1. Selective inhibitor of Janus tyrosine kinase 3, PNU156804, prolongs allograft survival and acts synergistically with cyclosporine but additively with rapamycin
by Stanislaw M. Stepkowski, Rebecca A. Erwin-Cohen, Fariba Behbod, Mou-Er Wang, Xienui Qu, Neelam Tejpal, Zsuzsanna S. Nagy, Barry D. Kahan, and Robert A. Kirken
Blood
Volume 99(2):
January 15, 2002
©2002 by American Society of Hematology

2. PNU disrupts cell growth of activated, but not of unactivated, human T cells in a dose-dependent manner.Proliferation of quiescent PHA-activated human T cells (5.0 × 104 cells/well) in the presence of 1 nM IL-2 was examined after treatment with increa...
PNU disrupts cell growth of activated, but not of unactivated, human T cells in a dose-dependent manner.Proliferation of quiescent PHA-activated human T cells (5.0 × 104 cells/well) in the presence of 1 nM IL-2 was examined after treatment with increasing concentrations of PNU (●) or inactive control PNU (♦) for 16 hours at 37°C. Conversely, Jurkat cells were treated in an identical fashion with PNU (▴). All cells were then pulsed with [3H]-thymidine (0.5 μCi [ MBq]/200 μL) for 4 hours, incorporated into a radiolabeled probe, and plotted on the abscissa expressed as total cpm (n = 6).
Stanislaw M. Stepkowski et al. Blood 2002;99:
©2002 by American Society of Hematology

3. PNU inhibits the signal 3 mediator Jak3 but not the signal 1 transducer p56Lck.(A) Antiphosphotyrosine (αPY) immunoblot of PHA-activated T cells stimulated with anti-CD3 in the presence of PNU
PNU inhibits the signal 3 mediator Jak3 but not the signal 1 transducer p56Lck.(A) Antiphosphotyrosine (αPY) immunoblot of PHA-activated T cells stimulated with anti-CD3 in the presence of PNU T cells (5.0 × 107 cells/lane) were treated with DMSO or with 20 μM PNU for 16 hours and then were stimulated in the presence or absence of 5 μg anti-CD3 for 5 minutes and subjected to cell lysis and clarification. Total cell lysates were separated by SDS-PAGE (lanes a-d) or were immunoprecipitated with antibodies to p56Lck (lanes e-h) and separated on 10% SDS-PAGE and then subjected to antiphosphotyrosine Western blot (WB) analysis. The blot was then stripped and reblotted with anti-Lck to verify equivalent loading (indicated in the lower panel). Arrow denotes migration of p56Lck. (B) PHA-activated human T cells were treated with DMSO or increasing concentrations of PNU (0-10 μM; lanes a-j) or inactive control PNU (lanes k-n) and were stimulated in the absence (−) or presence (+) of IL-2 (100 nM) for 10 minutes at 37°C. Cells were lysed, clarified, and immunoprecipitated (IP) with anti-Jak3 (αJak3), separated on 10% SDS-PAGE, transferred to PVDF membrane, and Western blotted with antiphosphotyrosine. The blot was stripped and reprobed with Jak3 antibody. (C) For antiphosphotyrosine immunoblot of Jak3 autokinase assay, PHA-activated T cells were stimulated for 10 minutes with (+) or without (−) 100 nM IL-2 and then immunoprecipitated with anti-Jak3 and treated for 15 minutes on ice with DMSO control (lanes a-d) or 10 μM PNU156 804 (lanes e-h). Jak3 was incubated for 20 minutes at 37°C in the absence (−) or presence (+) of 15 μM unlabeled ATP, separated by SDS-PAGE, transferred to PVDF membrane, and Western blotted with antiphosphotyrosine antibodies (upper panel). The same blots were reblotted with α-Jak3 to verify equal loading of enzyme (lower panel). Ratio of Tyr-phosphorylated Jak3 to total Jak3 protein was analyzed by densitometry and plotted as the stimulatory index (SI) value in arbitrary units.
Stanislaw M. Stepkowski et al. Blood 2002;99:
©2002 by American Society of Hematology

4. PNU inhibits IL-2–inducible Tyr phosphorylation of Stat5a/b in human T cells.PHA-activated human T cells were pretreated with DMSO, 10 μM in active analogue PNU159744, or 10 μM PNU for 16 hours and then stimulated for 10 minutes with (+) or with...
PNU inhibits IL-2–inducible Tyr phosphorylation of Stat5a/b in human T cells.PHA-activated human T cells were pretreated with DMSO, 10 μM in active analogue PNU159744, or 10 μM PNU for 16 hours and then stimulated for 10 minutes with (+) or without (−) 100 nM IL-2 for 10 minutes at 37°C. Cells were lysed and immunoprecipitated with anti-Stat5a (A) or anti-Stat5b (B) and were blotted with monoclonal antiphosphotyrosine Stat5 (Y701, upper panel), polyclonal phosphoserine Stat5 (S726-Stat5a or S731 Stat5b, middle panel), and reblotted with monoclonal pan-Stat5 (lower panel). WB indicates Western blot; PY, .
Stanislaw M. Stepkowski et al. Blood 2002;99:
©2002 by American Society of Hematology

5. PNU156804 disrupts IL-2–mediated p44/42 ERK1/2 phosphorylation
PNU disrupts IL-2–mediated p44/42 ERK1/2 phosphorylation.Quiescent PHA-activated T cells were treated with DMSO (control; lanes a-b) or increasing concentrations of PNU for 16 hours and were stimulated in the presence of 100 nM IL-2 at 37°C for ...
PNU disrupts IL-2–mediated p44/42 ERK1/2 phosphorylation.Quiescent PHA-activated T cells were treated with DMSO (control; lanes a-b) or increasing concentrations of PNU for 16 hours and were stimulated in the presence of 100 nM IL-2 at 37°C for 10 minutes. Cells were lysed, and total cell lysate was separated on 10% SDS-PAGE, transferred to PVDF membrane, Western blotted with antiphospho–p44/42 Erk1/2 (upper panel), stripped, and reprobed with pan-Erk antibody (lower panel). Arrows indicate the location of p44/42 Erk1/2.
Stanislaw M. Stepkowski et al. Blood 2002;99:
©2002 by American Society of Hematology

6. PNU selectively inhibits Jak3-γc-cytokine–mediated cell proliferation compared with Jak2 growth factors.(A) Proliferation of quiescent PHA-activated human T cells (5.0 × 104 cells/well) were cultured in the absence or presence of 1 nM human IL-2 (●), ...
PNU selectively inhibits Jak3-γc-cytokine–mediated cell proliferation compared with Jak2 growth factors.(A) Proliferation of quiescent PHA-activated human T cells (5.0 × 104 cells/well) were cultured in the absence or presence of 1 nM human IL-2 (●), IL-4 (○), IL-7 (▾), or IL-15 (▿) with increasing concentrations of PNU (ordinate) for 16 hours at 37°C. Cells were then pulsed with [3H]-thymidine (0.5 μCi [ 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 [○]) or Jak3 activator (1 nM IL-2 [●]), with increasing concentrations of PNU (ordinate, μM) for 16 hours at 37°C. Cells were pulsed with [3H]-thymidine (0.5 μCi [ MBq]/200 μL) during the final 4 hours of the assay, and the DNA-incorporated radiolabeled probe was plotted on the abscissa and expressed as percentage inhibition of total cpm from DMSO-treated sample sets (n = 6). (inset) Antiphosphotyrosine immunoblot of Jak2-Jak3–activated signaling pathway. Nb2-11c cells treated with PNU or DMSO (as described in Figure 1A) were stimulated with 100 nM IL-2 (lanes a-f) or 100 nM PRL (lanes g-l). Jak3 or Jak2 (upper panel), Stat5a (middle panel), and Stat5b (lower panel) were immunoprecipitated from lysates, separated by SDS-PAGE, transferred to PVDF membrane, and blotted with specific antiphosphotyrosine mAb.
Stanislaw M. Stepkowski et al. Blood 2002;99:
©2002 by American Society of Hematology

7. PNU prolongs the survival of heart allografts alone and acts synergistically with CsA but not RAPA.WF (donor, RT11) to BUF (recipient, RT1a) rats received alternate-day oral gavage for 14 days (6 total) of 40, 80, or 120 mg/kg PNU alone or in co...
PNU prolongs the survival of heart allografts alone and acts synergistically with CsA but not RAPA.WF (donor, RT11) to BUF (recipient, RT1a) rats received alternate-day oral gavage for 14 days (6 total) of 40, 80, or 120 mg/kg PNU alone or in combination with CsA at ratios of 1:1 to 1:64 or in combination with RAPA at ratios of 40:1 to 160:1. Some recipients received various amounts of CsA or RAPA, alone as indicated within the table. Graft survival was evaluated daily; the last day of a heartbeat was considered the day of rejection. CI values were calculated by the median effect analysis: CI < 1 showed synergistic interactions, CI > 1 showed antagonistic interactions, and CI = 1 showed additive interactions. See “Materials and methods” for details.
Stanislaw M. Stepkowski et al. Blood 2002;99:
©2002 by American Society of Hematology

8. PNU blocks allograft damage to myocytes and reduces infiltration of polymorphonuclear cells.BUF heart allografts were harvested and examined on day after grafting from WF recipients that received alternate-day oral gavage of DMSO vehicle alone (A) or ...
PNU blocks allograft damage to myocytes and reduces infiltration of polymorphonuclear cells.BUF heart allografts were harvested and examined on day after grafting from WF recipients that received alternate-day oral gavage of DMSO vehicle alone (A) or vehicle with 80 mg/kg PNU (B). The presented sections were obtained after uniform histologic analysis of 3 hearts for each experimental group, with 12 horizontal cuts made on each heart. Each section was scored using the Society of Heart and Lung Transplantation system. See “Materials and methods” for details. Original magnification, × 200.
Stanislaw M. Stepkowski et al. Blood 2002;99:
©2002 by American Society of Hematology