Severe B cell deficiency in mice lacking the tec kinase family members Tec and Btk.

The cytoplasmic protein tyrosine kinase Tec has been proposed to have important functions in hematopoiesis and lymphocyte signal transduction. Here we show that Tec-deficient mice developed normally and had no major phenotypic alterations of the immune system. To reveal potential compensatory roles of other Tec kinases such as Bruton's tyrosine kinase (Btk), Tec/Btk double-deficient mice were generated. These mice exhibited a block at the B220(+)CD43(+) stage of B cell development and displayed a severe reduction of peripheral B cell numbers, particularly immunoglobulin (Ig)M(lo)IgD(hi) B cells. Although Tec/Btk(null) mice were able to form germinal centers, the response to T cell-dependent antigens was impaired. Thus, Tec and Btk together have an important role both during B cell development and in the generation and/or function of the peripheral B cell pool. The ability of Tec to compensate for Btk may also explain phenotypic differences in X-linked immunodeficiency (xid) mice compared with human X-linked agammaglobulinemia (XLA) patients.

Estrogen inhibits bone resorption by directly inducing apoptosis of the bone-resorbing osteoclasts.

Estrogen deficiency causes bone loss, which can be prevented by estrogen replacement therapy. Using a recently developed technique for isolation of highly purified mammalian osteoclasts, we showed that 17 beta-estradiol (E2) was able to directly inhibit osteoclastic bone resorption. At concentrations effective for inhibiting bone resorption, E2 also directly induced osteoclast apoptosis in a dose- and time-dependent manner. ICI164,384 and tamoxifen, as pure and partial antagonists, respectively, completely or partially blocked the effect of E2 on both inhibition of osteoclastic bone resorption and induction of osteoclast apoptosis. These data suggest that the protective effects of estrogen against postmenopausal osteoporosis are mediated in part by the direct induction of apoptosis of the bone-resorbing osteoclasts by an estrogen receptor- mediated mechanism.

Fabrication of nanostripe surface structure by multilayer film deposition combined with micropatterning.

Top-down fabrication processes for nanostructures are superior to bottom-up processes from the aspect of long-range order, but have limitations in their processing time and/or material selection. Here we developed a nanopatterning method for 'nanostripes' that incorporates deposition of a multilayer film on a microscale slope array and mechanical polishing. This method is used to fabricate a nanostripe structure consisting of two kinds of materials to form a stripe array on a silicon substrate. Although this nanopatterning method is categorized as a top-down fabrication process, the fabrication efficiency is quite high, because the number of nanostripes is 'multiplied' by the number of multilayered films. Another feature of the nanostripe is renewability; even if the nanostripe surface is damaged, the underlying nanostructure can be exposed and form a similar nanostripe by polishing. The nanostripe structure can be easily applied to a wide range of fields due to its ease of production.

Chondroprotective effect of the bioactive peptide prolyl-hydroxyproline in mouse articular cartilage in vitro and in vivo.

OBJECTIVE: To investigate the direct effect of prolyl-hydroxyproline (Pro-Hyp) on chondrocytes under in vivo and in vitro conditions in an attempt to identify Pro-Hyp as the bioactive peptide in collagen hydrolysate (CH). METHODS: The in vivo effects of CH and Pro-Hyp intake on articular cartilage were studied by microscopic examination of sections of dissected articular cartilage from treated C57BL/6J mice. In this study, mice that were fed diets containing excess phosphorus were used as an in vivo model. This mouse line showed loss of chondrocytes and reduced thickness of articular cartilage, with abnormality of the subchondral bone. The in vitro effects of CH, Pro-Hyp, amino acids and other peptides on proliferation, differentiation, glycosaminoglycan content and mineralization of chondrocytes were determined by MTT activity and staining with alkaline phosphatase, alcian blue and alizarin red. Expression of chondrogenesis-specific genes in ATDC5 cells was determined by semiquantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR). RESULTS: In vivo, CH and Pro-Hyp inhibited the loss of chondrocytes and thinning of the articular cartilage layer caused by phosphorus-induced degradation. In the in vitro study, CH and Pro-Hyp did not affect chondrocyte proliferation but inhibited their differentiation into mineralized chondrocytes. A combination of amino acids such as proline, hydroxyproline and prolyl-hydroxyprolyl-glycine did not affect chondrocyte proliferation or differentiation. Moreover, CH and Pro-Hyp caused two and threefold increases, respectively, in the staining area of glycosaminoglycan in the extracellular matrix of ATDC5 cells. RT-PCR indicated that Pro-Hyp increased the aggrecan mRNA level approximately twofold and decreased the Runx1 and osteocalcin mRNA levels by two-thirds and one-tenth, respectively. CONCLUSION: Pro-Hyp is the first bioactive edible peptide derived from CH to be shown to affect chondrocyte differentiation under pathological conditions.

A specific role of phosphatidylinositol 3-kinase gamma. A regulation of autonomic Ca(2)+ oscillations in cardiac cells.

Purinergic stimulation of cardiomyocytes turns on a Src family tyrosine kinase-dependent pathway that stimulates PLCgamma and generates IP(3), a breakdown product of phosphatidylinositol 4,5-bisphosphate (PIP2). This signaling pathway closely regulates cardiac cell autonomic activity (i.e., spontaneous cell Ca(2+) spiking). PIP2 is phosphorylated on 3' by phosphoinositide 3-kinases (PI3Ks) that belong to a broad family of kinase isoforms. The product of PI3K, phosphatidylinositol 3,4,5-trisphosphate, regulates activity of PLCgamma. PI3Ks have emerged as crucial regulators of many cell functions including cell division, cell migration, cell secretion, and, via PLCgamma, Ca(2+) homeostasis. However, although PI3Kalpha and -beta have been shown to mediate specific cell functions in nonhematopoietic cells, such a role has not been found yet for PI3Kgamma. We report that neonatal rat cardiac cells in culture express PI3Kalpha, -beta, and -gamma. The purinergic agonist predominantly activates PI3Kgamma. Both wortmannin and LY294002 prevent tyrosine phosphorylation, and membrane translocation of PLCgamma as well as IP(3) generation in ATP-stimulated cells. Furthermore, an anti-PI3Kgamma, but not an anti-PI3Kbeta, injected in the cells prevents the effect of ATP on cell Ca(2+) spiking. A dominant negative mutant of PI3Kgamma transfected in the cells also exerts the same action. The effect of ATP was observed on spontaneous Ca(2+) spiking of wild-type but not of PI3Kgamma(2/2) embryonic stem cell-derived cardiomyocytes. ATP activates the Btk tyrosine kinase, Tec, and induces its association with PLCgamma. A dominant negative mutant of Tec blocks the purinergic effect on cell Ca(2+) spiking. Tec is translocated to the T-tubes upon ATP stimulation of cardiac cells. Both an anti-PI3Kgamma antibody and a dominant negative mutant of PI3Kgamma injected or transfected into cells prevent the latter event. We conclude that PI3Kgamma activation is a crucial step in the purinergic regulation of cardiac cell spontaneous Ca(2+) spiking. Our data further suggest that Tec works in concert with a Src family kinase and PI3Kgamma to fully activate PLCgamma in ATP-stimulated cardiac cells. This cluster of kinases provides the cardiomyocyte with a tight regulation of IP(3) generation and thus cardiac autonomic activity.

A genomic analysis of adult T-cell leukemia.

Adult T-cell leukemia (ATL) is an intractable malignancy of CD4+ T cells that is etiologically associated with infection by human T-cell leukemia virus-type I. Most individuals in the chronic stage of ATL eventually undergo progression to a highly aggressive acute stage. To clarify the mechanism responsible for this stage progression, we isolated CD4+ cells from individuals in the chronic (n=19) or acute (n=22) stages of ATL and subjected them to profiling of gene expression with DNA microarrays containing >44,000 probe sets. Changes in chromosome copy number were also examined for 24 cell specimens with the use of microarrays harboring approximately 50,000 probe sets. Stage-dependent changes in gene expression profile and chromosome copy number were apparent. Furthermore, expression of the gene for MET, a receptor tyrosine kinase for hepatocyte growth factor (HGF), was shown to be specific to the acute stage of ATL, and the plasma concentration of HGF was increased in individuals in either the acute or chronic stage. HGF induced proliferation of a MET-positive ATL cell line, and this effect was blocked by antibodies to HGF. The HGF-MET signaling pathway is thus a potential therapeutic target for ATL.

Autocrine and/or paracrine growth of aggressive ATLL cells caused by HGF and c-Met.

Adult T-cell leukemia/lymphoma (ATLL) is a neoplasia characterized by the massive invasion of various organs by tumor cells. Previously, we found that expression of the gene for c-Met, a receptor tyrosine kinase for hepatocyte growth factor (HGF), was specific to the acute type among 41 patients with ATLL by microarray. First in the present study, we analyzed the survival of the patients in relation to expression of c-Met and HGF in ATLL cells. Expression of the former but not the latter was associated with poor prognosis. Then, we analyzed the growth of ATLL cells caused by HGF and c-Met. c-Met was expressed in 0/7 chronic ATLLs, 12/14 acute ATLLs, 1/1 IL-2-independent ATLL cell line and 1/7 IL-2-dependent ATLL cell lines as assessed by flow cytometry. HGF induced the proliferation of primary cells from most acute cases examined as well as the c-Met-positive KK1 cell line in contrast to c-Met-negative cells. HGF induced autophosphorylation of c-Met in c-Met-positive cells from an acute case and KK1 cells. The plasma level of HGF was elevated in acute as compared to chronic cases. The levels of HGF and/or IL-6 which induces the production of HGF by stromal cells, were elevated in the supernatant of short-term cultured cells from certain patients with acute or chronic disease. Finally, infiltrated ATLL cells and adjacent stromal cells in liver were shown to be positive for c-Met/HGF and HGF, respectively, in acute cases. Autocrine and/or paracrine growth caused by HGF and c-Met was suggested in aggressive ATLL cells secreting HGF and/or IL-6, respectively.

Divergent cytotoxic effects of PKC412 in combination with conventional antileukemic agents in FLT3 mutation-positive versus -negative leukemia cell lines.

FMS-like tyrosine kinase-3 (FLT3) is a new therapeutic target for acute myelocytic leukemia (AML), because FLT3 mutations are the most common genetic alterations in AML and are directly related to leukemogenesis. We studied cytotoxic interactions of a FLT3 inhibitor, PKC412, with eight conventional antileukemic agents (cytarabine, doxorubicin, idarubicin, mitoxantrone, etoposide, 4-hydroperoxy-cyclophosphamide, methotrexate and vincristine) using three leukemia cell lines carrying FLT3 mutations (MOLM13, MOLM14 and MV4-11) and five leukemia cell lines without FLT3 mutations (KOPB-26, THP-1, BALL-1, KG-1 and U937). PKC412 showed synergistic effects with all agents studied except methotrexate for FLT3-mutated cell lines in isobologram analysis. In contrast, PKC412 was rather antagonistic to most drugs, except for 4-hydroperoxy-cyclophosphamide and vincristine, in leukemia cell lines without FLT3 mutations. Cell-cycle analysis revealed that PKC412 induced G1 arrest in leukemia cell lines carrying FLT3 mutations, whereas it arrested cells in G2/M phase in the absence of FLT3 mutations, which may underlie the divergent cytotoxic interactions. These results suggest that the simultaneous administration of PKC412 and other agents except methotrexate is clinically effective against FLT3 mutation-positive leukemias, whereas it would be of little benefit for FLT3 mutation-negative leukemias. Our findings may be of help for the design of PKC412-based combination chemotherapy.

Epigenetic silencing of AXIN2 in colorectal carcinoma with microsatellite instability.

Mutation or epigenetic silencing of mismatch repair genes, such as MLH1 and MSH2, results in microsatellite instability (MSI) in the genome of a subset of colorectal carcinomas (CRCs). However, little is yet known of genes that directly contribute to tumor formation in such cancers. To characterize MSI-dependent changes in gene expression, we have now compared transcriptomes between fresh CRC specimens positive or negative for MSI (n=10 for each) with the use of high-density oligonucleotide microarrays harboring >44,000 probe sets. Correspondence analysis of the expression patterns of isolated MSI-associated genes revealed that the transcriptome of MSI+ CRCs is clearly distinct from that of MSI- CRCs. Such MSI-associated genes included that for AXIN2, an important component of the WNT signaling pathway. AXIN2 was silenced, apparently as a result of extensive methylation of its promoter region, specifically in MSI+ CRC specimens. Forced expression of AXIN2, either by treatment with 5'-azacytidine or by transfection with AXIN2 cDNA, resulted in rapid cell death in an MSI+ CRC cell line. These data indicate that epigenetic silencing of AXIN2 is specifically associated with carcinogenesis in MSI+ CRCs.

Tec kinase associates with c-kit and is tyrosine phosphorylated and activated following stem cell factor binding.

Stem cell factor (SCF) plays a crucial role in hematopoiesis through its interaction with the receptor tyrosine kinase c-kit. However, the signaling events that are activated by this interaction and involved in the control of growth or differentiation are not completely understood. We demonstrate here that Tec, a cytoplasmic, src-related kinase, physically associates with c-kit through a region that contains a proline-rich motif, amino terminal of the SH3 domain. Following SCF binding, Tec is tyrosine phosphorylated and its in vitro kinase activity is increased. Tyrosine phosphorylation of Tec is not detected in the response to other cytokines controlling hematopoiesis, including colony-stimulating factor-1 (CSF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3). Conversely, the cytoplasmic kinase JAK2 is activated by IL-3 but not by SCF stimulation. The activation of distinct cytoplasmic kinases may account for the synergy seen in the actions of SCF and IL-3 on hematopoietic stem cells.

Of the GATA-binding proteins, only GATA-4 selectively regulates the human interleukin-5 gene promoter in interleukin-5-producing cells which express multiple GATA-binding proteins.

Interleukin-5 (IL-5) is produced by T lymphocytes and known to support B-cell growth and eosinophilic differentiation of the progenitor cells. Using ATL-16T cells which express IL-5 mRNA, we have identified a region within the human IL-5 gene promoter that regulates IL-5 gene transcription. This cis-acting sequence contains the core binding motif, (A/T)GATA(A/G), for GATA-binding family proteins and thus suggests the involvement of this family members. In this report, we describe the cloning of human GATA-4 (hGATA-4) and show that hGATA-4 selectively interacts with the -70 GATA site within the IL-5 proximal promoter region. By promoter deletion and mutation analyses, we established this region as a positive regulatory element. Cotransfection experiments revealed that both hGATA-4 and phorbol-12-myristate-13-acetate (PMA)-A23187 stimulation are necessary for IL-5 promoter activation. The requirement for another regulatory element called CLE0, which lies downstream of the -70 GATA site, was also demonstrated. ATL-16T cells express mRNAs of three GATA-binding proteins, hGATA-2, hGATA-3, and hGATA-4, and each of them has a potential to bind to the consensus (A/T)GATA(G/A) motif. However, using ATL-16T nuclear extract, we demonstrated that GATA-4 is the only GATA-binding protein that forms a specific DNA-protein complex with the -70 GATA site. An electrophoretic mobility shift assay with extracts of COS cells expressing GATA-binding proteins showed that GATA-4 has the highest binding affinity for the -70 GATA site among the three GATA-binding proteins. When the transactivation abilities were compared among the three, GATA-4 showed the highest activity. These results demonstrate the selective role of GATA-4 in the transcriptional regulation of the IL-5 gene in a circumstance where multiple members of the GATA-binding proteins are expressed.

Tec family of protein-tyrosine kinases: an overview of their structure and function.

The Tec family is a recently emerging subfamily of non-receptor protein-tyrosine kinases (PTKs) represented by its first member, Tec. This family is composed of five members, namely Tec, Btk. Itk/Emt/Tsk, Bmx and Txk/Rlk. The most characteristic feature of this family is the presence of a pleckstrin homology (PH) domain in their protein structure. The PH domain is known to bind phosphoinositides; on this basis, Tec family PTKs may act as merge points of phosphotyrosine-mediated and phospholipid-mediated signaling systems. Many Tec family proteins are abundantly expressed in hematopoietic tissues, and are presumed to play important roles in the growth and differentiation processes of blood cells. Supporting this, mutations in the Btk gene cause X chromosome-linked agammaglobulinemia (XLA) in humans and X chromosome-linked immunodeficiency (Xid) in mice, indicating that Btk activity is indispensable for B-cell ontogeny. In addition, Tec family kinases have been shown to be involved in the intracellular signaling mechanisms of cytokine receptors, lymphocyte surface antigens, heterotrimeric G-protein-coupled receptors and integrin molecules. Efforts are being made to identify molecules which interact with Tec kinases to transfer Tec-mediated signals in vivo. Candidates for such second messengers include PLC-gamma2, guanine nucleotide exchange factors for RhoA and TFII-I/BAP-135. This review summarizes current knowledge concerning the input and output factors affecting the Tec kinases.

Paraneoplastic syndrome of hypercalcemia and leukocytosis caused by squamous carcinoma cells (T3M-1) producing parathyroid hormone-related protein, interleukin 1 alpha, and granulocyte colony-stimulating factor.

Previously we reported that a clonal squamous cell carcinoma cell line (T3M-1) derived from a lower jaw cancer of a patient with marked leukocytosis and hypercalcemia produced factors containing a potent bone-resorbing activity (BRA) (Mr 15,000-20,000) and a colony-stimulating activity. To elucidate the pathogenesis of this humoral hypercalcemia, BRA and colony-stimulating activity in both the conditioned medium and cells were characterized. The conditioned medium, when eluted at neutral pH, contained colony-stimulating activity and thymocyte proliferation-stimulating activity, the latter of which comigrated with BRA. Upon elution with acetic acid (pH 2.0), the conditioned medium contained no interleukin 1-like activity but potent parathyroid hormone-like activity, which comigrated with BRA. Northern blot hydridization analysis revealed that T3M-1 cells produced constitutively mRNA for parathyroid hormone-related protein and granulocyte colony-stimulating factor. Furthermore, primer extension analysis revealed that the cells also produced mRNA for interleukin 1 alpha (IL-1 alpha). Since parathyroid hormone-related protein and IL-1 alpha (osteoclast-activating factor) synergistically increase the concentration of serum calcium, and since IL-1 alpha (hemopoietin 1) potentiates granulocyte colony-stimulating factor-induced granulocytopoiesis, we speculate that parathyroid hormone-related protein, granulocyte colony-stimulating factor, and IL-1 alpha are synergistically involved in a paraneoplastic syndrome of hypercalcemia and leukocytosis, at least in some patients with solid tumors.

Interleukin 3 and erythropoietin induce association of Vav with Tec kinase through Tec homology domain.

Although hematopoietic cytokine receptors lack tyrosine kinase domains, the binding of their ligands to the receptors induce rapid tyrosine phosphorylation of various cellular target proteins. The specific tyrosine kinases which phosphorylate these substrates, however, have not been identified, other than that JAK kinases which phosphorylate STAT proteins and the receptors. We found that the c-vav proto-oncogene product, Vav, is rapidly and transiently tyrosine-phosphorylated in response to erythropoietin and IL3 stimulations and that Tec kinase is also transiently activated by these cytokines. Immunoprecipitation experiments demonstrated that Tec kinase binds to Vav upon these cytokine stimulations and that Grb2 constitutively associates with Vav. In vitro binding assays showed that erythropoietin and IL3 stimulation induce the specific binding of Vav to Tec kinase through Tec homology domains. We therefore concluded that Tec kinase is one of the key enzymes in Epo and IL3 receptor-mediated signaling pathways and that Vav plays an important role in the cytokine receptor-mediated signal transduction.

Tec protein-tyrosine kinase directly associates with Lyn protein-tyrosine kinase through its N-terminal unique domain.

Most of non-receptor-type protein-tyrosine kinases share common structures, such as N-terminal unique domains, Src homology region (SH)-2 domains, SH-3 domains and kinase domains. Although vast effort has brought some information about the in vivo roles of SH-2, -3 and kinase domains, little is still understood about the function of N-terminal unique domain. By utilizing the glutathione S-transferase (GST)-fusion system, we have investigated the role of N-terminal unique domain of the Tec protein-tyrosine kinase in a mouse IL-3-dependent myeloid cell line. We could reveal that the C-terminal half of the Tec N-terminal unique domain (NTec2 region) can bind to a set of tyrosine-phosphorylated cellular proteins in vitro in an IL-3-dependent manner. Surprisingly, p56/53Lyn constitutively binds to the NTec2 region. Among the NTec2-bound Lyn proteins, only the p56 form seems to be inducibly tyrosine-phosphorylated in response to IL-3. Binding domain of Lyn to the NTec2 region was localized to its SH-3 domain. Tec was also shown to make a stable complex with Lyn in vivo. This is the first report demonstrating the direct association between distinct cytoplasmic protein-tyrosine kinases, especially through N-terminal unique domain.

A novel protein-tyrosine kinase, tec, is preferentially expressed in liver.

To identify protein-tyrosine kinases which play an important role in the process of hepatocarcinogenesis, we have screened a murine liver cDNA library with v-fps kinase domain as a probe. Using low stringency screening, we could isolate cDNAs of a putative protein-tyrosine kinase, tec (tyrosine kinase expressed in hepatocellular carcinoma). Nucleotide sequences of the cDNAs show that the C-terminal domain of its predicted protein has significant homology with that of the members of the src family. The tec gene is expressed mainly in liver and faintly in heart, kidney and ovary. Northern analysis further shows that in 2 out of 4 cell lines of human hepatocellular carcinoma (HCC) the tec gene is highly expressed compared to normal human liver. This is the first report showing a protein-tyrosine kinase which may be specifically involved in the cell growth of hepatocytes or in the step of hepatocarcinogenesis.

Tec is involved in G protein-coupled receptor- and integrin-mediated signalings in human blood platelets.

Tec is a non-receptor type tyrosine kinase which is tyrosine phosphorylated and activated upon stimulation of hematopoietic cells with various cytokines. The role of Tec in G protein-coupled receptor- and integrin-mediated signalings has not been elucidated. We therefore investigated the regulation of Tec in human blood platelets. Tec was rapidly tyrosine phosphorylated in response to platelet agonists which activate G protein-coupled receptors such as thromboxane A2 analog (U46619), thrombin, and thrombin receptor activating peptide (TRAP). TRAP-induced phosphorylation in Tec was significantly reduced under the conditions which abrogate fibrinogen binding to GP IIb-IIIa and subsequent platelet aggregation. However, TRAP induced significant levels of the phosphorylation even under these conditions and also in thrombasthenic platelets which lack functional GP IIb-IIIa molecules, suggesting that activation of G-protein-coupled receptor causes the phosphorylation. To clarify whether integrin engagement by itself causes the phosphorylation in Tec, we examined the state of the phosphorylation in platelets activated by integrin engagement. Platelet adhesion to immobilized fibrinogen or collagen induced significant levels of the phosphorylation. Furthermore, Tec was translocated to cytoskeleton in response to TRAP in a manner dependent on platelet aggregation, suggesting that Tec can be a component of integrin-mediated signalings. These results collectively indicate that Tec is involved in G protein-coupled receptor- and integrin-mediated signalings in human blood platelets.

Expression of a novel form of Tec kinase in hematopoietic cells and mapping of the gene to chromosome 5 near Kit.

The Tec kinase was initially identified as a novel cytoplasmic protein tyrosine kinase that is preferentially expressed in the liver and is highly homologous to the Drosophila Dsrc28C src-related tyrosine kinase. In screening of interleukin 3 (IL-3)-dependent myeloid leukemia cells for protein tyrosine kinases, we observed that all cell lines examined expressed high levels of Tec transcripts. However, characterization of Tec cDNAs indicated that they differed significantly from the published sequence. Most strikingly, an insertion of 41 bp in the 5' region affects the initiation codon and results in replacing the published 13 amino acid amino-terminal sequences with 94 amino acids. Using polymerase chain reaction (PCR) analysis, only the form containing the insertion was detected in hematopoietic cells. In addition, we found an in-frame insertion of 66 bp that introduces an additional 22 amino acids into the SH3 domain. This insertion restores conserved SH3 sequences that are found in the src gene family and in the Dsrc28C gene. By PCR analysis, approximately equal levels of Tec transcripts containing the intact SH3 domain and containing the 22 amino acid deletion were found in hematopoietic cells. Lastly, by interspecies backcross analysis, we show that the Tec gene is tightly linked to the c-Kit gene on mouse chromosome 5.

The C-terminal SH3 domain of the mouse c-Crk protein negatively regulates tyrosine-phosphorylation of Crk associated p130 in rat 3Y1 cells.

We have isolated the mouse c-crk cDNA from a mouse liver cDNA library. It encodes 304 amino acids and consists mainly of SH2/SH3 regions. In Northern blot analysis, the mouse c-crk mRNA is expressed ubiquitously in every tissue and organ, suggesting that the c-Crk protein may be a common signal transducing molecule among tissues. In contrast to the v-Crk protein, which has a single SH3 domain, the c-Crk protein contains two, the more N-terminal SH3(1) domain and the C-terminal SH3(2) domain. To elucidate functions of these SH3 domains, we have constructed two c-crk mutants, B-crk and D-crk, which lack the SH3(2) and the SH3(1) domain, respectively. These mutants were expressed in rat 3Y1 cells, and examined for their transforming ability in terms of morphological phenotypes and for tyrosine phosphorylation profiles of cells expressing the mutant proteins. Morphological alteration and increased tyrosine phosphorylation of 130-140 kDa proteins, the major component of which is the Crk-associated p130, were observed in cells expressing B-Crk as well as those expressing v-Crk, but little in cells expressing c-Crk even at a similar level of expression. Although a highly tyrosine-phosphorylated form of the p130 was coimmunoprecipitated with c-Crk as well as B-Crk, the relative level of tyrosine phosphorylation of the p130, which is normalized to the amount of Crk protein immunoprecipitated, was 10 to 20 times higher in B-Crk-expressing cells than in c-Crk- or D-Crk-expressing cells. The present results indicate that the SH3(2) domain of mouse c-Crk protein negatively regulates tyrosine phosphorylation of the p130, and that lack of the SH3(2) domain in B-Crk and v-Crk may contribute, at least partly, to their morphological alteration or transforming ability through increasing tyrosine phosphorylation of the p130.

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.