Recent advances in gene therapy technologies have enabled the treatment of congenital disorders and cancers and facilitated the development of innovative methods, including induced pluripotent stem ...cell (iPSC) production and genome editing. We recently developed a novel non-transmissible and non-integrating measles virus (MV) vector capable of transferring multiple genes simultaneously into a wide range of cells through the CD46 and CD150 receptors. The MV vector expresses four genes for iPSC generation and the GFP gene for a period of time sufficient to establish iPSCs from human fibroblasts as well as peripheral blood T cells. The transgenes were expressed differentially depending on their gene order in the vector. Human hematopoietic stem/progenitor cells were directly and efficiently reprogrammed to naive-like cells that could proliferate and differentiate into primed iPSCs by the same method used to establish primed iPSCs from other cell types. The novel MV vector has several advantages for establishing iPSCs and potential future applications in gene therapy.
This new non-transmissible and non-integrating measles virus vector, which can transfer multiple genes simultaneously into a wide range of cells through the CD46 and CD150 receptors and induce primed or naive-like pluripotent stem cells from hematopoietic cells in the same condition, will definitely contribute to the gene and cell therapy.
Objective：Docosahexaenoic acid (DHA) is known as a chemopreventive substance for cancers. Previously we reported that DHA induces apoptosis in HL-60 cells. The aim of this study was to clarify the ...role of phosphatidylinositol 3-kinase (PI3-kinase)/Akt signaling during DHA-induced apoptosis in HL-60 cells. Methods：The inhibitory effects of dibutyryl cAMP (db-cAMP) or LY294002 (a specific inhibitor of the PI3-kinase/Akt pathway) on DHA-induced apoptosis in HL-60 cells were evaluated by the appearance of apoptosis, and from the activities of caspases (3 and 8), the phospholylation of Akt, and cleavage of Bid using DNA indexes, emzymatic measurement of fragmented substrates, and Western blotting, respectively. Results：The pre-incubation of db-cAMP reduced the activation of caspasses (3 and 8) during the occurrence of DHA-induced apoptosis in HL-60. However, the inhibition of PI3-kinase/Akt signaling by LY294002 resulted in recovery of the caspases' activities, appearance of apoptotic cells, and cleavage of the Bid molecule when LY294002 was co-treated with db-cAMP before the occurrence of DHA-induced apoptosis in HL-60. It was also confirmed that LY294002 strongly inhibited phospholylation of Akt during db-cAMP induced-reduction of DHA-induced apoptosis in HL-60. Conclusion：We demonstrated that DHA-induced apoptosis was sensitive to the modulation of PI3-kinase activity by treatment with db-cAMP or LY294002. These results may provide new insights into the mechanisms of the anti-cancer activity of DHA.
Abstract
A plethora of proteins and processes regulate proper mitosis and are yet to be fully understood. Since mitotic cells are an emerging target for cancer therapy, it is of utmost importance to ...decipher the mechanisms that regulate mitosis. The purpose of our study is to identify the role CUL3 plays in regulating mitosis. Specifically, it is our aim to identify CUL3 interacting partners that play roles in spindle assembly, in order to understand the function of CUL3 in constructing the mitotic spindle. The CUL3 protein is an ubiquitin E3 ligase scaffold. By means of ubiquitination, CUL3 can regulate an array of proteins. We have performed CUL3 RNAi experiments, followed by cell cycle analysis using flow cytometry and immunostaining of DNA, α-tubulin and γ-tubulin, to identify the importance of CUL3 in regulating mitosis. Furthermore, we have performed CUL3 immunoprecipitation (IP) experiments in X.laevis mitotic egg extracts and in HeLa cells synchronized at mitosis, to identify the binding partners of CUL3 which orchestrate mitosis. Using treatment with either nocodazole (promotes microtubule depolymerization) or monastrol (causes microtubule stabilization), we performed interaction studies in HeLa cells to determine the effect of microtubule polymerization on the interaction of CUL3 with its mitotic binding partners. Similarly, in X.laevis egg extracts, we have used warm (for microtubule depolymerization) and cold (for microtubule stabilization) IP conditions to determine the effects of microtubule status on CUL3 interaction with its binding partners. Our results have shown that CUL3-depleted cells display a dramatic mitotic phenotype which includes: defective spindles, multiple centrosomes, enlarged cells and nuclei, G2/M accumulation and delayed cytokinesis. To understand how CUL3 may participate in the formation of the mitotic spindle, we determined the binding partners of CUL3 that are involved in spindle assembly. CUL3 IP results from both X.laevis egg extracts and HeLa cells indicate that CUL3 interacts with the kinesin motor protein, Eg5. Eg5 is an important spindle assembly factor, which is essential in mitosis for proper formation of a bipolar spindle. Interestingly, targeting Eg5 is emerging as an attractive means to inhibit the proliferation of cancer cells. Thus, better characterizing the interaction of CUL3 and Eg5 is critical to understanding the function of CUL3 as well as Eg5 in mitosis. Further characterization of the interaction between CUL3 and Eg5 demonstrated that this interaction is present only in the presence of depolymerized microtubules. Our results culminate to the following conclusions: CUL3 is required for cells to properly undergo mitosis, especially the correct formation of spindle apparatus. CUL3 interacts with the spindle assembly factor Eg5 in the presence of depolymerized microtubules, specifically during mitosis. We suspect that CUL3-Eg5 interaction is important for mitosis and in particular for bipolar spindle formation.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-288. doi:1538-7445.AM2012-LB-288
Asbestos causes malignant tumors such as lung cancer and malignant mesothelioma (MM). To determine whether asbestos exposure causes reduction of antitumor immunity, we established an in vitro T-cell ...line model of low-dose and continuous exposure to asbestos using an human adult T-cell leukemia virus-1 immortalized human polyclonal T-cell line, MT-2, and revealed that MT-2 cells exposed continuously to asbestos showed resistance to asbestos-induced apoptosis. In addition, the cells presented reduction of surface CXCR3 chemokine receptor expression and IFN-γ production. In this study, to confirm that these findings are suitable for clinical translation, surface CXCR3 and IFN-γ expression were analyzed using freshly isolated human CD4(+) T cells derived from healthy donors and patients with pleural plaque (PP) or MM. The results revealed that CXCR3 and IFN-γ expression in the ex vivo model were reduced in some cases. Additionally, CXCR3 expression in CD4(+) T cells from PPs and MMs was significantly reduced compared with that from healthy donors, and CD4(+) T cells from patients with MMs exhibited a marked reduction in IFN-γ mRNA levels after stimulation in vitro. Moreover, CD4(+)CXCR3(+) T cells in lymphocytes from MMs showed a tendency for an inverse correlation with its ligand CXCL10/IP10 in plasma. These findings show reduction of antitumor immune function in asbestos-exposed patients and indicate that CXCR3, IFN-γ, and CXCL10/IP10 may be candidates to detect and monitor disease status.
Because patients with silicosis who are chronically exposed to silica particles develop not only pulmonary fibrosis, but also complications involving autoimmune diseases such as rheumatoid arthritis ...and systemic sclerosis, exposure to asbestos may affect the human immune system. This immunologic effect may impair antitumor immune function because cancer complications such as lung cancer and malignant mesothelioma are found in patients exposed to asbestos. To elucidate the antitumor immune status caused by CD4(+) T cells exposed to asbestos, an in vitro T-cell model of long-term and low-level exposure to chrysotile asbestos was established from a human adult T-cell leukemia virus-1-immortalized human polyclonal T cell line, MT-2, and the resulting six sublines showed resistance to asbestos-induced apoptosis after more than 8 months of continuous exposure. The results of DNA microarray analysis showed that the expression of 139 genes was altered by long-term and low-level exposure to asbestos, and the profile was almost similar among the six sublines when compared with the original MT-2 cells that had never been exposed to asbestos. Pathway and network analysis indicated a down-regulation of IFN-γ signaling and expression of CXC chemokine receptor 3 (CXCR3) in the sublines, whereas ELISA and flow cytometry analysis demonstrated a reduction in Th1-related IFN-γ production and cell-surface CXCR3 expression. These findings suggest that chronic exposure to asbestos may reduce antitumor immune status in CD4(+) T cells, and that an in vitro T-cell model may be useful in identifying molecules related to the impairment of antitumor immune function.
Erythropoiesis is the process of proliferation, differentiation, and maturation of erythroid cells. Understanding these steps will help to elucidate the basis of specific diseases associated with ...abnormal production of red blood cells. In this study, we continued our efforts to identify genes involved in erythroid proliferation. Lentivirally transduced UT-7/Epo erythroleukemic cells expressing ribosomal protein L11 (RPL11) or retinol dehydrogenase 11 (RDH11) could proliferate in the absence of erythropoietin, and their cell-cycle profiles revealed G0 /G1 prolongation and low percentages of apoptosis. RPL11-expressing cells proliferated more rapidly than the RDH11-expressing cells. The antiapoptotic proteins BCL-XL and BCL-2 were expressed in both cell lines. Unlike the parental UT-7/Epo cells, the expression of hemoglobins (Hbs) in the transduced cells had switched from adult to fetal type. Several signal transduction pathways, including STAT5, were highly activated in transduced cells; furthermore, expression of the downstream target genes of STAT5, such as CCND1 , was upregulated in the transduced cells. Taken together, the data indicate that RPL11 and RDH11 accelerate erythroid cell proliferation by upregulating the STAT5 signaling pathway with phosphorylation of Lyn and cyclic AMP response element-binding protein (CREB).
Indoor air conditions may have an impact on human health. Among the various factors affecting indoor air conditions, very few published reports deal with negatively-charged particles. In this review, ...we detail the biological effects of negatively-charged indoor air conditions, particularly on the psycho-neuro-endocrino-immune network. Short-term (2.5 hours) and medium-term (2 weeks) abidance in a room dominated by negatively-charged air particles resulted in activation of natural killer cell activity induced by recurrent transient activation of interleukin 2. These results indicate that negatively-charged indoor air conditions are better for immune status and may improve our daily lives. (Accepted on August 5, 2009)
Background
For the diagnosis and treatment of adult T‐cell leukemia/lymphoma (ATLL) caused by human T‐lymphotropic virus type 1 (HTLV‐1) are required therapeutic modalities urgently. Non‐human ...primate models for ATLL would provide a valuable information for clinical studies. We did a pilot study to establish an ATLL non‐human primate model using common marmosets (Callithrix jacchus).
Methods
We inoculated HTLV‐1–producing MT‐2 cells into 9‐month‐old marmosets, either intraperitoneally or intravenously. We next administrated MT‐2 cells into 13‐month‐old marmosets under cyclosporine A (CsA) treatment to promote infection. HTLV‐1 infection was determined by measuring HTLV‐1 antibody titer in the common marmosets.
Results
The HTLV‐1 antibody titer increased in the intraperitoneally inoculated marmoset with or without CsA treatment, and it kept over five 5 years though proviral copy number (proviral load, PVL) remained low throughout the study.
Conclusion
We obtained HTLV‐1 asymptomatic carriers of common marmosets by inoculating MT‐2 cells.
Silicosis patients (SILs) and patients who have been exposed to asbestos develop not only respiratory diseases but also certain immunological disorders. In particular, SIL sometimes complicates ...autoimmune diseases such as systemic scleroderma, rheumatoid arthritis (known as Caplan syndrome), and systemic lupus erythematoses. In addition, malignant complications such as lung cancer and malignant mesothelioma often occur in patients exposed to asbestos, and may be involved in the reduction of tumor immunity. Although silica-induced disorders of autoimmunity have been explained as adjuvant-type effects of silica, more precise analyses are needed and should reflect the recent progress in immunomolecular findings. A brief summary of our investigations related to the immunological effects of silica/asbestos is presented. Recent advances in immunomolecular studies led to detailed analyses of the immunological effects of asbestos and silica. Both affect immuno-competent cells and these effects may be associated with the pathophysiological development of complications in silicosis and asbestos-exposed patients such as the occurrence of autoimmune disorders and malignant tumors, respectively. In addition, immunological analyses may lead to the development of new clinical tools for the modification of the pathophysiological aspects of diseases such as the regulation of autoimmunity or tumor immunity using cell-mediated therapies, various cytokines, and molecule-targeting therapies. In particular, as the incidence of asbestos-related malignancies is increasing and such malignancies have been a medical and social problem since the summer of 2005 in Japan, efforts should be focused on developing a cure for these diseases to eliminate nationwide anxiety.
Since sphingosine 1-phosphate (Sph-l-P) is stored in abundant amounts in blood platelets and released extracellularly upon stimulation, it is important to clarify the effects of this bioactive ...lysophospholipid on vascular endothelial cells from the viewpoint of platelet-endothelial cell interactions. In this study, we investigated the effects of Sph-l-P on the cytoskeletal remodeling of human umbilical vein endothelial cells (HUVECs). Of a focal adhesion kinase (FAK) family of non-receptor protein-tyrosine Mnases, HUVECs were found to express FAK, but scarcely proline-rich tyrosine kinase 2. Sph-l-P induced FAK tyrosine phosphorylation, myosin light chain phosphorylation, and the formation of stress fibers in HUVECs. The specific Rho rnactivator C3 transferase from Clostridium botulinum abolished all of these cytoskeletal responses induced by Sph-l-P, while pertussis toxin only partly inhibited FAK tyrosine phosphorylation, and hardly affected myosin light chain phosphorylation and stress fiber formation. In contrast, Sph-l-P-induced intracellular Ca2+ mobilization was suppressed by pertussis toxin, but not at all by C3 exoenzyme. Our results suggest that Sph-l-P, a bioactive lipid released from activated platelets, induces endothelial cell cytoskeletal reorganization, mainly through Rho-mediated signaling pathways
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