小澤 敬也

Flow cytometric, immunochemical and molecular studies were performed on leukemic blasts from a patient with minimally differentiated erythroleukemia (AML-M6v). The blasts expressed CD36 and CD71 but not lymphoid antigens, myeloid antigens, CD41 or glycophorin A. Analysis of carbohydrate antigens showed that the blasts expressed the sialyl-Tn antigen. Immunochemistry revealed that the blasts had neuron-specific enolase (NSE). Serum sialyl-Tn and NSE levels were markedly increased. Finally, an erythroid lineage was confirmed in the presence of alpha-globin messages in the blasts. Sialyl-Tn and NSE expression in leukemic blasts may be useful to identify AML-M6v.

Vascular endothelial growth factor (VEGF)-induced angiogenesis is involved in the etiology of some cardiovascular diseases. The soluble form of VEGF receptor, FLT-1 (sFLT-1), is a potent antagonist of VEGF. Therefore, we investigated whether transfection with the sFLT-1 gene could inhibit VEGF-induced angiogenesis. Human embryonic kidney (HEK)-293 cells were transfected with plasmids containing VEGF and sFLT-I (pCMV-VEGF and pCMV-sFLT-1) by the calcium-phosphate co-precipitation method. VEGF- and/or sFLT-1-transfected HEK-293 cells were incubated for 24 h, and then conditioned medium was collected. The effects of conditioned medium on angiogenesis were tested by incorporation of [3H]thymidine into human umbilical vein endothelial cells (HUVECs). Expression of VEGF protein was determined by Western blotting. The conditioned medium from sFLT-1 gene-transfected HEK-293 cells significantly inhibited recombinant VEGF-induced increase in [3H]thymidine incorporation by HUVECs. VEGF gene-transfected HEK-293 cells secreted VEGF protein into conditioned medium. This conditioned medium increased [3H]thymidine incorporation by HUVECs, which was significantly inhibited by co-transfection of sFLT-1 gene with VEGF gene. These observations suggested that sFLT-1 gene transfer could inhibit VEGF-induced DNA synthesis of vascular endothelial cells.

Background: Recent studies have shown that several cytokines could induce apoptosis in vascular smooth muscle cells (VSMCs) via the induction of nitric oxide (NO). In the present study, we explored whether human recombinant erythropoietin (rHuEPO) has a modulatory effect of apoptosis on interleukin-1β (IL-1β) or NO donor sodium nitroprusside (SNP)-induced apoptosis in rat cultured VSMCs. Methods: The quantitation of apoptosis among VSMCs was assessed by nuclear morphological analysis with fluorescent DNA- binding dye Hoechst 33258. Apoptotic changes were also confirmed by the detection of DNA fragmentation. The expression of EPO receptor (EpoR), cellular protein tyrosine phosphorylation, including EpoR and Janus kinase (JAK) 2, and the association of p85 subunit of phosphatidylinositol 3 kinase (PI3-kinase) to tyroSine-phosphorylated proteins, including EpoR, were explored by using Western blotting analysis combined in part with immunoprecipitation. Results: rHuEPO inhibited the apoptosis induced by IL- 1β or SNP in a dose- and time-dependent manner. The anti-apoptotic effects of rHuEPO were diminished in the presence of a tyrosine kinase (TK) inhibitor genistein or anti-EpoR antibody. After stimulation with rHuEPO, EpoR and JAK 2 were tyrosine phosphorylated, and p85 subunits were associated with EpoR. Also, rHuEPO induced phosphorylation of Akt through a PI3-kinase-dependent pathway. The phosphorylation of Akt and the anti-apoptotic effects of rHuEPO were diminished in the presence of a PI3-kinase inhibitor, wortmannin. Conclusion: Our present study demonstrates that rHuEPO inhibites IL-1β or SNP-induced VSMC apoptosis. The TK-dependent pathway, particularly the PI3- kinase-dependent pathway, seems to be critical to the countervailing effect of rHuEPO on IL-1β and SNP-induced VSMC apoptosis.

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that stimulates endothelial cell proliferation, increases endothelial permeability, and promotes collateral vessel formation. We transferred human VEGF gene into rat cardiac myocytes using adeno-associated virus (AAV) vectors and investigated whether VEGF secreted from the transduced cardiac myocytes promoted proliferation of endothelial cells. We produced VEGF-expressing AAV vectors (AAV-VEGF) by the adenovirus-free method. Immunoblotting revealed VEGF protein expression in AAV-VEGF-transduced rat cardiac myocytes. More than 60% of cardiac myocytes were stained positively on immunohistochemical staining using anti-VEGF antibody. Concentration of VEGF in the culture medium of AAV-VEGF-transduced myocytes was increased in a vector dose-dependent manner, and VEGF secretion from the transduced myocytes persisted for ≥14 days. Thymidine incorporation into human vascular endothelial cells was significantly increased by incubation with the conditioned medium from AAV-VEGF-transduced myocytes. This increased thymidine uptake was significantly inhibited by anti-VEGF antibody. We demonstrated here that AAV-mediated VEGF gene transfer into cardiac myocytes induces the secretion of functional VEGF.

A patient with a Philadelphia chromosome (Ph)-positive acute mixed-lineage leukemia (AMLL) expressing both major and minor BCR/ABL mRNA transcripts is described. Phenotypic analysis of the leukemic blasts revealed positivity for both myeloid and B-cell lineages. Southern blot analysis showed a rearrangement of the immunoglobulin heavy chain (IgH) gene. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed the expression of both major and minor BCR/ABL mRNA transcripts. To our knowledge, this is the first report of AMLL expressing both major and minor BCR/ABL mRNA transcripts and rearrangement of the IgH gene. (C) 2000 Wiley-Liss, Inc.

We explored a novel approach to the functional regulation of nuclear proteins altering their subcellular localization. To anchor a nuclear protein, β-galactosidase with the nuclear localization signal of SV40 (nβ-gal), within the cytoplasm, nβ-gal was fused to the transmembrane domain of granulocyte colony-stimulating factor receptor (G-CSFR), a membrane protein. To liberate the nβ-gal portion from the fusion protein, we used a protease derived from a plant virus, whose recognition sequence was inserted between the G-CSFR and nβ-gal. Western analysis showed that the chimeric protein was cleaved in the presence of the protease in 293 cells and that the fusion protein without the recognition sequence remained intact. This chimeric protein was localized exclusively in the cytoplasm as visualized by X-gal staining and immunofluorescence microscopy. In contrast, when expressed together with the protease, β-gal was predominantly detected in the nuclei. Moreover, we isolated 293-cell clones constitutively expressing the protease, indicating that this protease is not cytotoxic. These results suggest that the viral protease-mediated alteration of subcellular localization can potentially regulate the function of nuclear proteins.

The function of the Fanconi anemia complementation group A (FANCA) protein remains unclear. To investigate possible protein-protein interactions, we performed yeast two-hybrid screening using a FANCA fragment as bait. Sorting nexin 5 (SNX5), a new member of the human SNX family, was identified as a putative FANCA-binding protein. The interaction between FANCA and SNX5 was confirmed by immunoprecipitation studies. All members of the SNX family have a characteristic amino acid region termed the phox homology (PX) domain. Deletion mutant analysis indicated that the PX domain is not required for binding to FANCA. The SNX proteins are thought to play an important role in receptor trafficking between organelles. We found that overexpression of SNX5 increased FANCA protein levels. Northern blot analysis of SNX5 showed the presence of alternatively spliced transcripts and different expression patterns in various human cancer cell lines and normal tissues. Further studies are needed to elucidate the functional significance of FANCA and SNX5 binding however, we speculate that FANCA may affect SNX5, traffic with cell surface receptors.

Tec, Btk, Itk, Bmx, and Txk constitute the Tec family of protein tyrosine kinases (PTKs), a family with the distinct feature of containing a pleckstrin homology (PH) domain. Tec acts in signaling pathways triggered by the B cell antigen receptor (BCR), cytokine receptors, integrins, and receptor-type PTKs. Although upstream regulators of Tec family kinases are relatively well characterized, little is known of the downstream effectors of these enzymes. The yeast two-hybrid system has identified several proteins that interact with the kinase domain of Tec, one of which is now revealed to be a previously unknown docking protein termed BRDG1 (BCR downstream signaling 1). BRDG1 contains a proline-rich motif, a PH domain, and multiple tyrosine residues that are potential target sites for Src homology 2 domains. In 293 cells expressing recombinant BRDG1 and various PTKs, Tec and Pyk2, but not Btk, Bmx, Lyn, Syk, or c-Abl, induced marked phosphorylation of BRDG1 on tyrosine residues. BRDG1 was also phosphorylated by Tec directly in vitro. Efficient phosphorylation of BRDG1 by Tec required the PH and SH2 domains as well as the kinase domain of the latter. Furthermore, BRDG1 was shown to participate in a positive feedback loop by increasing the activity of Tec. BRDG1 transcripts are abundant in the human B cell line Ramos, and the endogenous protein underwent tyrosine phosphorylation in response to BCR stimulation. BRDG1 thus appears to function as a docking protein acting downstream of Tec in BCR signaling.

We have developed a novel system for expansion of transduced hematopoietic cells. This system involves 'selective amplifier genes' encoding fusion proteins between the granulocyte colony-stimulating factor receptor (Gcr) and the estrogen receptor (Er). The GcrEr chimeric gene conferred estrogen-dependent growth ability on murine hematopoietic cells. Here, we constructed a modified 'selective amplifier gene' to circumvent possible concerns with the Er/estrogen switching system. The bacterial gyrase B (Gyr) gene was fused to the Gcr gene, and the GcrGyr fusion construct was introduced into interleukin-3 (IL-3)-dependent Ba/F3 cells. The dimeric antibiotic coumermycin induced IL-3-independent growth in Ba/F3 cells expressing GcrGyr. This stimulatory effect was antagonized by an excess amount of novobiocin, a monomeric form of coumermycin. These results suggest the feasibility of using Gyr as a molecular switch to regulate a growth signal in hematopoietic cells.

We have proposed a novel concept, ie selective expansion of transduced cells, to overcome the low efficiency of gene transfer into hematopoietic stem cells. Previously, a fusion gene encoding a chimeric receptor (ΔGCRER) between the mouse granulocyte colony-stimulating factor receptor (G-CSFR) and the hormone-binding domain of rat estrogen receptor was constructed as a 'selective amplifier gene'. Although the chimeric gene conferred estrogen-inducible proliferation on the transduced Ba/F3 cells, it also mediated differentiation of the retrovirally transduced 32D cells upon estrogen treatment. Since only a growth signal is required for our purposes, we further modified the ΔGCRER gene to attenuate its differentiation signal. Based on the observation that tyrosine-703 in wild-type G-CSFR plays a pivotal role in transmitting the differentiation signal, phenylalanine was substituted for this residue in ΔGCRER. When the resultant selective amplifier gene (ΔGCRER gene) was expressed in 32D cells, sustained growth was supported by estrogen, while differentiation was suppressed. These cells ceased to grow upon estrogen withdrawal and differentiated with G-CSF treatment. The present findings suggested that ΔY703F-GCRER may have desirable properties as a selective amplifier for hematopoietic stem cell expansion and gene therapy.

The E2A-HLF fusion gene transforms human pro-B lymphocytes by interfering with an early step in apoptotic signaling. In a search for E2A-HLF-responsive genes, we identified a zinc finger transcription factor, SLUG, whose product belongs to the Snail family of developmental regulatory proteins. Importantly, SLUG bears close homology to the CES-1 protein of C. elegans, which acts downstream of CES-2 in a neuron-specific cell death pathway. Consistent with the postulated role of CES-1 as an antiapoptotic transcription factor, SLUG was nearly as active as Bcl-2 or Bcl-x(L) in promoting the survival of IL-3-dependent murine pro-B cells deprived of the cytokine. We conclude that SLUG is an evolutionarily conserved transcriptional repressor whose activation by E2A-HLF promotes the aberrant survival and eventual malignant transformation of mammalian pro-B cells otherwise slated for apoptotic death.

C-KIT, TIE and HKT expression on leukemic cells from patients were simultaneously analyzed using flow cytometry. Consistent with previous reports, leukemic cells from most patients with de novo acute myeloid leukemia (AML) were C-KIT-positive (28/35), while those from patients with B-lineage acute lymphoid leukemia (B-ALL) were C-KIT-negative (0/9). In the B-ALL patients, leukemic cells trom seven patients had one or more myeloid antigen such as CD13, CD15 and CD33. In contrast to C-KIT expression, leukemic cells from only one patient with acute monocytic leukemia were TIE-positive. Similarly, leukemic cells from only two patients (one, B-ALL with t(4:11)(q21:q23) and one, essential thrombocythemia in myeloblastic transformation (ET-MBT)) were HTK-positive. These results suggest that among the three receptor tyrosine kinases, C-KIT is the most useful marker for identifying AML.

Glial cell line-derived neurotrophic factor (GDNF) is known as a potent neurotrophic factor for dopaminergic neurons. Since adeno-associated virus (AAV) vector is a suitable vehicle for gene transfer into neurons, rat E14 mesencephalic cells were transduced with an AAV vector expressing GDNF. When compared with mock transduction, a larger number of dopaminergic neurons survived in AAV-GDNF-transduced cultures (234% and 325% of controls at 1 and 2 weeks, respectively P &lt 0.01). Furthermore, the dopaminergic neurons in the latter cultures grew more prominent neurites than those in the former. These findings suggest that AAV vector-mediated GDNF gene transfer may prevent dopaminergic neuron death, and is therefore a logical approach for the treatment of Parkinson's disease.

Background: Tec is a member of the recently emerging subfamily among nonreceptor protein-tyrosine kinases (PTKs). Although many members of this family have been shown to be involved in a wide range of cytokine-mediated signalling systems, the molecular mechanism by which they exert in vivo effects remains obscure. To gain insights into the downstream pathways of Tec, we here looked for Tec-interacting proteins (TIPs) by using the yeast two-hybrid screening. Results: One of TIPs turned out to be Grb10/GrbIR, which carries one pleckstrin homology domain and one Src homology 2 domain. Grb10/GrbIR was known to bind receptor PTKs in a ligand-dependent fashion, but not to be phosphorylated on tyrosine residues. In a transient expression system in human kidney 293 cells, however, Grb10/GrbIR becomes profoundly tyrosine-phosphorylated by Tec, but not by Syk, Jak2 or insulin receptor. We also reveal that expression of Grb10/GrbIR suppresses the cytokine-driven and Tec-driven activation of the c-fos promoter. Conclusion: Our results indicate a novel role of Grb10/GrbIR as an effector molecule to a subset of nonreceptor PTKs.

Adeno-associated virus (AAV) vectors, derived from a non-pathogenic parvovirus, are considered to be an appropriate gene transfer vehicle for both dividing and non-dividing cells. In the present study, we investigated whether the rat heart could be efficiently transduced with AAV vectors. Rat cardiac myocytes (CM) were infected with AAV-lacZ vector containing β-galactosidase (β-gal) gene in vitro and the expression of β-gal in CM was evaluated by X-gal staining and β-gal ELISA. With increasing multiplicities of infection (MOI), more than 60% of CM were stained positively with X-gal, and the β-gal expression increased to 31.1 ± 4.6 ng/mg protein in a MOI-dependent manner (MOI: 104 to 106 particles/cell). The β-gal expression was also increased in an incubation period-dependent manner (1-24 h). β-gal expression was maximal at day 3 and then gradually decreased with time. However, β-gal expression at day 14 was almost the same level as that at day 1 (45.5 ± 5.9 v 55.2 ± 6.2 ng/mg protein). Excised rat right ventricular papillary muscles were also infected with AAV-lacZ ex vivo. When the β-gal expression was evaluated by X-gal staining, more than 80% of CM in the papillary muscles were stained positively, indicating efficient gene transfer into CM using AAV vectors. These findings suggest that AAV vectors are promising for cardiac gene therapy.

Objectives: Recombinant viral vectors based on the nonpathogenic parvovirus, adeno-associated virus (AAV), have a number of attractive features for gene therapy, including the ability to transduce non-dividing cells and its long-term transgene expression. In this study, an AAV vector containing bacterial β-galactosidase gene (lacZ) was used to transduce cultured rat vascular smooth muscle cells (VSMC) in vitro and rat thoracic aortas ex vivo. Methods: VSMC were transduced with AAV-lacZ at multiplicities of infection (MOI) ranging from 5.0 x 105 to 1.0 x 107. Expression of β- galactosidase (β-gal) in VSMC was evaluated by X-gal staining and a β-gal ELISA method. Excised rat aortas were incubated with medium containing AAV- lacZ. Expression of β-gal in the aortic segments was evaluated by X-gal staining. Results: With increasing MOI, up to 50% of cultured VSMC were positive by X-gal staining and the β-gal expression increased up to 15 ng/mg protein. The expression gradually decreased during the culture but was detectable for at least 1 month. In the ex vivo study, AAV vectors transducer endothelial and adventitial cells in rat aortic segments, while no expression was seen in medial VSMC. Conclusions: AAV vectors can efficiently transduce rat VSMC in vitro. AAV-mediated ex vivo gene transfer into the normal aorta resulted in efficient gene transfer into endothelial and adventitial cells but not into medial VSMC. These findings suggest that AAV-based vectors are promising for use in cardiovascular gene therapy.

Tec is the prototype of a recently emerging subfamily among nonreceptor type protein-tyrosine kinases and is known to become tyrosine-phosphorylated and activated by a wide range of cytokine stimulations in hematopoietic cells. Although Tec was recently shown to be involved in the cytokine-driven activation mechanism of c-fos transcription, it is yet obscure how Tec relays the signals from cell surface receptors to the nucleus. To identify signaling molecules acting downstream of Tec, we have looked for Tec-interacting proteins (TIPs) by using the yeast two-hybrid system. Here we report the identification and characterization of a novel protein, TIP3, which has been simultaneously identified by other groups as SOCS-1, JAB, or SSI-1. TIP3 carries one Src homology 2 domain with a sequence similarity to that of CIS. In 293 cells, TIP3 associates with Tec and suppresses its kinase activity. Interestingly, TIP3 can also down-regulate the activity of Jak2 but not that of Lyn. We propose that SOCS-1/JAB/SSI-1/TIP3 is a novel type of negative regulator to a subset of protein-tyrosine kinases.

The cellular transformation by v-Src or v-Crk induces tyrosine phosphorylation of a common substrate molecule, p130(Cas), which tightly binds these oncoproteins in vivo. From its structure, Cas is deduced to be an ideal substrate for Src family kinases and Abl kinase. The tyrosine kinase activity associated with Cas was analysed using mouse variant fibroblasts lacking at least one of tyrosine kinases. In normal fibroblasts, Cas is associated with a significant level of tyrosine kinase activity which efficiently phosphorylates Cas in vitro. The Cas-associated tyrosine kinase activity was remarkably elevated in Csk(-/-) cells, which resulted in hyperphosphorylation of cellular Cas. The associated kinase activity was slightly increased in Src(-/-) cells whereas not significantly changed in Abl(-/-) nor Fak(-/-) cells. On the contrary, the Cas-associated kinase activity was remarkably decreased in Fyn(-/-) cells. At the same time, association of Cas with Fyn kinase in vitro was most obviously detected in normal fibroblasts as well as Csk(-/-) cells. Transient expression of v-Crk induced elevation of the Cas-associated kinase activity in all of these cell lines except the primary culture of Fyn(-/-) fibroblasts. These results indicate that Fyn kinase plays an essential role in v-Crk-mediated phosphorylation of Cas.

G-CSF (granulocyte colony-stimulating factor) is known to specifically stimulate the production and the functional activation of neutrophils. To investigate the intracellular signaling pathway of myeloid cells stimulated by G-CSF, we isolated new genes whose expression was induced by G-CSF. First of all, we constructed αgt10 cDNA library from G-CSF-stimulated mononuclear cells (MNC) of a chronic myelogeneous leukemia (CML) patient (CML-MNC) and screened the cDNA library by a differential hybridization method. The 24 candidate clones which specifically hybridized with G-CSF-stimulated CML-MNC cDNA probes, but not with unstimulated CML-MNC cDNA probes, were obtained after 8×104 individual clones had been screened. One of these clones, GIG-1 (G-CSF-induced gene-1), was further characterized. The size of the GIG-1 mRNA was about 0.9kb. The GIG-1 mRNA was expressed mainly in the myeloid leukemic cell lines. © 1994 Academic Press, Inc.

Stromal cells are known to produce granulocyte colony-stimulating factor (G-CSF) and interleukin 6 (IL-6). To assess the function of bone marrow Stromal cells in the pathological state, we studied their in vitro ability to produce G-CSF and IL-6 in 9 patients with aplastic anemia and 5 normal volunteers, using an enzyme immunoassay method. Constitutive production of IL-6, but not G-CSF, was detected in almost all cases. Interestingly, the inducible production of G-CSF and IL-6 by stromal cells after stimulation with Interleukin-1 and/or lipopolysaccharides was severely depressed in 3 patients with aplastic anemia. Our results may reflect poor hematopoietic activity in the bone marrow in some cases of aplastic anemia. © 1993, Tohoku University Medical Press. All rights reserved.

We constructed a series of murine stem cell factor (mSCF) cDNAs which were sequentially truncated at the 3′ termini. The resultant six mutant cDNA encode N-terminal 183, 179, 162, 149, 142 and 133 amino acid residues of the mature mSCF protein fused to the heterogeneous C-terminal peptides derived from the linker sequences. Each mutant cDNA was transiently expressed in COS cells, and the cultured supernatant was assayed for its ability to support the growth of a human factor-dependent cell line, TF-1 and to enhance colony formation by murine hematopoietic progenitor cells . The results showed that as few as N-terminal 142 but not 133 amino acid residues of mSCF remained biologically active in vitro, suggesting that the region of 9 aminoacids from Asp134 to Ser142 containing aCys138-mediated disulfide bond may contribute to the C-terminal end of the active subdomain of mSCF. © 1992.

Previous studies demonstrated that human circulating monocytes can proliferate in vitro when incubated with lectin-induced factor(s) from lymphocytes [(1985) Biochem. Biophys. Res. Commun., in press]. This study shows that human monocytes were induced to proliferate when incubated with 1α,25-dihydroxy vitamin D3 (calcitriol) at physiological concentrations. The optimal dose was about 10 nM. Proliferative activity was examined both by measuring the [su3H]thymidine incorporation and by counting cell nuclei. Among other derivatives of vitamin D3, 1α,24 R-dihydroxyvitamin D3 and 1α,24R,25-trihydroxyvitamin D3 stimulated mitotic activity of monocytes. Addition of both calcitriol and lectin-stimulated lymphocyte-conditioned medium to the monocyte culture had an additional effect on the mitotic activity of monocytes. © 1985.