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Previous Article | Next Article 
J Virol, April 1998, p. 3138-3145, Vol. 72, No. 4
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
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- 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.
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- 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.
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
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
. 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.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(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.
-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.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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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).
|
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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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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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 |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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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 G1 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 PGE2
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 G1 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
PGE2. 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
p56lck (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-XL. 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 G1 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 |
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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).
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FOOTNOTES |
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* 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.
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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