded FLIP
prevents
apoptosis induced by death receptors (
50). This study,
however, shows that the expression of HVS-
flip as well as
HVS-
bcl-2 is restricted to cultures with viral replication.
Transcripts
of these genes were not detected in transformed human T
cells
that lack viral replication.
Further analysis of the TNF-R superfamily revealed that TNF-RII
continued to be expressed at the same level whereas CD30 was
induced by
HVS in transformed T cells. CD30 was originally described
as a marker
for Hodgkin's lymphoma, but it is also found on some
non-Hodgkin's
lymphoma cell lines and on a subpopulation of activated
native T cells
(
20). Interaction of CD30 with its ligand may
result in
proliferation, differentiation, or cell death. Direct
comparison of
HVS-transformed T-cell clones with their native
progenitors revealed
that CD30 was induced after transformation
by HVS. CD30 has been found
to interact with TRAF-1 and TRAF-2
(
31), signal-transducing
molecules that also interact with LMP1,
the EBV-encoded oncoprotein
(
16). Since HVS-transformed T cells
expressed both CD30 and
CD30 ligand, a mutual activation of HVS-transformed
T cells via this
receptor-ligand pair seems possible. Further
studies are required to
evaluate a potential role of CD30-mediated
signaling in the stable
growth of HVS-transformed T cells.
HVS-transformed T cells, but not their uninfected parental clones,
constitutively expressed CD95 ligand on their surface.
CD95
ligand-expressing tumor cells can kill attacking cytotoxic
T cells
(
22). In vitro, the simultaneous expression of CD95
ligand
and of its receptor CD95 by HVS-transformed T cells might
contribute to
the spontaneous cell death we observed. In vivo,
the expression of CD95
ligand by HVS-transformed T cells may protect
these transformed T cells
from attack by HVS-specific cytotoxic
T cells, which could facilitate
spread of the virus and development
of lymphoma. The amount of CD95
ligand expression on HVS-transformed
T cells that we detected was
similar to that found on malignant
cells of patients with large
granular lymphocyte leukemia (
49).
Native and HVS-transformed T cells showed a similar sensitivity to
apoptosis mediated by oxygen radicals and to the effects
of irradiation
and dexamethasone. Dexamethasone induced a shift
of the cell cycle
distribution toward G
1 and a minor degree of
cell death
only at very high concentrations. These experiments
demonstrated that
the reactivity of HVS-transformed T cells to
glucocorticoids resembles
the reactivity of mature T cells but
is different from the reactivity
of immature thymocytes or murine
T-cell hybridomas, which are highly
sensitive to glucocorticoid-induced
apoptosis.
To further evaluate a potential modification of the T-cell regulation
after transformation with HVS, the effects of CsA and
PGE
2
on proliferation, cell cycle distribution, and development
of cell
death were analyzed. Both substances reduced the spontaneous
proliferation of HVS-transformed T-cell clones by inducing a cell
cycle
block at the G
1 phase without causing significant cell
death.
This indicates that proliferation and cell death are regulated
independently in HVS-transformed T cells and suggests that the
corresponding regulatory pathways remain functional. The T-cell
leukemia cell line Jurkat showed a different reactivity; the
autocrine
growth of Jurkat cells was not inhibited by CsA or
PGE
2. Importantly,
the spontaneous growth of
HVS-transformed T cells and the antigen-driven
proliferation of native
T-cell clones showed a similar sensitivity
to reagents that interfere
with T-cell activation. This supports
the hypothesis that HVS
transforms human T cells by using normal
cellular activation pathways.
This concept is also supported by
the observation that HVS-transformed
T cells activate each other
via CD2-CD58 interactions and continue to
express a functionally
intact T-cell receptor (
11,
52). The
interactions of the viral
proteins saimiri transformation-associated
protein of C strains
(Stp-C) and tyrosine kinase-interacting protein
(Tip) with the
signal transducing molecules ras (
28) and
p56
lck (
5,
28) also support the
concept that HVS uses cellular
activation pathways for transformation.
Our conclusion that the
transformation by HVS does not induce
resistance to apoptosis
is also supported by the observed spontaneous
cell death of HVS-transformed
T cells and the finding that
HVS-transformed T cells readily undergo
cell death upon ligation of
their T-cell receptor (
10).
Comparison of human lymphocytes transformed by HVS, EBV, and
HTLV-1.
HVS, EBV, and human T-cell leukemia virus type 1 (HTLV-1)
have different effects on the regulation of proliferation and
apoptosis. HTLV-1 infected T cells become resistant to CsA-mediated
inhibition (26). By contrast, the spontaneous growth of
HVS-transformed T cell clones is as sensitive to CsA as the
antigen-induced proliferation of the native parental T-cell clones.
Protection from apoptosis by Tax, the transforming gene of HTLV-1, has
been suggested to favor the survival of HTLV-1-infected cells
(14). Tax reduces the expression of Bax and might thereby
suppress apoptosis in HTLV-1-infected cells (9). The present
study demonstrates that human HVS-transformed T cells continue to
express high levels of the pro-apoptotic protein Bax.
When looking at the effects of EBV on cellular regulation, both
differences from and similarities to HVS become obvious. The
EBV-encoded proteins EBNA-2 and LMP1 induce the expression of
Bcl-2
after transfection into EBV-negative Burkitt's lymphoma
cells
(
19,
23). Fractionated B cells from peripheral blood
constitutively express Bcl-2. EBV infection does not upregulate
Bcl-2,
neither in these cells nor in their immortalized progeny
(
35). Induction of EBV latent genes in Burkitt's lymphoma
cells
renders these B cells resistant to some stimuli that induce
apoptosis
(
21). Our data argue that transformation with HVS
does not induce
resistance to apoptosis and does not modulate the
expression level
of Bcl-2 or Bcl-X
L. Both HVS and EBV code
for a
bcl-2 homolog,
whereas only HVS contains a
flip gene. The EBV-encoded Bcl-2 homolog
is expressed
predominantly during lytic infection, but expression
in latently
infected B lymphocytes has also been reported (
2).
Deletion
of this gene does not affect the ability of EBV to transform
B cells
(
33). Expression of HVS-
bcl-2 is restricted to
cultures
with lytic replication. Transcripts of HVS-
bcl-2
could not be
detected in growth-transformed human T cells. A reduced
glucocorticoid-mediated
signal transduction was described in
EBV-positive Burkitt's lymphomas
(
48). The present study
indicates that HVS does not alter the
sensitivity to the glucocorticoid
dexamethasone. Both EBV-transformed
B cells and HVS-transformed T cells
are very sensitive to DNA
damage. Upon DNA damage by cisplatin,
EBV-transformed B cells
die by apoptosis whereas normal activated B
cells undergo growth
arrest (
1). Upon irradiation,
HVS-transformed T cells arrest
in the G
1 phase of the cell
cycle and some of them die.
In summary, we used three experimental approaches to study effects of
HVS on apoptosis in T-cell transformation. First, analysis
of virus
encoded antiapoptotic genes showed that expression of
HVS-
bcl-2 and HVS-
flip was confined to cultures
with viral replication
and was not associated with T-cell
transformation. Second, analysis
of cellular regulators of apoptosis
demonstrated that HVS neither
upregulated cellular antiapoptotic
effectors nor downregulated
proapoptotic molecules. Constitutive
surface expression of CD95
ligand by HVS-transformed T cells may
contribute to immune evasion
in primate species that are susceptible to
HVS-induced lymphomas.
Third, HVS-transformed T-cell clones were
compared with their
uninfected parental T-cell clones. Both cell types
were equally
sensitive to apoptosis mediated by death receptors or by
disturbances
of intracellular homoestasis. The spontaneous growth of
HVS-transformed
T cells and the T-cell receptor-mediated proliferation
of native
T-cell clones showed a similar sensitivity to inhibitors of
T-cell
activation, while the T-cell leukemia cell line Jurkat showed
a
different response. These findings indicate that HVS transforms
human T
cells to stable growth without inducing resistance to
apoptosis but,
rather, by using normal cellular activation pathways.
| |
ACKNOWLEDGMENTS |
We thank S. Wittmann for technical assistance and P. Rohwer for
help with the cell cycle analysis. We are grateful to B. Biesinger and
B. Bröker for valuable discussions.
This work was supported by the Deutsche Forschungsgemeinschaft (SFB
466), the Bayerische Forschungsstiftung, the Bundesministerium für Bildung und Forschung, and the EU-Biomed 2 Program
(Immunoregulatory aspects of T cell autoimmunity in multiple
sclerosis).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Klinische und Molekulare Virologie, Schlossgarten 4, D-91054
Erlangen, Germany. Phone: 49-9131-853786. Fax: 49-9131-856493. E-mail:
ermeinl{at}viro.med.uni-erlangen.de.
| |
REFERENCES |
| 1.
|
Allday, M. J.,
A. Sinclair,
G. Parker,
D. H. Crawford, and P. J. Farrell.
1995.
Epstein-Barr virus efficiently immortalizes human B cells without neutralizing the function of p53.
EMBO J.
14:1382-1391[Medline].
|
| 2.
|
Austin, P. J.,
E. Flemington,
C. N. Yandava,
J. L. Strominger, and S. H. Speck.
1988.
Complex transcription of the Epstein-Barr virus BamHI fragment H rightward open reading frame 1 (BHRF1) in latently and lytically infected B lymphocytes.
Proc. Natl. Acad. Sci. USA
85:3678-3682[Abstract/Free Full Text].
|
| 3.
|
Bertin, J.,
R. C. Armstrong,
S. Ottilie,
D. A. Martin,
Y. Wang,
S. Banks,
G. H. Wang,
T. G. Senkevich,
E. S. Alnemri,
B. Moss,
M. J. Lenardo,
K. J. Tomaselli, and J. I. Cohen.
1997.
Death effector domain-containing herpesvirus and poxvirus proteins inhibit both Fas- and TNFR1-induced apoptosis.
Proc. Natl. Acad. Sci. USA
94:1172-1176[Abstract/Free Full Text].
|
| 4.
|
Biesinger, B.,
I. Müller Fleckenstein,
B. Simmer,
G. Lang,
S. Wittmann,
E. Platzer,
R. C. Desrosiers, and B. Fleckenstein.
1992.
Stable growth transformation of human T lymphocytes by herpesvirus saimiri.
Proc. Natl. Acad. Sci. USA
89:3116-3119[Abstract/Free Full Text].
|
| 5.
|
Biesinger, B.,
A. Y. Tsygankov,
H. Fickenscher,
F. Emmrich,
B. Fleckenstein,
J. B. Bolen, and B. M. Bröker.
1995.
The product of the herpesvirus saimiri open reading frame 1 (tip) interacts with T cell-specific kinase p56lck in transformed cells.
J. Biol. Chem.
270:4729-4734[Abstract/Free Full Text].
|
| 6.
|
Bodmer, J. L.,
K. Burns,
P. Schneider,
K. Hofmann,
V. Steiner,
M. Thome,
T. Bornand,
M. Hahne,
M. Schroter,
K. Becker,
A. Wilson,
L. E. French,
J. L. Browning,
H. R. MacDonald, and J. Tschopp.
1997.
TRAMP, a novel apoptosis-mediating receptor with sequence homology to tumor necrosis factor receptor 1 and Fas(Apo-1/CD95).
Immunity
6:79-88[Medline].
|
| 7.
|
Boise, L. H.,
A. J. Minn,
P. J. Noel,
C. H. June,
M. A. Accavitti,
T. Lindsten, and C. B. Thompson.
1995.
CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-XL.
Immunity
3:87-98[Medline].
|
| 8.
|
Boumpas, D. T.,
E. D. Anastassiou,
S. A. Older,
G. C. Tsokos,
D. L. Nelson, and J. E. Balow.
1991.
Dexamethasone inhibits human interleukin 2 but not interleukin 2 receptor gene expression in vitro at the level of nuclear transcription.
J. Clin. Invest.
87:1739-1747.
|
| 9.
|
Brauweiler, A.,
J. E. Garrus,
J. C. Reed, and J. K. Nyborg.
1997.
Repression of Bax gene expression by the HTLV-I Tax protein: implications for suppression of apoptosis in virally infected cells.
Virology
231:135-140[Medline].
|
| 10.
|
Bröker, B. M.,
M. S. Kraft,
U. Klauenberg,
F. Le Deist,
J.-P. de Villartay,
B. Fleckenstein,
B. Fleischer, and E. Meinl.
1997.
Activation induces apoptosis in herpesvirus saimiri-transformed T cells independent of CD95 (Fas, APO-1).
Eur. J. Immunol.
27:2774-2780[Medline].
|
| 11.
|
Bröker, B. M.,
A. Y. Tsygankov,
I. Müller Fleckenstein,
A. H. Guse,
N. A. Chitaev,
B. Biesinger,
B. Fleckenstein, and F. Emmrich.
1993.
Immortalization of human T cell clones by herpesvirus saimiri. Signal transduction analysis reveals functional CD3, CD4, and IL-2 receptors.
J. Immunol.
151:1184-1192[Abstract].
|
| 12.
|
Chinnaiyan, A. M.,
C. G. Tepper,
M. F. Seldin,
K. O'Rourke,
F. C. Kischkel,
S. Hellbardt,
P. H. Krammer,
M. E. Peter, and V. M. Dixit.
1996.
FADD/MORT1 is a common mediator of CD95 (Fas/APO-1) and tumor necrosis factor receptor-induced apoptosis.
J. Biol. Chem.
271:4961-4965[Abstract/Free Full Text].
|
| 13.
|
Cohen, J. J.,
R. C. Duke,
V. A. Fadok, and K. S. Sellins.
1992.
Apoptosis and programmed cell death in immunity.
Annu. Rev. Immunol.
10:267-293[Medline].
|
| 14.
|
Copeland, K. F.,
A. G. Haaksma,
J. Goudsmit,
P. H. Krammer, and J. L. Heeney.
1994.
Inhibition of apoptosis in T cells expressing human T cell leukemia virus type I Tax.
AIDS Res. Hum. Retroviruses
10:1259-1268[Medline].
|
| 15.
|
Cory, S.
1995.
Regulation of lymphocyte survival by the bcl-2 gene family.
Annu. Rev. Immunol.
13:513-543[Medline].
|
| 16.
|
Devergne, O.,
E. Hatzivassiliou,
K. M. Izumi,
K. M. Kaye,
M. F. Kleijnen,
E. Kieff, and G. Mosialos.
1996.
Association of TRAF1, TRAF2, and TRAF3 with an Epstein-Barr virus LMP1 domain important for B-lymphocyte transformation: role in NF- B activation.
Mol. Cell. Biol.
16:7098-7108[Abstract].
|
| 17.
|
Fickenscher, H.,
B. Biesinger,
A. Knappe,
S. Wittmann, and B. Fleckenstein.
1996.
Regulation of the herpesvirus saimiri oncogene stpC, similar to that of T-cell activation genes, in growth-transformed human T lymphocytes.
J. Virol.
70:6012-6019[Abstract].
|
| 18.
|
Fickenscher, H.,
C. Bökel,
A. Knappe,
B. Biesinger,
E. Meinl,
B. Fleischer,
B. Fleckenstein, and B. M. Bröker.
1997.
Functional phenotype of transformed human alphabeta and gammadelta T cells determined by different subgroup C strains of herpesvirus saimiri.
J. Virol.
71:2252-2263[Abstract].
|
| 19.
|
Finke, J.,
R. Fritzen,
P. Ternes,
P. Trivedi,
K. J. Bross,
W. Lange,
R. Mertelsmann, and G. Dolken.
1992.
Expression of bcl-2 in Burkitt's lymphoma cell lines: induction by latent Epstein-Barr virus genes.
Blood
80:459-469[Abstract/Free Full Text].
|
| 20.
|
Gattei, V.,
M. Degan,
A. Gloghini,
A. De Iuliis,
S. Improta,
F. M. Rossi,
D. Aldinucci,
V. Perin,
D. Serraino,
R. Babare,
V. Zagonel,
H. J. Gruss,
A. Carbone, and A. Pinto.
1997.
CD30 ligand is frequently expressed in human hematopoietic malignancies of myeloid and lymphoid origin.
Blood
89:2048-2059[Abstract/Free Full Text].
|
| 21.
|
Gregory, C. D.,
C. Dive,
S. Henderson,
C. A. Smith,
G. T. Williams,
J. Gordon, and A. B. Rickinson.
1991.
Activation of Epstein-Barr virus latent genes protects human B cells from death by apoptosis.
Nature
349:612-614[Medline].
|
| 22.
|
Hahne, M.,
D. Rimoldi,
M. Schroter,
P. Romero,
M. Schreier,
L. E. French,
P. Schneider,
T. Bornand,
A. Fontana,
D. Lienard,
J. Cerottini, and J. Tschopp.
1996.
Melanoma cell expression of Fas(Apo-1/CD95) ligand: implications for tumor immune escape.
Science
274:1363-1366[Abstract/Free Full Text].
|
| 23.
|
Henderson, S.,
M. Rowe,
C. Gregory,
D. Croom Carter,
F. Wang,
R. Longnecker,
E. Kieff, and A. Rickinson.
1991.
Induction of bcl-2 expression by Epstein-Barr virus latent membrane protein 1 protects infected B cells from programmed cell death.
Cell
65:1107-1115[Medline].
|
| 24.
|
Herrmann, M.,
H. M. Lorenz,
R. Voll,
M. Grunke,
W. Woith, and J. R. Kalden.
1994.
A rapid and simple method for the isolation of apoptotic DNA fragments.
Nucleic Acids. Res.
22:5506-5507[Free Full Text].
|
| 25.
|
Hockenbery, D. M.,
Z. N. Oltvai,
X. M. Yin,
C. L. Milliman, and S. J. Korsmeyer.
1993.
Bcl-2 functions in an antioxidant pathway to prevent apoptosis.
Cell
75:241-251[Medline].
|
| 26.
|
Hollsberg, P.,
K. W. Wucherpfennig,
L. J. Ausubel,
V. Calvo,
B. E. Bierer, and D. A. Hafler.
1992.
Characterization of HTLV-I in vivo infected T cell clones. IL-2-independent growth of nontransformed T cells.
J. Immunol.
148:3256-3263[Abstract].
|
| 27.
| Hu, S., C. Vincenz, M. Buller, and V. M. Dixit. 1997. A novel family of viral death effector
domain-containing molecules that inhibit both CD-95- and tumor necrosis
factor-1-induced apoptosis. J. Biol. Chem. 9621-9624.
|
| 28.
|
Jung, J. U.,
S. M. Lang,
U. Friedrich,
T. Jun,
T. M. Roberts,
R. C. Desrosiers, and B. Biesinger.
1995.
Identification of Lck-binding elements in tip of herpesvirus saimiri.
J. Biol. Chem.
270:20660-20667[Abstract/Free Full Text].
|
| 29.
|
Kitson, J.,
T. Raven,
Y. P. Jiang,
D. V. Goeddel,
K. M. Giles,
K. T. Pun,
C. J. Grinham,
R. Brown, and S. N. Farrow.
1996.
A death-domain-containing receptor that mediates apoptosis.
Nature
384:372-375[Medline].
|
| 30.
|
Knappe, A.,
C. Hiller,
M. Thurau,
S. Wittmann,
H. Hofmann,
B. Fleckenstein, and H. Fickenscher.
1997.
The superantigen-homologous viral immediate-early gene ie14/vsag in herpesvirus saimiri-transformed human T cells.
J. Virol.
71:9124-9133[Abstract].
|
| 31.
|
Lee, S. Y.,
C. G. Park, and Y. Choi.
1996.
T cell receptor-dependent cell death of T cell hybridomas mediated by the CD30 cytoplasmic domain in association with tumor necrosis factor receptor-associated factors.
J. Exp. Med.
183:669-674[Abstract/Free Full Text].
|
| 32.
|
Levine, B.,
Q. Huang,
J. T. Isaacs,
J. C. Reed,
D. E. Griffin, and J. M. Hardwick.
1993.
Conversion of lytic to persistent alphavirus infection by the bcl-2 cellular oncogene.
Nature
361:739-742[Medline].
|
| 33.
|
Marchini, A.,
B. Tomkinson,
J. I. Cohen, and E. Kieff.
1991.
BHRF1, the Epstein-Barr virus gene with homology to Bc12, is dispensable for B-lymphocyte transformation and virus replication.
J. Virol.
65:5991-6000[Abstract/Free Full Text].
|
| 34.
|
Marsters, S. A.,
J. P. Sheridan,
C. J. Donahue,
R. M. Pitti,
C. L. Gray,
A. D. Goddard,
K. D. Bauer, and A. Ashkenazi.
1996.
Apo-3, a new member of the tumor necrosis factor receptor family, contains a death domain and activates apoptosis and NF-kappa B.
Curr. Biol.
6:1669-1676[Medline].
|
| 35.
|
Martin, J. M.,
D. Veis,
S. J. Korsmeyer, and B. Sugden.
1993.
Latent membrane protein of Epstein-Barr virus induces cellular phenotypes independently of expression of Bcl-2.
J. Virol.
67:5269-5278[Abstract/Free Full Text].
|
| 36.
|
Meinl, E.,
B. A. 't Hart,
R. E. Bontrop,
R. M. Hoch,
A. Iglesias,
R. de Waal Malefyt,
H. Fickenscher,
I. Müller Fleckenstein,
B. Fleckenstein,
H. Wekerle,
R. Hohlfeld, and M. Jonker.
1995.
Activation of a myelin basic protein-specific human T cell clone by antigen-presenting cells from rhesus monkeys.
Int. Immunol.
7:1489-1495[Abstract/Free Full Text].
|
| 37.
|
Meinl, E.,
H. Fickenscher,
R. M. Hoch,
R. de Waal Malefyt,
B. A. 't Hart,
H. Wekerle,
R. Hohlfeld, and B. Fleckenstein.
1997.
Growth transformation of antigen-specific T cell lines from rhesus monkeys by herpesvirus saimiri.
Virology
229:175-182[Medline].
|
| 38.
|
Meinl, E.,
R. Hohlfeld,
H. Wekerle, and B. Fleckenstein.
1995.
Immortalization of human T cells by herpesvirus saimiri.
Immunol. Today
16:55-58[Medline].
|
| 39.
|
Mittrücker, H. W.,
I. Müller Fleckenstein,
B. Fleckenstein, and B. Fleischer.
1992.
CD2-mediated autocrine growth of herpes virus saimiri-transformed human T lymphocytes.
J. Exp. Med.
176:909-913[Abstract/Free Full Text].
|
| 40.
|
Nava, V. E.,
E. H.-Y. Cheng,
M. Veliuona,
S. Zou,
R. J. Clem,
M. L. Mayer, and J. M. Hardwick.
1997.
Herpesvirus saimiri encodes a functional homolog of the human bcl-2 oncogene.
J. Virol.
71:4118-4122[Abstract].
|
| 41.
|
Pan, G.,
J. Ni,
Y.-F. Wei,
G.-L. Yu,
R. Gentz, and V. M. Dixit.
1997.
An antagonist decoy receptor and a death domain-containing receptor for TRAIL.
Science
277:815-818[Abstract/Free Full Text].
|
| 42.
|
Pan, G.,
K. O'Rourke,
A. M. Chinnaiyan,
R. Gentz,
R. Ebner,
J. Ni, and V. M. Dixit.
1997.
The receptor for the cytotoxic ligand TRAIL.
Science
276:111-113[Abstract/Free Full Text].
|
| 43.
|
Peter, M. E.,
F. C. Kischkel,
C. G. Scheuerpflug,
J. P. Medema,
K. M. Debatin, and P. H. Krammer.
1997.
Resistance of cultured peripheral T cells towards activation-induced cell death involves a lack of recruitment of FLICE (MACH/caspase 8) to the CD95 death-inducing signaling complex.
Eur. J. Immunol.
27:1207-1212[Medline].
|
| 44.
|
Schneider, P.,
J. L. Bodmer,
M. Thome,
K. Hofmann,
N. Holler, and J. Tschopp.
1997.
Characterization of two receptors for TRAIL.
FEBS Lett.
416:329-334[Medline].
|
| 45.
|
Screaton, G. R.,
X.-N. Xu,
A. L. Olsen,
A. E. Cowper,
R. Tan,
A. J. McMichael, and J. I. Bell.
1997.
LARD: a new lymphoid-specific death domain containing receptor regulated by alternative pre-mRNA splicing.
Proc. Natl. Acad. Sci. USA
94:4615-4619[Abstract/Free Full Text].
|
| 46.
|
Sheridan, J. P.,
S. A. Marsters,
R. M. Pitti,
A. Gurney,
M. Skubatch,
D. Baldwin,
L. Ramakrishnan,
C. L. Gray,
K. Baker,
W. I. Wood,
A. D. Goddard,
P. Godowski, and A. Ashkenazi.
1997.
Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors.
Science
277:818-821[Abstract/Free Full Text].
|
| 47.
|
Sigal, N. H., and F. J. Dumont.
1992.
Cyclosporin A, FK-506, and rapamycin: pharmacologic probes of lymphocyte signal transduction.
Annu. Rev. Immunol.
10:519-560[Medline].
|
| 48.
|
Sinclair, A. J.,
M. G. Jacquemin,
L. Brooks,
F. Shanahan,
M. Brimmell,
M. Rowe, and P. J. Farrell.
1994.
Reduced signal transduction through glucocorticoid receptor in Burkitt's lymphoma cell lines.
Virology
199:339-353[Medline].
|
| 49.
|
Tanaka, M.,
T. Suda,
K. Haze,
N. Nakamura,
K. Sato,
F. Kimura,
K. Motoyoshi,
M. Mizuki,
S. Tagawa,
S. Ohga,
K. Hatake,
A. H. Drummond, and S. Nagata.
1996.
Fas ligand in human serum.
Nat. Med.
2:317-322[Medline].
|
| 50.
|
Thome, M.,
P. Schneider,
K. Hofmann,
H. Fickenscher,
E. Meinl,
F. Neipel,
C. Mattmann,
K. Burns,
J.-L. Bodmer,
M. Schröter,
C. Scaffidi,
P. H. Krammer,
M. E. Peter, and J. Tschopp.
1997.
Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors.
Nature
386:517-521[Medline].
|
| 51.
|
Vercammen, C., and J. L. Ceuppens.
1987.
Prostaglandin E2 inhibits human T-cell proliferation after crosslinking of the CD3-Ti complex by directly affecting T cells at an early step of the activation process.
Cell. Immunol.
104:24-36[Medline].
|
| 52.
|
Weber, F.,
E. Meinl,
K. Drexler,
A. Czlonkowska,
S. Huber,
H. Fickenscher,
I. Müller Fleckenstein,
B. Fleckenstein,
H. Wekerle, and R. Hohlfeld.
1993.
Transformation of human T-cell clones by herpesvirus saimiri: intact antigen recognition by autonomously growing myelin basic protein-specific T cells.
Proc. Natl. Acad. Sci. USA
90:11049-11053[Abstract/Free Full Text].
|
| 53.
|
Zheng, L.,
G. Fisher,
R. E. Miller,
J. Peschon,
D. H. Lynch, and M. J. Lenardo.
1995.
Induction of apoptosis in mature T cells by tumour necrosis factor.
Nature
377:348-351[Medline].
|
J Virol, April 1998, p. 3138-3145, Vol. 72, No. 4
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
