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,§
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Cytokine Molecular Mechanisms Section, Laboratory of Molecular Immunoregulation, Division of Basic Sciences, and
Laboratory of Experimental Immunology, National Cancer Institute, Frederick, MD 21702;
Intramural Research Support Program, Science Applications International Corporation (SAIC)-Frederick, Frederick Cancer Research and Development Center, Frederick, MD 21702; and
§
Department of Integrative Biology, Pharmacology, and Physiology, University of Texas, Houston, TX 77030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The propagation of IL-2-mediated signal transduction occurs following
binding of the ligand to the high affinity IL-2 receptor complex
consisting of two members of the hematopoietin receptor superfamily,
designated IL-2Rß and IL-2R
(7, 8, 9). IL-2-induced
heterodimerization of IL-2Rß and IL-2R results in intermolecular
transphosphorylation of their corresponding receptor-associated Janus
kinase 1 (JAK1)4 and JAK3, respectively
(10, 11). The JAKs often catalyze the activation of two distinct
signaling pathways; for IL-2 it includes the
Shc/Ras/Raf/mitogen-activated protein kinase (MAPK) cascade, while the
other is comprised of a family of gene-regulating transcription
factors, known as STATs (12, 13, 14, 15). These signaling proteins regulate
gene transcription, ultimately controlling cell growth,
differentiation, and immune responsiveness (12). Moreover, JAK3
provides a key role in T cell development and activation. Patients
unable to activate this kinase, as well as JAK3-deficient mice, display
SCID (16).
AG-490 is a recent addition to the synthetically derived tyrphostin family of tyrosine kinase inhibitors (17, 18, 19). At relatively low concentrations, AG-490 has been shown to block hyperactive forms of JAK2 found in B cell precursors of acute lymphoblastic leukemia (ALL) patients (19) and in genetically constructed variants (9). AG-490 has also been shown to inhibit cytokine-induced activation of JAK2 in eosinophils stimulated with granulocyte-macrophage CSF (20) and in vascular smooth muscle cells and cardiac myocytes activated by angiotensin II (21, 22). A recent study has demonstrated that AG-490 effectively blocks STAT3 activation in mycosis fungoides-derived T cell lymphoma cells (23). While many kinase inhibitors are often promiscuous in the enzymes they target, AG-490 is unique in that it does not inhibit other lymphocytic tyrosine kinases such as Lck, Lyn, Btk, Syk, Src, JAK1, or Tyk2 (19).
In this study, our results suggest that this tyrphostin potently and effectively inhibits IL-2-mediated T cell responses. To define the mechanism of action for this inhibitor, we investigated putative molecular targets in the IL-2 signal transduction pathway. We provide evidence that JAK3 and its downstream substrates, STAT and MAPK signaling pathways may indeed be targets of AG-490. Taken together, this data suggests that AG-490 or its analogues may represent previously unrecognized therapeutic agents that can be used to treat a variety of IL-2-derived T cell disorders by inhibiting JAK3, STATs, and MAPK signaling pathways.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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AG-490 was obtained from Calbiochem (San Diego, CA). Tissue
culture materials were purchased from Life Technologies (Gaithersburg.
MD). IL-1 was obtained from PeproTech (Rock Hill, NJ), and IL-2 was
from Hoffmann-LaRoche (Nutley, NJ). Con A was purchased from Sigma (St.
Louis. MO). STAT1 Ab was purchased from Santa Cruz Biotechnology (Santa
Cruz, CA) and STAT3, -5a, and -5b from R&D Systems (Minneapolis, MN).
The rabbit anti-Shc, anti-JAK1, -2, -3, and Tyk2 and monoclonal
antiphosphotyrosine Abs were obtained from Upstate Biotechnology (Lake
Placid, NY), rabbit antiphospho-extracellular signal-related kinase 1
and 2 (ERK1/2) were from New England BioLabs (Beverly, MA), and the
monoclonal pan-ERK from Transduction Laboratories (Lexington, KY).
Protein A-Sepharose beads were purchased from Pharmacia (Piscataway,
NJ). Immobilion-P (polyvinylidene difluoride (PVDF)) membrane was
purchased from Millipore (Bedford, MA). TRIzol reagent was purchased
from Life Technologies. Phycoerythrin-labeled anti-CD25 for
IL-2R
, anti-CD122 for IL-2Rß, anti-common
(c)-chain or control mAb, and all probes for
ribonuclease protection assays (RPA) were purchased from PharMingen
(San Diego, CA).
Cell culture and treatment
The IL-2-dependent T cell line, D10, kindly provided by Dr. Li-Weber (DKFZ Cancer Research Center, Heidelberg, Germany), was maintained in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine and penicillin-streptomycin (50 IU/ml and 50 µg/ml, respectively), IL-1 (2 U/ml), recombinant human IL-2 (25 U/ml), Con A (2 µg/ml), 35 µM ß-mercaptoethanol, and 6 mM HEPES. CTLL-2 cells were maintained in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine and penicillin-streptomycin (50 IU/ml and 50 µg/ml, respectively), and 25 U/ml of recombinant human IL-2 (25 U/ml). Cells were treated with varying concentrations of AG-490, as described in figure legends. All reactions with AG-490 were kept in the dark to avoid inactivation of the tyrphostin. Cells were stimulated with 100 nM of IL-2 at 37°C, as indicated in the corresponding figure legends. Cell pellets were frozen at -70°C.
Proliferation assays
Quiescent cells (50 x 103/well) were plated in flat-bottom 96-well microtiter plates in 200 µl of growth medium (described above), employing 5% FCS in the presence or absence of IL-2 (1 nM). Cells were treated for 16 h with AG-490 as above, pulsed for the remaining 4 h of the assay with [3H]thymidine (0.5 µCi/200 µl), and harvested onto glassfiber filters. [3H]thymidine incorporation was analyzed by liquid scintillation counting (24).
Flow cytometric analysis
D10 cells were stained with phycoerythrin-labeled anti-CD25
for IL-2R, anti-CD122 for IL-2Rß,
anti-c-chain or control mAb, respectively. FACS
analysis of indirect cell immunofluorescence of IL-2 receptor chains
was performed on a flow cytometer (Becton Dickinson, San Jose, CA).
Data are presented as histograms of cell number (y-axis) vs
fluorescence intensity (log scale, x-axis), as corrected
mean fluorescence after subtraction of nonbinding isotype-matched
control mAb.
Solubilization of membrane proteins, immunoprecipitation, and Western blot analysis
Frozen cell pellets were thawed on ice and solubilized in lysis
buffer (108 cells/ml) containing 10 mM Tris-HCl (pH 7.6), 5
mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride,
200 mM sodium ortho-vanadate, 1% Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 1 µg/ml pepstatin
A, and 2 µg/ml leupeptin. Cell lysates were rotated end over end at
4°C for 60 min, and insoluble material was pelleted at 12,000 x
g for 20 min. Depending on the experiment, supernatants were
incubated with 5 µg/ml polyclonal rabbit antisera raised against
JAK3, STAT5a, STAT5b, anti-Shc, or monoclonal antiphosphotyrosine
Abs for 2 h at 4°C. Abs were captured by incubation for 30 min
with protein A-Sepharose beads. Precipitated material was eluted by
boiling in SDS-sample buffer for 4 min and subjected to 7.5% SDS-PAGE
under reducing conditions. For MAPK analysis, 
20 µg of total cell
lysate was dissociated in SDS-sample buffer and separated on SDS-PAGE,
as described above. All proteins were transferred to PVDF membrane as
previously described (10). Blots were Westerned with above Abs, rabbit
antiphospho-ERK1/2 and monoclonal pan-ERK were diluted 1:1000 in
blocking buffer, as previously described (10).
Electrophoretic mobility shift assay (EMSA)
AG-490 and control (DMSO)-treated D10 cells were washed with 5
vol of the hypotonic buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM
MgCl2, and 0.5 mM DTT), then lysed in the same buffer
supplemented with 1% Nonidet P-40 and incubated for 20 min on ice. The
nuclei-containing pellet was resuspended in equal volumes of low-salt
buffer (10 mM HEPES, 25% glycerol, 1.5 mM MgCl2, 20 mM
KCl, 0.5 mM DTT, and 0.2 mM EDTA) and high-salt buffer (high-salt
buffer containing 800 mM KCl). The nuclear extract was centrifuged at
4°C for 10 min. Supernatants were saved as nuclear protein extract
and stored at -70°C. For the EMSA, end-labeled
[32P]-oligonucleotides probes corresponding to the
ß-casein gene sequence (5'-AGATTTCTAGGAATTCAATCC-3') were used to
detect STAT5a and STAT5b binding, m67 serum-inducible response
element (SIE) gene sequence
(5'-AGCTTGTCGACATTTCCCGTAAATCGTCGAG-3') to identify STAT3 and
STAT1, and activating protein-1 (AP-1) oligonucleotide
(5'-CGCTTGATGAGTCAGCCGGAA-3') to monitor Fos/Jun binding. Each probe
was then incubated with 5 µg of nuclear extracted proteins in 15 µl
of binding mixture (50 mM Tris-Cl (pH 7.4), 25 mM MgCl2,
0.5 mM DTT, and 50% glycerol) at 4°C for 2 h. For supershift
assays, nuclear extracts were preincubated with 1 µg of either normal
rabbit serum or antisera specific to STAT1, -3, -5a, -5b, Jun, or Fos
(Figs. 4
, 6) at 4°C for 20 min. Samples were then incubated
for an additional 15 min at room temperature. The DNA-protein complexes
were resolved on a 5% polyacrylamide gels containing 0.25x TBE that
were prerun in 0.25x TBE buffer for 1 h at 100 V. After loading
of samples, gels were electrophoresed at room temperature for 2 h at
140 V. Gels were then dried by heating under vacuum and exposed to
x-ray film at -70°C overnight.
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D10 cells were treated as described above and then pelleted by centrifugation (20,000 x g) for 1 min at 4°C and subsequently washed in PBS. Total RNA was isolated using TRIzol from parallel sets of treated cells. Receptor RNA-message was examined by RPA using 20 µg RNA hybridized with 1 x 105 cpm of each 33P-labeled probes corresponding to mCR-1 probes set overnight at 56°C. Unhybridized RNA probes were digested with RNase T1 and RNase A for 45 min at 30°C. The RNase-treated samples were then digested with proteinase K for 15 min at 37°C. After phenol/chloroform extraction and sodium acetate/ethanol precipitation, hybridized RNA probes were denatured at 90°C for 3 min and electrophoresed on a 5% polyacrylamide gel. The dried gels were exposed to x-ray film.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Utilizing a murine IL-2-dependent T cell line, D10, we first
sought to identify an effective pharmacological agent that could block
IL-2-mediated cell proliferation. The results demonstrating the
efficacy of AG-490 are presented in Fig. 1 (upper
panel). D10 cells were cocultured with increasing concentrations
of AG-490 for 16 h. AG-490 abolished IL-2-inducible
[3H]thymidine incorporation in a dose-dependent manner,
displaying an IC50 of 25 µM. Overall, these cells were
judged to be >85% viable based on trypan blue dye exclusion and FACS
analysis using propidium iodide and Annexin V staining as apoptotic
indicators (data not shown). This effect was not limited to D10 cells
since similar results were also observed for the IL-2-dependent T cell
line, CTLL-2 (Fig. 1, lower panel). These findings suggest
that AG-490 potently inhibits IL-2-mediated proliferation in T cells,
results distinct from previous studies that showed this agent induced
apoptosis in ALL cells while exerting apparently no effects on the
growth of mitogen-stimulated normal T cells (19). To identify putative
targets of AG-490, we next investigated known IL-2R-activated signaling
proteins.
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Since the initial step in IL-2 signaling requires competent
activation of its cognate receptor chains, we examined whether
AG-490-mediated inhibitory effects were due to reduced expression of
IL-2R, -ß, or - chains. For this analysis, total mRNA was
isolated from two different sets of control (DMSO) or AG-490-treated
cells and hybridized against 33P-labeled receptor probes
(Fig. 2A). RNase-protected probes were
electrophoretically separated by PAGE, dried, and subjected to
autoradiography. Receptor message for DMSO (lanes a
and b) or AG-490 (lanes c and
d)-treated D10 cells failed to show a significant change in
IL-2R, -ß, and - mRNA expression, compared with the control
housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Next, we addressed whether surface expression of receptor proteins was
compromised by AG-490. FACS analysis for IL-2R, -ß, and -
chains showed no change following drug treatment (Fig. 2B).
From this data, it could be concluded that loss of IL-2-mediated cell
growth by AG-490 was not due to a significant loss of IL-2R expression
and suggested that its site of action was distal to the receptor.
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Following cytokine-induced oligomerization of the receptor chains,
JAKs are believed to phosphorylate themselves, their respective
receptor, and signaling molecules that are directed to the activated
complex (14). Since one cytokine can often show a unique pattern of JAK
activation in a cell-dependent manner, we investigated which JAKs were
activated by IL-2 in D10 cells. The results shown in Fig. 3A suggest that JAK3 is the most potently
activated JAK based on tyrosine phosphorylation (lanes
c and d), similar to previously published findings that
were observed for many other cell lines (10, 24, 25). We did not
observe detectable levels of activated JAK1, JAK2, or Tyk2, which is
weakly expressed in this cell line. We next examined the dose-dependent
inhibition of JAK3 by AG-490 over a 16-h period. As shown in Fig. 3B, AG-490 indeed blocked IL-2 activation of JAK3 at
concentrations as low as 50 µM (lane d) and showed
nearly complete inhibitory at 100 µM (lane f).
Reblots of JAK3 (lower panel) confirmed equivalent
loading (Fig. 3B).
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B). Reblotting for both STAT5a and STAT5b
showed equivalent loading and also showed that changes in tyrosine
phosphorylation were not due to a loss in protein expression
(lower panel). Quantification of decreased tyrosine
phosphorylation levels was performed by normalizing densitometric
traces against total JAK3, STAT5a, or STAT5b protein (Fig. 3C). AG-490 clearly inhibited tyrosine phosphorylation of
STAT5a, as well as STAT5b, at approximately IC50 (50–75
µM) following drug treatment. STAT1 and STAT3 also displayed a
similar dose-dependent sensitivity to AG-490 (data not shown). The
reason AG-490 displayed a greater inhibitory effect on STAT5a compared
with STAT5b is not readily apparent, since reprobing of these blots
with anti-STAT5a and -STAT5b confirmed equivalent loading. However,
we have previously demonstrated in PHA-activated human T cells and
IL-2Rß-transfected murine BA/F3 cells that IL-2 preferentially
induces more pronounced tyrosine and serine phosphorylation of STAT5b.
This may be due to additional phosphorylation sites not present in
STAT5a (20), thus accounting for the differences observed in Fig. 3, B and C. AG-490 inhibits IL-2-induced STAT1, -3, -5a, and -5b DNA binding
JAK-regulated STAT tyrosine phosphorylation is a prerequisite for
STAT dimerization, nuclear translocation, and gene transcription (12, 14, 33). Since AG-490 blocked tyrosine phosphorylation of STATs, we
used gel EMSAs to confirm a predicted concomitant loss in STAT-DNA
binding activity. For this analysis, STAT5a and STAT5b was examined for
its ability to bind to the prolactin-response element of the ß-casein
gene promoter, while STAT1/STAT3 binding was assessed using an
oligonucleotide probe corresponding to the SIE-element (Fig. 4). Nuclear extracts obtained from AG-490-treated cells
(Fig. 4A, lanes g-l) showed reduced ß-casein
DNA binding activity as compared with equivalent protein samples (5
µg) obtained from DMSO control-treated cells (Fig. 4A,
lanes a-f). Moreover, these IL-2-inducible DNA complexes
could be partially supershifted with anti-STAT5a (lane
c), STAT5b (lane d), or completely supershifted
with both Abs (lane e) confirming their identity.
Densitometric analysis of DMSO and AG-490-treated cells showed
IL-2-inducible DNA binding was reduced by 78, 65, and 65% for STAT5a
and STAT5b, STAT1, and STAT3, respectively. The findings above suggest
that AG-490 can act as a potent inhibitor of JAK3 and its downstream
gene-regulating transcription factors STAT1, -3, -5a, and -5b.
AG-490 inhibits Shc and ERK1/2 phosphorylation in a dose-dependent manner
It is generally accepted that activation of the MAPK cascade represents a key signal required for cellular proliferation. IL-2 potently activates the Shc/Ras/Raf/MAPK pathway via the adapter protein, Shc, which binds to Tyr338 of the IL-2R ß-chain (13). Mutations or deletions of this residue result in a significant loss of cytokine-inducible growth (13, 34) and strengthen the hypothesis that this pathway is at least partially responsible for IL-2-mediated cell proliferation. Thus, we investigated whether the loss in T cell growth was due to disruption of the Shc/Ras/Raf/MAPK cascade.
To answer this question we first examined activation of the Shc/ERK1/2
pathway using Abs that recognize activated forms of these signaling
molecules. Shc tyrosine phosphorylation was completely blocked at 50
µM AG-490 (Fig. 5A), while Abs to activated
phospho-ERK1/2 (Thr202/Tyr204) displayed a
parallel dose-dependent inhibition. Immunoblotting of Shc and ERK1/2
(indicated beneath phosphorylation blots) verified equivalent loading.
Densitometric analysis indicated that loss of tyrosine phosphorylation,
normalized to nonphosphorylated protein, displayed an IC50
of 50 µM for all three proteins (Fig. 5B). These data
support our earlier conclusions that demonstrated deletional mutation
of the acidic domain of the IL-2R ß-chain, Tyr338, or
inactivation of Shc/MAPK inhibits IL-2-mediated cell growth (35).
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AP-1 is a heterodimeric complex comprised of fos and jun oncogene
family members (36). Moreover, it is generally accepted that ERKs
activate AP-1, which promotes gene transcription (37). Therefore, we
examined whether AG-490 treatment of T cells modified AP-1 DNA binding
activity as measured by gel mobility shift assays. As shown in Fig. 6, nuclear extracts obtained from AG-490-treated cells
(lanes f-j) showed reduced DNA binding as compared
with equivalent protein samples (5 µg) obtained from DMSO-treated
cells (lanes a-e). Densitometric analysis revealed an
inhibition of >83% in AP-1 DNA binding of AG-490-treated cells
(lane g, 10.1) in comparison to DMSO treatment
(lane b, 59.7). In our hands, we observed a
significant reduction of AP-1 complex with both anti-Jun and Fos
Abs as demonstrated by the densitometric relative units in each lane
(lane b, 59.7; lane c, 50.5; and
lane d, 28.2). Furthermore, the anti-Jun Ab induced
supershift of the band. Interestingly, AG-490 greatly reduced basal
levels of AP-1 DNA binding (lane f, 10.8), as well as
its IL-2-induction, as compared with control samples (lane
a, 34.8). This data suggests that the inability of T cells to
adequately respond to IL-2 may be due to the inhibitory effects of
AG-490 on the JAK3/STAT1, -3, -5a, -5b, JAK3/AP-1, and
JAK3/Shc/Ras/Raf/MAPK cascades.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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JAK3 is recognized to be predominantly expressed in leukocytes, including monocytes, T, B, and NK cells, and is required for their development and function (16). The importance of JAK3 in IL-2 signaling pathways has been substantiated by recent findings that demonstrate that homozygous-point or -deletion mutations in JAK3 are found in autosomal recessive T-B+ severe combined immunodeficiency patients (38, 39). Moreover, JAK3 knockout mice are also immunodeficient (40) and have served as an excellent model to define the signaling pathways necessary for discerning T and B cell development and their function. Current models for IL-2 signaling suggest that JAK3 functions to phosphorylate specific tyrosine residues within its cognate IL-2R ß-chain, thereby allowing for the recruitment of substrates to the activated receptor complex. In this model, JAK3 would then phosphorylate other proteins, including STATs, that would then disengage from the receptor and mediate gene transcription (41).
In D10 cells, the inhibition of IL-2-mediated growth observed with
AG-490 suggests that it is due to inhibition of JAK3. We demonstrated
in Fig. 3 that IL-2 only activates JAK3 in these cells and not JAK1,
JAK2, or Tyk2. JAK3 also displays dose-dependent sensitivity to AG-490
(Fig. 3). Likewise, STAT5a and STAT5b tyrosine phosphorylation were
also blocked by AG-490 (Fig. 3), presumably due to inactive JAK3, which
would be required to catalyze this event. Similarly, AG-490 effectively
inhibited STAT1, -3, -5a, and -5b DNA binding (Fig. 4). In addition to
tyrosine phosphorylation of STATs, serine phosphorylation has also been
proposed to be required for nuclear localization and DNA binding
activity (42, 43). Emerging evidence suggests that serine
phosphorylation of STATs may be mediated by MAPK-dependent and
-independent pathways (32, 44). Since we showed in Fig. 5 that
activation of MAPK pathway (p44/p42 ERK1/2) was greatly suppressed by
AG-490 in a dose-dependent manner, we cannot rule out the possibility
that STAT serine phosphorylation was also disrupted by AG-490 and may
account for this loss in DNA binding. Similarly, reduced AP-1 DNA
binding activity (Fig. 6) may also be due to inhibition of its upstream
activator, MAPK. Collectively, these findings suggest that inhibition
of JAK3 activity by AG-490 leads to a loss in STAT and AP-1
transcriptional activity and their consequent ability to transcribe
proliferative response elements, as was clearly observed in the
thymidine-incorporation assay for D10 and CTLL-2 cells (Fig. 1). It is
of interest to note that AG-490 reduced basal levels of STAT/AP-1 DNA
binding (Figs. 4A and 6). Moreover, we have found no change
in basal levels of DNA binding components not believed to be activated
by the JAK3 signaling pathway, including c-Maf or C/EBPß in
AG-490-treated cells (data not shown). These suggest that AG-490 may
have a therapeutic function in constitutively activated JAK3 signaling
pathways, such as those reported by Meydan et al. (19). Ongoing studies
in the laboratory are exploring such issues discussed as below.
While AG-490 has previously been considered to be a JAK2-specific inhibitor, these conclusions were primarily drawn from ALL cells that lack readily detectable levels of JAK3 (19). Herein, we have provided evidence that suggests AG-490 effectively inhibits IL-2 signaling pathway in D10 cells that fail to activate JAK1, JAK2, or Tyk2. Interestingly, this drug has been shown not to affect other lymphocytic tyrosine kinases (i.e., Lck and Src) nor the more ubiquitously expressed JAKs, JAK1 and Tyk2 (19). This may also illustrate why AG-490 is well-tolerated by mice, findings that support the notion that it is not a general kinase inhibitor (19). The ability of AG-490 to selectively block JAK2 and JAK3 may be explained by the high level of homology shared between these two closest related members of the JAK family. Indeed, AG-490 displays similar micromolar inhibitory concentrations required to block JAK2 and JAK3 activity (19). It seems plausible to expect that generation of AG-490 analogues may refine and discriminate between not only JAK2 and JAK3 kinases, but JAK1 and Tyk2 as well.
From these findings, it therefore seems reasonable to expect that AG-490 may ablate or diminish certain lymphoid-dependent pathologies such as graft-vs-host disease or HTLV-1 based adult T cell leukemia, which is characterized by hyperactivated forms of JAK3 and constitutively active STAT proteins (45). Similarly, recent work by Nielson et al. (23) has shown that AG-490 can block constitutive activation of STAT3 and growth of mycosis fungoides-derived T cell lymphoma cells. Moreover, we have also observed that AG-490 effectively inhibits constitutive growth of the NK-like tumor line, YT, completely blocking tyrosine phosphorylated forms of JAK3 and STAT5a and STAT5b (R.A.K. and W.L.F., unpublished observation) suggesting that certain tumors may be more (or less) susceptible to AG-490. Recently, we have found that AG-490 can also block activation of JAK3/STAT6 phosphorylation induced by IL4 and IL13 (L.H.W. and W.L.F., unpublished observation), cytokines that may play a pivotal role in various type 2 disease states including Leishmaniasis, leprosy, allergy, and viral infection (46). Interestingly, AG-490 reduced the production of the Th2 cytokine, IL4, yet had no effect on other Th2 cytokine. Whether or not AG-490 may also impair lymphocyte recruitment and activation that characterizes a number of autoimmune diseases awaits further investigation.
In conclusion, we have provided evidence that AG-490 effectively inhibits IL-2-mediated T cell growth and JAK3, STAT, AP-1, and MAPK activation by IL-2. These findings suggest that this tyrphostin may prove therapeutically beneficial by suppressing immunopathological states such as graft-vs-host disease, autoimmune disorders and perhaps viral infections that compromise T cell function and may have far-reaching applications in addition to treating ALL patients with hyperactive forms of JAK2.
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Acknowledgments |
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Footnotes |
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2 The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government. By acceptance of this article, the publisher or recipient acknowledge the right of the U.S. government to retain nonexclusive, royalty-free license in and to any copyright covering the article.
3 Address correspondence and reprint requests to Dr. William L. Farrar, National Cancer Institute, P. O. Box B, Building 560, Room 31-68, Frederick, MD 21702. E-mail address:
4 Abbreviations used in this paper: JAK, Janus kinase; ALL, acute lymphoblastic leukemia; AP-1, activating protein-1; PVDF, polyvinylidene difluoride; c, common ; EMSA, electrophoretic mobility shift assay; MAPK, mitogen-activated protein kinase; RPA, ribonuclease protection assay; ERK, extracellular signal-related kinase; SIE, serum-inducible response element.
Received for publication September 16, 1998. Accepted for publication December 28, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain between receptors for IL-2 and IL-4. Science 262:1874. chain: a functional component of the interleukin-4 receptor. Science 262:1880. c chains with Jak1 and Jak3: implications for XSCID and XCID. Science 266:1042. and interleukin-4 receptor-. Biochem. J. 319:865.
. J. Biol. Chem. 270:9630. chain: a functional component of the interleukin-7 receptor. Science 262:1877.-induced cytoplasmic protein tyrosine kinase(s). Cell 70:323.[Medline]
- and interferon ß-stimulated gene expression through STAT proteins. Science 269:1721.This article has been cited by other articles:
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 |
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  D. Carbonnelle, M. Duflos, P. Marchand, C. Chauvet, J.-Y. Petit, and F. Lang A Novel Indole-3-propanamide Exerts Its Immunosuppressive Activity by Inhibiting JAK3 in T Cells J. Pharmacol. Exp. Ther., November 1, 2009; 331(2): 710 - 716. [Abstract] [Full Text] [PDF]
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|
 A. C. Breggia, D. M. Wojchowski, and J. Himmelfarb JAK2/Y343/STAT5 signaling axis is required for erythropoietin-mediated protection against ischemic injury in primary renal tubular epithelial cells Am J Physiol Renal Physiol, December 1, 2008; 295(6): F1689 - F1695. [Abstract] [Full Text] [PDF]
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A. K. Salahudeen, N. Haider, J. Jenkins, M. Joshi, H. Patel, H. Huang, M. Yang, and H. Zhe Antiapoptotic properties of erythropoiesis-stimulating proteins in models of cisplatin-induced acute kidney injury Am J Physiol Renal Physiol, June 1, 2008; 294(6): F1354 - F1365. [Abstract] [Full Text] [PDF]
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 J. W. Tyner, D. K. Walters, S. G. Willis, M. Luttropp, J. Oost, M. Loriaux, H. Erickson, A. S. Corbin, T. O'Hare, M. C. Heinrich, et al. RNAi screening of the tyrosine kinome identifies therapeutic targets in acute myeloid leukemia Blood, February 15, 2008; 111(4): 2238 - 2245. [Abstract] [Full Text] [PDF]
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 Y. Fujinaka, K. Takane, H. Yamashita, and R. C. Vasavada Lactogens Promote Beta Cell Survival through JAK2/STAT5 Activation and Bcl-XL Upregulation J. Biol. Chem., October 19, 2007; 282(42): 30707 - 30717. [Abstract] [Full Text] [PDF]
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 S. Kim, H. S. Kim, K. W. Chung, S. H. Oh, J. W. Yun, S.-H. Im, M.-K. Lee, K.-W. Kim, and M.-S. Lee Essential Role for Signal Transducer and Activator of Transcription-1 in Pancreatic {beta}-Cell Death and Autoimmune Type 1 Diabetes of Nonobese Diabetic Mice Diabetes, October 1, 2007; 56(10): 2561 - 2568. [Abstract] [Full Text] [PDF]
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|
 C. Ratthe, M. Pelletier, S. Chiasson, and D. Girard Molecular mechanisms involved in interleukin-4-induced human neutrophils: expression and regulation of suppressor of cytokine signaling J. Leukoc. Biol., May 1, 2007; 81(5): 1287 - 1296. [Abstract] [Full Text] [PDF]
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 L. Bao, S. Devi, J. Bowen-Shauver, S. Ferguson-Gottschall, L. Robb, and G. Gibori The Role of Interleukin-11 in Pregnancy Involves Up-Regulation of {alpha}2-Macroglobulin Gene through Janus Kinase 2-Signal Transducer and Activator of Transcription 3 Pathway in the Decidua Mol. Endocrinol., December 1, 2006; 20(12): 3240 - 3250. [Abstract] [Full Text] [PDF]
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 E. Mansour, F. G. Pereira, E. P. Araujo, M. E. C. Amaral, J. Morari, N. R. Ferraroni, D. S. Ferreira, I. Lorand-Metze, and L. A. Velloso Leptin Inhibits Apoptosis in Thymus through a Janus Kinase-2-Independent, Insulin Receptor Substrate-1/Phosphatidylinositol-3 Kinase-Dependent Pathway Endocrinology, November 1, 2006; 147(11): 5470 - 5479. [Abstract] [Full Text] [PDF]
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 M. Samardzija, A. Wenzel, S. Aufenberg, M. Thiersch, C. Reme, and C. Grimm Differential role of Jak-STAT signaling in retinal degenerations FASEB J, November 1, 2006; 20(13): 2411 - 2413. [Abstract] [Full Text] [PDF]
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 E. R. Gross, A. K. Hsu, and G. J. Gross The JAK/STAT pathway is essential for opioid-induced cardioprotection: JAK2 as a mediator of STAT3, Akt, and GSK-3beta Am J Physiol Heart Circ Physiol, August 1, 2006; 291(2): H827 - H834. [Abstract] [Full Text] [PDF]
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 M. Maggio, J. M. Guralnik, D. L. Longo, and L. Ferrucci Interleukin-6 in aging and chronic disease: a magnificent pathway. J. Gerontol. A Biol. Sci. Med. Sci., June 1, 2006; 61(6): 575 - 584. [Abstract] [Full Text] [PDF]
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Y. Yu, H. Zeng, M. Vijay-Kumar, A. S. Neish, D. Merlin, S. V. Sitaraman, and A. T. Gewirtz STAT Signaling Underlies Difference between Flagellin-induced and Tumor Necrosis Factor-{alpha}-induced Epithelial Gene Expression J. Biol. Chem., August 20, 2004; 279(34): 35210 - 35218. [Abstract] [Full Text] [PDF]
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S. Xi, Q. Zhang, K. F. Dyer, E. C. Lerner, T. E. Smithgall, W. E. Gooding, J. Kamens, and J. R. Grandis Src Kinases Mediate STAT Growth Pathways in Squamous Cell Carcinoma of the Head and Neck J. Biol. Chem., August 22, 2003; 278(34): 31574 - 31583. [Abstract] [Full Text] [PDF]
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L. L. Molinero, M. B. Fuertes, L. Fainboim, G. A. Rabinovich, and N. W. Zwirner Up-regulated expression of MICA on activated T lymphocytes involves Lck and Fyn kinases and signaling through MEK1/ERK, p38 MAP kinase, and calcineurin J. Leukoc. Biol., June 1, 2003; 73(6): 815 - 822. [Abstract] [Full Text] [PDF]
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 L. H. Wang, X. Y. Yang, X. Zhang, K. Mihalic, W. Xiao, and W. L. Farrar The cis Decoy against the Estrogen Response Element Suppresses Breast Cancer Cells via Target Disrupting c-fos not Mitogen-activated Protein Kinase Activity Cancer Res., May 1, 2003; 63(9): 2046 - 2051. [Abstract] [Full Text] [PDF]
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J. V. Stein, S. F. Soriano, C. M'rini, C. Nombela-Arrieta, G. G. de Buitrago, J. M. Rodriguez-Frade, M. Mellado, J.-P. Girard, and C. Martinez-A. CCR7-mediated physiological lymphocyte homing involves activation of a tyrosine kinase pathway Blood, January 1, 2003; 101(1): 38 - 44. [Abstract] [Full Text] [PDF]
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 L. Burdelya, R. Catlett-Falcone, A. Levitzki, F. Cheng, L. B. Mora, E. Sotomayor, D. Coppola, J. Sun, S. Sebti, W. S. Dalton, et al. Combination Therapy with AG-490 and Interleukin 12 Achieves Greater Antitumor Effects than Either Agent Alone Mol. Cancer Ther., September 1, 2002; 1(11): 893 - 899. [Abstract] [Full Text] [PDF]
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 P. An, G. W. Nelson, L. Wang, S. Donfield, J. J. Goedert, J. Phair, D. Vlahov, S. Buchbinder, W. L. Farrar, W. Modi, et al. Modulating influence on HIV/AIDS by interacting RANTES gene variants PNAS, July 23, 2002; 99(15): 10002 - 10007. [Abstract] [Full Text] [PDF]
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B. F. Skinnider and T. W. Mak The role of cytokines in classical Hodgkin lymphoma Blood, May 29, 2002; 99(12): 4283 - 4297. [Abstract] [Full Text] [PDF]
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X. Y. Yang, L. H. Wang, K. Mihalic, W. Xiao, T. Chen, P. Li, L. M. Wahl, and W. L. Farrar Interleukin (IL)-4 Indirectly Suppresses IL-2 Production by Human T Lymphocytes via Peroxisome Proliferator-activated Receptor gamma Activated by Macrophage-derived 12/15-Lipoxygenase Ligands J. Biol. Chem., February 1, 2002; 277(6): 3973 - 3978. [Abstract] [Full Text] [PDF]
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S. M. Stepkowski, R. A. Erwin-Cohen, F. Behbod, M.-E. Wang, X. Qu, N. Tejpal, Z. S. Nagy, B. D. Kahan, and R. A. Kirken Selective inhibitor of Janus tyrosine kinase 3, PNU156804, prolongs allograft survival and acts synergistically with cyclosporine but additively with rapamycin Blood, January 15, 2002; 99(2): 680 - 689. [Abstract] [Full Text] [PDF]
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K. Kirito, K. Nakajima, T. Watanabe, M. Uchida, M. Tanaka, K. Ozawa, and N. Komatsu Identification of the human erythropoietin receptor region required for Stat1 and Stat3 activation Blood, January 1, 2002; 99(1): 102 - 110. [Abstract] [Full Text] [PDF]
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S. B. Han, S. H. Park, Y. J. Jeon, Y. K. Kim, H. M. Kim, and K. H. Yang Prodigiosin Blocks T Cell Activation by Inhibiting Interleukin-2Ralpha Expression and Delays Progression of Autoimmune Diabetes and Collagen-Induced Arthritis J. Pharmacol. Exp. Ther., November 1, 2001; 299(2): 415 - 425. [Abstract] [Full Text] [PDF]
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J. Frasor, U. Barkai, L. Zhong, A. T. Fazleabas, and G. Gibori PRL-Induced ER{alpha} Gene Expression Is Mediated by Janus Kinase 2 (Jak2) While Signal Transducer and Activator of Transcription 5b (Stat5b) Phosphorylation Involves Jak2 and a Second Tyrosine Kinase Mol. Endocrinol., November 1, 2001; 15(11): 1941 - 1952. [Abstract] [Full Text] [PDF]
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 J. Y. Lee and K. E. Sullivan Gamma Interferon and Lipopolysaccharide Interact at the Level of Transcription To Induce Tumor Necrosis Factor Alpha Expression Infect. Immun., May 1, 2001; 69(5): 2847 - 2852. [Abstract] [Full Text] [PDF]
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X. Sun, J. E. Layton, A. Elefanty, and G. J. Lieschke Comparison of effects of the tyrosine kinase inhibitors AG957, AG490, and STI571 on BCR-ABL-expressing cells, demonstrating synergy between AG490 and STI571 Blood, April 1, 2001; 97(7): 2008 - 2015. [Abstract] [Full Text] [PDF]
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 F. Behbod, R. A. Erwin-Cohen, M.-E. Wang, B. W. Trawick, X. Qu, R. Verani, B. D. Kahan, S. M. Stepkowski, and R. A. Kirken Concomitant Inhibition of Janus Kinase 3 and Calcineurin-Dependent Signaling Pathways Synergistically Prolongs the Survival of Rat Heart Allografts J. Immunol., March 15, 2001; 166(6): 3724 - 3732. [Abstract] [Full Text] [PDF]
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M. B. Lilly, M. Zemskova, A. E. Frankel, J. Salo, and A. S. Kraft Distinct domains of the human granulocyte-macrophage colony-stimulating factor receptor {alpha} subunit mediate activation of Jak/Stat signaling and differentiation Blood, March 15, 2001; 97(6): 1662 - 1670. [Abstract] [Full Text] [PDF]
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A. Prigent-Tessier, U. Barkai, C. Tessier, H. Cohen, and G. Gibori Characterization of a Rat Uterine Cell Line, UIII Cells: Prolactin (PRL) Expression and Endogenous Regulation of PRL-Dependent Genes; Estrogen Receptor {{beta}}, {{alpha}}2-Macroglobulin, and Decidual PRL Involving the Jak2 and Stat5 Pathway Endocrinology, March 1, 2001; 142(3): 1242 - 1250. [Abstract] [Full Text] [PDF]
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E. Chklovskaia, C. Nissen, L. Landmann, C. Rahner, O. Pfister, and A. Wodnar-Filipowicz Cell-surface trafficking and release of flt3 ligand from T lymphocytes is induced by common cytokine receptor {gamma}-chain signaling and inhibited by cyclosporin A Blood, February 15, 2001; 97(4): 1027 - 1034. [Abstract] [Full Text] [PDF]
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R. A. Kirken, R. A. Erwin, L. Wang, Y. Wang, H. Rui, and W. L. Farrar Functional Uncoupling of the Janus Kinase 3-Stat5 Pathway in Malignant Growth of Human T Cell Leukemia Virus Type 1-Transformed Human T Cells J. Immunol., November 1, 2000; 165(9): 5097 - 5104. [Abstract] [Full Text] [PDF]
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 J. J O'Shea, R. Visconti, T. P Cheng, and M. Gadina Jaks and Stats as therapeutic targets Ann Rheum Dis, November 1, 2000; 59(90001): i115 - 118. [Abstract] [Full Text] [PDF]
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L. H. Wang, R. A. Kirken, X. Y. Yang, R. A. Erwin, L. DaSilva, C.-R. Yu, and W. L. Farrar Selective disruption of interleukin 4 autocrine-regulated loop by a tyrosine kinase inhibitor restricts activity of T-helper 2 cells Blood, June 15, 2000; 95(12): 3816 - 3822. [Abstract] [Full Text] [PDF]
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T. Chen, L. H. Wang, and W. L. Farrar Interleukin 6 Activates Androgen Receptor-mediated Gene Expression through a Signal Transducer and Activator of Transcription 3-dependent Pathway in LNCaP Prostate Cancer Cells Cancer Res., April 1, 2000; 60(8): 2132 - 2135. [Abstract] [Full Text]
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X. Y. Yang, L. H. Wang, T. Chen, D. R. Hodge, J. H. Resau, L. DaSilva, and W. L. Farrar Activation of Human T Lymphocytes Is Inhibited by Peroxisome Proliferator-activated Receptor gamma (PPARgamma ) Agonists. PPARgamma CO-ASSOCIATION WITH TRANSCRIPTION FACTOR NFAT J. Biol. Chem., February 18, 2000; 275(7): 4541 - 4544. [Abstract] [Full Text] [PDF]
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S.-i. Kagami, H. Nakajima, K. Kumano, K. Suzuki, A. Suto, K. Imada, H. W. Davey, Y. Saito, K. Takatsu, W. J. Leonard, et al. Both Stat5a and Stat5b are required for antigen-induced eosinophil and T-cell recruitment into the tissue Blood, February 15, 2000; 95(4): 1370 - 1377. [Abstract] [Full Text] [PDF]
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C. S. Zong, J. Chan, D. E. Levy, C. Horvath, H. B. Sadowski, and L.-H. Wang Mechanism of STAT3 Activation by Insulin-like Growth Factor I Receptor J. Biol. Chem., May 12, 2000; 275(20): 15099 - 15105. [Abstract] [Full Text] [PDF]
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A. S. Kristof, J. Marks-Konczalik, and J. Moss Mitogen-activated Protein Kinases Mediate Activator Protein-1-dependent Human Inducible Nitric-oxide Synthase Promoter Activation J. Biol. Chem., March 9, 2001; 276(11): 8445 - 8452. [Abstract] [Full Text] [PDF]
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W. Xiao, L. Wang, X. Yang, T. Chen, D. Hodge, P. F. Johnson, and W. Farrar CCAAT/Enhancer-binding Protein beta Mediates Interferon-gamma -induced p48 (ISGF3-gamma ) Gene Transcription in Human Monocytic Cells J. Biol. Chem., June 22, 2001; 276(26): 23275 - 23281. [Abstract] [Full Text] [PDF]
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L. H. Wang, X. Y. Yang, K. Mihalic, W. Xiao, D. Li, and W. L. Farrar Activation of Estrogen Receptor Blocks Interleukin-6-inducible Cell Growth of Human Multiple Myeloma Involving Molecular Cross-talk between Estrogen Receptor and STAT3 Mediated by Co-regulator PIAS3 J. Biol. Chem., August 17, 2001; 276(34): 31839 - 31844. [Abstract] [Full Text] [PDF]
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