Transforming growth factor-␤1 (TGF-␤1

The transcription factor RUNX3, which mediates apoptosis and cell growth inhibition in gastric epithelial cells, is a candidate tumor suppressor that is frequently lost in gastric cancer cells. Here, we found that restoration of RUNX3 expression in the cell line not expressing RUNX3 induced apoptosis and that it physically interacted with the Forkhead transcription factor FoxO3a/FKHRL1, known to be an important regulator of apoptosis and the cell cycle. Active unphosphorylated FoxO3a/FKHRL1 was expressed in the gastric cancer cell lines. RUNX3-induced apoptosis depended on the expression of Bim, a proapoptotic BH3-only protein, and both RUNX3 and FoxO3a/FKHRL1 were required for induction of Bim expression. Furthermore, we showed that interaction of RUNX3 and FoxO3a/FKHRL1 was also indispensable for Bim expression and apoptosis in mouse embryonic fibroblasts. In the Bim promoter, RUNX3 bound to two conserved RUNX-binding elements (RBE1 and RBE2), with RBE1 being immediately downstream of a FoxO-binding element. The physical interaction of RUNX3 and FoxO3a/FKHRL1 on the Bim promoter activated transcription of Bim. These findings show that RUNX3 cooperates with FoxO3a/FKHRL1 to participate in the induction of apoptosis by activating Bim and may play an important role in tumor suppression in gastric cancer.Runx transcription factors are ␣ subunits of the polyomavirus enhancer-binding protein 2 (PEBP2) 2 /core-binding factor (CBF), which consists of ␣ and ␤ subunits. They have a highly conserved Runt domain responsible for DNA binding and heterodimer formation with the ␤ subunit (PEBP2␤/CBF␤) (1). Three Runx transcription factors, Runx1, Runx2, and Runx3, have been identified in mammals. Although all these ␣ subunits have closely related structures and biochemical properties and bind the same DNA-binding motifs (1), they possess distinct biological functions in vivo. Runx1 is essential for definitive hematopoiesis, Runx2 plays critical roles in osteoblast maturation and osteogenesis, and Runx3 is ubiquitously expressed and involved in a variety of biological activities including development of gastrointestinal tract, neurogenesis, and lineage specification of thymocytes (1). Abnormalities in human Runx (RUNX) genes have been linked with some diseases (1, 2). RUNX1/AML1 is an important translocation breakpoint in acute leukemias and heterozygous loss of RUNX2 causes cleidocranial dysplasia syndrome. RUNX3 is implicated as a tumor suppressor gene in gastric cancer.Runx3-deficient mice exhibit hyperplasias in gastric mucosa due to reduced apoptosis and stimulated proliferation of gastric epithelial cells. Gastric epithelial cells are less sensitive to the proapoptotic and growth inhibitory effects of transforming growth factor-␤ (TGF-␤) (3). Furthermore, RUNX3 is inactivated in 40% of early stage and in nearly 90% of advanced stage gastric carcinomas by hemizygous deletion and hypermethylation of its promoter (3). Among the three Runx transcription factors, Runx3 is predominantly induced by TGF-␤ to bind and activate th...

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The role of Ras and MAP kinases (MAPKs) in the regulation of erythroid di erentiation was studied using a cell line (SKT6) derived from Friend virus (Anemic strain)-induced murine erythroleukemia. This cell line undergoes di erentiation in vitro in response to erythropoietin (EPO) or other chemical inducers such as dimethylsulfoxide (DMSO). When a constitutively active ras mutant (ras12V) was expressed in SKT6 cells, EPO-induced di erentiation was inhibited. Conversely, a dominant negative ras mutant (ras17N) induced di erentiation even in the absence of EPO, suggesting that the basal Ras activity is essential for the maintenance of the undi erentiated phenotype and proliferative potential in this cell line. Rapid inactivation of ERK was observed after expression of ras17N. Slow but signi®cant inactivation of ERK was also observed during EPOinduced di erentiation. Furthermore, overexpression of a constitutively active mutant of ERK-activating kinase (MAPKK) was found to suppress erythroid di erentiation, while pharmacological inhibition of MAPKK induced di erentiation. These ®ndings suggest that down-regulation of Ras/ERK signaling pathway may be an essential event in EPO-induced erythroid di erentiation in this system. Oncogene (2000) 19, 1500 ± 1508.

Large-scale extravasation of lymphocytes takes place in vivo under physiological conditions in lymph nodes at very specialized vascular segments called high endothelial venules (HEV). When circulating lymphocytes leave the blood, they first bind to endothelial cells of HEV (HE cells) and subsequently enter lymph nodes by crossing the endothelial lining of HEV. Although the lymphocyte-HEV interaction has recently been the subject of intense research by many laboratories, it has been studied almost exclusively by the use of the lymphocyte-binding assay in which lymphocyte binding is examined on nonviable HEV present on frozen sections and, hence, no dynamic interaction between HE cells and lymphocytes has been studied. We report herein that endothelial cells of rat HEV can be grown in vitro and that the lymphocyte-HEV interaction can be studied dynamically using viable cells in culture vessels. The identification of the cultured line, termed Ax, as HE cells was based on their phenotypic, morphological, cytochemical and biochemical characteristics, and most importantly on its in vitro behavior, particularly in terms of its specific ability to interact with mature lymphocytes. Phenotypic analysis demonstrated that not only did monoclonal antibodies, known to react with HE cells, recognize the Ax but also a monoclonal antibody raised against the Ax specifically recognized HE cells in vivo, as determined by an immunoperoxidase staining of frozen sections, supporting the notion that the cell strain, Ax, is derived from HEV. This Ax, even after long-term culture (greater than 50 passages), allowed mature, but not immature, lymphocytes to bind to the cell surface and subsequently transport bound cells underneath their cytoplasm. This phenomenon was inhibited in a dose-dependent manner by various reagents known to inhibit lymphocyte recirculation in vivo. The cultured line derived from HE cells should provide a means to investigate the biochemical nature of lymphocyte-HEV interaction, and to understand the molecular mechanisms underlying the large-scale lymphocyte traffic taking place in vivo.

Abnormal erythropoietin (EPO)-independent cell growth is induced after infection of erythroid progenitor cells with a polycythemic strain of Friend virus (FV p ). Binding of its Env-related glycoprotein (gp55) to the EPO receptor (EPOR) mimics the activation of the EPOR with EPO. We investigated the gp55-EPORErythropoietin (EPO) is a hematopoietic cytokine which regulates erythrocyte production, acting on proliferation, differentiation, and apoptosis of erythroid progenitor cells (25). The EPO receptor (EPOR) is a type I cytokine receptor, one of the four subtypes of the cytokine receptor superfamily (10, 45). Following EPO stimulation, the EPOR, which has no catalytic activity as do other members of the cytokine receptor superfamily, associates with and activates JAK2 tyrosine kinase, a member of the Janus kinase (JAK) family (32,52). This activation of JAK2 induces rapid tyrosine phosphorylation of the EPOR and STATs (signal transducers and activators of transcription) and association of the EPOR with a variety of cellular proteins possessing the Src homology 2 (SH2) domain, such as Shc (9, 29), hematopoietic protein tyrosine phosphatase 1 (22, 56), and phosphatidylinositol 3-kinase (PI3-K) (8,14,31).The JAKs are responsible for a variety of cytokine signaling. By using mutant cell lines defective in their response to interferons (IFNs), JAKs were first demonstrated to be involved in IFN signaling mediated by the IFN receptors, the type II cytokine receptors (46). JAK1 and Tyk2 are required for alpha/ beta IFN (IFN-␣/␤) signaling, and JAK1 and JAK2 are required for IFN-␥ signaling. The interleukin-2 (IL-2) receptor ␤ chain (␤c) and ␥c associate with JAK1 and JAK3, respectively, and activate each JAK in IL-2 signaling of T cells (3,20,33,53). NIH 3T3 cell-derived transfectants expressing the reconstituted IL-2 receptors become responsive to IL-2 only after expression of JAK3, indicating that JAK3 plays a critical role in IL-2 signaling (33). The STATs are cytoplasmic transcription factors possessing SH2 and SH3 domains and undergo rapid tyrosine phosphorylation after cytokine stimulation of the receptor. The tyrosine-phosphorylated STATs then become dimerized and translocate to the nucleus, where they regulate the expression of their target genes by binding to specific DNA sequences, such as the IFN-␥ activation site (GAS)-related sequence and the IFN-stimulated response element (12,15,27). Recent evidence indicates that specific JAKs and STATs are constitutively tyrosine phosphorylated in cells transformed with human T-cell lymphotropic virus type I (30), v-src (57), v-abl (11), or v-mpl (36), suggesting that activation of the JAK/STAT pathway participates in transformation of the cells by viral oncoproteins.A replication-defective Friend spleen focus-forming virus (F-SFFV) of the polycythemic strain of Friend virus complex (FV p ) causes erythroleukemia in adult mice (5). Although it has been demonstrated that a glycoprotein of 55 kDa (gp55) encoded by an env-related gene of F-SFFV binds to the EPOR and make...

Erythropoietin receptor (EPOR) and interleukin‐2 receptor beta chain (IL‐2R beta) belong to the same cytokine receptor superfamily and have highly conserved sequences in their intracellular signaling domain. However, common downstream signaling pathways of these receptors have not been demonstrated. In the present study, we introduced and expressed the murine EPOR in murine IL‐2‐, IL‐3‐ and IL‐5‐dependent cell lines and analyzed their growth response to EPO. We found that the expression of EPOR induced EPO dependence in IL‐3‐dependent BAF‐B03 and IL‐5‐dependent Y16 cells but not in IL‐2‐dependent CTLL‐2 cells, although the EPOR‐expressing CTLL‐2 cell lines could bind and internalize EPO as efficiently as the BAF‐B03‐derived cell lines. Additional expression of AIC2B, a common signal transducer for IL‐3R, IL‐5R and GM‐CSFR, made no difference to the EPO responsiveness of the EPOR‐expressing CTLL‐2 cell lines. These results suggest that the cellular components required for the transduction of EPOR signal and IL‐2R signal are at least partially different, and this difference cannot be explained solely by the absence of AIC2B.

The Monte Carlo simulation code acat−gas has been developed in order to simulate the whole system of a planar magnetron sputtering discharge. The atomic collisions in the cathode, which lead to sputtering and reflection, are simulated by the acat routine of the acat−gas code; the thermalization of emitted atoms in the background gas within the a planar sputtering discharge is simulated by the Monte Carlo technique, where the Thomas–Fermi–Morse potential is used to describe collisions between ejected atoms and background gas atoms. The simulated average energy of sputtered atoms at the midpoint between the cathode and the substrate is in good agreement with experimental results. It is found that various physical quantities such as the velocity distributions of sputtered atoms and reflected atoms, the transmission rate of sputtered atoms and reflected atoms, the average energies in the background gas, and the normalized energy of arriving reflected atoms at the substrate depends strongly on the discharge voltage at the low pressure (<2 Pa).