Yoshitsugu Kubota

Src directly tyrosine-phosphorylates STAT5 on its activation site and is involved in erythropoietin-induced signaling pathway

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 tyrosine-phosphorylate 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 differentiation 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 identified 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 C-terminal-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 EPO-induced signal transduction via STAT5.

Src transduces erythropoietin-induced differentiation signals through phosphatidylinositol 3-kinase.

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.

Constitutive activation of PI3K is involved in the spontaneous proliferation of primary acute myeloid leukemia cells: direct evidence of PI3K activation.

Constitutive activation of PI3K is involved in the spontaneous proliferation of primary acute myeloid leukemia cells: direct evidence of PI3K activation

Singar1, a novel RUN domain-containing protein, suppresses formation of surplus axons for neuronal polarity

Although neuronal functions depend on their robust polarity, the mechanisms that ensure generation and maintenance of only a single axon remain poorly understood. Using highly sensitive two-dimensional electrophoresis-based proteomics, we identified here a novel protein, single axon-related (singar)1/KIAA0871/RPIPx/RUFY3, which contains a RUN domain and is predominantly expressed in the brain. Singar1 expression became up-regulated during polarization of cultured hippocampal neurons and remained at high levels thereafter. Singar1 was diffusely localized in hippocampal neurons and moderately accumulated in growth cones of minor processes and axons. Overexpression of singar1 did not affect normal neuronal polarization but suppressed the formation of surplus axons induced by excess levels of shootin1, a recently identified protein located upstream of phosphoinositide-3-kinase and involved in neuronal polarization. Conversely, reduction of the expression of singar1 and its splicing variant singar2 by RNA interference led to an increase in the population of neurons bearing surplus axons, in a phosphoinositide-3-kinase-dependent manner. Overexpression of singar2 did not suppress the formation of surplus axons induced by shootin1. We propose that singar1 ensures the robustness of neuronal polarity by suppressing formation of surplus axons.

Automatic detection of immature platelets for decision making regarding platelet transfusion indications for pediatric patients

Immature or reticulated platelets are known as a clinical marker of thrombopoiesis. Recently, an automatic method was established to detect reticulated platelets as immature platelet fraction (IPF) by means of hematology analyzer XE-2100. We assessed the effects of IPF detection after chemotherapy for various pediatric malignant disorders of 16 patients. Our results indicate that IPF should be considered a useful marker of imminent platelet recovery so that unnecessary platelet transfusion can be avoided.

The immature platelet fraction is a useful marker for predicting the timing of platelet recovery in patients with cancer after chemotherapy and hematopoietic stem cell transplantation.

Prediction of the timing of platelet recovery after chemotherapy and hematopoietic stem cell transplantation (HSCT) allows for optimal platelet transfusion. We assessed the clinical utility of the percentage value of the immature platelet fraction (IPF%) monitored using an XE‐2100 automated hematology analyzer to predict the timing of platelet recovery after chemotherapy and HSCT. The IPF% was serially monitored in 31 patients with cancer who received 66 courses of chemotherapy and HSCT. In patients with cancer undergoing chemotherapy and HSCT, a transient increase in IPF% was observed 1–11u2003days prior to platelet recovery (>30u2003×u2003109/l). In patients undergoing chemotherapy with a peak IPF% >10%, platelet recovery occurred significantly earlier than in those with IPF% peak values ≤10% (median periods were 2 and 5u2003days; Pu2003<u20030.05). Platelet recovery appears to occur earlier in patients undergoing HSCT with a peak IPF% >10% than in those with IPF% peak values ≤10% (median periods were 2 and 6u2003days). Thus, the IPF% peak value is a useful parameter for predicting the timing of platelet recovery after chemotherapy and HSCT and has the potential to facilitate optimal platelet transfusion.

Multivariate analysis of factors influencing oral mucositis in allogeneic hematopoietic stem cell transplantation

Little information is available regarding the effect of oral intervention on the outcome of hematopoietic stem cell transplantation (HSCT). We retrospectively analyzed the incidence of oral mucositis after allogeneic HSCT with or without oral intervention among 96 consecutive patients in our hospital between January 1988 and March 2006. We combined two oral intervention strategies: cryotherapy and oral health care. The former was applied beginning in 2003 for patients being treated with melphalan, and the latter, which was the study’s main strategy, was applied to all HSCT recipients beginning in 2004. Oral mucositis was evaluated according to NCI CTCAE v3.0. The incidence of oral mucositis was 30.9% (17/55) in reduced-intensity stem cell transplantation (RIST), which was significantly lower than the 90.2% (37/41) in conventional stem cell transplantation (CST; Pu2009<u20090.001). Among these 96 patients, severe oral mucositis was observed in 19 (46.3%) CST cases and in 6 (10.9%) RIST cases (Pu2009<u20090.001). The occurrence of oral mucositis apparently decreased after oral health care instructions were given. Multiple logistic analysis revealed that the conditioning regimen and oral health care were independent risk factors for the incidence of oral mucositis. The cryotherapy did not exert enough potency to prevent oral mucositis in patients who had undergone CST or RIST. We concluded that oral health care improved tissue damage due to an overall upgrade in oral hygiene during chemotherapy.

THROMBOPOIETIN MODULATES PLATELET ACTIVATION IN VITRO THROUGH PROTEIN-TYROSINE PHOSPHORYLATION

To determine the roles of thrombopoietin (TPO) in platelet function in vitro, we examined the effects of TPO on platelet aggregation. Although several proteins in platelets were tyrosine‐phosphorylated by TPO treatment, TPO alone was unable to induce platelet aggregation. However, the secondary wave of platelet aggregation induced by adenosine diphosphate (ADP) was enhanced by TPO in a dose‐dependent manner. TPO in conjunction with ADP augmented tyrosine phosphorylation of platelet proteins, including tyrosine‐phosphorylated proteins induced by TPO alone. Genistein inhibited protein‐tyrosine phosphorylation in platelets induced by TPO with ADP and suppressed TPOenhanced platelet aggregation. Moreover, tyrosine phosphorylation of MAP‐kinases induced by TPO alone and TPO with ADP was consistent with TPO‐enhanced platelet aggregation. These findings in the present study suggest that signal transduction involved in TPO‐enhanced platelet aggregation is mediated in part by tyrosine‐phosphorylated proteins, including MAP‐kinases, in platelets through TPO‐stimulated c‐Mpl, TPO receptor.

Intravascular large B-cell lymphoma with FDG accumulation in the lung lacking CT/67gallium scintigraphy abnormality

Intravascular large B‐cell lymphoma (IVLBCL) is a rare lymphoma characterized by the presence of large tumour cells within the blood vessels. It has been considered that IVLBCL is a highly malignant disease with poor prognosis. However, it has been shown that a therapeutic effect resembling that of conventional B‐cell lymphomas may be obtained with the application of systemic chemotherapy at the early stage of this disease. Although involvement in the lung is often detected at autopsy, early diagnosis is quite difficult. In this report, we present a case of IVLBCL with pulmonary involvement where 18‐fluoro‐deoxyglucose positron emission tomography (FDG‐PET) was useful in the early diagnosis. Neither computed tomography (CT) nor 67gallium scintigraphy could reveal the presence of disease in the lung. Histological evidence of IVLBCL was obtained by TBLB after FDG uptake in the lung was confirmed by FDG‐PET. The patient exhibited a good response to the subsequent combination chemotherapy. We propose that FDG‐PET is a powerful tool for the early diagnosis of IVLBCL with pulmonary involvement, if the possibility of this disease presents in the patient with respiratory symptoms without abnormal findings by CT and 67gallium scintigraphy. Copyright

Constitutively activated phosphatidylinositol 3-kinase primes platelets from patients with chronic myelogenous leukemia for thrombopoietin-induced aggregation.

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.