Biomedical subjects

Chu-Tse Wu

Publications and source records attributed to Chu-Tse Wu.

15 recordsLinked to original sources

PubMed2006

Adenoviral-mediated gene expression of hepatocyte growth factor prevents postoperative peritoneal adhesion in a rat model.

BACKGROUND: Mesothelial cell proliferation and migration play important roles in reducing formation of postoperative peritoneal adhesions. Hepatocyte growth factor (HGF) is a multifunctional cytokine that stimulates proliferation and migration of various cell types, including mesothelial cells. METHODS: We investigated the effect of adenovirus-mediated HGF gene expression on the proliferation and migration of mesothelial cells and evaluated its preventive effects on postoperative formation of peritoneal adhesions. Rat mesothelial cells were isolated and characterized by expression of cytokeratin and vimentin. RESULTS: Immunohistochemical staining showed that these cells expressed c-Met, the receptor for HGF. Adenoviral-mediated HGF gene transfer into mesothelial cells resulted in high expression of HGF and enhanced migration. To evaluate the preventive effects of adenoviral-mediated HGF gene transfer on the formation of postoperative peritoneal adhesion, we employed a rat model of cecum abrasion-induced adhesion formation in which 80% of the rats developed postoperative peritoneal adhesions. Local application of recombinant adenovirus carrying the HGF gene reduced adhesion formation in 16 of 20 control rats compared with 7 of 20 treated rats in this model. CONCLUSIONS: These results suggest that adenoviral-mediated HGF gene transfer may be a novel strategy for preventing postoperative peritoneal adhesions.

Adenoviridae↗

PubMed2006

Shp-2 tyrosine phosphatase is required for hepatocyte growth factor-induced activation of sphingosine kinase and migration in embryonic fibroblasts.

Shp-2, a ubiquitously expressed protein tyrosine phosphatase containing two Src homology 2 domains, plays an important role in integrating signaling from the cell surface receptors to intracellular signaling mechanisms. Previous studies have demonstrated that the Shp-2 is involved in hepatocyte growth factor (HGF)-induced cell scattering. Here we report that Shp-2 is required for the HGF-induced activation of sphingosine kinase-1 (SPK1), a highly conserved lipid kinase that plays an important role in cell migration. Loss-of-function mutation of Shp-2 did not affect the expression of SPK1, but resulted in its inactivation and the blockage of HGF-induced migration in embryonic fibroblasts. Reintroduction of functional wild type (WT) Shp-2 into the mutant cells partially restored SPK1 activation, and overexpression of SPK1 in these mutant cells enhanced HGF-induced cell migration. Inhibition of expression or activity of SPK1 in WT cells markedly decreased intracellular S1P levels and HGF-induced cell migration. Furthermore, we found that Shp-2 co-immunoprecipitated with SPK1 and c-Met in embryonic fibroblasts. These studies suggest that Shp-2 is an SPK1-interacting protein and that it plays an indispensable role in HGF-induced SPK1 activation.

Animals↗

PubMed2006

Efficient gene transfer into hematopoietic cells by a retargeting adenoviral vector system with a chimeric fiber of adenovirus serotype 5 and 11p.

OBJECTIVE: Adenoviral vectors (Ad) were widely used in gene therapy and study of gene function, but the commonly used serotype 5 adenovirus-based vectors (Ad5) could poorly transduce hematopoietic cells because of low expression of viral receptors on these cells. To overcome this limitation, we developed a retargeting adenovector with a chimeric fiber of Ad5 and Ad11p (Ad5F11p) and evaluated its gene transfer ability in hematopoietic cells. MATERIALS AND METHODS: An Ad11p fiber pseudotyped Ad5 vector was generated by modifying the fiber gene of pAdEasy-1 backbone plasmid. Ad5F11p-GFP encoding enhanced green fluorescence protein (GFP) gene was transferred into human leukemic cell lines, primary leukemic cells, and CD34(+) hematopoietic cells. The gene transduction efficiency was determined by fluorescence-activated cell sorting assay. RESULTS: More than 90% of U937 or K562 cells could be infected by Ad5F11p-GFP at a moderate multiplicity of infection (MOI). Ad5F11p-GFP is also significantly more effective than control Ad5-GFP in infection of primary myeloid leukemic cells. At 200 MOI, GFP-positive percentages of Ad5F11p-GFP transduced myeloid leukemic cells range from 10.58% to 92.63% with a median of 28.65%. Ad5F11p-GFP could transduce about 50% human hematopoietic stem/progenitor (CD34(+)) cells, while Ad5-GFP could transduce <15% at 200 MOI. CD46 was reported to be the receptor of Ad11p. Our data suggest that CD46 participates in the process of Ad5F11p-GFP infection but is not the unique molecule determining its gene transfer efficiency of host cells. CONCLUSION: We established a retargeting adenovector system, which could infect hematopoietic cells effectively and would benefit research work on Ad tropism.

Adenoviridae↗

PubMed2006

Genetically modified myeloma cell vaccine inducing antitumor immune response in vivo.

This study was aimed to evaluate the in vivo antitumor effect of genetically modified myeloma cell vaccine on human myeloma xenografts implanted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Human immune system was established in NOD/SCID mice by intraperitoneal injection of human peripheral blood lymphocytes (PBLs). After being inoculated subcutaneously with irradiated myeloma cell line sko-007, adenovirally transferred with GFP or p53, granulocyte-macrophage colony-stimulating factor (GM-CSF) and B7-1 genes, huPBL-NOD/SCID mice were challenged by subcutaneous injection of non-transferred sko-007 cells. The results indicated that Ad-p53/GM-CSF/B7-1-infected sko-007 cell vaccination significantly reduced local tumor growth compared with controls. Histopathological and immunohistochemical analysis showed that tumor tissues increasingly displayed diffuse necrosis, mainly caused by apoptosis, accompanied with significant fibroplasias and blood vessel hyperplasia, and human T cells infiltrated into the tumor tissues. It is concluded that transgenic p53, GM-CSF and B7-1 expression produces an immune response against myeloma cells and may be of therapeutic value for multiple myeloma in human being.

Adenoviridae↗

PubMed2005

Adenoviral-mediated transfer of human wild-type p53, GM-CSF and B7-1 genes results in growth suppression and autologous anti-tumor cytotoxicity of multiple myeloma cells in vitro.

Multiple myeloma (MM) remains incurable despite the use of high-dose chemotherapy and stem cell transplantation. However, immunotherapy is expected to offer long-term disease control, or even possibly a cure. We have previously demonstrated the suppressive effect of a recombinant adenovirus carrying human wild-type p53, granulocyte-macrophage colony-stimulating factor, and B7-1 genes (Ad-p53/GM-CSF/B7-1) on the growth of laryngeal cancer cells. In the present study, we evaluated the effects of an Ad-p53/GM-CSF/B7-1-modified myeloma cell vaccine strategy aimed to induce apoptosis and to augment the immunogenicity of MM cells. Both MM cell lines and purified primary myeloma cells were infected with Ad-p53/GM-CSF/B7-1. High expression levels of these three genes were confirmed separately by Western blot, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. When wild-type p53, GM-CSF and B7-1 genes were introduced, the growth of MM cells was inhibited via enhanced apoptosis and the immunogenicity of tumor cells was augmented. The combinatorial effect of these three genes on inducing cytotoxic T lymphocytes (CTLs) was more evident than that of p53 individually or any combinations of two (p53 plus GM-CSF or p53 plus B7-1). Furthermore, significant proliferation of autologous peripheral blood lymphocytes (PBLs) and specific cytotoxicity against autologous primary MM cells were induced in vitro. These results suggest that myeloma cell vaccination co-transferred with p53, GM-CSF and B7-1 genes may be a promising immunotherapeutic approach against MM.

Adenoviridae↗

PubMed2005

[Membrane microparticles and their roles in the regulation of hematopoiesis - review].

Membrane microparticles are shed from the plasma membrane of most eukaryotic cells when these cells were undergone activation or apoptosis, and released into the extracellular environment. Their composition depends on the cellular origin and processes triggering their formation. Several lines of evidence suggest that membrane microparticles might be able to facilitate cell-cell cross-talk and play an important roles in the regulation of survival, proliferation, differentiation, adhesion and chemotaxis of hematopoietic cells. Here, the components, mechanism of formation and the regulatory roles of membrane microparticles in hematopoiesis were reviewed.

Caveolae↗

PubMed2004

Sphingosine kinase activation regulates hepatocyte growth factor induced migration of endothelial cells.

Hepatocyte growth factor (HGF)-induced migration of endothelial cells is critical for angiogenesis. Sphingosine kinase (SPK) is a key enzyme catalyzing the formation of sphingosine-1-phosphate (S1P), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through both intracellular and extracellular mechanisms. The aim of this study was to investigate whether activation of SPK is involved in the migration of endothelial cells induced by HGF. The biological functions of HGF are mediated through the activation of its high-affinity tyrosine kinase receptor, c-met protooncogene. In the present study, Treatment of ECV304 endothelial cells with HGF resulted in tyrosine phosphorylation of c-Met and activation of SPK in a concentration-dependent manner. Either Ly294002 or PD98059, specific inhibitor of the PI3K and ERK/MAPK pathways, respectively, blocked the HGF-induced activation of SPK. HGF stimulation significantly increased intracellular S1P level, but no detectable secretion of S1P into the cell culture medium was observed. Treatment of ECV304 cells with pertussis toxin (PTX) has no effect on the HGF-induced migration, indicating extracellular S1P is dispensable for this process. Overexpression of wild-type SPK gene in ECV 304 cells increased the intracellular S1P and enhanced the HGF-induced migration, whereas inhibition of cellular SPK activity by N,N-dimethylsphingosine (DMS), a potent inhibitor of SPK, or by expression of a dominant-negative SPK (DN-SK) blocked the HGF-induced migration of ECV 304 cells. It is suggested that PI3K and ERK/MAPK mediated the activation of SPK and would be involved in the HGF-induced migration of endothelial cells. These results elucidate a novel mechanism by which intracellularly generated S1P mediates signaling from HGF/c-Met to the endothelial cell migration.

Cell Line↗

PubMed2004

[Suppressive effect of Notch signal activation on apoptosis of multiple myeloma cells].

The proliferation and apoptosis of multiple myeloma (MM) cells were regulated by bone marrow microenvironments in which Notch signal plays important role in mediating cell-cell communication. However, the regulatory effect of Notch signal on the proliferation and apoptosis of multiple myeloma cells remains unclear. In this study, regulatory effect of Notch signal on the apoptosis of MM cells induced by DMS (N, N-dimethylsphingosine) was investigated. RT-PCR was used to identify the expression of Notch receptor and related molecules such as Dll-1, Jagged-1, Deltex-1 in MM cell lines. The intracellular domain of Notch (ICN), active form of Notch, was transferred into MM cells by retrovirus. The apoptosis of MM cells was determined by trypan blue exclusion tests and TdT-mediated dUTP nick end labeling (TUNEL) assay. The results showed that multiple myeloma cells expressed the Notch-1 and its related molecules. Notch activated multiple myeloma cell lines were obtained. Activation of Notch protected the multiple myeloma cells from the apoptosis induced by DMS,which was determined by cell viability and TUNEL assay. In conclusion, Notch signal suppressed the apoptosis of multiple myeloma cells and would possibly be a novel therapeutic target.

Apoptosis↗

PubMed2004

[Progress in Notch signaling].

Notch signaling plays a vital role in cell fate decisions in both invertebrate and vertebrate development. Signals exchanged between neighboring cells through the Notch receptor amplify and consolidate molecular differences, which eventually dictate cell fates. In this review, the details of notch signaling will be discussed, with a focus on Lin/Notch repeats (CSL) independent modes.

Carrier Proteins↗

PubMed2003

Treatment of myocardial ischemia with bone marrow-derived mesenchymal stem cells overexpressing hepatocyte growth factor.

Mesenchymal stem cells could differentiate into cardiomyocytes in vitro and have been shown to reconstitute the impaired myocardium in vivo. Hepatocyte growth factor, a recognized angiogenic factor and endothelial cell chemoattractant, has been applied in the treatment of myocardial ischemia. In this study, we used a ligation model of proximal left anterior descending coronary artery of rats to evaluate the effect of mesenchymal stem cells overexpressing hepatocyte growth factor in the treatment of myocardial ischemia. Bone marrow-derived mesenchymal stem cells were isolated, expanded, characterized, and infected with adenovirus carrying human hepatocyte growth factor cDNA (Ad-HGF). Mesenchymal stem cells infected by Ad-HGF released soluble HGF protein at a high level, which was maintained at least for 2 weeks. Implantation of mesenchymal stem cells overexpressing hepatocyte growth factor into left anterior descending risk areas improved the functions of impaired myocardium, including diminishing the area of ischemia, increasing the number of capillaries, and reducing collagen content. By using the sry gene as a marker, we also demonstrated that the engrafted cells or their progeny incorporated into ischemic cardiac muscle. These results showed that treatment of myocardial ischemia with bone marrow-derived mesenchymal stem cells overexpressing hepatocyte growth factor could be a novel strategy that can both restore local blood flow and regenerate lost cardiomyocytes.

Adenoviridae↗

PubMed2003

[Cultivation of human mesenchymal stem cells on macroporous CultiSpher G microcarriers].

Adult human bone marrow-derived mesenchymal stem cells (MSCs) were cultured on microcarriers in spinner flasks and were compared with those in conventional culture in 12-well plates. For the production of adherently growing MSCs, macroporous CultiSpher G gelatin microcarriers were used in concentration of 1 g/L. The cells were seeded in a density of 5 x 10(4) cells/ml in both spinner culture and conventional stationary culture. The result showed that after 7 days of cultivation a maximum viable cell concentration of 5.15 x 10(5) cells/ml was obtained in spinner culture. Whereas the cell density increased to a maximum of 1.675 x 10(5) cells/ml on day 5 in conventional stationary culture. Lactate was produced up to 12.06 mmol/L in spinner culture and up to 13.10 mmol/L in stationary culture, and glucose was consumed up to 7.38 mmol/L and 5.37 mmol/L respectively. The average lactate yield on glucose consumption in spinner culture was only 1.63, lower than that in stationary culture 2.44. This indicated that the energy metabolism in spinner culture was significantly more efficient than that in conventional culture. After spinner culture for 12 days, the MSCs maintain the characteristics of stem cells. It is concluded that the microcarrier culture system is a suitable way to expand the seeding cells for tissue engineering.

Adult↗

PubMed2003

[The extracellular domain of human delta-like-1 expressed and purified from CHO cells promotes expansion of hematopoietic progenitor cells].

Notch signal path plays important roles in the regulation of proliferation and differentiation of hematopoietic stem cells. An extracellular domain of human Delta-like-1 (hDll-1(ext)), one of Notch ligands, was cloned and expressed in CHO cells, and the effect of hDll-1(ext) on expansion of hematopoietic stem/progenitor cells was investigated in this study. Total RNA was isolated from human marrow mononuclear cells. hDll-1(ext) was amplified by RT-PCR and cloned to T vector, then the gene was sequenced and subcloned to pcDNA3.1/Myc-His(+)A expression vector. The constructed plasmid was transfected into CHO cells with lipofectin and the expression of secreted hDll-1(ext) in G418-resistant clones was assayed by Western blot. hDll-1(ext) high-expressed clone was cultured to collect supernatant. Fusion protein hDll-1(ext) was purified from the supernatant by immobilized metal affinity chromatography (IMAC). The results showed that expression of Notch-1 receptor was detected in cord blood-derived CD34(+) cells by RT-PCR. Human umbilical blood CD34(+) cells were cultured in serum-free medium containing SCF, IL-3, VEGF, and with or without purified hDll-1(ext) for 4 or 8 days. Effect of hDll-1(ext) on the expansion of progenitor cells was analyzed then by clonogenic assays. The number of CFU-Mix and HPP-CFC generated from the culture system containing hDll-1(ext) was 1.5 times of that from the control. In conclusion, the recombinant hDll-1(ext) promotes the expansion of primitive hematopoietic progenitors.

Animals↗

PubMed2003

Effect of human hepatocyte growth factor on promoting wound healing and preventing scar formation by adenovirus-mediated gene transfer.

OBJECTIVE: To evaluate the effects of hepatocyte growth factor (HGF) on the prevention of scar formation and the promotion of wound healing by gene transfer. METHODS: A total of 12 female New Zealand rabbits were used in this study. Rabbits were anesthetized with an intravenous injection of sodium pentobarbital, and identical wounds were made over the ventral surface of each ear. Five circular wounds, 7 mm in diameter, were created in each ear by excision through the skin to the underlying cartilage using sterile technique. After the surgical procedures, 10 of the rabbits were randomly allocated to five groups, with 2 rabbits in each group: Ad-HGF group 1, Ad-HGF group 2, Ad-HGF group 3, Ad-GFP (a reporter gene) group and the solvent group. Immediately after surgery, 6 x 10(7) pfu Ad-HGF, 6 x 10(8) pfu Ad-HGF, 6 x 10(9) pfu of Ad-HGF, 6 x 10(9) pfu of Ad-GFP, or same volume of solvent (PBS, pH 7.2) was applied once to each wound in groups 1 to 5, respectively. One additional rabbit was used to evaluate the transfer efficiency of the adenovirus vector by transferring Ad-GFP (6 x 10(9) pfu) into its wounds. Ice slides of wounds from this animal were observed under fluorescence microscopy. Another additional rabbit was used to evaluate the expression of HGF and TGFbeta1 after transferring Ad-HGF (6 x 10(9) pfu) into each of its wound. Immunohistochemistry was used for detection. RESULTS: The effect of HGF on reducing excessive dermal scarring was observed by adenovirus-mediated gene transfer. Transfection of the human HGF cDNA into skin wounds through an adenoviral vector suppressed the over-expression of TGFbeta1, which plays an essential role in the progression of dermal fibrogenesis. Application of HGF to the wounds significantly enhanced wound healing and inhibited over scarring. CONCLUSION: HGF gene therapy could be a new approach for preventing excessive dermal scarring in wound healing.

Animals↗

PubMed2002

Purging effect of dibutyl phthalate on leukemia cells involves fas independent activation of caspase-3/CPP32 protease.

We previously found that dibutyl phthalate (DBP) had a pharmacological activity in eliminating tumor cells and could be used as a purging agent in autologous bone marrow transplantation. In this study, we show that DBP can induce apoptosis in MO7e and U937 leukemia cell lines. Treatment of these cells with DBP up-regulates cellular activity of caspase-3/CPP32 and causes apoptosis rapidly as determined by cell viability and dUTP nick end labeling assay. Activation of caspase-3/CPP32 and cleavage of poly(ADP-ribose) polymerase were determined in DBP-induced apoptosis of leukemia cells. However, DBP treatment did not induce the expression of fas. Two caspase inhibitors, z-VAD-fmk and N-acetyl-Asp-Glu-Val-Asp-aldehyde partly blocked the cell death of MO7e cells induced by DBP. These results suggest that fas-independent activation of caspase-3 protease plays important roles in the purging effect of DBP on leukemia cells.

Amino Acid Chloromethyl Ketones↗

PubMed2002

[Stem cell factor enhances the adhesion of hematopoietic cells to fibronectin].

Adhesion to extracellular matrix plays important roles in the regulation of survival, proliferation, differentiation and homing of hematopoietic cells and is regulated by a wide variety of growth factors, adhesion receptors and other ligands that mediate the cell to matrix and cell to cell interaction. Stem cell factor (SCF) plays important roles in the regulating growth and self-renewal of hematopoietic stem/progenitor cells. In the report, the effects of stem cell factor on the adhesion of hematopoietic cells to fibronectin were observed by using a hematopoietic growth factor dependent cell line-Mo7e. Results showed that Mo7e cells express the very late antigen VLA-4 (beta1 alpha4) and VLA-5 (beta1 alpha5) integrins. The expression of the SCF receptor (c-kit) was also detected in the Mo7e cells. SCF enhances the adhesion of Mo7e cells to fibronectin in a concentration dependent manner. SCF enhanced adhesion of Mo7e cells to fibronectin was blocked by anti-beta1, alpha4 and alpha5 antibodies. Addition of PI-3 kinase inhibitors also blocked the adhesion of Mo7e cells to fibronectin induced by SCF. It was concluded that SCF enhances the adhesion of Mo7e cells to fibronectin, and this process is mediated by integrins and PI-3 kinase pathway.

Antibodies, Monoclonal↗