Signal transducers and activators of transcription (STAT) proteins are transcription factors activated by phosphorylation on tyrosine residues after cytokine stimulation. In erythropoietin receptor (EPOR)-mediated signaling, STAT5 is tyrosine-phosphorylated by EPO stimulation. Although Janus Kinase 2 (JAK2) is reported to play a crucial role in EPO-induced activation of STAT5, it is unclear whether JAK2 alone can tyrosinephosphorylate STAT5 after EPO stimulation. Several studies indicate that STAT activation is caused by members of other families of protein tyrosine kinases such as the Src family. We previously reported that reduction of Src by induction of antisense src RNA expression suppressed EPO-promoted erythroid dierentiation in K562 cells. In the present study, we explored the function of Src downstream of the EPOR-initiated signaling. Reduction of Src diminished tyrosine phosphorylation of STAT5 in K562 cells regardless of EPO treatment. The tyrosine phosphorylation level of STAT5 induced by EPO in F-36P cells was reduced in the presence of PP1 or PP2 selective Src inhibitor. In addition, the expression of dominant negative Src in F-36P cells reduced the tyrosine phosphorylation of STAT5. When Src and STAT5 were co-expressed in COS7 cells, tyrosine phosphorylation of STAT5 was observed, and tyrosine residue 694 (Tyr 694) of STAT5A was identi®ed as the major phosphorylation site by Src. In vitro kinase assay revealed that GST-STAT5 fusion protein with the conserved C-terminal, but not the Cterminal-truncated mutant which lacks Tyr 694, was tyrosine-phosphorylated by Src. Src can thus directly tyrosine-phosphorylate the activation site of STAT5 (Tyr 694 in STAT5A), and Src may contribute to EPOinduced signal transduction via STAT5. Oncogene (2001) 20, 6643 ± 6650.

In this study, we examined the molecular mechanism of erythropoietin‐initiated signal transduction of erythroid differentiation through Src and phosphatidylinositol 3‐kinase (PI3‐kinase). Antisense oligonucleotides against src but not lyn inhibited the formation of erythropoietin‐dependent colonies derived from human bone marrow cells and erythropoietin‐induced differentiation of K562 human erythroleukaemia cells. Antisense p85α oligonucleotide or LY294002, a selective inhibitor of PI3‐kinase, independently inhibited the formation of erythropoietin‐dependent colonies. In K562 cells, Src associated with PI3‐kinase in response to erythropoietin. Antisense src RNA expression in K562 cells inhibited the erythropoietin‐induced activation of PI3‐kinase and its association with erythropoietin receptor. PP1, a selective inhibitor of the Src family, reduced erythropoietin‐induced tyrosine phosphorylation of erythropoietin receptor and its association with PI3‐kinase in F‐36P human erythroleukaemia cells. The coexpression experiments and in vitro kinase assay further demonstrated that Src directly tyrosine‐phosphorylated erythropoietin receptor, and associated with PI3‐kinase. In vitro binding experiments proved that glutathione S‐transferase–p85α N‐ or C‐terminal SH2 domains independently bound to erythropoietin receptor, which was tyrosine‐phosphorylated by Src. Taken together, Src transduces the erythropoietin‐induced erythroid differentiation signals by regulating PI3‐kinase activity.

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In this study, we examined the effect of thrombopoietin (TPO) on the aggregation of platelets from 40 patients with myeloproliferative disorders (MPDs), including 17 patients with chronic myelogenous leukemia in the chronic phase (CML-CP), 10 with polycythemia vera, 10 with essential thrombocythemia, and three with myelofibrosis. TPO by itself dose-dependently induced the aggregation of platelets from patients with CML-CP but not from those with other MPDs or with CML-CP in cytogenetical complete remission. The expression of CD63 in CML-CP platelets was induced by TPO treatment. Phosphatidylinositol 3-kinase (PI3-kinase) was constitutively activated in CML-CP platelets. Pretreatment with PI3-kinase inhibitors (wortmannin and LY294002) dose-dependently inhibited TPO-induced aggregation of CML-CP platelets. The Abl kinase inhibitor imatinib mesylate and the Jak inhibitor AG490 suppressed TPO-induced aggregation of CML-CP platelets. Pretreatment with imatinib mesylate, but not with AG490, inhibited the activity of PI3-kinase in CML-CP platelets. In addition, tyrosine phosphorylation of Jak2 was undetected in CML-CP platelets before TPO treatment. These findings indicate that the constitutive activation of PI3-kinase primes CML-CP platelets for the aggregation induced by TPO, and that Bcr-Abl, but not Jak family protein tyrosine kinases, are involved in the constitutive activation of PI3-kinase in CML-CP platelets.

This is the first report of the successful treatment of advanced biliary tract cancer with gemcitabine single-agent chemotherapy in combination with Juzen-taiho-to (JTT), a Japanese traditional herbal medicine. An 84-year-old woman was referred to our hospital with general fatigue and appetite loss; she was diagnosed with advanced biliary tract cancer and accompanying colonic invasion and hepatic metastasis. The patient's response to combination chemotherapy was extremely good, and her tumors disappeared. Recent studies have confirmed the occurrence of spontaneous and induced antitumor immune responses, carried out by tumor-infiltrating lymphocytes in the tumor microenvironment. The availability, antigen presentation, and proliferation of these immune cells are increased by cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-2. Recently, JTT has gained recognition as a biological response modifier that has stimulatory effects on systemic immune responses such as enhancement of cytokine expression (GM-CSF, IL-2, etc.). In addition, some chemotherapy agents, such as anthracyclines and gemcitabine, are effective boosters of the immune response through tumor-specific antigen overexpression after apoptotic tumor cell destruction. These findings suggest that JTT enhances the antitumor effects of gemcitabine, in particular its tumor-specific effects on immune response, and these drugs are a good combination for advanced biliary tract cancer therapy.