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*
Department of Internal Medicine I, Eberhard-Karls University, Tübingen, Germany;
Division of Cellular Immunology, Tumor Immunology Program, German Cancer Research Center, Heidelberg, Germany; and
Tularik Inc., San Francisco, CA 94080
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B, which is a major target of reactive
oxygen intermediates. The long-term exposure of Jurkat cells to hormone
eventually results in a selection of cell clones that have lost Tax
activity. A subsequent transfection of these apparently
"nonresponsive" clones allows the recovery of Tax responses in
these cells. Our observations indicate that changes in the
intracellular redox status may be a determining factor in
Tax-mediated DNA damage, apoptosis, and selection against the long-term
expression of Tax function. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit, several lymphokines such as IL-4 and IL-6, and a
potent radical-scavenging and reducing agent known as adult T cell
leukemia-derived factor/human thioredoxin (ADF) (reviewed in 4 .
HTLV-I carries its own potential oncogene, Tax, which has no
cellular counterpart (5, 6) and has been implicated in the transforming
properties of the virus (reviewed in 7 . Tax protein has been
shown to promote the transcriptional transactivation of a wide range of
growth-associated genes. In particular, it has been suggested that a
Tax-induced expression of the IL-2R subunit promotes autocrine
stimulation and consequently the transformation of HTLV-I-infected T
cells (8, 9, 10, 11). Thus far, however, only a single report has described
the immortalization of human T cells through the expression of Tax
alone. In this study, the immortalized T cells were particularly
sensitive to activation via the CD3/TCR complex; this complex promoted
their mitosis under conditions in which IL-2R function was presumably
blocked (12).
A previous study using Jurkat T cells expressing a conditional version of Tax, which was generated by the fusion of the Tax protein of HTLV-I and the hormone-binding domain of the human estrogen receptor (ER-Tax), documented an inhibition of proliferation and an induction of apoptotic cell death upon the hormone-dependent activation of Tax (13). Moreover, we were able to demonstrate that caspases mediate Tax-induced apoptotic T cell death and that the CD95-signaling pathway plays a critical role in this Tax-triggered apoptotic process in our recent work using this hormone-inducible system (14). The antiproliferative effects were dependent upon the duration of ER-Tax activity and were significantly enhanced when the CD3/TCR complex was simultaneously activated (13). These observations corroborated the report of apoptotic cell death in Tax-transformed Rat-1 fibroblasts after serum starvation (15). Furthermore, the cooperative action of Tax and CD3 in T cells supports the reported overlap of the mode of action of Tax with the CD28 T cell costimulatory pathway (16).
The transcriptional complex that is activated by CD28 is closely
related to NF-B. Both play crucial roles in the TCR-independent,
cyclosporin A-insensitive pathways of T cell activation. Both
are known to respond to reactive oxygen intermediates (ROIs) and
to be selectively suppressed by antioxidants (17, 18). Interestingly,
the potent activation of NF-B by HTLV-I Tax is, at least in
transient transfection assays, also blocked by antioxidants (19). Thus,
it seems likely that Tax may induce prooxidant conditions. Such redox
changes are expected to function in a cooperative manner, with redox
changes induced through Ag or mAb stimulation of the CD3/TCR complex
and leading to apoptosis in ER-Tax-expressing Jurkat T cells.
To test the above possibilities, we have generated ER-Tax-expressing Jurkat T cells. A hormone-dependent activation of Tax leads to the trans-activation of Tax-responsive promoters in an inducible manner. We report that a hormone-dependent posttranslational induction of ER-Tax leads to rapid changes in the intracellular levels of H2O2 and glutathione (GSH), promoting an intracellular prooxidant state. Concomitant activation of the CD3/TCR pathway exaggerates the oxidative stress and causes DNA fragmentation in live cells. Antioxidants block the Tax-mediated DNA damage. A long-term exposure of the cells to active ER-Tax selects for or renders surviving cells unresponsive to Tax and allows an apparent recovery of the intracellular redox status. These observations indicate that changes in the intracellular redox state may be a determining factor in Tax-mediated DNA damage, apoptosis, and unresponsiveness.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Jurkat T cells that had been stably transfected to express
ER-Tax or ER-
Tax fusion constructs were maintained in culture as
described previously (13). The ER-Tax construct carried the
ligand-binding domain of the human ER at the 5' end of Tax; the
ER-Tax construct encoded an inactive fusion protein that was missing
the first 12 aa of Tax. Transient transfections and chloramphenicol
acetyltransferase (CAT) assays were performed by the
DEAE-dextran method as described previously (13) using 3 µg of the
CAT reporter plasmids p(HTLV-I)LTR-CAT or p(NF-B)4-CAT
and 1 µg of transactivator (ER-Tax-expressing plasmid) (13). For the
induction of Tax activity, cells were treated with 1 µM of the
hormone 17ß-estradiol (Sigma, Deisenhofen, Germany). For the
stimulation of NF-B activity (positive control), cells were
incubated with 20 ng/ml of PMA (Sigma) and 1 µg/ml of PHA (Sigma) for
1 h. Recombinant ADF was produced in Escherichia coli
and purified as described previously (20).
Electrophoretic mobility shift assay (EMSA)
Total cell extracts were prepared using a high-salt detergent
buffer (Totex) as described previously (21). In brief, the cells
were harvested by centrifugation; washed once in ice-cold PBS; and
resuspended in 4 cell volumes of Totex buffer containing 20 mM HEPES
(pH 7.9), 350 mM NaCl, 20% (w/v) glycerol, 1% (w/v) of Nonidet P-40,
1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT, 0.1%
PMSF, and 1% aprotinin. The cell lysate was incubated on ice for 20
min and then centrifuged for 5 min at 13,000 x g at
4°C. The protein content of the supernatant was determined, and equal
amounts of protein (10–15 µg) were added to the binding reaction
mixture containing 20 µg of BSA (Sigma), 2 µg of poly(dI-dC)
(Boehringer, Mannheim, Germany), 2 µl of a buffer (20 mM HEPES (pH
7.9), 20% glycerol, 100 mM KCl, 0.5 mM EDTA, 1% of Nonidet P-40, 2 mM
DTT, and 0.1% PMSF), 4 µl of a buffer (20% Ficoll 400, 100 mM
HEPES, 300 mM KCl, 10 mM DTT, and 0.1% PMSF), and 10,000 cpm
(Cerenkov) of a 32P-labeled NF-B oligonucleotide
in a final volume of 20 µl. An NF-B oligonucleotide with a
high-affinity NF-B-binding motif (Promega, Heidelberg, Germany) was
labeled with [
-32P]ATP (3000 Ci/mmol; Amersham,
Braunschweig, Germany) and T4 polynucleotide kinase (Promega). The
reaction mixture was incubated at room temperature for 25 min. Native
4% polyacrylamide gels were run in 45 mM Tris-borate, 1 mM
EDTA. NF-B-binding activity was determined by the
beta-imaging of native gels.
Apoptosis assay
The induction of apoptosis was measured using a modified version of a method that has been described previously (22). Briefly, cells were loaded with 1 µg/ml of Hoechst 33342 (Molecular Probes, Eugene, OR) for 5 min at room temperature and then transferred on ice while protected from light before measurement. After transferring the cells to flow cytometer-compatible tubes, propidium iodide (PI) was added to a final concentration of 0.5 µg/ml. FACS Vantage (Becton Dickinson, Heidelberg, Germany) was used for sample acquisition and data analysis. Hoechst 33342 was excited at 360 nm, and emission was measured at 450 nm. PI was excited at 488 nm, and emission was detected at 610 nm. The cell population that was bright for Hoechst 33342 staining but still negative for PI was considered to be undergoing apoptosis.
Intracellular GSH measurement
The measurement of intracellular GSH was performed according to Rice et al. (23) with modifications. Cells that had been kept in culture medium were loaded with 20 µM of monochlorobimane (MCB) (Molecular Probes) for 10 min at 37°C. The reaction was stopped by the addition of ice- cold PBS. Cells were subsequently harvested, washed through cold FCS followed by two additional washes with cold PBS, and analyzed with a FACS Vantage flow cytometer. The 351- to 364-nm bandpass filter was used for excitation, and emission was detected at 450 nm. Dead cells were excluded by forward/side scatter gating. The mean values of the fluorescence signal from duplicate samples were considered for data analysis.
Fluorescent measurement of intracellular peroxides with 2',7'-dichlorofluorescein diacetate (DCFH)
Intracellular peroxides were measured according to Royall et al. (24). A stock solution of DCFH (10 mM; Molecular Probes) was prepared under nitrogen in DMSO and stored at -20°C. Cells were loaded with 5 µM of DCFH in culture medium at 37°C for 45 min and then placed on ice before measurement. Loaded cells were collected into FACS-compatible tubes, supplemented with PI (1 µg/ml) (Sigma), and measured using a FACScan (Becton Dickinson).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ER-Tax fusion proteins were shown to transactivate several
Tax-responsive reporter plasmids in a hormone-dependent manner (13).
Here, we generated Jurkat T cells stably expressing inducible Tax
(ER-Tax) or an inactive form of Tax (ER-Tax). The transactivation
potential of the stably transfected, ER-Tax-expressing Jurkat cells was
determined in a time-course experiment using (HTLV-I) long terminal
repeat (LTR)- and NF-B-dependent reporter plasmids. Cells were
preincubated for different lengths of time in medium with or without
estradiol and then transfected with the Tax-responsive reporter
plasmids. The transiently transfected cells were cultivated for another
2 days in the presence or absence of estradiol before CAT reporter gene
assays were performed. A hormone-dependent transactivation of the
(HTLV-I)LTR- and NF-B-controlled promoters was observed in the cells
that were cultured for 2 days with estradiol immediately following the
transfections (Fig. 1
, day 0). Comparable
increases in CAT activity were detected with cells that had been
preincubated with estradiol for 3 days (data not shown) and 7 days
before transfection (Fig. 1, day -7). Thus, stably transfected ER-Tax
Jurkat cells exhibit estradiol-dependent Tax-activity.
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To test the long-term effects of constitutive Tax-activity, cells
were preincubated for 30 days with or without estradiol before being
transfected with the Tax-responsive plasmids. Preincubating the cells
for 30 days with hormone led to unresponsiveness to Tax, which reduced
the expression of the (HTLV-I)LTR- and NF-B-driven reporter gene
plasmids almost to the basal level of uninduced cells (Fig. 1
, day
-30). Cells that had lost responsiveness to Tax due to 1 mo of
continuous culture in the presence of estradiol responded to a new
transient transfection of exogenous ER-Tax. In these cells, a
hormone-dependent transactivation of LTR- and NF-B-dependent
reporter plasmids could be detected (Fig. 1, day -30 plus ER-Tax).
Exposing control cells expressing an inactive (truncated) form of Tax
(ER-Tax) to estradiol for the same time periods did not result in
the transactivation of transiently transfected NF-B- or
LTR-CAT constructs (data not shown).
The DNA-binding activity of NF-B was examined by EMSA for different
timepoints after the activation of ER-Tax. Treating ER-Tax-expressing
Jurkat cells with hormone resulted in a time-dependent, marked increase
of the NF-B-specific DNA-binding activity for 
5 days (Fig. 2). After estradiol treatment of the
cells for 5 days, a 50-fold increase of the NF-B-specific band
compared with control cells was observed. Thus, in contrast to most
other activators of NF-B, Tax-induced NF-B activation is
long-lasting and is stable for 
5 days. In agreement with the CAT
assays, NF-B activation was no longer detected after a prolonged
exposure (1 mo) of the cells to hormone (Fig. 2). Control
ER-Tax-expressing cells were not able to induce NF-B-binding
activity at the various timepoints tested (Fig. 2 and data not shown).
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We subsequently analyzed the viability of the Jurkat cells that
had been exposed to Tax activity or treated with moderate
concentrations (1 µg/ml) of anti-CD3 mAb. As shown in Figure 3, treating ER-Tax-expressing Jurkat
cells with anti-CD3 mAb alone or for 30 h with estradiol alone
resulted in little or no changes in apoptosis (DNA fragmentation) as
measured by Hoechst 33342 staining. However, the combination of these
two stimuli was strongly synergistic, leading to a nearly threefold
increase in the total number of cells undergoing apoptosis compared
with the background level of spontaneous apoptosis in these cells.
While the induction of apoptosis by a combination of anti-CD3 and
hormone was already detectable in ER-Tax Jurkat cells at 8 h after
treatment, no significant changes in the number of apoptotic cells were
observed for 30 h posttreatment in control ER-Tax cells (data not
shown). Thus, the apoptosis observed in TCR-stimulated cells could
clearly be attributed to Tax activity.
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). Pyrrolidine dithiocarbamate (PDTC), which is an
iron chelator and radical scavenger, was slightly less effective.
In ADF-treated cells, viability was even moderately increased compared
with control cells. Thus, antioxidants can block Tax-induced apoptotic
cell death. Tax induces a prooxidant state in T cells
Since antioxidants can prevent Tax-induced apoptosis, we examined
whether Tax has an effect on the cellular redox state. Jurkat cells
stably expressing a hormone-inducible Tax fusion protein (ER-Tax) were
treated for various times with estradiol. Untransfected cells or cells
expressing ER-Tax were used as controls. The activation of the
ER-Tax fusion protein resulted in an immediate (15 min postinduction)
drop in the intracellular amount of GSH and in considerably reduced
levels within 2 h after Tax activation (Fig. 4). A slow recovery of the GSH levels
approaching the levels seen for control cells was seen at 5 days
after the beginning of estradiol treatment. In contrast, untransfected
or control ER-Tax Jurkat cells that had been treated with estradiol
did not show any decrease in intracellular GSH levels (Fig. 4). ER-Tax
Jurkat cells that were cultured in the presence of hormone for a
prolonged period of time (1 mo) showed normal levels of GSH (data not
shown).
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Tax Jurkat cells had been treated with hormone. The
treatment of ER-Tax Jurkat cells with estradiol led to a marked
increase in the intracellular levels of H2O2
and a concomitant decrease of GSH (Fig. 5, A and D). The
Tax-mediated prooxidant state was inhibited by pretreating the cells
with ADF or PDTC (Fig. 5, A and D). Thus, the
Tax-induced prooxidant state as well as apoptosis can be blocked by
antioxidants. These observations indicate that enhanced prooxidant
conditions play a crucial role in the induction of T cell apoptosis by
HTLV-I Tax.
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The CD3/TCR complex and Tax reportedly cooperate in inducing
activation-associated events in T cells as well as activation-induced
apoptosis (11, 12, 13). Thus, the individual and combined effects of these
two independent stimuli on the intracellular redox state of Jurkat T
cells were examined. In response to treatment with immobilized
anti-CD3 mAb alone, both ER-Tax- or control ER-Tax-expressing
cells exhibited a comparable increase in the intracellular
concentration of H2O2 (Fig. 5, B and
C). As expected, estradiol alone enhanced the intracellular
H2O2 in the ER-Tax cells, while no such
increase was seen in the ER-Tax Jurkat cells. Treatment with
anti-CD3 mAb and estradiol led to an additive enhancement of
intracellular H2O2 in ER-Tax Jurkat cells, but
no enhancement was observed in the control ER-Tax Jurkat cells (Fig. 5, B and C). While only transient changes
(lasting for a few hours) were observed in the intracellular levels of
H2O2 when anti-CD3 mAb was used, the
changes that were induced by hormone activation of ER-Tax were
sustained and lasted for several days (data not shown). This
observation highlights the chronic nature of the activation events that
are mediated by Tax in contrast to the transient effects that are seen
in response to TCR stimulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activity could not be detected in the
nucleus following a long-term expression of Tax in Jurkat T lymphocytes
(27). In this case, the ability of mitogens and cytokines to induce
NF-B activation was also blocked. It seems that the
NF-B-signaling pathway was impaired in the latter study, since Tax
was active and the induction of other transcription factors, such as
Fos and Jun, was unaffected. However, our results suggest a selection
against Tax activity following the prolonged activation of this protein
rather than a suppression of NF-B activation. Taken together, these
observations indicate that T cells may not be able to tolerate the
intracellular constitutive expression of Tax for prolonged time
periods. This intolerance may explain the apparent shut down of
Tax expression in HTLV-I-infected leukemic T cells.
Our posttranslational inducible system allowed a careful kinetic
analysis both of the Tax-mediated effects and of NF-B activation. We
have shown previously that one of the early effects of Tax function is
activation-induced T cell apoptosis (13). The observations that we
report here indicate that HTLV-I Tax mediates oxidative stress, which
in turn may subsequently activate NF-B, a prooxidant-inducible
transcription factor. The activation of NF-B has been recently
linked to apoptosis. NF-B plays either an antiapoptotic or a
proapoptotic role, and the role that is chosen may depend upon the cell
type, subunit composition, or duration of the NF-B response
(reviewed in 28 . In our study, apoptotic cell death in Jurkat
cells that had been induced by Tax was shown to be accompanied by
NF-B activation. The duration of Tax stimulation determines the
intensity of T cell activation and oxidative stress and the severity of
the antiproliferative effect. This finding may be related in part to
the persistence of a prooxidative state and to a prolonged activation
of NF-B. Transactivation and DNA-binding studies revealed that Tax
activity induces a long-lasting induction of NF-B, rather than a
transient one as has been shown for several chemical and physiologic
stimuli of NF-B (27, 29). The strong NF-B activation was entirely
due to the induced Tax protein and not to a direct response of the
cells to hormone, as control ER-Tax-expressing cells did not show
any NF-B activation after being exposed to estradiol. Therefore, as
previously proposed for apoptosis triggered by serum withdrawal (30),
the unusually persistent Tax-induced activation of NF-B may exert a
proapoptotic function in our experimental system.
A short-term hormone induction of ER-Tax in Jurkat cells together with
the stimulation of TCR promotes apoptotic cell death (13). The
apoptosis-promoting effect of Tax is particularly evident when low
concentrations of anti-CD3 are used. Tax is also able to induce
apoptosis in Rat-1 fibroblasts, and this apoptosis is blocked by the
Bcl-2 protein (15). Interestingly, Bcl-2 has been shown to
function in an antioxidant pathway to prevent apoptosis (31). Moreover,
intracellular prooxidant conditions reportedly induce apoptosis in
several instances (reviewed in 32 . In the present study, we
provide evidence that the mediator of apoptosis, Tax, can induce
prooxidant conditions in the cell. This is revealed by a decrease in
the intracellular GSH level that occurs rapidly (within 15 min) and
lasts for several days. The decrease in the intracellular GSH level is
accompanied by a Tax-mediated increase of intracellular
H2O2 levels. The long-lasting, Tax-induced
prooxidant state correlated with the observed apoptosis and NF-B
activation. Tax-induced NF-B activation can be blocked by
antioxidants (19). In addition, Tax-induced apoptosis can be prevented
by radical scavengers.
The fact that Tax activity induces a prooxidant state in the cell shows
that Tax uses common pathways to induce cellular activation and
apoptosis. Since ROIs are involved in the induction of CD95 ligand
(CD95L) mRNA expression in some cases (33, 34), this involvement
could explain the mechanism underlying the Tax-induced up-regulation of
CD95L (14). Moreover, it has been recently shown that
prooxidant-induced NF-B activation may be involved in the
transcriptional activation of the CD95L gene (34, 35).
Tax can transactivate the antioxidative stress protein ADF/thioredoxin (36). ADF is a stress-inducible protein that is secreted from cells; this protein plays an essential role in cellular protection against oxidative stress and cell death and is itself induced by various oxidative agents (37).
The present work provides direct evidence for the production of ROIs by Tax and the involvement of these intermediates in Tax-induced apoptotic T cell death. Our findings suggest that changes in the intracellular redox status may be a critical factor in Tax-mediated DNA damage and apoptosis and in the apparent shut down of Tax expression in HTLV-I-infected cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Katerina Chlichlia, Division of Cellular Immunology, Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. E-mail address:
3 Abbreviations used in this paper: HTLV, human T cell leukemia virus; ER, estrogen receptor; GSH, glutathione; ADF, adult T cell leukemia-derived factor/human thioredoxin; CAT, chloramphenicol acetyltransferase; PDTC, pyrrolidine dithiocarbamate; EMSA, electrophoretic mobility shift assay; PI, propidium iodide; MCB, monochlorobimane; ROI, reactive oxygen intermediate; DCFH, 2',7'-dichlorofluorescein diacetate; CD95L, CD95 ligand; LTR, long terminal repeat.
Received for publication March 12, 1998. Accepted for publication May 19, 1998.
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-chain promoter: activation of a nonfunctional promoter by the transactivator gene of HTLV-I. Cell 49:47.[Medline]
B activation through CD28 requires reactive oxygen production by 5-lipoxygenase. EMBO J. 14:3731.[Medline]
B transcription factor and HIV-1. EMBO J. 10:2247.[Medline]
B by human T-cell leukemia virus type I Tax protein. J. Virol. 66:6288.B and AP-1. Proc. Natl. Acad. Sci. USA 91:1672.B: a specific inhibitor of the NF-B transcription factor. Science 242:540.B in cells constitutively expressing HTLV-I Tax protein. New Biol. 2:894.[Medline]
B or die?. Curr. Biol. 7:R94.[Medline]
immunoglobulin enhancer-binding protein NF-B by a posttranslational mechanism. Cell 47:921.[Medline]
B is induced upon apoptosis. J. Cell Biol. 134:13.This article has been cited by other articles:
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  A. Krueger, S. C. Fas, M. Giaisi, M. Bleumink, A. Merling, C. Stumpf, S. Baumann, D. Holtkotte, V. Bosch, P. H. Krammer, et al. HTLV-1 Tax protects against CD95-mediated apoptosis by induction of the cellular FLICE-inhibitory protein (c-FLIP) Blood, May 15, 2006; 107(10): 3933 - 3939. [Abstract] [Full Text] [PDF]
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T. Sanchez Alcaraz, P. Kerkhofs, M. Reichert, R. Kettmann, and L. Willems Involvement of Glutathione as a Mechanism of Indirect Protection against Spontaneous Ex Vivo Apoptosis Associated with Bovine Leukemia Virus J. Virol., June 15, 2004; 78(12): 6180 - 6189. [Abstract] [Full Text] [PDF]
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A. Tripp, Y. Liu, M. Sieburg, J. Montalbano, S. Wrzesinski, and G. Feuer Human T-Cell Leukemia Virus Type 1 Tax Oncoprotein Suppression of Multilineage Hematopoiesis of CD34+ Cells In Vitro J. Virol., November 15, 2003; 77(22): 12152 - 12164. [Abstract] [Full Text] [PDF]
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S. Kawata, Y. Ariumi, and K. Shimotohno p21Waf1/Cip1/Sdi1 Prevents Apoptosis as Well as Stimulates Growth in Cells Transformed or Immortalized by Human T-Cell Leukemia Virus Type 1-Encoded Tax J. Virol., July 1, 2003; 77(13): 7291 - 7299. [Abstract] [Full Text] [PDF]
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) was activated by the addition of 1 µM of
estradiol. Untransfected control cells (
) and ER-
) were similarly treated. Cells were harvested and analyzed
for intracellular GSH content at the indicated time intervals. Next,
cells were loaded with MCB and subjected to FACS analysis. The mean
values obtained from four independent experiments are shown together
with the variation from the mean. The fluorescence intensity of the
unstimulated cells is given as 100%.