From the Institut für Klinische und Molekulare
Virologie, Universität Erlangen-Nürnberg,
Schloßgarten 4, D-91054 Erlangen, Federal Republic of Germany and
§ Molecular Virology Section, NIAID, National Institutes of
Health, Bethesda, Maryland 20892-0460
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ABSTRACT |
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The human T-cell leukemia virus type 1 (HTLV-1)
transactivator (Tax) has been shown to interfere with regulated
cellular proliferation. Many studies have focused on the ability of Tax
to transform rodent fibroblasts; however, none has defined the
molecular requirements for Tax transformation of human lymphoid cells.
We show here that tax induces permanent growth of human
primary T-lymphocytes by using a transformation/immortalization
defective rhadinovirus vector. The cells phenotypically resemble
HTLV-immortalized lymphocytes and contain episomally persisting
recombinant rhadinoviral sequences, which stably express functional Tax
protein. As Tax can activate major cellular signal transducing pathways
including NF-B and cAMP-responsive element binding protein (CREB),
we asked for the relevance of these routes in the immortalization of
human lymphocytes. By using Tax mutants that either activate
exclusively CREB/activating transcription factor or are defective in
activating this signaling pathway, we delineated that Tax can induce
immortalization of primary human T-lymphocytes through a mechanism
independent of NF-
B activation.
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INTRODUCTION |
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HTLV-1,1 a complex human retrovirus (1), is the etiological agent for adult T-cell leukemia (ATL) (2, 3) and a chronic neurodegenerative disease (4, 5). The virus has little cytotoxic effect on T-lymphocytes, its target cells, but changes their growth properties. In contrast to normal T-cells, HTLV-1-infected lymphocytes can proliferate permanently without antigen stimulation (for review see Ref. 6). Cells freshly immortalized by HTLV-1 initially require exogenous interleukin (IL)-2 for proliferation (7-9); however, prolonged cultivation of these cells eventually results in IL-2 independent growth and loss of T-cell receptor and T-cell functions (7, 8). The IL-2 independent growth can be caused by a constitutive stimulation of the IL-2 receptor-associated Jak-Stat pathway (10, 11).
The HTLV-1-encoded transactivator protein p40tax
has many properties typical for oncoproteins. (i) It immortalizes
primary rodent fibroblasts and cooperates with the ras
oncogene in the transformation of these cells (12); in addition it
transforms established rat fibroblast lines (13, 14). (ii) It induces
leukemia and neurofibromas in transgenic mice (15, 16). (iii) It has a
potential for immortalizing primary human T-lymphocytes (17-20). Tax
potently stimulates viral transcription and acts on an array of
cellular promoters. Mechanistically, Tax can stimulate transcription
either directly through an intrinsic activation domain (21) or through activation of signal transducing pathways such as NF-B. Despite much
investigation, it currently still remains unclear whether NF-
B
activation by Tax is linked with its property to transform human
lymphocytes.
To define the Tax-stimulated pathway, which mediates the
immortalization of human T-cells, we expressed two types of Tax mutants in primary lymphocytes. One mutant activates CREB/ATF but not NF-B.
The second mutant activates neither CREB nor NF-
B. Here we report
that the transduction of a Tax mutant that is active for CREB/ATF but
inactive for NF-
B resulted in permanent growth of human primary
T-lymphocytes. These results, although not directly addressing the role
of NF-
B activation in lymphocyte growth, show that the NF-
B
activating function of Tax is dispensable for the immortalization of
primary human T-cells.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and Transfection--
The T-cell lines Kati-40,
TRI-1, and TAXI-1 (17, 18) were immortalized in vitro from
cord blood by rhadinoviral transduction of Taxwt. Mondi, an
interleukin (IL)-2-dependent HTLV-1-infected T-cell line,
was derived from peripheral blood lymphocytes of a South African
tropical spastic paraparesis patient (kindly provided by Dr. Walter B. Becker, University of Stellenbosch). Mondi, Kati-40, TRI-1, TAXI-1, and
NATI-1-5 were grown in 40% RPMI 1640 and 40% CG medium (Vitromex,
Vilshofen, Germany) supplemented with 20% fetal calf serum and 20 units/ml IL-2 (Boehringer Mannheim). The same medium was used for the
HTLV-1-negative human IL-2-dependent T-cell line SS8BPT
(22). The HTLV-1-negative T-cell lines Jurkat, Molt-4, and HuT-78 and
the HTLV-1-infected lines HuT-102, C91PL, and MT-2 were cultured with
RPMI 1640 supplemented with 10% fetal calf serum. To determine Tax
activity, lymphoid cultures were transfected with chloramphenicol
acetyltransferase (CAT) indicator plasmids containing the HTLV-1-LTR
(17) or four NF-B binding sites fused to a minimal promoter (23). To
discriminate between NF-
B and IL-2 effects on the IL-2R
promoter,
CAT indicator plasmids pmIL2R
PrCAT1 and pmIL2R
PrTKCAT8 (24) were
obtained from Dr. Markus Nabholz (Epalinges, Switzerland). Plasmid
pmIL2R
PrCAT1 contains a complete IL-2R
promoter (
2470 to +162).
The plasmid pmIL2R
PrTKCAT8 contains the thymidine kinase of herpes
simplex virus basal promoter fused to a segment of the IL2-R
promoter containing the IL-2 response elements. Plasmid DNA (10 µg)
was electroporated, and CAT reactions were performed as described (25,
26). Results were quantified by phosphorimaging (BAS2000, Fuji-X). As
an internal standard, luciferase activity from a co-transfected plasmid
pRSVluc (5 µg) was determined.
Fluorescence-activated Cell Sorter Analysis--
Flow cytometric
analyses were done as described (25). Antibodies recognizing CD2
(Leu5-b), CD3 (LeuTM-4), CD4 (LeuTM-3a), CD8
(LeuTM-2), CD19 (LeuTM-12), CD25
(Anti-IL-2R), CD45 (Anti-HLe-1), CD69(LeuTM-23), CD95,
HLA-DR (HLA-DR), and B7-1 (BB1) were purchased from Becton Dickinson
(Heidelberg). The CD28 (9.3) and CD58 (AICD58) antibodies were obtained
from Squibb (Seattle) and Dianova (Hamburg), respectively.
Construction of Recombinant Rhadinoviruses and Transduction of
Primary Human Lymphocytes--
Recombinant viruses were generated as
described previously (17, 18). The DNAs of the Tax mutants M7 (27) and
S258A (28) were assembled into the same expression cassette (pHISL)
used previously for the construction of Taxwt-expressing
rhadinoviruses (17, 18). Upon transfection these open reading frames
yielded the same amount of protein as wild type, if they were expressed under the control of the same promoter (data not shown and Ref. 27).
This indicated similar stabilities of wild type and mutant Tax
proteins. The phenotypes of the Tax mutants were verified in
co-transfection experiments using LTR-CAT and NF-B CAT indicator plasmids. The expression cassettes were inserted into a recombination plasmid containing a neor selection marker and flanking
rhadinoviral sequences. These sequences permitted recombination with
the rhadinovirus Herpesvirus saimiri A11S4 (29) and directed
the Tax expression cassettes into the right junction of unique and
repetitive sequences of its genome. The recombinant rhadinoviruses were
purified by geneticin selection and subsequently by two plaque
isolations. The presence of the mutations was verified in the purified
recombinant viral stocks by Southern blot analysis and sequencing of
PCR products. For immortalization assays, primary lymphocytes were
isolated from cord blood by Ficoll-Hypaque density gradient
centrifugation, stimulated by phytohemagglutinin and infected as
described (18).
DNA and RNA Analyses--
To detect episomally persisting
recombinant rhadinovirus DNA, cells were lysed in situ on
top of an agarose gel; the DNA was separated by electrophoresis into
chromosomal, episomal, and linear fractions (18). The Southern blot was
analyzed with a Tax-specific probe. To verify unrearranged
tax sequences, total cellular DNA was subjected to PCR
analyses using tax-specific primers (sense 5'-CAGCCCACTTCCCAGGGTTTGGAC-3' or 5'-CCAATCACTCATACAACCCCCA-3' and
antisense 5'-GTGTGAGAGTAGAAATGAGGGGT-3'). These primers resulted in PCR
products of 627 and 880 nucleotides. To confirm the presence of the
mutation within tax, purified PCR products were sequenced. Cytokine RNAs were detected by standard reverse transcriptase-PCR using
primers described in Platzer et al. (30). Tax-, IB-
-, IL-1
-, and IL-2R
-RNA expression was detected by Northern blot analyses as described previously (18). Hybridization signals were
quantified by phosphorimaging.
Protein Analyses--
Tax was detected on Western blots with
monoclonal mouse antibodies using the ECL system (Amersham Corp.,
Braunschweig, Germany). The Tax-specific hybridomas 168B17-46-34/50
(contributor, Beatrice Langton) and the polyclonal anti-rabbit
antiserum (contributor, Bryan Cullen) were obtained through the AIDS
Research and Reference Reagent Program, Division of AIDS, NIAID,
National Institutes of Health. To determine the IB-
protein
half-life, lymphocytes were cultured in the presence of 150 µg/ml
cycloheximide (Sigma, Deisenhofen, Germany). Cells were lysed in 50 mM Tris (pH 800), 150 mM NaCl, 1% Nonidet
p-40, 2 mM EDTA, 0.2% SDS, 1 mM
Na3OV4, aprotinin (10 µg/ml), leupeptin (10 µg/ml), and 5 mM NaF. The protein extract (50 µg/lane)
was subjected to immunoblot analysis using anti-I
B-
/MAD-3
antibodies (C15, Santa Cruz, Heidelberg, Germany).
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RESULTS |
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Immortalization of Cord Blood Cells by a Tax Variant Active for
CREB/ATF and Deficient for NF-B Signaling--
To investigate
whether the Tax-mediated activation of CREB/ATF or NF-
B
transcription is important for the immortalizing phenotype, we tested
two distinctive Tax mutants using rhadinovirus transduction. The
capacity of Tax to activate NF-
B can be selectively abolished by
point mutagenesis, without affecting its activation of the CREB/ATF
path (27, 31). This selectivity has been described for the
TaxS258A mutant, which contains a single amino acid change at position 258 from serine to alanine. The S258A mutation within Tax
abolishes NF-
B activation. However, it leaves the protein activity
on CREB/ATF-dependent transcription unaltered.
TaxS258A was incapable of stimulating NF-
B
site-dependent transcription using indicator plasmids (23)
(data not shown) but retains its stimulatory effect on the HTLV-1 LTR.
The NF-
B phenotype of TaxS258A has also been extensively
characterized in a previous study; no NF-
B activation above basal
levels could be detected (31). A second Tax mutant M7 (27) is negative
for both NF-
B and CREB-dependent transcriptional
stimulation. A rhadinovirus vector derived from the A11 strain of the
New World primate agent H. saimiri, which has been clearly
shown not to transform human lymphocytes (6, 32), was used for the
transduction of the tax mutants. This vector is a
non-transforming apathogenic deletion variant of the A11 strain (17,
18, 29); it persists in human T-cells without detectable expression of
herpesvirus genes (33). This ensures that biological effects elicited
from this approach are a consequence of Tax expression.
Cells Transduced with Tax Resemble HTLV-1-immortalized Cells-- To verify if immortalization by Tax transduction may be a biologically important model for HTLV-1 transformation/ATLL, the immortalized cells were characterized in detail. First the cells were checked for an absence of infectious H. saimiri. None of the human CBL-derived cell lines (NATI) produced infectious rhadinovirus, which was concluded from co-cultivation experiments with permissive OMK-637 cells. Futhermore it was confirmed that each human CBL line contained the expected tax mutant as a part of episomally persisting recombinant rhadinoviral DNA and not as linear DNA, which would be characteristic for virus particles. Additionally, PCR with two tax-specific primer pairs detected intact configuration of tax sequences. The preservation of the tax mutations 8-12 months after transduction was verified by direct sequencing of PCR products. The expression of Tax could be quantitated by detection of specific transcripts in Northern blots (Fig. 1A) and of Tax protein in Western blots (Fig. 1B). The amounts of Tax protein in the cells immortalized by the mutant taxS258A were roughly equal to the amounts observed in cell lines immortalized previously by the transduction of the taxwt gene (not shown).
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Reduced Expression of IL-2R in Cells Immortalized with the
Mutant Tax--
Tax stimulates the IL-2R
promoter via NF-
B (34).
To determine whether the reduced surface expression of IL-2R
could
be due to a loss of promoter transactivation by Tax, we investigated the IL-2R
transcription in cells without Tax, with
Taxwt, and with mutant Tax. The mRNAs were detected by
Northern blots and normalized to an internal standard mRNA (GAPDH).
Fig. 2B shows the average from three independent
experiments. As a result the content of IL-2R
transcripts was
clearly reduced in cells containing the Tax mutant thus accounting for
the diminished surface expression of CD25. These data suggested that
the TaxS258A-expressing cells have lower NF-
B activity
compared with the wild type counterpart.
Reduced NF-B Activity in T-cells Immortalized by
TaxS258A--
To check whether autocrine activation of
NF-
B exists in Tax-immortalized CBLs, the state of NF-
B
activation was analyzed. This was measured using the expression level
of RNAs initiated from NF-
B site-dependent IL-1
promoter (36). The mRNA content of cells without Tax, with
Taxwt, and with mutant Tax was quantitated by Northern
blots, which were normalized to an internal standard mRNA (GAPDH).
Fig. 3 shows the average from three
independent experiments. A clear difference was seen between those
cells expressing wild type and mutant Tax. IL-1
transcripts were
strongly reduced in the lymphocytes transduced with mutant
TaxS258A. These data suggest that the
TaxS258A-expressing cells have lower NF-
B activity compared with its wild type counterpart. This finding likely reflects an intrinsic property of TaxS258A since four out of four
independently immortalized cell lines show the same phenotype.
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DISCUSSION |
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How HTLV-1 Tax transforms cells is controversial. Although it is
agreed that Tax can activate CREB/ATF and NF-B, there is disagreement about the pathway correlating with transformation (14,
39). Thus when immortalized rodent cells were used each of these
pathways have been linked to transformation. Because immortalized
rodent cells are an artificial model for the development of adult
T-cell leukemia, we asked what is required of Tax in the first step of
transformation (i.e. immortalization) of primary human
lymphocytes. The experiments described here indicate that a Tax mutant
which activates CREB/ATF but cannot activate NF-
B can immortalize
human primary T-lymphocytes. We verified that the immortalizing
phenotype was not due to fortuitous activation of NF-
B since these
cells were found to contain greatly reduced levels of NF-
B activity.
Our findings support an NF-
B-independent pathway for Tax
immortalization of human T-lymphocytes.
Although the NF-B phenotype of the mutant S258A was thoroughly
established by two groups, it is formally not possible to exclude that
some unmeasurable low effect on NF-
B could have contributed to the
T-cell immortalization. The residual IL-2R
transcription in the
mutant immortalized cells does not indicate residual NF-
B activity,
since the promoter is activated by its IL-2 response elements. By using
many independent criteria, we found that cells immortalized by the
rhadinovirus tax hybrids resemble HTLV-1-immortalized
lymphocytes. These cells express virtually identical cytokine patterns
and have similar patterns of surface markers. Like HTLV-1-transformed
lymphocytes, Tax-immortalized lymphocytes can lose the CD-3 surface
marker expression (8). This finding is unique to HTLV-1-associated
immortalization since this does not occur with H. saimiri
immortalized lymphocytes (32). Three facts support that Tax induced the
immortalization of the primary T-cells and that rhadinovirus genes did
not contribute to the immortalized phenotype. 1) The only transcription
found in human T-cells originated from the genomic region, which is deleted in the vector strain used (40). 2) No herpes viral transcripts were found in T-cells infected and immortalized by the recombinants expressing the Taxwt gene (41). 3) The cells infected with
vector expressing TaxS258A did not synthesize infectious
herpesvirus particles. These findings, besides the observation that the
transduction with the H. saimiri vector produces no
immortalization, indicate that the cellular growth changes are a direct
consequence of HTLV-Tax expression.
The human primary lymphocytes transduced by the Tax mutant S258A are
immortalized since they have an extended life span, and they
proliferate in the absence of antigen presenting cells. The cells
resemble HTLV-1-infected cell cultures derived from patients both in
surface marker expression and growth factor requirements. These
findings underline the proximity of this model system to ATL
lymphocytes. We are not persuaded that rodent fibroblast models for Tax
transformation have similar physiological relevance. In this regard we
note that in other systems Tax proteins of HTLV-1 and BLV have been
shown to exert an immortalizing effect on primary rat fibroblasts (12,
42) and transforming activity in established rat fibroblast cell lines.
Thus Yamaoka and colleagues (14) showed that Tax, which is deficient in
NF-B activation, does not transform the Rat-1 fibroblast line.
Surprisingly, a different group showed that the Rat-2 cell line could
be morphologically transformed by a Tax mutant, which was deficient for
NF-
B activation (39). Confusion about the impact of Tax mutations on
fibroblast transformation probably stems from system to system
variations (i.e. Tax mutants used) and cell type
differences. These differences suggest cautiousness in
extrapolating results about the role of Tax in transformation from
rodent fibroblast cell line systems to human primary lymphocytes.
We believe that our current findings contribute toward the development
of a relevant human primary T-cell system for studying HTLV-1-induced
ATL. Unlike the findings in rodent cell lines, in primary human
T-cells, NF-B activation by Tax is evidently not a critical
component of immortalization. This observation agrees with the finding
from Smith and Greene (39). However, our results do not exclude that in
other settings activation of NF-
B can be a potent modulator/inductor
of cellular proliferation.
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ACKNOWLEDGEMENTS |
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We thank the AIDS Research and Reference Reagent Program (Division of AIDS, NIAID, National Institutes of Health). We also thank Patrick Baeuerle, Brigitte Biesinger-Zwosta, Bryan Cullen, Werner Falk, Warner C. Greene, Beatrice Langton, Edgar Meinl, Markus Nabholz, and Thomas Stamminger for providing reagents and Bernhard Fleckenstein for permanent support.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft Grant SFB-466 (Lymphoproliferation und Virale Immundefizienz) and Wilhelm-Sander Stiftung.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Dept. of Microbiology, University of Virginia, Jordan Hall 7-88, Charlottesville, VA 22908.
To whom correspondence should be addressed. Tel.:
49-9131-856784/853013; Fax: 49-9131-852101/856493; E-mail:
grassmann{at}viro.med.uni-erlangen.de.
1 The abbreviations used are: HTLV-1, human T-cell leukemia virus type 1; ATL, adult T-cell leukemia; IL-2, interleukin 2; IL-2R, IL-2 receptor; CREB, cAMP-responsive element binding protein; ATF, activating transcription factor; CAT, chloramphenicol acetyltransferase; wt, wild type; PCR, polymerase chain reaction; LTR, long terminal repeat; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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REFERENCES |
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