PURPOSE: Although follicular thyroid cancer (FTC) has a relatively fair prognosis, distant metastasis sometimes results in poor prognosis and survival. There is little understanding of the mechanisms contributing to the aggressiveness potential of thyroid cancer. We showed that hypoxia inducible factor-1alpha (HIF-1alpha) induced aggressiveness in FTC cells and identified the underlying mechanism of the HIF-1alpha-induced invasive characteristics. MATERIALS AND METHODS: Cells were cultured under controlled hypoxic environments (1% O2) or normoxic conditions. The effect of hypoxia on HIF-1alpha, and epithelial-to-mesenchymal transition (EMT) related markers were evaluated by quantitative real-time PCR, Western blot analysis and immunocytochemistry. Invasion and wound healing assay were conducted to identify functional character of EMT. The involvement of HIF-1alpha and Twist in EMT were studied using gene overexpression or silencing. After orthotopic nude mouse model was established using the cells transfected with lentiviral shHIF-1alpha, tissue analysis was done. RESULTS: Hypoxia induces HIF-1alpha expression and EMT, including typical morphologic changes, cadherin shift, and increased vimentin expression. We showed that overexpression of HIF-1alpha via transfection resulted in the aforementioned changes without hypoxia, and repression of HIF-1alpha with RNA interference suppressed hypoxia-induced HIF-1alpha and EMT. Furthermore, we also observed that Twist expression was regulated by HIF-1alpha. These were confirmed in the orthotopic FTC model. CONCLUSION: Hypoxia induced HIF-1alpha, which in turn induced EMT, resulting in the increased capacity for invasion and migration of cells via regulation of the Twist signal pathway in FTC cells. These findings provide insight into a possible therapeutic strategy to prevent invasive and metastatic FTC.
Adenocarcinoma, Follicular/*genetics/metabolism ; Animals ; Anoxia/*genetics ; Cadherins/genetics ; Epithelial-Mesenchymal Transition/*genetics ; Gene Expression Regulation, Neoplastic ; Hypoxia-Inducible Factor 1, alpha Subunit/*genetics/metabolism ; Lymphokines ; Mice ; Neoplasm Invasiveness ; Phenotype ; Real-Time Polymerase Chain Reaction ; Signal Transduction/drug effects ; Thyroid Neoplasms/*genetics/metabolism ; Transcriptional Activation ; Twist Transcription Factor/*genetics/metabolism ; Vimentin/metabolism

Mesenchymal stem cells (MSCs) can inhibit T cell proliferation; however, the underlying mechanisms are not clear. In this study, we investigated the mechanisms of the immunoregulatory activity of MSCs on T cells. Irradiated MSCs co-cultured with either naive or pre-activated T cells in a mixed lymphocyte reaction (MLR) significantly suppressed T cell proliferation in a dose-dependent manner, irrespective of allogeneic disparity between responders and MSCs. Transwell assays revealed that the suppressive effect was primarily mediated by soluble factors that induced apoptosis. Splenocytes stimulated with alloantigen in the presence of the MSC culture supernatant (CS) produced a significant amount of IL-10, which was attributed to an increase in the number of IL-10 secreting cells, confirmed by an ELISPOT assay. The blockade of IL-10 and IL-10 receptor interaction by anti-IL-10 or anti-IL-10-receptor antibodies abrogated the suppressive capacity of MSC CS, indicating that IL-10 plays a major role in the suppression of T cell proliferation. The addition of 1-methyl-DL-tryptophan (1-MT), an indoleamine 2,3-dioxygenase (IDO) inhibitor, also restored the proliferative capacity of T cells. In conclusion, we demonstrated that soluble mediators from culture supernatant of MSCs could suppress the proliferation of both naive and pre-activated T cells in which IL-10 and IDO play important roles.
Animals ; Cell Proliferation ; Cells, Cultured ; Indoleamine-Pyrrole 2,3,-Dioxygenase/antagonists & inhibitors/metabolism ; Interleukin-10/*biosynthesis ; *Lymphocyte Activation ; Lymphokines/pharmacology ; Mesenchymal Stem Cells/cytology/*metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Receptors, Interleukin-10/metabolism ; T-Lymphocytes/cytology/*immunology/metabolism ; Tryptophan/analogs & derivatives/pharmacology

Mesenchymal stem cells (MSCs) can inhibit T cell proliferation; however, the underlying mechanisms are not clear. In this study, we investigated the mechanisms of the immunoregulatory activity of MSCs on T cells. Irradiated MSCs co-cultured with either naive or pre-activated T cells in a mixed lymphocyte reaction (MLR) significantly suppressed T cell proliferation in a dose-dependent manner, irrespective of allogeneic disparity between responders and MSCs. Transwell assays revealed that the suppressive effect was primarily mediated by soluble factors that induced apoptosis. Splenocytes stimulated with alloantigen in the presence of the MSC culture supernatant (CS) produced a significant amount of IL-10, which was attributed to an increase in the number of IL-10 secreting cells, confirmed by an ELISPOT assay. The blockade of IL-10 and IL-10 receptor interaction by anti-IL-10 or anti-IL-10-receptor antibodies abrogated the suppressive capacity of MSC CS, indicating that IL-10 plays a major role in the suppression of T cell proliferation. The addition of 1-methyl-DL-tryptophan (1-MT), an indoleamine 2,3-dioxygenase (IDO) inhibitor, also restored the proliferative capacity of T cells. In conclusion, we demonstrated that soluble mediators from culture supernatant of MSCs could suppress the proliferation of both naive and pre-activated T cells in which IL-10 and IDO play important roles.
Animals ; Cell Proliferation ; Cells, Cultured ; Indoleamine-Pyrrole 2,3,-Dioxygenase/antagonists & inhibitors/metabolism ; Interleukin-10/*biosynthesis ; *Lymphocyte Activation ; Lymphokines/pharmacology ; Mesenchymal Stem Cells/cytology/*metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Receptors, Interleukin-10/metabolism ; T-Lymphocytes/cytology/*immunology/metabolism ; Tryptophan/analogs & derivatives/pharmacology

The nomenclature "embryonic lymphoid tissue inducer (LTi) cell" reflects the fundamental role of the cell in secondary lymphoid tissue organization. In addition, it is equally important in primary lymphoid tissue development as it regulates central tolerance to self-antigens in the thymus. An adult LTi cell constitutively expresses two sets of tumor necrosis factor (TNF) family members, whereas its embryonic counterpart expresses only one. The first set is lymphotoxin (LT)alpha, LTbeta, and TNFalpha, which are essential for the secondary lymphoid organogenesis during embryogenesis and for maintaining an organized secondary lymphoid structure during adulthood. The second set is OX40- and CD30-ligands, which are critical for memory T cell generation. Adult LTi cells regulate adaptive immune responses by providing LTbetaR signals to stromal cells to maintain secondary lymphoid tissue structure, and determine adaptive immune responses by providing OX40 and CD30 survival signals to activated T cells in memory T cell generation. Along with the consideration of the roles of embryonic LTi cells in primary and secondary lymphoid tissues, this review highlights the roles of adult LTi cells in secondary lymphoid tissue function.
Adult ; Animals ; Humans ; Lymphoid Tissue/cytology/embryology/*immunology ; Lymphokines/immunology/metabolism ; T-Lymphocytes, Helper-Inducer/cytology/*immunology/metabolism ; Thymus Gland/cytology/embryology/*immunology

Animals ; Fatty Liver ; metabolism ; pathology ; Liver ; pathology ; Liver Cirrhosis, Experimental ; metabolism ; pathology ; Lymphokines ; metabolism ; Male ; Platelet-Derived Growth Factor ; metabolism ; Rats ; Rats, Sprague-Dawley

To investigate the effect of arsenic trioxide (As(2)O(3)) on vascular endothelial growth factor (VEGF) expression in different courses of chronic myeloid leukemia (CML), VEGF level was measured with ELISA in the cultural supernatants of bone marrow mononuclear cells from CML patients. The results showed that supernatants of cultured bone marrow cells from 35 CML patients (20 chronic, 8 accelerated and 7 blast crisis phases) contained significantly higher VEGF levels (649.16 +/- 382.20 pg/ml, 560.27 +/- 409.14 pg/ml and 587.18 +/- 415.28 pg/ml, respectively) than that in 15 normal control samples (152.16 +/- 150.09 pg/ml; P < 0.01), but no significant differences were found in VEGF levels among different phases of CML. The bone marrow cells treated with As(2)O(3) (5 x 10(-6)mol/L) for 72 hours resulted in significant reduction of VEGF levels (down to 396.66 +/- 257.47 pg/ml, 363.42 +/- 239.85 pg/ml and 407.47 +/- 219.38 pg/ml, respectively) (P < 0.05). In conclusion, abnormal high expression of VEGF plays a role in the pathogenetic course of CML and it is probably an additional anticancer mechanism for As(2)O(3) to inhibit VEGF expression of leukemic cells.
Adolescent ; Adult ; Aged ; Arsenicals ; pharmacology ; Bone Marrow Cells ; drug effects ; metabolism ; Cells, Cultured ; Child ; Culture Media, Conditioned ; chemistry ; Down-Regulation ; drug effects ; Endothelial Growth Factors ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; blood ; pathology ; Lymphokines ; metabolism ; Male ; Middle Aged ; Oxides ; pharmacology ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

By using AdEasy system, which is based on the homologous recombination in bacteria, an EGFP labled recombinant adenovirus vector containing hVEGF(165) was generated quickly and its property was studied in vitro. First, hVEGF(165) coding sequence was subcloned into the shuttle plasmid pAdTrack-CMV, then linearized and cotransferred with adenoviral backbone vector pAdEasy-1 into E. coli strain BJ(5183). After positive kanamycin-resistant colony was picked up, the recombinant adenoviral plasmid was identified by restriction analysis with PacI and transfected into HEK 293 cells to assembly replication-defective adenovirus Ad-EGFP/hVEGF(165). The further amplified recombinant adenoviruses were purified by CsCl banding at 32,000 rpm for 18 to 24 hours. Electron microscopy and PCR were performed for testing the recombinant adenovirus. The results showed that the purified particles were homogenous hexagon with a high titer up to 2 x 10(12)pfu/ml. An amplified band of 540 bp fragment demonstrated the successful insert of hVEGF(165). Under fluorescence microscopy, the expression of EGFP was easily detected in HEK 293 and other target cells. The maximal stimulating effect on the proliferation of hUVEC was obtained when the given multiplicity of infection (MOI) of Ad-EGFP/hVEGF(165) was 100. The rate of EGFP positive mouse bone marrow mononuclear cells analysed by flow cytometry was 27.3% after 24 hour-incubation with Ad-EGFP/hVEGF(165) (MOI = 100), and the expression of hVEGF(165) protein in the conditioned medium was 1385 +/- 332 pg/10(6) cells. It is concluded that the construction of adenovirus vector by homologous recombination in bacteria using AdEasy system can be quickly and easily performed, and the prepared high titer of Ad-EGFP/hVEGF(165) is an efficient helpful vector to transfer genes into target cells, all of which make the further in vivo experiments with VEGF(165) possible.
Adenoviridae ; genetics ; ultrastructure ; Animals ; Cell Division ; genetics ; Cell Line ; Cells, Cultured ; Cloning, Molecular ; methods ; Endothelial Growth Factors ; genetics ; metabolism ; Endothelium, Vascular ; cytology ; metabolism ; Genetic Vectors ; genetics ; ultrastructure ; Green Fluorescent Proteins ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Leukocytes, Mononuclear ; cytology ; metabolism ; Luminescent Proteins ; genetics ; metabolism ; Lymphokines ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Microscopy, Electron ; Microscopy, Fluorescence ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

To investigate the effect of vascular endothelial growth factor (VEGF) on beta1 integrin (VLA-4 and VLA-5) activation ability in K562 cells transfected with antisense VEGF121 cDNA, K562 cells were transfected with antisense (As), sense (S) and vector (V, pcDNA(3)). Flow cytometry was used to evaluate the expression rate of VLA-4 (CD49d/CD29) and VLA-5 (CD49e/CD29) and beta1 integrin activation ability in the transfected K562 cells. The results showed that the expression rates of VLA-4 and VLA-5 were more than 92% in the transfected K562 cells and there was no difference among the K562/V, K562/S and K562/As cells. However, beta1 integrin activated 9EG7 expression rate in K562/As cells was higher than that in K562/V cells [(75.6 +/- 10.5)% vs (41.2 +/- 2.1)%, P < 0.01)] after activation with beta1 integrin activator 8A2. It is concluded that function of beta1 integrin to be activated is restored in K562 cells transfected with antisense VEGF121 cDNA.
DNA ; genetics ; DNA, Antisense ; genetics ; Endothelial Growth Factors ; genetics ; metabolism ; Flow Cytometry ; Humans ; Integrin alpha4beta1 ; biosynthesis ; Integrin alpha5beta1 ; biosynthesis ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; K562 Cells ; Lymphokines ; genetics ; metabolism ; Transfection ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

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 ; Antigens, CD34 ; immunology ; Binding Sites ; genetics ; CHO Cells ; Cell Division ; drug effects ; physiology ; Colony-Forming Units Assay ; Cricetinae ; Endothelial Growth Factors ; pharmacology ; Fetal Blood ; cytology ; immunology ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; Glycoproteins ; genetics ; pharmacology ; physiology ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Interleukin-3 ; pharmacology ; Lymphokines ; pharmacology ; Membrane Proteins ; genetics ; RNA ; genetics ; metabolism ; Receptor, Notch1 ; Receptors, Cell Surface ; Recombinant Proteins ; isolation & purification ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cell Factor ; pharmacology ; Transcription Factors ; Transfection ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo explore the response of nasal mucosa epithelial cells to hypoxia in terms of formation of nasal polyps (NP).

METHODSEpithelial cells of NP and inferior turbinate (IT) were cultured serum-free under normal oxygen and hypoxic circumstances with stimulation of IL-1 beta and TNF alpha. The vascular endothelial growth factor (VEGF) mRNA and VEGF protein levels of the cultured cells were detected using in situ hybridization and ELISA, respectively.

RESULTSThe expression of VEGF mRNA was significantly higher in epithelial cells of NP than in IT exposed to pro-inflammatory cytokines or hypoxia (P < 0.01). VEGF levels were higher in NP epithelial cells than those of IT (P < 0.01) under hypoxia.

CONCLUSIONVEGF-induced by hypoxia is very important for the early stages of forming polyps.

Cell Hypoxia ; physiology ; Cells, Cultured ; Endothelial Growth Factors ; genetics ; Enzyme-Linked Immunosorbent Assay ; Erythropoietin ; genetics ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; Interleukin-1 ; pharmacology ; Lymphokines ; genetics ; Nasal Mucosa ; metabolism ; Nasal Polyps ; etiology ; metabolism ; RNA, Messenger ; analysis ; Tumor Necrosis Factor-alpha ; pharmacology ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors