ABSTRACT
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.
Subject(s)
Animals , Mice , Adenoviridae , Genetics , B7-1 Antigen , Genetics , Allergy and Immunology , Cancer Vaccines , Allergy and Immunology , Genes, p53 , Allergy and Immunology , Genetic Vectors , Granulocyte-Macrophage Colony-Stimulating Factor , Genetics , Allergy and Immunology , Immunotherapy , Mice, Inbred NOD , Mice, SCID , Multiple Myeloma , Allergy and Immunology , Neoplasm TransplantationABSTRACT
<p><b>AIM</b>To assess whether hepatocyte growth factor recruits bone marrow-derived endothelial progenitor cells into blood circulation to participate in postnatal angiogenesis and endothelium repair.</p><p><b>METHODS</b>The adenovirus vector encoding HGF gene (Ad-HGF) were intravenous administrated into BALB/c mice, and then serum HGF was determined by enzyme-linked immunosorbent assay, the number of CD34+ cells in peripheral blood was assayed by flow cytometry, and the nucleated cells in peripheral blood were isolated, cultured and the endothelial cell colonies were characterized by staining with antibodies against tie-2, vWF. The carbon tetrachloride-induced liver damage model of female mice was established. The peripheral blood nucleated cells of Ad-HGF treated male mice were intravenous administrated into these mice, and 4 weeks later, in situ hybridization for the sry gene was used to identify the implanted cells in the damaged tissues.</p><p><b>RESULTS</b>Intravenous administration of Ad-HGF resulted in significant elevation of serum hepatocyte growth factor level and induced profoundly increase of endothelial progenitor cells in the peripheral blood, which were characterized by their ability to form endothelial cell colonies in culture and expression of CD34, tie-2, and vW factor. HGF-mobilized endothelial progenitors could incorporate into sites of neovascularization in a liver regeneration model.</p><p><b>CONCLUSION</b>Hepatocyte growth factor could markedly recruit bone marrow-derived endothelial progenitor cells into blood circulation.</p>
Subject(s)
Animals , Female , Male , Mice , Bone Marrow Cells , Cell Biology , Endothelial Cells , Cell Biology , Hematopoietic Stem Cell Mobilization , Hepatocyte Growth Factor , Pharmacology , Mice, Inbred BALB C , Stem Cells , Cell BiologyABSTRACT
<p><b>AIM</b>To establish the methods for determining the activity of sphingosine kinase(SPK) and the content of sphingosine 1-phosphate (S1P) in biological samples.</p><p><b>METHODS</b>The ECV304 cells were transfected with pcDNA3 vector encoding Flag-labeled SPK gene. The expression of SPK was measured by Western blot assay and the activity of SPK was determined by enzymatic reaction, isotope incorporation and thin-layer chromatography methods. The S1P in biological samples was extracted, digested by alkaline phosphatase and then catalyzed by SPK. The S1P contents were determined according to the amounts of products.</p><p><b>RESULTS</b>SPK gene transfection could enhance the expression and activity of SPK in cells markedly, and the cellular S1P was also increased obviously. HGF stimulation could increase the activity of SPK and cellular S1P in ECV304 cells.</p><p><b>CONCLUSION</b>Methods for determining the activity of SPK and the content of SPK in biological samples were established.</p>
Subject(s)
Humans , Cell Line , Cytophotometry , Isotope Labeling , Lysophospholipids , Metabolism , Phosphotransferases (Alcohol Group Acceptor) , Metabolism , Sphingosine , MetabolismABSTRACT
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.
Subject(s)
Humans , Apoptosis , Cell Division , Multiple Myeloma , Drug Therapy , Pathology , Receptor, Notch1 , Receptors, Cell Surface , Physiology , Signal Transduction , Transcription Factors , PhysiologyABSTRACT
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.
Subject(s)
Animals , Cricetinae , Humans , Antigens, CD34 , Allergy and Immunology , Binding Sites , Genetics , CHO Cells , Cell Division , Physiology , Colony-Forming Units Assay , Endothelial Growth Factors , Pharmacology , Fetal Blood , Cell Biology , Allergy and Immunology , Metabolism , Gene Expression , Genetic Vectors , Genetics , Glycoproteins , Genetics , Pharmacology , Physiology , Hematopoietic Stem Cells , Cell Biology , Intercellular Signaling Peptides and Proteins , Pharmacology , Interleukin-3 , Pharmacology , Lymphokines , Pharmacology , Membrane Proteins , Genetics , RNA , Genetics , Metabolism , Receptor, Notch1 , Receptors, Cell Surface , Recombinant Proteins , Pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Stem Cell Factor , Pharmacology , Transcription Factors , Transfection , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
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.