Hiroshi Kamano

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.

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.

Antisense src expression inhibits tyrosine phosphorylation of Shc and its association with Grb2 and Sos which leads to MAP kinase activation in U937 human leukemia cells

We constructed a recombinant plasmid which expresses antisense src RNA after dexamethasone (Dexa) treatment, and transfected it into U937 human monoblastic leukemia cells (U937-ASRC). Induction of antisense src RNA expression diminished the amounts of c-Src and its protein tyrosine kinase (PTK) activity in U937-ASRC cells. The declines in c-Src and its PTK activity subsequently reduced the proliferation of U937-ASRC cells. To elucidate the growth signal transduction pathway downstream of c-Src, tyrosine phosphorylation of Shc was examined in U937-ASRC cells treated with Dexa. The decline in c-Src by induction of antisense src RNA expression decreased the level of tyrosine phosphorylation of Shc. Immunoprecipitated c-Src directly phosphorylated immunoprecipitated Shc on tyrosine residues in vitro. The amounts of Grb2 and Sos co-immunoprecipitated with Shc were decreased after Dexa treatment. However, the amount of Sos co-immunoprecipitated with Grb2 was apparently not affected by Dexa treatment. These results indicate that Grb2 and Sos constitutively associate with each other in U937 cells. Furthermore, the level of phosphorylation on tyrosine (204) essential for MAP kinase activation was decreased after Dexa treatment. Taken together with all these findings, it is suggested that c-Src directly phosphorylates Shc on tyrosine residues, which in turn binds to Grb2 constitutively associated with Sos to form a Shc-Grb2-Sos complex, and that the complex formation is coupled with MAP kinase activation mediated by Ras activation in U937 cells.

p75c-myb expression in leukemia-lymphoma cells correlated with proliferation and differentiation

Monoclonal antibodies (McAbs) against a part of v-myb gene product were prepared for the detection of human c-myb gene product (p75c-myb). Western blotting analyses with these McAbs were performed on human leukemia-lymphoma cells. All T-cell lines were positive in p75c-myb expression. B-cell lines were variable, myeloid and erythroid cells were positive although the amount of expressed p75c-myb was less than the T-cell lines. Cells isolated from patients were positive in expression except for cells from acute myeloblastic leukemia with maturation (AML M2), acute hypergranular promyelocytic leukemia (AML M3) and erythroleukemia (AML M6) developed from myelodysplastic syndromes. Differences in p75c-myb expression seemed to depend upon the differentiation stage and distinctive lineage from which each cell line had been established. The p75c-myb expression in HL60 (acute promyelocytic leukemia cell line) showed remarkably high at logarithmic growth. When examined with HL60, p75c-myb expression significantly decreased during the differentiation induced by 12-O-tetradecanoylphorbol-13-acetate or retinoic acid. These results suggest that p75c-myb expression plays a crucial role in hematopoietic cell proliferation and differentiation and that multiple mechanisms including aberrant expression of p75c-myb is involved in leukemogenesis.

A Trial Design of e-Healthcare Management Scheme with IC-Based Student ID Card, Automatic Health Examination System and Campus Information Network

A Health Education Support System has been being developed for Students and university staffs of Kagawa University. The system includes an IC card reader(writer), several types of physical measuring devices (height meter, weight meter, blood pressure monitor, etc. for health examination), a special-purpose PC, distributed information servers and campus network environment. We have designed our prototype of a Health Education Support System as follows; Students and/or university staffs can utilize the above system for their health education and/or healthcare whenever they want anywhere in university. They can use IC-based ID cards for user authentication, operate the physical measuring devices very much simply, and maintain their physical data periodically. Measured data can be obtained at any point of university by means of measuring devices connected with the system on-line, transferred through campus network environment, and finally cumulated into a specific database of secured information servers. We have carried out some experiments to design our system and checked behaviour of each subsystem in order to evaluate whether such a system satisfy our requirements to build facilities to support health education described above. In this paper, we will introduce our design concepts of a Health Education Support System, illustrate some experimental results and discuss perspective problems as our summaries.

Induction of erythroid differentiation of K562 cells by 4-carbamoylimidazolium 5-olate (SM-108)

The effects of 4-carbamoylimidazolium 5-olate (SM-108), an antipurine compound, on a human leukemia cell line, K562, were studied. Treatment with SM-108 induced erythroid differentiation of K562 cells. During a 6-day culture with 100 microM SM-108, the cell number decreased to 37% of the control number, 77% of the cells became benzidine-positive, and the hemoglobin content increased from 2.1 +/- 0.2 to 10.6 +/- 1.3 pg/cell. Cell differentiation was associated with reduction of IMP dehydrogenase activity and intracellular GTP content to 25 and 36%, respectively, of the control values within 1.5 hr. The differentiation and decrease in the GTP pool induced by SM-108 were blocked by the presence of 25 microM guanine or guanosine. SM-108 also induced erythroid differentiation of K562 subline cells transfected with pMSG (K562/pMSG), which have an additional salvage pathway for GMP production from xanthine. The addition of 100 microM xanthine prevented erythroid differentiation of this subline and restored the GTP pool. These findings suggest that the induction of erythroid differentiation of K562 cells by SM-108 may be due to an early decrease in IMP dehydrogenase activity and a subsequent decrease in GTP content in the cells. Thus, purine metabolism may have an important role in SM-108-induced differentiation.

Photodynamic purging of leukemia cells by high-brightness light emitting diode and gallium-metal porphyrin

We applied a high-brightness light emitting diode (LED) and gallium-metal porphyrin (Ga-C10-P) for leukemia cell purging. The blue, blue-green, green, yellow-green, yellow, orange and red color LEDs were constructed in photo-irradiation equipment (BIOLED). We set up the BIOLED in a CO/sub 2/ incubator and cultured human chronic myelocytic leukemia cell line K562 in RPMI1640. We irradiated 525 nm green light, 612 nm orange light or 660 nm red fight on to the K562 cells in the presence of 0.1 /spl mu/M or 1 /spl mu/M Ga-C10-P. Surprisingly, the 525 nm photo-irradiation induced the suppression of proliferation of leukemia cells in the presence of 1 /spl mu/M Ga-C10-P. Our results suggest a possibility of eliminating leukemia cells in autologous bone marrow transplantation by photodynamic purging using LED light and Ga-C1O-P.

Novel applications of high-brightness LED in biomedical technology-development of photodynamic therapy of leukemia by LED

Light irradiation onto leukemia cells by LED were performed at 37 /spl deg/C using several sets of BIOLED placed in a CO/sub 2/ incubator under a fixed light intensity. We investigated the effect of light wavelength on the cell division. Irradiations onto cells were done for two kind of culture media: (1) with an addition of metal porphyrin which is used in photodynamic therapy (PDT) of cancers, and (2) without any additive.

Development of an automatic health screening system for Student Health Education of University

A Health Education Support System has been designed and being implemented for our students of Kagawa University in Japan. This paper describes the detail of An Automatic Health Screening System, which can play the former part role of our Health Education Support System, is described in detail in this paper. Major characteristics of our screening system are using IC card for user identification, interfacing several kinds of physical measuring devices like as height meter, weight meter, blood pressure monitor, etc. and retrieving measured data through Web-DB system. Human errors and incorrect identification can be reduced by means of automatic identification with IC card. Speedup of screening can be realized through interfacing between physical measuring devices and computers. And efficient retrieval of measured data can be performed with Web-DB system and Distributed campuses network environment in our University. With our automatic health screening system, almost students can easily participate in University-level health screening and investigate/retrieve their proper physical data for their healthcare through dedicated Web-DB system on the network environment.