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TRANSPLANTATION
From the Department of Surgery/Division of Immunology
and Organ Transplantation and the Department of Integrative Biology and
Pharmacology, University of Texas Medical School at Houston, and the
Department of Biochemistry and Molecular Biology, Medical Health
Science Center, University of Debrecen, Hungary.
Janus kinase 3 (Jak3) is a cytoplasmic tyrosine (Tyr) kinase
associated with the interleukin-2 (IL-2) receptor common gamma chain
( Complete activation of T cells requires 3 threshold-limited sequential signals.1 Signal 1, delivered
by antigens that engage a specific T-cell receptor (TCR), is followed
by signal 2, delivered by a B7-CD28 interaction. Within seconds to
minutes of TCR engagement, the CD3 Current clinical immunosuppressive regimens are dominated by CaN
inhibitors (cyclosporine [CsA] or FK50612) that block
T-cell progression through the early G1 stages of the cell
cycle.12,13 However, ubiquitous expression of CaN in many
different tissues contributes to several adverse side effects,
including nephrotoxicity and neurotoxicity.14,15 One
therapeutic approach aimed at improving immunosuppression while
simultaneously reducing CsA-induced toxicity is to develop combination
therapy. Rapamycin (RAPA), an agent recently approved by the United
States Food and Drug Administration for use in clinical
transplantation, disrupts late G1 and early S cell-cycle
progression by inhibition of the serine (Ser)-threonine (Thr) kinase,
the mammalian target of rapamycin (mTOR).16 Our extensive
preclinical and recent clinical studies have documented that a
combination of CsA and RAPA produces potent synergistic interactions
allowing for a 50% reduction of CsA trough blood levels.17,18 Although RAPA is not nephrotoxic alone,
however, the ubiquitous tissue distribution of mTOR leads to other
toxicities such as myelosuppression and
hyperlipidemia.18-22 The current study explores the
hypothesis that the inhibition of molecular targets unique to
lymphocytes, particularly those activated by TCGFs, would provide a
novel and selective means to block T-cell function and allograft
rejection when used alone or in combination with CaN inhibitors. In
particular, we seek to identify antagonists of Jak3, thereby inhibiting
an entire family of TCGF-dependent pathways. Indeed, Jak3 (primarily
expressed in T, B, and natural killer [NK] cells) is activated
through the A recently published report28 revealed that PNU156804, an
analogue of the toxic parent compound undecylprodigiosin, blocks IL-2-induced T-cell proliferation in the late G1 phase of
the cell cycle. On the molecular level, these authors showed that PNU156804 inhibits the activation of NF- Cell culture and treatment
Proliferation assays
Solubilization of membrane proteins, immunoprecipitation, and Western blot analysis Frozen cell pellets were thawed on ice and solubilized in lysis buffer (108 cells/mL) as previously described.29 For human T cells, supernates were incubated, rotating end-over-end for 2 hours at 4°C with either 5 µL/mL polyclonal rabbit antisera raised against peptides derived from the unique carboxyl group (COOH) termini of Jak3 (amino acid [aa] 1104-1124) or carboxyl termini of human Stat5a (aa 775-794) or Stat5b (aa 777-787), and phosphoserine Stat5 rabbit polyclonal antibodies (pAbs) were generated against a phosphopeptide surrounding S725 of human Stat5a.30 For rat Nb2-11cc and T cells, Jak3, Jak2, p56Lck, and antiphosphotyrosine antibodies were obtained from Upstate Biotechnology (Lake Placid, NY). Proteins bound to antibodies were captured by incubation for 30 minutes with Protein A-Sepharose beads (Pharmacia, Piscataway, NJ), sedimented for purification, and eluted by boiling in 2× sodium dodecyl sulfate (SDS) sample buffer (20% glycerol, 10% 2-mercaptoethanol, 4.6% SDS, 0.004% bromophenol blue in 0.125 M Tris, pH 6.8) for 4 minutes. For phospho-MAPK assays, approximately 25 µg total cell lysate was dissociated in SDS sample buffer and was separated on 10% (all others on 7.5%) SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions. Proteins were transferred to polyvinylidene difluoride (PVDF) (Immobilon; catalog no. 1PVH 00010; Millipore, Bedford, MA) as previously described.11 Western blot analysis was performed with either pAbs, murine antiphosphotyrosine monoclonal antibodies (mAbs) (4G10; catalog no. 05-321; Upstate Biotechnology, Lake Placid, NY), or phospho p44/42 MAPK (catalog no. 9101; New England Biolabs, Beverly, MA). Western blots with the above antibodies, rabbit antiphospho-Erk1/2 (extracellular regulated kinase 1/2), and monoclonal pan-Erk (catalog no. E17120; PharMingen, San Diego, CA) were diluted 1:1000 in blocking buffer and were used as previously described.11Rat heart transplantations Wistar Furth (WF; RT1u) and Buffalo (BUF; RT1b) rats (each weighing 160-200 g) obtained from Harlan Sprague-Dawley (Indianapolis, IN) were cared for according to the guidelines of the Animal Welfare Committee. Rats were housed in light- and temperature-controlled quarters and given chow and water ad libitum. Heterotopic heart transplantation was performed using a standard microsurgical technique of end-to-side anastomoses to recipient aorta and vena cava.31 Cold ischemia times were less than 30 minutes. Graft survival time was defined as the last day of transabdominally palpable cardiac contractions. Recipients remained untreated or were treated by oral gavage every other day for 14 days (6 treatments) with 40, 80, or 120 mg/kg PNU156804 alone or in combination with daily oral gavage of 1.25, 2.5, or 5 mg/kg CsA or 0.25, 0.5, or 1 mg/kg RAPA. Some recipients were treated with CsA or RAPA alone. The results, presented as mean survival time ± standard deviation, were assessed for statistical significance by the Gehan survival test. In addition, the interaction between PNU156804 and CsA or RAPA was evaluated by the median effect analysis.32,33 Computer software was used to calculate combination index (CI) values: CI < 1 showed synergistic interactions, CI > 1 showed antagonistic interactions, and CI = 1 showed additive interactions.33Histopathologic evaluation WF recipients of BUF heart allografts were treated as described above with vehicle (n = 3) or vehicle containing 80 mg/kg PNU156804 (n = 3). At day 7 after transplantation, heart allografts were placed in Bouin fixative (Poly Scientific R&D, Bay Shore, NY). Each heart was sectioned in an identical fashion consisting of single horizontal cut followed by 3 consecutive incisions used to generate slides. Another dissection was made, and 3 more consecutive slices and slides were generated. Twelve slides per heart were stained with hematoxylin and eosin, as described previously.34 Rejection was graded in accordance with the standards established by the International Society of Heart and Lung Transplantation35: grade 0, no evidence of rejection; grade 1A, focal perivascular infiltration; grade 1B, diffused interstitial infiltration; grade 2 for moderate infiltration with uni-focal myocyte damage; grade 3A, moderate infiltration with severe focal myocyte damage; grade 3B, moderate infiltration with severe diffused myocyte damage; and grade 4, severe infiltration with ongoing severe myocyte damage.
PNU156804 blocks IL-2-mediated growth and Jak3 autophosphorylation To investigate the effects of PNU156804 on T-cell proliferation, T cells that had been activated for 72 hours with PHA were then made quiescent and admixed with ascending PNU156804 concentrations (1-100 µM) for 16 hours in the absence or presence of 1 nM IL-2. As shown in Figure 1, PNU156804 completely abolished IL-2-induced [3H]-thymidine incorporation at 25 µM (IC50 of approximately 7.5 µM). In contrast, the inactive analog PNU159744 showed minimal effect on T-cell proliferation at similar concentrations of drug, with only 20% inhibition observed at 100 µM (Figure 1). This inhibitory effect was not caused by cell death from toxicity because cell viability was typically greater than 85% based on a trypan blue dye exclusion test at this time point (16 hours), which was measured at the completion of each experiment (data not shown). Moreover, the effect of the drug appeared to be confined to activated T cells because PNU156804 did not inhibit actively growing non-Jak3-expressing Jurkat cells, a model for unactivated T cells (Figure 1).
PNU156804 fails to inhibit anti-CD3 activation of p56Lck in primary human T cells The above experiments suggest PNU156804 inhibits activated but not unactivated T cells by disrupting the signal 3 pathway. Earlier work by Mortellaro et al28 supports this model because the expression of IL-2 and its affinity-conferring -chain, both dependent on signal 1 and 2, were not affected. To test this notion further, effects of PNU156804 on the TCR-activated pathways were investigated by stimulating T cells by anti-CD3 activation and measuring changes in p56Lck and other Tyr-phosphorylated proteins. For
this assay, primary human T cells were treated with 20 mM active drug
(PNU156804) for 16 hours and were stimulated without or with 5 µg/mL
(µM) anti-CD3 antibody for 5 minutes at 37°C (Figure 2A). Cell lysates were clarified, and 30 µg total cell lysate was separated on SDS-PAGE from vehicle (lanes
a-b) or from PNU156804-treated samples (lanes c-d). Alternatively,
lysates were immunoprecipitated with antibodies to p56Lck (lanes e-h).
Proteins were Western blotted with antiphosphotyrosine antibodies.
Overall, no changes were observed in total Tyr-phosphorylated protein
or TCR activation of the p56Lck Tyr kinase. Striping and reprobing of
the blot confirmed an equivalent loading of p56Lck.
Catalytically active Jak3 is required for IL-2-driven Tyr phosphorylation of Jak1 and Stat5a/b.11,26,27,36,37 To determine whether the blockade of T-cell proliferation was caused by a loss of Jak3 activity, PHA-activated T cells (72 hours) were treated with ascending concentrations of PNU156804 (0-10 µM) for 16 hours, followed by a 10-minute stimulation with 100 nM IL-2. Lysed cells were immunoprecipitated with Jak3 pAb and then Western blotted with antiphosphotyrosine mAb (Figure 2B). Tyr phosphorylation of Jak3 was notably reduced at 5 µM (lane i) and almost completely inhibited at 10 µM (lane j) concentrations. Five separate experiments confirmed the loss of Jak3 Tyr phosphorylation between 5 and 20 µM PNU156804. Inactive PNU159744 control was ineffective in blocking Jak3 activation (Figure 2B, lanes k-n). The same samples reblotted with anti-Jak3 pAb to measure total Jak3 confirmed equivalent protein levels (Figure 2B, lower panel). Thus, PNU156804 disrupts Tyr phosphorylation of Jak3. Because an earlier study demonstrated that the lack of cell
proliferation in the presence of PNU156804 was not attributed to the
loss of IL-2R PNU156804 disrupts IL-2-mediated Stat5a/b Tyr/Ser phosphorylation Given that both Stat5a and Stat5b are downstream of Jak3 and that T cells from Stat5a/b gene-deficient mice failed to proliferate in response to IL-2 stimulation,6 we examined whether PNU156804 inhibits IL-2-induced activation of Stat5a/b. For this assay, PHA-activated quiescent human T cells were treated with 20 µM PNU156804 or inactive control PNU159744 for 16 hours, followed by IL-2 stimulation. PNU156804 blocked Stat5a (Figure 3) and Stat5b (Figure 3B) Tyr phosphorylation. In addition to Tyr phosphorylation, Stat5a/b transcription factors are (like other Stats) Ser phosphorylated.6 Indeed, interferon/ (IFN-/)
or IFN- -mediated Ser phosphorylation of Stat1 or IL-6 of Stat3
is believed necessary for its function, including maximal nuclear
translocation, DNA binding, transcriptional activation, and cell cycle
progression.43-45 We previously mapped a Ser
phosphorylation site in Stat5a (Ser726) that is conserved in Stat5b (Ser731).30,46,47 As shown in
Figure 3A-B, PNU156804 inhibited IL-2-induced Stat5a/b Ser kinase
activity. In particular, neither site was inducibly Ser phosphorylated
in the presence of cytokine and PNU156804, as measured by
phosphoserine-specific Stat5a/b pAb (middle panels). The same samples
reblotted with anti-Stat5a/b mAb confirmed equivalent protein levels
(panels A and B). These results suggest that PNU156804 inhibits Jak3
from mediating Tyr and Ser phosphorylation of Stat5. In contrast, the inactive PNU159744 analogue showed no effect on either Tyr or Ser
kinase activity. Because Jak-regulated Stat Tyr/Ser phosphorylation is
required for dimerization, nuclear translocation, and gene transcription,25 we conclude that IL-2-Stat5a/b-mediated
gene transcription critical for IL-2-mediated cell cycle progression is one explanation for the loss of IL-2-inducible T-cell proliferation observed in Figure 1.
PNU156804 inhibits Erk1/2 Tyr/Thr phosphorylation IL-2 potently activates the Shc/Ras/Raf/MAPK pathway by the adapter protein SHC, which binds to Tyr338 of the IL-2R
chain ultimately to drive T-cell proliferation.48,49 To
investigate whether PNU156804 disrupts this signaling pathway, PHA-activated T cells were treated with vehicle alone (Figure 4, lanes a-b) or with ascending PNU156804
concentrations. Total cell lysates separated on 10% SDS-PAGE were
blotted with phospho-Erk1/2 pAb that recognize activated
Thr202 and Tyr204 sites on both enzymes. As
shown in the representative experiment, Erk1 and Erk2 were completely
inhibited at 10 µM PNU156804 (lane i). Two additional experiments
confirmed the loss of active Erk1/2 proteins observed at 10 to 20 µM
PNU156804 concentrations. Immunoblotting with a pan-Erk1/2 (indicated
beneath phosphorylation blots) verified equivalent loading, though with
only weak p44 for Erk1. These data support the conclusion that the
inhibition of Jak3 by PNU156804 disrupts Erk1/2 (p44/42) activity in
IL-2-mediated signaling pathways.
PNU156804 preferentially inhibits Jak3- rather than Jak2-dependent cell proliferation Because Jak3 is recruited by IL-2 and other TCGFs,6 we tested the effect of PNU156804 on PHA-activated human T cells stimulated with either 1 nM IL-2, -4, -7, or -15 (Figure 5). [3H]-thymidine incorporation, plotted as percentage inhibition of total incorporated radiolabel versus increasing concentrations of PNU156804, displayed similar efficacy in disrupting T-cell growth stimulated by various TCGFs (Figure 5A). Thus,c-Jak-dependent T-cell
proliferation is equally inhibited in response to IL-2, -4, -7, or
-15.
To test the selectivity of the drug, PNU156804 was added to T cells
undergoing Jak3-versus Jak2-dependent proliferation. For this assay,
the rat Nb2-11c cell line was chosen because it responds to either PRL
(Jak2) or IL-2 (Jak3) stimulation.50 As depicted in Figure
5B, Nb2-11c cells treated with ascending concentrations of PNU156804
showed nearly 2-fold greater inhibition for the IL-2-Jak3-dependent pathway than for the PRL-Jak2-dependent pathway. Moreover, though 20 µM PNU156804 reduced cell proliferation to basal levels ( In vivo effect of PNU156804 alone or in combination with CsA or RAPA on heart allograft survival We also examined whether the Jak3 inhibitor PNU156804 displays immunosuppressive activity in vivo. As shown in Figure 6, WF (RT1u) recipients of BUF (RT1b) heart allografts oral gavaged on alternate days for 14 days with DMSO alone showed a mean survival time of 6.3 ± 0.5 days (Figure 6). Alternate-day oral gavage of ascending doses of PNU156804 (40, 80, or 120 mg/kg) significantly extended heart allograft survival in a dose-dependent fashion (all P < .01). The signal 1 inhibitor CsA, delivered daily alone by oral gavage for 14 days (1.25, 2.5, or 5.0 mg/kg), produced in vivo effects similar to those produced by PNU156804. However, combinations of PNU156804 and CsA administered at ratios ranging from 64:1 to 1:1 exhibited much better effects than either drug alone, with many transplanted hearts surviving more than 100 days (Figure 6). To determine the quality of interaction between PNU156804 and CsA, results were evaluated by the median effect analysis to calculate CI values. Although all 2-drug ratios tested were synergistic (CI, 0.2-0.8), optimal results were produced by the PNU156804-CsA ratio of 4:1 (CI, 0.22).
Next, we examined the interaction of PNU156804 with another signal 3 inhibitor, RAPA, which targets the 256-kd Ser/Thr kinase and regulator of protein translation, mTOR. RAPA given alone daily by oral gavage for 7 days (0.25, 0.5, or 1.0 mg/kg) resulted in modest, albeit significant, prolongation of heart allograft survival. Combinations of PNU156804 and RAPA produced only additive effects (CI, 0.9-1.0). These results showed that selective inhibition of the Jak3-dependent signal 3 pathway blocks allograft rejection. This effect is synergistic with a signal 1 inhibitor but only additive with another signal 3 inhibitor. Inhibition of Jak3 blocks graft damage and reduces leukocyte cell infiltration For histologic examination, heart allografts were obtained from recipients that had received alternate-day oral gavage of DMSO alone or DMSO combined with 80 mg/kg PNU156804. Each heart was uniformly cut horizontally, and 12 hematoxylin-eosin-stained sections per heart were scored as described in "Materials and methods." Untreated heart allografts examined on day 7 after grafting displayed maximum grade 4 damage with extensive myocardial necrosis involving more than 50% of the complete cross-section. In the areas of necrosis, the myocardial fibers were totally destroyed up to the epicardium. The remaining areas of the samples without necrosis showed intense infiltration with polynuclear and mononuclear cells (Figure 7A). In contrast, heart allografts from recipients treated with PNU156804 showed only grade 1B changes with mild infiltration of epicardium, myocardium, and endocardium, but without any evidence of myocyte damage (Figure 7B). Given that similar changes were observed on all sections in each group, we conclude that selective inhibition of Jak3 blocks graft damage and reduces infiltration of leukocytes.
The current results demonstrate that PNU156804 inhibits TCGF-induced T-cell growth (IC50, approximately 7.5 µM; Figure 1A) by the disruption of Jak3 autokinase activity (Figure 2B-C). Consequently, PNU156804 (but not the inactive control compound PNU159744) blocks the activation of Jak3 substrates, namely, Stat5a and Stat5b, as assessed by phosphotyrosine and phosphoserine Western blots (Figure 3). In fact, PNU156804 completely disrupts not only Jak3-dependent downstream activation of Stat5a and Stat5b Ser kinases (Figure 3), it also disrupts the downstream Ser/Thr kinases, p44/Erk1 and p42/Erk2 (Figure 4). Although the inhibitory activity of PNU156804 was equally effective in blocking Jak3-driven T-cell proliferation by either IL-2, -4, -7, or -15 (Figure 5A), the drug was 2-fold less efficient in inhibiting growth by PRL through a closely homologous Tyr kinase, Jak2 (Figure 5B). Last, PNU156804 alone significantly extends cardiac allograft survival and acts synergistically in combination with CsA (CI, 0.2-0.8) and additively in combination with RAPA (CI, 1.0; Figure 6). Thus, PNU156804 represents a selective Jak3 inhibitor with sufficient potency to block allograft rejection. Because presently used clinical immunosuppressants act in a ubiquitous fashion, they produce potent side effects. For example, an active metabolite of 6-mercaptopurine, 6-thioinosinic acid incorporates into nucleic acids causing DNA and RNA breakage in many cells, thereby causing severe bone marrow depression.51,52 Although glucocorticoids inhibit the production of many cytokines within different cell types, they produce multiorgan side effects, such as Cushingoid features, growth retardation, poor wound healing, and many others deleterious effects.52 CsA or FK506 can disrupt the Ser-Thr phosphatase activity of CaN in several non-T-cell types, thereby contributing to nephrotoxicity and neurotoxicity.53 Moreover, mTOR inhibition of RAPA ablates not only cytokine-mediated growth of T and B cells but many other cells, which can result in myelosuppression and hyperlipidemias.18,19,21,22 Given that the fundamental problem for all available immunosuppressants is the ubiquitous distribution of their targets, we must seek highly specific agents that inactivate molecules uniquely expressed in resting T cells (eg, Zap70) or in activated T and B cells (eg, Jak3). Several recent studies have revealed that the Jak3 Tyr kinase enzyme is
an essential signaling intermediate for the development and function of
T and B cells and of NK cells.23,24 Indeed, the retroviral
introduction of Jak3 enzyme into Jak3-deficient mice restores normal
T-cell development.54 Although understanding of the
signaling pathways activated by Jak3 (directly or indirectly) is
incomplete, Jak3 signaling by Stat5a/b is necessary to regulate genes
required for cellular proliferation.26 As shown here, PNU156804 abolishes IL-2-dependent T-cell proliferation by the inhibition of Jak3-mediated autokinase activity and Stat5a/b Ser/Tyr phosphorylation. Consequently, PNU156804 prevents Stat5a/b dimerization by their SH2 domains and by Stat5a/b translocation to the nucleus. Given the limited pattern of Jak3 expression, the
We have recently reported that AG-490 blocks T-cell proliferation by the inhibition of Jak3 autokinase activity and Stat5a/b Tyr phosphorylation.29,55 However, AG-490 is equally potent in inhibiting Jak2 activation, including eosinophils stimulated by granulocyte-macrophage colony-stimulating factor and in vascular smooth muscle cells and cardiac myocytes activated by angiotensin 2.56-58 Our most recent study showed that AG-490 inhibits IL-2-dependent proliferation of PHA-stimulated human T cells with an IC50 of approximately 20 µM.59 In contrast, PNU156804 is at least twice as effective in inhibiting human T cells when performed under identical experimental conditions by displaying an IC50 of approximately 7.5 µM for IL-2 and other T-cell growth factors that activate the Jak3 cascade. Furthermore, PNU156804 shows 2-fold greater specificity to block Jak3-dependent T-cell proliferation in response to IL-2 compared to Jak2-dependent cell proliferation (Figure 5). Whether in vivo comparison with both drugs will support the hypothesis that PNU156804 is more efficacious remains to be determined. In addition to Tyr phosphorylation, Stats undergo Ser phosphorylation
on a conserved Ser residue that plays a critical role in
cytokine-induced nuclear translocalization, maximal gene transcription, and cell cycle progression.43-45,60-62 Earlier evidence
has been presented that shows minichromosome maintenance-5 protein is
required for full DNA replication by binding to phosphorylated
Ser727 on Stat1 A recently published report found that PNU156804 inhibits IL-2-induced
proliferation of human T cells without affecting the expression of
IL-2R Our current findings document that the blockade of Jak3 in vivo
prevents allograft rejection (Figure 6). Furthermore, histologic examination of allografts from PNU156804-treated hosts showed reduced
intragraft cellular infiltration of mononuclear cells without myocyte
damage (Figure 7). Lack of clonal proliferation might limit the
generation of a sufficient number of effector T cells The combination of PNU156804 and CsA results in potent synergistic interaction (CI, 0.2-0.8), extending allograft survival beyond that produced by monotherapy with each drug alone (Figure 6). Similarly, CsA and RAPA act synergistically to prolong organ allograft survival in animal models17 and in humans.67 Rats treated intravenously with combinations of RAPA (0.04-0.8 mg/kg per day) and of CsA (0.5-2.0 mg/kg per day) displayed potent synergistic interactions on heart and kidney allograft survival, as documented by CI values of 0.001 to 0.2.67 We have shown in this model that even subtherapeutic CsA doses reduced the expression of IL-2 mRNA at the graft site, thereby facilitating inhibition by RAPA of IL-2-dependent growth T cells and producing synergistic interaction. A similar mechanism might explain the synergistic effects of CsA in combination with PNU156804 produced at all tested PNU156804-CsA ratios (1:1-64:1), as documented by CI values of 0.2 to 0.8. Optimal synergism was produced by a 4:1 PNU156804-CsA ratio (CI, 0.22). Oral delivery of CsA and RAPA resulted in synergism with an almost identical range of CI values between 0.1 and 0.6.18 In contradistinction, a combination of PNU156804 with RAPA yielded an additive effect (CI, 1). These results suggest that the sequential inhibition of signal 1 by CsA, followed by the inhibition of signal 3 by PNU156804 or RAPA, produces synergism, whereas the concomitant inhibition of signal 3 by PNU156804 and RAPA results only in an additive immunosuppressive effect. However, given that RAPA produces myelosuppressive and lipotoxic side effects because of the role of mTOR in non-cytokine-activated pathways,22 we postulate that a combination of CsA and PNU156804 would produce similarly potent immunosuppression without myelosuppressive and lipotoxic side effects. We propose that because mTOR lies downstream of Jak3 in T, B, and NK cells, TCGF signaling pathways could be selectively inhibited by PNU156804 without affecting mTOR signaling pathways in non-Jak3-expressing cell types. In conclusion, PNU156804 preferentially disrupts Jak3 (compared with
Jak2 autokinase activity), thereby selectively inhibiting
We thank Natasha M. Teixeira, Dwavalon Young, and Scott Homes for skilled preparation of the manuscript and the figures.
Submitted January 26, 2001; accepted June 13, 2001.
Supported by grants from the National Institute of Diabetes and Digestive Kidney Diseases (NIDDK 38016-12), the National Institutes of Heart and Lung Diseases (NHL69723), and the Roche Foundation (862506002).
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: Robert A. Kirken, Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, 6431 Fannin, MSB Rm 4.218, Houston, TX 77030; e-mail: robert.a.kirken{at}uth.tmc.edu.
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Chan AC, Kadlecek TA, Elder ME, et al.
ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency.
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1994;264:1599-1601 4. Appleby MW, Gross JA, Cooke MP, Levin SD, Qian X, Perlmutter RM. Defective T cell receptor signaling in mice lacking the thymic isoform of p59fyn. Cell. 1992;70:751-763[CrossRef][Medline] [Order article via Infotrieve]. 5. Kuo CT, Leiden JM. Transcriptional regulation of T lymphocyte development and function. Annu Rev Immunol. 1999;17:149-187[CrossRef][Medline] [Order article via Infotrieve]. 6. Leonard WJ, O'Shea JJ. JAKs and STATs: biological implications. Annu Rev Immunol. 1998;16:293-322[CrossRef][Medline] [Order article via Infotrieve]. 7. Kondo M, Takeshita T, Ishii N, et al. Sharing of the interleukin-2 (IL-2) receptor gamma chain between receptors for IL-2 and IL-4. Science. 1993;262:1874-1877 |
Top
Abstract
Introduction
chain is tyrosine (Tyr)
phosphorylated during the autoactivation of Zap70, Lck, and Fyn protein
Tyr kinases.
a necessary step for NFAT to translocate to the nucleus and
bind discrete DNA-binding elements within the promoter of the
interleukin-2 (IL-2) gene.
B and AP-1 transcription factors, the expression of cyclins D2 and E and cyclin-dependent kinases 2 and 4, and the hyperphosphorylation of the retinoblastoma protein. In contrast, IL-2 receptor (IL-2R) messenger RNA (mRNA) expression of 
70°C.
) or inactive control PNU159744 (
) for 16 hours at 37°C.
Conversely, Jurkat cells were treated in an identical fashion with
PNU156804 (
). All cells were then pulsed with
[3H]-thymidine (0.5 µCi [0.0185 MBq]/200 µL) for 4 hours, incorporated into a radiolabeled probe, and plotted on the
abscissa expressed as total cpm (n = 6).
), IL-7 (
), or IL-15
(
) with increasing concentrations of PNU156804 (ordinate) for 16 hours at 37°C. Cells were then pulsed with
[3H]-thymidine (0.5 µCi [0.0185 MBq]/200 µL) for 4 hours, and the incorporated radiolabeled probe plotted on the abscissa
was expressed as percentage inhibition of total cpm from DMSO-treated
sample sets (n = 6). (B) Quiescent rat T cells
(5.0 × 104 cells/well) were cultured in the presence of
the Jak2 activator (1 nM PRL [
20% of
total [3H]-thymidine incorporation), PRL-mediated growth
was retained by 2-fold or inhibited by less than 50% (Figure 
