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NEOPLASIA
From the Institut für Klinische und Molekulare
Virologie, Erlangen, Germany; Ludwig Institute for Cancer Research,
Brussels Branch, Université Catholique de Louvain, Belgium; and
Department of Clinical Hematology, Hôpital Necker, Paris, France.
Adult T-cell leukemia (ATL) is an aggressive malignancy of
CD4+ T cells caused by the human T-cell leukemia virus type
1 (HTLV-1). The viral leukemogenesis is critically dependent on its
oncoprotein Tax because the protein as well as the virus can
immortalize primary human lymphocytes to permanent growth. As a
transcriptional transactivator, Tax can stimulate the expression of
distinct cellular genes. Alterations in the expression levels of
unknown growth-relevant genes may contribute to the changed growth
properties of Tax-immortalized and leukemic cells. To identify genes
that are linked to Tax transformation and ATL leukemogenesis, this
study systematically compared the gene expression of cultured cells
from patients with acute ATL with that of stimulated peripheral blood T
lymphocytes. Several overexpressed RNAs that encode signal transduction
functions were identified. These include a dual-specific protein
phosphatase (PAC1), an interferon-inducible factor (ISG15), a basic
helix-loop-helix transcription factor (DEC-1), and the secreted
antiapoptotic chemokine I-309. The ATL cell culture supernatants
contained an antiapoptotic activity that could be specifically
inhibited by antibodies directed against I-309. Inhibition of I-309
receptor (CCR8) signaling by pertussis toxin increased the apoptosis
rate of ATL cell cultures in the presence and absence of external
apoptotic stimuli. Both the I-309-specific antiapoptotic activity and
the proapoptotic effect of inhibitors of I-309 signaling suggest the
existence of an antiapoptotic autocrine loop in ATL cells. Thus, the
overexpression of this chemokine may inhibit apoptosis in ATL cells and
could substantially contribute to their growth.
(Blood. 2001;98:1150-1159) Adult T-cell leukemia (ATL) is an aggressive
leukemic disorder of CD4+ T cells caused by the human
T-cell leukemia virus type 1 (HTLV-1).1,2 It develops in
1% to 3% of infected individuals after more than 2 decades of viral
persistence.3,4 The contribution of the virus to the
pathology of the disease is believed to involve 2 mechanisms. First,
the virus directly stimulates the proliferation of the infected T cells
independently of antigen.5 Second, the HTLV-1 infection
leads to chromosomal abnormalities and an increased mutation rate in
infected cells.6-8 In addition to its oncogenic
properties, HTLV-1 induces a chronic neurologic disorder, termed
HTLV-1-associated myelopathy or tropical spastic paraparesis
(HAM/TSP),9-11 which primarily develops in patients with a
specific HLA subtype.12
As a complex retrovirus, HTLV-1 encodes the regulatory proteins Tax and
Rex in addition to structural proteins. Rex is essential for the
expression of the viral structural proteins,13-15 and Tax acts as a transactivator of the viral promoter.16-18 In
addition, Tax influences multiple cellular functions including gene
expression and proliferation and it increases the mutation
rate.19 Several lines of evidence indicate that the HTLV-1
regulatory protein p40tax is responsible for the
leukocyte transforming features of the virus.20-23 The
growth of primary human lymphocytes conditionally immortalized by Tax
depends on Tax expression. The proliferation of these immortalized
cells was reversibly arrested in the G1 phase of the cell
cycle by suppression of tax transcription, thus demonstrating that Tax stimulates the G1-to-S phase
transition in immortalized T lymphocytes.24
The mechanism by which Tax influences G1-to-S phase
transition and the growth of transformed primary human T cells is not well understood and different Tax functions may cooperate in the stimulation of cell proliferation. This includes the property of Tax to
directly interfere with the function of cell cycle-controlling proteins.25-28 For instance, Tax inhibits the
transactivating functions of the tumor suppressor p5329,30
and activates cyclin-dependent kinases CDK4 and CDK6; these CDKs are
essential for the control of the G1 phase
progression.31,32 Tax has also been shown to interfere
with DNA repair and in this way it possibly contributes to increase the
cellular mutagenesis rate.33 Thus, also this Tax function
may be crucial for leukemogenesis. Tax-induced mutations, which
accumulate during the decades of viral persistence, might affect
growth-relevant genes and as a consequence of dysregulated signaling
result in overexpressed genes.8
Its function as a modulator of cellular transcription is believed to
play a pivotal role in the stimulation of host cell proliferation, because Tax affects the gene expression of a variety of growth-relevant genes. It activates genes encoding proto-oncogenes,34,35
the In an attempt to identify genes that are linked to Tax transformation
or ATL leukemogenesis, we systematically compared the gene expression
patterns of cultured ATL cells with normal T lymphocytes using
subtractive hybridization.48 By this approach multiple overexpressed messenger RNAs (mRNAs) coding for signal transduction proteins were identified, among them the antiapoptotic chemokine I-309.
As reported here, the ATL cell cultures secreted an antiapoptotic activity and expressed the I-309 receptor CCR8. The Fas-mediated apoptosis rate of ATL cells was significantly increased when I-309 signaling was blocked. Taken together our data provide compelling evidence for an antiapoptotic autocrine loop in ATL cells.
Cell culture and stimulation of primary human T cells
Subtractive hybridization
Northern blotting For the verification and quantification of RNA expression, Northern blot analyses were performed. Total RNA was prepared from various cells, separated (10 µg/lane) on 1% formaldehyde denaturing agarose gels, and blotted as previously described.14 For the generation of probes the cDNA inserts of pT-Adv vectors were recovered by EcoRI digestion, purified by gel electrophoresis, and extracted from the agarose gel by silica-gel particles (Qiagen, Hilden, Germany). DNA fragments were radioactively labeled with [32P]-dATP (deoxyadenosine
triphosphate) by the random priming method. The hybridized mRNA was
quantified by phosphorimaging using the BAS2000 bioimaging
analyzer (Fuji, Tokyo, Japan). Signal analysis was done using the
program TINA2.0 (Raytest, Straubenhardt, Germany).
Cell proliferation assays The antiapoptotic activity of ATL cell culture supernatants was tested using BW5147C and DO-Cep4 cells, which were treated to undergo apoptosis. These and all other apoptosis- and cell proliferation-related experiments were done in triplicate in microtiter plates. Cells (2 × 103 cells/well) were incubated with 62.5 or 250 nM apoptosis-inducing dexamethasone (DEX; Sigma) and with serial dilutions of ATL cell culture supernatants in a final volume of 200 µL. Alternatively, apoptosis was induced using cisplatin (Sigma) at final concentrations ranging from 50 pg/mL to 5 ng/mL. To selectively block I-309 receptor signaling, 2.5 ng/mL pertussis toxin (Biomol, Hamburg, Germany) was added. To test the specificity of the antiapoptotic activity in the supernatants, sodium azide-free anti-I-309 antibody (R&D Systems, Wiesbaden, Germany) or a nonspecific control antibody (anti-human CD95; Becton Dickinson, Heidelberg, Germany) was added at final concentrations ranging from 1.25 ng/mL to 5 µg/mL. Purified I-309 (30 pg/mL to 20 ng/mL) and IL-9 (25 pg/mL to 2 ng/mL)50 served as controls. To assess cell viability and activity under the conditions above, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide; Sigma) dye assays were carried out. After 48 hours, 250 µg/mL MTT was added and cells were incubated for further 4 hours. In viable cells MTT is converted into formasan dye granules. Cells were lysed by the addition of 180 µL 0.01 N HCl in isopropanol and the formasan absorbance was determined in a microplate reader set at 630 nm. To calculate total cell survival, absorbance in the presence of ATL cell culture supernatant and DEX (ODDEX+sup) was standardized to the background (absorbance in the presence of DEX only: ODDEX), the latter being arbitrarily set 1. Additional cell survival (ACS) was determined by subtraction of the background (ODDEX).
Analysis of apoptosis One method for the detection of apoptotic cells was annexin V staining. Briefly, ATL-derived and control cells were cultured under the conditions described above in 48-well plates (4 × 103 cells/well in 400 µL). After 48 hours, the cells were harvested, washed in phosphate-buffered saline (PBS), and incubated with fluorescein isothiocyanate (FITC)-conjugated recombinant annexin V (Bendermedsystems Vienna, Austria) for 15 minutes. After staining, the cells were washed in PBS and measured by flow cytometry (FACScalibur; Becton Dickinson). Alternatively, cell death rate was measured using a modified propidium iodide (PI) staining assay, which allows the distinction between apoptotic, necrotic, and viable cells.53 Cells were treated with 25 µg/mL PI (Sigma) in 2% Triton-X100 and 0.05% sodium citrate and subjected to multiparameter flow cytometry analysis.Enzyme-linked immunosorbent assay The I-309 concentration in cell culture supernatants was determined by antigen capture enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-I-309 capture and biotinylated anti-I-309 detection antibodies according to the manufacturer's recommendations (R&D Systems). Briefly, 96-well microtiter plates were coated overnight with capture antibodies. The plates were incubated with serially diluted cell culture supernatants and biotinylated anti-I-309 detection antibody (R&D Systems). A standard curve was obtained using serial dilutions of recombinant human I-309 (R&D Systems). To detect the chemokine, streptavidin-conjugated horseradish peroxidase (HRP; Zymed, San Francisco, CA) and substrate solution (H2O2:tetramethylbenzidine = 1:1; Kirkegaard & Perry Laboratories, Gaithersburg, MD) were added. After terminating the reaction with 1 M H2SO4, the optical density was determined using a microplate reader set to 540 nm and 570 nm.
Identification of differentially expressed cDNAs in ATL-derived cells Dysregulated gene expression frequently indicates or causes aberrant activation of growth-relevant signal transduction pathways. In ATL cells such changes may result from transactivation of cellular promoters mediated by viral proteins or mutagenesis or both. To identify genes linked to the malignant growth, the gene expression pattern of acute ATL cells was systematically compared with that of nontransformed normal postmitotic T lymphocytes using the PCR-based SSH. This approach resulted in the cloning of cDNAs derived from transcripts, which are overexpressed in the cultured leukemia cells. In contrast to gene array techniques,46 SSH allows the isolation of novel sequences derived from unknown genes. In total, the nucleotide sequences of 346 cDNA clones were determined. The subsequent database analysis revealed that about one third of the sequences were derived from genes involved in gene regulation and signal transduction pathways. Two sequences were novel (GenBank accession numbers AJ276510 and AJ272002) and many were homologous to uncharacterized cDNAs from the expressed sequence tag (EST) database (Table 1). A variety of cDNAs corresponded to HTLV-1 or genes that have already been shown to be up-regulated in HTLV-1-infected cells due to Tax transactivation. These included the IL-2 receptor-chain and the CDK inhibitor
p21WAF1/CIP1.38,43 The genes for the
transferrin receptor and thymosin  -4 have previously been found to
be considerably overexpressed in ATL cells.54,55 The
isolation of known HTLV-1-up-regulated genes confirmed that our
experiment had resulted in cloning of differentially expressed
sequences. To quantify the RNA expression, Northern blots were
performed with following genes selected according to their biologic
function in transcription control and cellular signaling. The basic
helix-loop-helix transcription factor DEC-1 and the tumor necrosis
factor- (TNF-)-inducible B94 protein are putatively associated
with differentiation.56,57 The dual-specific protein
phosphatase PAC1 and the interferon-induced ISG15 gene were
selected because of their contribution to cellular
signaling.58,59 Calcylin, a calcium-binding protein, is
expressed in a cell cycle-specific manner and dysregulated in various
malignancies.60 The ferritin L chain and the chemokine
I-309 play a role in cell proliferation and apoptosis control,
respectively.50,61
These expression analyses revealed that the ATL-derived JuanaW cells
contained increased mRNA levels of various transcription factor and
signal transduction genes compared to PHA-stimulated T cells
(PBMCPHA). To determine the fold overexpression of these genes, the specific radioactive signals were normalized to the GAPDH
signals. A significant to strong overexpression could be found for the
basic helix-loop-helix transcription factor DEC-1 (4-fold), the dual
specific phosphatase PAC1 (7-fold), the calcium ion-binding protein
calcyclin (3-fold), the ferritin L-subunit (3-fold), the TNF-inducible
protein B94 (3-fold), and the interferon (IFN)-induced ISG15
gene (9-fold) as well as for the antiapoptotic chemokine I-309 (more
than 40-fold) (Figures 1 and
2A). Further Northern analyses were
carried out to investigate whether the up-regulation observed in JuanaW
cells is specific for HTLV-1-infected or Tax-expressing T cells. The
RNA samples used were extracted from HTLV-1-transformed cell lines
(C91-PL, MT-2), a Tax-transformed cell line (TAXI-1), ATL cells
(HuT-102, JuanaW), HTLV
Strong overexpression of the chemokine I-309 in ATL-derived cells To discriminate whether the overexpression of I-309 was an individual feature of cultured JuanaW ATL cells or was due to its leukemic origin, permanently growing cultures from 3 other patients with acute ATL were established and checked for I-309 expression. Subsequent Northern blot analyses showed a huge overexpression of the I-309 gene in the ATL-derived cultures Champ, PaBe, JuanaW (Figure 2A), and StEd (data not shown). In contrast, I-309 transcripts could be neither observed in the acute lymphoblastic leukemia (ALL)-derived CD4+ T-cell lines HuT-78 and Jurkat nor in PHA-treated PBMCs. Two specific bands corresponding to the expected mRNA sizes of approximately 550 and 2200 bases were detected. The most abundant transcript observed corresponds to the 550-base mRNA reported to code for the I-309 polypeptide.62 The larger transcript is of unknown function. To determine whether the high amount of RNA leads to the secretion of I-309 protein, cell culture supernatants were analyzed by antigen capture ELISA. As a result, I-309 was detected at concentrations ranging from about 35 ng/mL (PaBe) to more than 180 ng/mL (Champ) (Figure 2B). The same results were obtained after more than 6 months of permanent ATL cell culture. This observation indicates that I-309 overexpression is not due to uninfected activated T cells in the ATL cell preparation. In accordance with Northern blot results (data not shown), the supernatant of TAXI-1 cells, which are transformed by transduction of the Tax gene, contained only minimal amounts of I-309. Neither HAM-derived Mondi cells nor the HTLV-1-transformed MT-2 cells produced any I-309 protein. Because both cell lines are known to express the Tax protein, this suggests that the viral protein alone cannot account for the I-309 overexpression. Yet, the analysis of the I-309 promoter sequence with the MatInspector V2.2 (http://transfac.gbf.de/index.html) program63 revealed binding sites for both CREB and nuclear factor- B (NF-B) at
upstream positions 393 bp and 272 bp, which exactly match the
corresponding consensus sequences. Because Tax can stimulate the
transcription via these sites, the transactivator may contribute to the
activation of the I-309 gene in ATL cells. Taken together, these data suggest that the production and secretion of I-309 is a
characteristic of ATL-derived cells, which is neither shared by
HAM-derived nor by other HTLV-1- and Tax-expressing T cells.
Antiapoptotic activity of ATL cell supernatants is mediated by the chemokine I-309 To analyze whether the amount of I-309 secreted into the supernatant is sufficient to exert the antiapoptotic effect,50 ATL cell culture supernatants were titrated onto mouse thymoma BW5147C cells, which had been treated to undergo apoptosis. These cells have been shown to be highly susceptible to apoptosis induction by DEX.50 The addition of supernatants from 4 different ATL cultures (Champ, PaBe, JuanaW, StEd) protected the thymoma cells from DEX-induced apoptosis in a dose-dependent manner, whereas the supernatants of MT-2 and Jurkat had no antiapoptotic effect (Figure 3A). The addition of, for example, Champ supernatant increased cell survival by up to 100% compared with cell survival in the absence of ATL culture supernatant. This is consistent with ELISA results, which showed that Champ cells expressed the largest amounts of I-309 (Figure 2B). Flow cytometric analysis of the DNA content of individual nuclei confirmed that the increased cell survival is largely due to a reduced apoptosis rate (data not shown).
To confirm that the antiapoptotic activity was mediated by the I-309 receptor, CCR8, an inhibitor was added. Pertussis toxin interferes with the CCR8-associated Gi-protein signaling. As a result, the antiapoptotic effect of the ATL cell supernatant was dramatically reduced or even completely abolished in the presence of pertussis toxin (Figure 3B). The same result could be obtained with purified I-309 (Figure 3C). These observations suggested the involvement of a Gi-protein-coupled receptor like CCR8 in this pathway and corroborated the notion that I-309 is the factor in the supernatant that had prevented apoptosis. These data could be confirmed using 2 different methods (MTT and [3H]-thymidine incorporation assays) in 2 murine cell lines (BW5147C, DO-Cep4) (Figure 3 and data not shown). Culturing both tester cells with ATL culture supernatant also increased cell survival by up to 45% in the presence of cisplatin, a DNA intercalating drug leading to strand breaks. Under these conditions, cell viability was considerably reduced by pertussis toxin (data not shown). To add further proof that the I-309 chemokine is the factor mediating the antiapoptotic activity, DEX-treated BW5147C cells were incubated with JuanaW supernatant and increasing amounts of monoclonal antibodies specific to I-309. As shown in Figure 3D, the antiapoptotic effect could be abolished by anti-I-309 antibodies in a dose-dependent manner, whereas the isotypic control antibody had no effect on the growth of the cells. Hence, the I-309 present in the ATL supernatant is capable of preventing apoptosis of T cells. Autocrine antiapoptotic activity of I-309 in an ATL cell culture contributes to cell proliferation The demonstration of the antiapoptotic activity in the ATL cell supernatant led us to the hypothesis that I-309 may act on the producer cells to increase their resistance to apoptosis. This, however, would require the presence of the cognate receptor (CCR8), which is highly specific for I-309, on these cells. To test for CCR8 gene expression, further Northern blot analyses were performed. As Figure 4 shows, CCR8 transcripts are present in high abundance in all ATL-derived cells tested. To obtain further proof for an antiapoptotic autocrine action of I-309, the chemokine signaling was suppressed in PaBe ATL cells that had been treated with monoclonal anti-Fas antibodies to undergo apoptosis (Figure 5).64 Increasing the concentration of Fas antibodies resulted in reduced [3H]-thymidine incorporation in ATL (PaBe) and Tax-immortalized (TAXI-1) cells, which is due to an increased apoptosis rate within the culture.65 The level of apoptosis increased with antibody concentrations between 0 and 100 ng/mL and reached a plateau at more than 100 ng/mL, indicating a partial resistance of these cells to apoptosis. This is in good agreement with previous studies.66 At the highest anti-Fas concentration of 500 ng/mL, cell proliferation of both ATL and TAXI-1 cells was reduced by only 39% compared with untreated cells (Figure 5A). However, in the presence of anti-I-309 antibodies the proliferation of the ATL cells was further decreased to 55%, whereas the control (TAXI-1) was unaffected. Similar results were obtained with Champ ATL cells. In contrast, the addition of isotype-matched control antibody did not influence cell growth. The corresponding proliferation rate was identical with the one obtained in the absence of antibody. Interestingly, even in the absence of external apoptotic signals, the anti-I-309 antibodies alone diminished cell proliferation by some 6% (Figure 5A, 0 µg/mL anti-Fas, + anti-I-309 antibody). A triple combination of anti-Fas, anti-I-309, and pertussis toxin did not substantially change the rate of cell proliferation. The moderate additional reduction in cell proliferation is probably due to pertussis toxin-mediated inhibition of I-309 signaling triggered by intracellular ligand-receptor interaction. Thus, this observation also supports the notion that interference with the I-309 receptor and no other G-protein-coupled receptor had caused the reduced cell proliferation. To specifically determine apoptosis, annexin V staining was applied, which detects a marker of early apoptosis.67 This analysis revealed that Fas-induced apoptosis in Mondi and TAXI-1 control cells remained unchanged in the presence of I-309-specific antibodies (Figure 5B and data not shown). In contrast, the anti-I-309 antibodies increased early apoptosis in ATL (PaBe) cells (Figure 5B). Together these data corroborate an autocrine antiapoptotic effect of I-309 in the presence of external apoptotic stimuli.
The high level of I-309 might counteract not only external but also
internal proapoptotic stimuli. To elucidate this possibility, ATL cells
(JuanaW) were incubated with increasing concentrations of pertussis
toxin. As shown in Figure 6A the drug
inhibited the proliferation of JuanaW cells in a dose-dependent manner;
at the highest pertussis toxin concentration, cell proliferation was reduced by more than 50%. Similar results were obtained with PaBe and
Champ ATL-derived cells (data not shown). In contrast, the proliferation of both CD4+ control cell lines, HuT-78
(T-ALL) and MT-2 (HTLV-1-transformed), was not affected. This
indicates that pertussis toxin has no unspecific cytotoxic effect on
uninfected and HTLV-1-infected T cells. To verify that the reduced
proliferation was due to apoptosis, DNA contents of pertussis
toxin-treated cells were determined by a modified PI staining assay
that allows the simultaneous distinction of viable, apoptotic, and
necrotic cells (Figure 6B).53 These analyses showed that
pertussis toxin significantly increased apoptosis, whereas necrosis was
only minimally affected. These results were confirmed by annexin V
staining (Figure 6C). Hence, all 3 assays demonstrate that pertussis
toxin induced apoptosis in ATL cells and, therefore, suggest a role for
I-309 signaling in counteracting inherent apoptosis. Taken together,
the autocrine antiapoptotic CCR8/I-309 signaling may stimulate the
proliferation of ATL cells by rendering the leukemic cells less
susceptible to internal and external apoptotic signals.
To link lymphocyte genes to HTLV-1-induced leukemogenesis, we
have identified several overexpressed mRNAs in cultured ATL cells. Many
of the corresponding proteins have functions in signal transduction and
could, thus, play a role in deregulated proliferation of the malignant
cells. Up-regulated genes included the dual-specific protein
phosphatase PAC1, the bHLH-transcription factor DEC-1, and the TNF- The overexpression of the ISG15 and I-309 genes
could only be found in JuanaW and other ATL-derived cell cultures.
ISG15, a 15-kd protein encoded by an interferon Four of 4 ATL patient-derived cells expressed the protein at strongly
increased levels, but none of the in vitro HTLV-1-infected cell lines
did so. This restricted expression pattern suggests that I-309
overexpression is a characteristic of ATL cell cultures. Long-term
cultivation of one of the used lines indicated that I-309
overexpression can be maintained for a minimum of 2 years in
IL-2-dependent cultures. The overexpression of the I-309
gene exclusively in ATL cells indicates that viral functions including Tax alone are not sufficient to activate its transcription. However, Tax may contribute to the I-309 overexpression. This is supported by
the notion that Tax stimulates the IL-1 gene,73
which is required for I-309 gene expression.72
Furthermore, the murine homologue (TCA3) carries a NF- The chemokine I-309 and its murine homologue TCA3 were both initially identified in T cells that had been restimulated after a history of previous activation.62,75 I-309 is a member of the family of CC chemokines, which contains 2 disulfide bonds at the N terminus. As a structural peculiarity, I-309 displays a third well-separated disulfide bond.76 I-309, like many other chemokines, induces leukocyte migration. In contrast to all other members of the CC chemokine family, I-309 has an additional biologic function in apoptosis.50 In addition to I-309, all ATL-derived cultures expressed the gene for the cognate receptor, CCR8, a 7-transmembrane domain Gi-protein-coupled receptor.77-79 In contrast to most other chemokine receptors that exhibit a promiscuous binding property to various chemokines, only one high-affinity cellular ligand has been identified for CCR8 (I-309).80 Recently, several tumor viral proteins that bind to CCR8 have been identified.81-83 Both, the high selectivity to its cellular ligand and its function as a viral target indicate that this chemokine has a unique function. The I-309 protein secreted by our ATL cultures prevented apoptosis of heterologous T cells. The following observations suggest that it also acts on the producer cells and increases their resistance to apoptosis. First, the cognate receptor (CCR8) is expressed in these cells. Second, interference with I-309 signaling considerably augmented the proapoptotic Fas signals in ATL cell cultures. At least partially this autocrine I-309 signaling may be explained by intracellular binding of the ligand to its receptor, a phenomenon observed for various growth factors and cytokines previously.84-87 This is suggested by the moderate increase of apoptosis resulting from addition of pertussis toxin to anti-I-309 antibody-treated ATL cultures. The I-309-mediated counteraction to external proapoptotic stimuli may cooperate with the antiapoptotic function of the death antagonist Bcl-XL, which is up-regulated in ex vivo ATL samples,88 to increase the survival of leukemia cells.89 Both mechanisms may contribute to the high resistance of patients with ATL to chemotherapy. The large amounts of I-309 could not only prevent exogenously induced apoptosis but may also antagonize internal proapoptotic stimuli. This is suggested by our observation that pertussis toxin, an inhibitor of I-309 signaling, significantly increased apoptosis of ATL-derived cells. In summary, these data provide evidence that the CCR8/I-309 signaling may stimulate the proliferation of ATL cells by generating an antiapoptotic autocrine loop, which counteracts inherent apoptotic stimuli. In addition, CCR8/I-309 could render leukemic cells less susceptible to external apoptosis signals and could, hence, contribute to ATL progression.
The technical assistance of Elisabeth Derow and Daniel Romaker is greatly appreciated. We thank Kerstin Haller and Claudia Matteucci for helpful scientific discussions.
Submitted November 20, 2000; accepted April 9, 2001.
Supported by the Deutsche Forschungsgemeinschaft (SFB 466 Lymphoproliferation und virale Immundefizienz), by the Wilhelm Sander-Stiftung, and by HERN (HTLV European Research Network).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Ralph Grassmann, Institut für Klinische und Molekulare Virologie, Schlossgarten 4, D-91054 Erlangen, Germany; e-mail: grassmann{at}viro.med.uni-erlangen.de.
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Top
Abstract
Introduction
T-cell lines (Jurkat,
HuT-78, Molt-4), the HTLV-1+ T-cell lines (C91-PL, MT-2,
HuT-102), and the Tax-immortalized T-cell line (TAXI-1) were cultured
as previously described.
; Champ, 
; JuanaW, 
; StEd,
) in the BW5147C/DEX cells. MT-2 (×) and Jurkat (
) cell culture
supernatants served as negative controls. (B) Abrogation of the
antiapoptotic effect of the JuanaW cell culture supernatant in
BW5147C/DEX cells by the addition of pertussis toxin (2.5 ng/mL) 
/+
anti-Fas) and in the absence (
(IFN-