BTK gene targeting by homologous recombination using a helper-dependent adenovirus/adeno-associated virus hybrid vector.

X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies, which is caused by mutations in Bruton's tyrosine kinase (BTK) gene. To examine the possibility of using gene therapy for XLA, we constructed a helper-dependent adenovirus/adeno-associated virus BTK targeting vector (HD-Ad.AAV BTK vector) composed of a genomic sequence containing BTK exons 6-19 and a green fluorescence protein-hygromycin cassette driven by a cytomegalovirus promoter. We first used NALM-6, a human male pre-B acute lymphoblastic leukemia cell line, as a recipient to measure the efficiency of gene targeting by homologous recombination. We identified 10 clones with the homologous recombination of the BTK gene among 107 hygromycin-resistant stable clones isolated from two independent experiments. We next used cord blood CD34⁺ cells as the recipient cells for the gene targeting. We isolated colonies grown in medium containing cytokines and hygromycin. We found that the targeting of the BTK gene occurred in four of the 755 hygromycin-resistant colonies. Importantly, the gene targeting was also observed in CD19⁺ lymphoid progenitor cells that were differentiated from the homologous recombinant CD34⁺ cells during growth in selection media. Our study shows the potential for the BTK gene therapy using the HD-Ad.AAV BTK vector via homologous recombination in hematopoietic stem cells.

Amelogenin splice isoforms stimulate chondrogenic differentiation of ATDC5 cells.

OBJECTIVE: Amelogenins are the most abundant matrix proteins in enamel. Among the amelogenin isoforms, full-length amelogenin (M180) and leucine-rich amelogenin peptide (LRAP) are expressed in various tissues and are implicated as signalling molecules in mesenchymal cells. Here, we examined the effects of M180 and LRAP on a chondrogenic cell line, ATDC5, to investigate the role of amelogenins in chondrogenesis. MATERIALS AND METHODS: Recombinant mouse M180- or LRAP-protein-containing medium or control medium was mixed with a chondrogenesis-stimulating medium, and changes in the phenotype, gene expression levels and cell proliferation of cultured ATDC5 cells were analysed. RESULTS: The addition of amelogenins increased alkaline phosphatase activity and glycosaminoglycan secretion at 14 and 21 days of culture, respectively, as compared with the control. Quantitative PCR (Q-PCR) analysis revealed that LRAP increased the gene expression levels of Runx2, Col2a1 and Aggrecan at 7 days of differentiation. Moreover, both M180 and LRAP significantly increased the gene expression levels of ALP, Aggrecan, Col10a1 and osteopontin at 28 days of culture. Bromodeoxyuridine assay and Q-PCR analysis for Wnt signalling indicated that both M180 and LRAP reduced proliferation, but induced the cell differentiation possibly through altered non-canonical Wnt signalling. CONCLUSION: M180 and LRAP accelerate chondrogenic differentiation and maturation of ATDC5 cells.

Oral management with polaprezinc solution reduces adverse events in haematopoietic stem cell transplantation patients.

The aim of this study was to analyse the effects of gargling with and then swallowing PPAA (polaprezinc in polyacrylic acid solution), in addition to regular oral management, on patients with a haematopoietic neoplasm scheduled for haematopoietic stem cell transplantation (HSCT). A total of 120 patients scheduled for HSCT during the years 2006-2016 were recruited. Patient background, oral adverse events, the incidence and severity of systemic adverse events (sepsis/septic shock, acute graft-versus-host disease (GVHD) after transplantation), and outcomes (survival/death) were compared between groups treated with and without PPAA. The severities of oral adverse events (oral mucositis, oral pain, and dysgeusia) were significantly lower in patients treated with PPAA. There was no significant difference in the incidence of febrile neutropenia (P=0.622) or sepsis/septic shock (P=0.665) as systemic adverse events. The severity of allograft-induced acute graft-versus-host disease (GVHD) was significantly lower in the PPAA group (P=0.011). There was no significant difference in outcome between the two groups (P=0.285). Within the limitations of the study design, it may be concluded that oral management with PPAA reduces adverse events in HSCT. Oral management with concomitant use of PPAA decreased oral adverse events and reduced the systemic complication of GVHD.

Luminal hydrogen sulfide plays a pronociceptive role in mouse colon.

OBJECTIVE: Given recent evidence that hydrogen sulfide (H(2)S), a gasotransmitter, promotes somatic pain through redox modulation of T-type Ca(2+) channels, the roles of colonic luminal H(2)S in visceral nociceptive processing in mice were examined. METHODS: After intracolonic administration of NaHS, an H(2)S donor, visceral pain-like behaviour and referred abdominal allodynia/hyperalgesia were evaluated. Phosphorylation of extracellular signal-regulated protein kinase (ERK) in the spinal dorsal horn was determined immunohistochemically. The whole-cell recording technique was used to evaluate T-type Ca(2+) currents (T-currents) in cultured dorsal root ganglion (DRG) neurons. RESULTS: Like capsaicin, NaHS, administered intracolonically at 0.5-5 nmol per mouse, triggered visceral nociceptive behaviour accompanied by referred allodynia/hyperalgesia in mice. Phosphorylation of ERK in the spinal dorsal horn was detected following intracolonic NaHS or capsaicin. The behavioural effects of intracolonic NaHS were abolished by a T-type channel blocker or an oxidant, but not inhibitors of L-type Ca(2+) channels or ATP-sensitive K(+) (K(ATP)) channels. Intraperitoneal NaHS at 60 micromol/kg facilitated intracolonic capsaicin-evoked visceral nociception, an effect abolished by the T-type channel blocker, although it alone produced no behavioural effect. In DRG neurons, T-currents were enhanced by NaHS. CONCLUSIONS: These findings suggest that colonic luminal H(2)S/NaHS plays pronociceptive roles, and imply that the underlying mechanisms might involve sensitisation/activation of T-type channels probably in the primary afferents, aside from the issue of the selectivity of mibefradil.

Unbalanced translocation der(1;7)(q10;p10) defines a unique clinicopathological subgroup of myeloid neoplasms.

The unbalanced translocation, der(1;7)(q10;p10), is one of the characteristic cytogenetic abnormalities found in myelodysplastic syndromes (MDS) and other myeloid neoplasms. Although described frequently with very poor clinical outcome and possible relationship with monosomy 7 or 7q- (-7/7q-), this recurrent cytogenetic abnormality has not been explored fully. Here we analyzed retrospectively 77 cases with der(1;7)(q10;p10) in terms of their clinical and cytogenetic features, comparing with other 46 adult -7/7q- cases without der(1;7)(q10;p10). In contrast with other -7/7q- cases, where the abnormality tends to be found in one or more partial karyotypes, der(1;7)(q10;p10) represents the abnormality common to all the abnormal clones and usually appears as a sole chromosomal abnormality during the entire clinical courses, or if not, is accompanied only by a limited number and variety of additional abnormalities, mostly trisomy 8 and/or loss of 20q. der(1;7)(q10;p10)-positive MDS cases showed lower blast counts (P<0.0001) and higher hemoglobin concentrations (P<0.0075) at diagnosis and slower progression to acute myeloid leukemia (P=0.0043) than other -7/7q- cases. der(1;7)(q10;p10) cases showed significantly better clinical outcome than other -7/7q cases (P<0.0001). In conclusion, der(1;7)(q10;p10) defines a discrete entity among myeloid neoplasms, showing unique clinical and cytogenetic characteristics.

Triple synergism of human T-lymphotropic virus type 1-encoded tax, GATA-binding protein, and AP-1 is required for constitutive expression of the interleukin-5 gene in adult T-cell leukemia cells.

Accumulated evidence demonstrates that adult T-cell leukemia (ATL) is frequently associated with eosinophilia, and human T-lymphotropic virus type 1 (HTLV-1)-infected cells frequently express interleukin-5 (IL-5). However, the molecular mechanism of constitutive IL-5 expression in HTLV-1-infected cells remains unclear. To clarify the mechanism of aberrant IL-5 expression in HTLV-1-infected cells, we investigated the response of the human IL-5 promoter to the HTLV-1-encoded protein Tax. Cotransfection experiments using Jurkat cells revealed that Tax is incapable of activating the IL-5 promoter by itself but that it synergistically transactivates the promoter with GATA-binding protein (GATA-4) and 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation. By introducing a series of mutations within the IL-5 promoter, we found that conserved lymphokine element 0 (CLE0) is responsible for mediating the signal induced by Tax-TPA. A deletion construct of the promoter indicated that the -75 GATA element and CLE0 are sufficient to mediate synergistic activation of the IL-5 promoter. Electrophoretic mobility shift assays using Jurkat cell nuclear extracts demonstrated that TPA induces a transcription factor to bind CLE0, and an experiment using JPX-9 cell nuclear extracts showed that Tax enhances this binding activity. An antibody supershift experiment revealed that this band consists of c-Jun and JunD. However, among the Jun family members, only c-Jun is able to cooperate with Tax and GATA-4 to activate the IL-5 promoter. We have determined the minimum factors required for IL-5 gene activation by reconstituting the IL-5 promoter activity in F9 cells. This is the first report to demonstrate the functional involvement of Tax protein in IL-5 gene regulation and to suggest the functional triple synergism among Tax, GATA-4, and AP-1, which disrupts regulated control of the gene and leads to constitutive expression of the IL-5 gene.

Dual functions of the AML1/Evi-1 chimeric protein in the mechanism of leukemogenesis in t(3;21) leukemias.

The chromosomal translocation t(3;21)(q26;q22), which is found in blastic crisis in chronic myelogenous leukemias and myelodysplastic syndrome-derived leukemias, produces AML1/Evi-1 chimeric transcription factor and is thought to play important roles in acute leukemic transformation of hemopoietic stem cells. We report here the functional analyses of AML1/Evi-1. It was revealed that AML1/Evi-1 itself does not alter the transactivation level through mouse polyomavirus enhancer-binding protein 2 (PEBP2; PEA2) sites (binding site of AML1) but dominantly suppresses the transactivation by intact AML1, which is assumed to be a stimulator of myeloid cell differentiation. DNA-binding competition is a putative mechanism of such dominant negative effects of AML1/Evi-1 because it binds to PEBP2 sites with higher affinity than AML1 does. Furthermore, AML1/Evi-1 stimulated c-fos promoter transactivation and increased AP-1 activity, as Evi-1 (which is not normally expressed in hemopoietic cells) did. Experiments using deletion mutants of AML1/Evi-1 showed that these two functions are mutually independent because the dominant negative effects on intact AML1 and the stimulation of AP-1 activity are dependent on the runt domain (DNA-binding domain of AML1) and the zinc finger domain near the C terminus, respectively. Furthermore, we showed that AML1/Evi-1 blocks granulocytic differentiation, otherwise induced by granulocyte colony-stimulating factor, of 32Dcl3 myeloid cells. It was also suggested that both AML1-derived and Evi-1-derived portions of the fusion protein play crucial roles in this differentiation block. We conclude that the leukemic cell transformation in t(3;21) leukemias is probably caused by these dual functions of AML1/Evi-1 chimeric protein.

A novel interferon regulatory factor family transcription factor, ICSAT/Pip/LSIRF, that negatively regulates the activity of interferon-regulated genes.

We have isolated a novel cDNA clone encoding interferon (IFN) consensus sequence-binding protein in adult T-cell leukemia cell line or activated T cells (ICSAT); this protein is the human homolog of the recently cloned Pip/LSIRF. ICSAT is structurally most closely related to the previously cloned ICSBP, a member of the IFN regulatory factor (IRF) family of proteins that binds to interferon consensus sequences (ICSs) found in many promoters of the IFN-regulated genes. Among T-cell lines investigated, ICSAT was abundantly expressed in human T-cell leukemia virus type 1 (HTLV-1)-infected T cells. When the HTLV-1 tax gene was expressed or phorbol myristake acetate-A23187 stimulation was used, ICSAT expression was induced in Jurkat cells which otherwise do not express ICSAT. When the binding of ICSAT to four different ICSs was tested, the relative differences in binding affinities for those ICSs were determined. To study the functional role of ICSAT, we performed cotransfection experiments with the human embryonal carcinoma cell line N-Tera2. ICSAT was demonstrated to possess repressive function over the gene activation induced by IFN stimulation or by IRF-1 cotransfection. Such repressive function is similar to that seen in IRF-2 or ICSBP. However, we have found that ICSAT has a different repressive effect from that of IRF-2 or ICSBP in some IFN-responsive reporter constructs. These results suggest that a novel mechanism of gene regulation by "differential repression" is used by multiple members of repressor proteins with different repressive effects on the IFN-responsive genes.

The extracellular signal-regulated kinase pathway phosphorylates AML1, an acute myeloid leukemia gene product, and potentially regulates its transactivation ability.

AML1 (also called PEBP2alphaB, CBFA2, or CBFalpha2) is one of the most frequently disrupted genes in chromosome abnormalities seen in human leukemias. It has been reported that AML1 plays several pivotal roles in myeloid hematopoietic differentiation and other biological phenomena, probably through the transcriptional regulation of various relevant genes. Here, we investigated the mechanism of regulation of AML1 functions through signal transduction pathways. The results showed that AML1 is phosphorylated in vivo on two serine residues within the proline-, serine-, and threonine-rich region, with dependence on the activation of extracellular signal-regulated kinase (ERK) and with interleukin-3 stimulation in a hematopoietic cell line. These in vivo phosphorylation sites of AML1 were phosphorylated directly in vitro by ERK. Although differences between wild-type AML1 and phosphorylation site mutants in DNA-binding affinity were not observed, we have shown that ERK-dependent phosphorylation potentiates the transactivation ability of AML1. Furthermore the phosphorylation site mutations reduced the transforming capacity of AML1 in fibroblast cells. These data indicate that AML1 functions are potentially regulated by ERK, which is activated by cytokine and growth factor stimuli. This study provides some important clues for clarifying unidentified facets of the regulatory mechanism of AML1 function.

Analysis of abietic acid and dehydroabietic acid in food samples by in-tube solid-phase microextraction coupled with liquid chromatography-mass spectrometry.

A simple and sensitive method for the determination of abietic acid and dehydroabietic acid in food samples was developed using a fully automated method consisting of in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-mass spectrometry (LC/MS). These compounds were separated within 5min by HPLC using an ODS-3 column and 5mM ammonium formate/acetonitrile (10/90, v/v). Electrospray ionization conditions in the negative ion mode were optimized for MS detection of abietic acid and dehydroabietic acid. The optimum in-tube SPME conditions were 20draw/eject cycles of 40microL of sample using a Supel Q PLOT capillary column as an extraction device. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME LC/MS method, good linearity of the calibration curve (r>0.9998) was obtained in the concentration range from 0 to 50ng/mL, and the detection limits (S/N=3) of abietic acid and dehydroabietic acid were 2.9 and 2.1pg/mL, respectively. The in-tube SPME method showed above 75-fold greater sensitivity than the direct injection method (5microL injection). This method was applied successfully to analysis of food samples without interference peaks. The recoveries of abietic acid and dehydroabietic acid spiked into liquid samples were above 79%, and the relative standard deviations were below 6.6%. These compounds were detected at ng/mL or ng/g levels in various liquid or solid food samples contacted with paper.

Association of insulin receptor substrate proteins with Bcl-2 and their effects on its phosphorylation and antiapoptotic function.

Insulin receptor substrate (IRS) proteins are docking proteins that couple growth factor receptors to various effector molecules, including phosphoinositide-3 kinase, Grb-2, Syp, and Nck. Here we show that IRS-1 associates with the loop domain of Bcl-2 and synergistically up-regulates antiapoptotic function of Bcl-2. IRS-2 but not IRS-3 binds to Bcl-2, and IRS-1 associates with Bcl-XL but not with Bax or Bik. Overexpression of IRS-1 suppresses phosphorylation of Bcl-2 induced by stimulation with insulin, and the hypophosphorylation may lead to its enhanced antiapoptotic activity. The binding site for Bcl-2 is located on the carboxyl half-domain of IRS-1. IRS-3, which lacks the corresponding region, dominant-negatively abrogates the survival effects of IRS-1 and Bcl-2. For the antiapoptotic activity of IRS-1, binding to Bcl-2 is more critical than activating phosphoinositide-3 kinase. Our results indicate that IRS proteins transmit signals from the insulin receptor to Bcl-2, thus regulating cell survival probably through regulating phosphorylation of Bcl-2.

Establishment of a human megakaryoblastic cell line (T-33) from chronic myelogenous leukemia in megakaryoblastic crisis.

A megakaryoblastic cell line, termed T-33, was established from the peripheral blood of a patient with Philadelphia chromosome-positive chronic myelogenous leukemia in megakaryoblastic crisis. T-33 cells have been maintained in RPMI 1640 medium containing 10% fetal calf serum in a single cell suspension with a doubling time of 24-36 h for over 2 years. Giemsa-banded karyotypes were female hyperdiploid with a modal chromosomal number of 51, all cells including Philadelphia chromosome. The cells showed strong positivity for periodic acid-Schiff and alpha-naphthyl acetate esterase, and weak for alpha-naphthyl butyrate esterase, but were negative for myeloperoxidase. Flow cytometric analysis of cell surface markers showed the existence of HLA-DR, MY-7, MY-9, and a platelet antigen (Yukb), and no markers for T- or B-lymphocytes. Most of the cells fixed with acetone were positive for Factor VIII, platelet glycoprotein IIb-IIIa, IIIa (Yukb), and Ib, but negative for glycophorin A and hemoglobin. Ultrastructural platelet peroxidase was demonstrated in 2-3% of cells and the percentage of positive cells increased up to 20% after the treatment with 12-O-tetradecanoylphorbol-13-acetate. The cells contained small dense granules negative for platelet peroxidase, their number increasing threefold after 12-O-tetradecanoylphorbol-13-acetate treatment. Such treated cells frequently showed a complex of the demarcation membrane in the cytoplasm. T-33 responded thrombin to exhibit calcium influx. This cell line may be useful for the study of the early stage of megakaryocytic differentiation in human megakaryopoiesis.

Successful glycemic control with three times a week degludec injection by medical staff for an elderly hemodialysis patient with type 2 diabetes.

For elderly hemodialysis patients with diabetes, the treatment options are restricted, and insulin therapy plays an important role. However, sometimes it might be difficult for them to inject insulin by themselves because of technical and/or social problems. The recently introduced basal insulin analog degludec has a longer half life than the previous basal insulin analogs such as glargine or detemir. Here we report an elderly hemodialysis patient with type 2 diabetes who was successfully treated with insulin degludec injection by the medical staff at every hemodialysis clinic visit. Throughout this treatment, he did not experience any side effects due to degludec, including hypoglycemia. There are few reports of using degludec for HD patients. In addition, this is the first report showing the validity of a three-times-a-week degludec regimen as a basal supported oral therapy for a hemodialysis patient with diabetes who could not inject insulin by himself. The inferiority of the three-times-a-week degludec regimen compared with the once-a-day glargine regimen in non-hemodialysis patients has already been reported. Based on this, this three-times-a-week degludec regimen should also not be considered as a standard regimen in hemodialysis patients. However, this three-times-a-week degludec regimen may be useful as an alternative for hemodialysis patients who cannot inject insulin once a day by themselves to achieve good and safe glycemic control, improving the prognosis and avoiding problems with hyperglycemia.

The AML1/Evi-1 fusion protein in the t(3;21) translocation exhibits transforming activity on Rat1 fibroblasts with dependence on the Evi-1 sequence.

The t(3;21) (q26;q22) chromosomal translocation associated with blastic crisis of chronic myelogenous leukemia (CML) results in the formation of a chimeric protein fusing the amino-terminal DNA-binding domain encoded by the AML1 gene to the carboxyl-terminal-encoding portion of the Evi-1 gene. In order to evaluate transforming activity of this protein, AML1/Evi-1 was introduced into Rat1 fibroblasts. Cells expressing the fusion product formed macroscopic colonies in soft agar, indicating that AML1/Evi-1 is a transforming gene. It was also demonstrated that introduction of AML1/Evi-1 into the Rat1 clones harboring BCR/ABL also conferred enhanced capacity for anchorage independent growth. Analyses of deletion mutants of AML1/Evi-1 revealed that removal of the second zinc finger domain within the Evi-1 sequence totally abrogated the ability of AML1/Evi-1 to transform Rat1 cells. We showed that the transforming effect is correlated with the AP-1 activation induced by AML1/Evi-1. Furthermore, we demonstrated that c-jun is transcriptionally activated in Rat1 cells transformed by AML1/Evi-1, suggesting that c-jun expression is under control of AML1/Evi-1. These results indicate that the oncogenic effect of the t(3;21) translocation is caused by the generation of a chimeric transcriptional factor and that AML1/Evi-1 could perform a pivotal role in leukemic progression of CML.

Overexpression of the AML1 proto-oncoprotein in NIH3T3 cells leads to neoplastic transformation depending on the DNA-binding and transactivational potencies.

The AML1 gene encodes DNA-binding proteins that contain the runt homology domain and is found at the breakpoints of t(8;21) and t(3;21) translocations associated with myelogenous leukemias. From the AML1 gene, two representative forms of proteins, AML1a and AML1b, are generated by alternative splicing. Both forms have the runt homology domain that possesses the DNA-binding ability but, unlike AML1b, AML1a lacks a putative transcriptional activation domain downstream of the runt homology domain. By using retroviral infection, we demonstrated that AML1b causes neoplastic transformation of NIH3T3 cells. AML1b-expressing cells form macroscopic colonies in soft agar and induce tumors in nude mice, indicating that AML1 can be a transforming gene when overexpressed in fibroblasts. Both the runt homology domain and the transactivational domain were required to transform NIH3T3 cells. By analysis of deletion mutants, it was shown that an element determining the transactivational potency exists between amino acids 288 and 396 within the region downstream of the runt homology domain. Furthermore, we demonstrated that this region was also required for fibroblast transformation, indicating that the transforming activity of AML1 is correlated with the transactivational potencies. These results suggest a role of AML1 in the regulation of cellular proliferation, as well as myeloid cell differentiation.

Mutations of the Smad4 gene in acute myelogeneous leukemia and their functional implications in leukemogenesis.

The Smad family proteins are critical components of the transforming growth factor (TGF)-beta signaling pathway. TGF-beta is a multipotent cytokine that elicits many biological functions. In particular, TGF-beta exhibits effects on the cell cycle manifested by G1-phase arrest, differentiation, or apoptosis of several target cells, suggesting that disruption of TGF-beta signaling pathway could be involved in cancer formation. Here we show one missense mutation of the Smad4 gene in the MH1 domain (P102L) and one frame shift mutation resulting in termination in the MH2 domain (Delta(483 - 552)) in acute myelogeneous leukemia. Both of the mutated Smad4 proteins lack transcriptional activities. Concomitant expression of the P102L mutant with wild-type Smad4 inactivates wild-type Smad4 through inhibiting its DNA-binding ability. The Delta(483 - 552) mutant blocks nuclear translocation of wild-type Smad4 and thus disrupts TGF-beta signaling. This is the first report showing that mutations in the Smad4 gene are associated with the pathogenesis of acute myelogeneous leukemia and the obtained results should provide useful insights into the mechanism whereby disruption of TGF-beta signaling pathway could lead to acute myelogeneous leukemia. Oncogene (2001) 20, 88 - 96.

Structurally altered Evi-1 protein generated in the 3q21q26 syndrome.

Overexpression of the Evi-1 gene appears to be a consistent feature of the 3q21q26 syndrome, an association of myeloid leukemias/myelodysplastic syndrome with a specific chromosomal aberration involving both 3q21 and 3q26, such as t(3;3)(q21;q26) or inv(3)(q21q26). The rearrangement in 3q26 has been reported to occur near the Evi-1 locus, implicating that it is the critical gene deregulated in the 3q21q26 syndrome. Here we present a structural abnormality of Evi-1 protein in a case with the 3q21q26 syndrome. In this case carrying typical inv(3)(q21q26), the 3q26 breakpoint is located within an intron of the Evi-1 gene, and resulted in overexpression of normally unexpressed, an aberrant form of Evi-1 protein, in which the C-terminal 44 amino acids of wild-type Evi-1 protein were truncated and replaced by five amino acids. The truncated Evi-1 protein is shown to increase AP1 activity when expressed in NIH3T3 cells as its wild-type counterpart. We also show that the origin of this peculiar type of rearrangement of the Evi-1 gene is not an artifact during establishment of the cell line, but is the event that occurred in the primary leukemic cells. Our results strongly support that the primary target for the 3q21q26 syndrome is the Evi-1 gene, and provide the first evidence that the structurally altered Evi-1 gene may be involved in the 3q21q26 syndrome.

The phosphatidylinositol 3' kinase pathway is required for the survival signal of leukocyte tyrosine kinase.

Leukocyte tyrosine kinase (LTK) is a receptor tyrosine kinase which belongs to the insulin receptor superfamily and is mainly expressed in pre-B lymphocytes and neuronal tissues. Recently, we demonstrated that LTK utilizes Shc and IRS-1 as two major substrates and while both equally activate the Ras pathway, only IRS-1 suppresses apoptosis of hematopoietic cells, suggesting the existence of another unidentified signaling pathway downstream of IRS-1, which is relevant to the anti-apoptotic activity. In the present study, we found that wortmannin, a specific inhibitor of phosphatidylinositol 3' (PI3)-kinase, abolished the survival effects of LTK. Although c-Cbl is found to be phosphorylated by LTK and therefore is a second candidate linking LTK with the PI3-kinase pathway along with IRS-1, we found that the p85 subunit of PI3 kinase directly binds to tyrosine 753 of LTK, which is located within a YXXM motif, a consensus binding amino acid sequence for the SH2 domain of p85, but fails to bind to IRS-1 or c-Cbl. Ba/F3 cells which stably express the EGF receptor-LTK chimeric receptor carrying a mutation at tyrosine 753 fell into apoptotic death even in the presence of EGF, indicating that the PI3 kinase pathway is required for the survival effects of LTK.

Characterization of stage progression in chronic myeloid leukemia by DNA microarray with purified hematopoietic stem cells.

Chronic myeloid leukemia (CML) is characterized by the clonal expansion of hematopoietic stem cells (HSCs). Without effective treatment, individuals in the indolent, chronic phase (CP) of CML undergo blast crisis (BC), the prognosis for which is poor. It is therefore important to clarify the mechanism underlying stage progression in CML. DNA microarray is a versatile tool for such a purpose. However, simple comparison of bone marrow mononuclear cells from individuals at different disease stages is likely to result in the identification of pseudo-positive genes whose change in expression only reflects the different proportions of leukemic blasts in bone marrow. We have therefore compared with DNA microarray the expression profiles of 3456 genes in the purified HSC-like fractions that had been isolated from 13 CML patients and healthy volunteers. Interestingly, expression of the gene for PIASy, a potential inhibitor of STAT (signal transducer and activator of transcription) proteins, was down-regulated in association with stage progression in CML. Furthermore, forced expression of PIASy has induced apoptosis in a CML cell line. These data suggest that microarray analysis with background-matched samples is an efficient approach to identify molecular events underlying the stage progression in CML.