bluuebunny/crossref_metadata_2025_split huggingface dataset:
Co‐culture of endothelial cells and smooth muscle cells affects gene expression of angiogenic factors
Authors: Heydarkhan‐Hagvall Sepideh , Helenius Gisela , Johansson Bengt R. , Li Julie Y. , Mattsson Erney , Risberg Bo 
Published: July 2003

AbstractEndothelial cells (EC) are in contact with the underlying smooth muscle cells (SMC). The interactions between EC and SMC in the vessel wall are considered to be involved in the control of growth and function of blood vessels. A co‐culture system of EC and SMC and a method for separation of these cells was developed in order to investigate whether the presence of physical contact between EC and SMC affected the gene expression of angiogenic factors. Human EC and SMC were prepared from the great saphenous veins. Autologous EC were added on top of the confluent layer of SMC. After 72 h in co‐culture, the EC were magnetically separated from SMC with the use of superparamagnetic beads. RT‐PCR products for bFGF, bFGFR, VEGF, PDGF‐AA, PDGF‐BB, TGF‐β, and β‐actin were analyzed to study the mRNA expressions. The protein level of selected factors was studied by ELISA technique. In co‐cultured SMC there was a statistically significant higher gene expression of VEGF, PDGF‐AA, PDGF‐BB, and TGF‐β and significant lower gene expression of bFGF and its receptor than in single cultured SMC. The protein level of PDGF‐BB and TGF‐β was also significantly higher in co‐cultured SMC. In co‐cultured EC there were no significant differences in gene expression of PDGF‐AA, PDGF‐BB, and TGF‐β compared with single cultured EC. The gene expression and protein synthesis of VEGF was significantly higher in co‐cultured EC. The findings from the present study suggest that cell‐cell interactions of EC and SMC affect the gene and protein expression of angiogenic factors. © 2003 Wiley‐Liss, Inc.
Epiregulin is more potent than EGF or TGFα in promoting in vitro wound closure due to enhanced ERK/MAPK activation
Authors: Draper Bradley K. , Komurasaki Toshi , Davidson Mari K. , Nanney Lillian B. 
Published: July 2003

AbstractEpiregulin (EPR) is a broad specificity EGF family member that activates ErbB1 and ErbB4 homodimers and all possible heterodimeric ErbB complexes. We have previously shown that topical EPR enhances the repair of murine excisional wounds. The purpose of this study was to determine whether EPR was more effective than EGF or TGFα in promoting in vitro wound closure and to compare the EPR induced signal transduction pathways with those activated by EGF and TGFα. Normal human epidermal keratinocytes or A431 cells were scratch wounded and treated for 24 h with varying doses of EPR, EGF or TGFα. Five‐fold lower doses of EPR were significantly better than EGF or TGFα in stimulating in vitro wound closure. Mitomycin‐c reduced EPR induced wound closure by 59%, versus a 9% and 25% decrease in EGF and TGFα induced closure. The ERK/MAPK inhibitor PD‐98059 decreased EPR induced wound closure by 88%. By contrast, the PLC inhibitor U‐73122, only reduced the EPR induced response by 21%. Immunoblot analysis revealed that 2 nM EPR stimulated a six‐fold increase in p‐ERK1/2, whereas 10 nM EGF or TGFα stimulated only a 3‐ and 2.5‐fold increase in p‐ERK1/2. When compared with EGF or TGFα, EPR is a more potent and more effective inducer of in vitro wound closure due to its ability to promote significantly greater ERK/MAPK activation. © 2003 Wiley‐Liss, Inc.
Non‐apoptogenic killing of hela cervical carcinoma cells after short exposure to the alkylating agent N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG)
Authors: Węsierska‐Gądek Józefa , Gueorguieva Marieta , Schloffer Daniela , Uhl Maria , Wojciechowski Jacek 
Published: July 2003

AbstractWe examined the action of N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG) on HeLa cells and compared it with that of cisplatin (CP). MNNG directly killed a substantial number of cells within 1 hour and resulted in strong DNA‐damage as evidenced by Comet measurements. Despite appearance of DNA lesions, p53 protein was not activated. Analysis of HeLa cells treated with MNNG for 1h, 3h and 6h by flow cytometry and by Hoechst staining did not reveal any sub‐G1 cell population and chromatin condensation/fragmentation characteristic for apoptosis, respectively. Also, no biochemical changes typical for apoptosis such as activation of caspase‐3 or release of cytochrome C from mitochondria were detected. Inactivation of PARP‐1 reduced the direct cytotoxicity exerted by MNNG. Our results showing that despite appearance of severe DNA lesions after short exposure of HeLa cells to MNNG neither activation of p53 response nor induction of apoptosis occurred implicate that generation of strong DNA damage is not sufficient to stabilize p53 protein in HeLa cells. Our data unequivocally show that the conscientious determination of the type of cell death induced by genotoxic agents is necessary. The assessment of the changes based on at least a few independent criteria is required to discriminate between apoptosis and necrosis. Since the alkylating agents generate DNA strand breaks, the recruitment of methods based on determination of DNA cleavage such as DNA ladder or TUNEL assay for evaluation of apoptosis is not adequate. © 2003 Wiley‐Liss, Inc.
Down‐regulation of Flt‐1 gene expression by the proteasome inhibitor MG262
Authors: Mezquita J. , Mezquita B. , Pau M. , Mezquita C. 
Published: July 2003

AbstractThe mechanisms involved in the anti‐angiogenic actions of the proteasome inhibitors are poorly understood. Here, we report that the gene expression of the VEGF receptor Flt‐1 (vascular endothelial growth factor receptor 1) was down‐regulated by the reversible proteasome inhibitor MG262 in explant cultures of the developing chicken pecten oculi, a vascular organ consisting of endothelial cells, pericytes, and macrophages. In addition, the inhibitor prevented the induction of Flt‐1 by lipopolysaccharide (LPS) in macrophages and down‐regulated the expression of Flt‐1 after LPS induction. Flt‐1 gene expression was also down regulated by MG262 in cultures of human microvascular endothelial cells. Interestingly, a transcript of Flt‐1, coding for a soluble form of the receptor (sFlt‐1) with anti‐angiogenic properties, was not down‐regulated in the same extent. Only a small decrease in the expression of VEGF and Ang‐2 was detected in the pecten oculi upon inhibition of the proteasome, while no major changes were observed in the expression of other angiogenic molecules, such as KDR or Ang‐1. Since recent experiments have demonstrated the importance of anti‐Flt‐1 therapy in the inhibition of tumor angiogenesis, retinal angiogenesis, arthritis, and atherosclerosis (Luttun et al. [2002]: Nat Med 8:831–840), our observation on down‐regulation of Flt‐1 in microvascular endothelial cells and macrophages by MG262 supports the postulated role of the proteasome inhibitors as potential candidates for therapeutic modulation of angiogenesis and inflammation. © 2003 Wiley‐Liss, Inc.
Establishment of a T‐Ag and NF‐κB binding elements gene co‐transfected stable HUVECs cell line
Authors: Liu Y.X. , Cao W.S. , Irani K. , Li X.J. 
Published: July 2003

AbstractTo set up a T‐Ag gene transfected stable human umbilical venous endothelial cells (HUVECs) cell line and a T‐Ag, nuclear transcriptional factor kappa B (NF‐κB) binding elements linked with luciferase reporter gene co‐transfected stable HUVECs cell line. Cultured HUVECs were transfected with pCI‐neo‐T‐Ag and pRSV‐luc‐3XκB by lipofectin. The G418 selected monoclones were subcultured. The expression of marker protein, vWF and the characteristic of uptake of lipids were compared by Western blotting and immunocytochemistry in non‐transfected and transfected HUVECs. The reporter gene assay was done in the presence of TNF‐α. A T‐Ag gene transfected stable HUVECs cell line and a T‐Ag and NF‐κB binding elements linked with luciferase reporter gene co‐transfected stable HUVECs cell lines were set up. The expression of vWF of these cell lines was similar with those in non‐transformed HUVECs. The function of uptaking of lipids was preserved as well in transfected cell lines. Furthermore, TNF‐α, a typical cytokine increasing the activity of NF‐κB was used to treat the transfected cells O/N. The higher luciferase reporter gene activity was seen. A pCI‐neo‐T‐Ag and pRSV‐luc‐3X κB co‐transfected stable HUVECs cell line might be used to check reporter gene activity directly. It might be a useful tool to screen drugs acting on transcription level. © 2003 Wiley‐Liss, Inc.
Effect of overexpressing fibroblast growth factor 2 protein isoforms in osteoblastic ROS 17/2.8 cells
Authors: Xiao L. , Liu P. , Sobue T. , Lichtler A. , Coffin J.D. , Hurley M.M. 
Published: July 2003

AbstractFibroblast growth factor‐2 (FGF‐2) is made by osteoblasts and modulates their function. There are high molecular weight (HMW) protein isoforms of FGF‐2 that have nuclear localization sequences and a low molecular weight (LMW) 18 kDa FGF‐2 protein that is exported from cells. Since FGF‐2 is a trophic factor and potent mitogen for osteoblasts, the goal of this study was to utilize targeted overexpression of FGF‐2 as a novel means of assessing different FGF‐2 isoforms on osteoblastic cell viability and proliferation. Either LMW or HMW human Fgf2 cDNAs were cloned downstream of 3.6 kb α1(I)—collagen 5′ regulatory elements (Col 3.6). A set of expression vectors, called Col3.6‐Fgf2 isoforms‐IRES‐GFPsaph, capable of concurrently overexpressing either LMW or HMW FGF‐2 isoforms concomitant with GFPsaph from a single bicistronic mRNA were built. Viable cell number in ROS 17/2.8 cells stably transfected with Vector (Col3.6‐IRES‐GFPsaph) versus each of the Col3.6‐Fgf2‐IRES‐GFPsaph constructs were compared. In the presence of 1 or 10% serum, DNA synthesis was increased in cells expressing any isoform of FGF‐2 compared with vector. However, cells transfected with HMW isoform had augmented DNA synthesis in 1 or 10% serum compared with cells expressing either ALL or LMW FGF‐2 isoforms. A neutralizing FGF‐2 antibody significantly reduced the mitogenic response in cells harboring ALL or the LMW FGF‐2 isoforms but did not block the mitogenic effect of cells harboring the HMW isoforms. In summary, overexpression of any isoform of FGF‐2 protein increased viable cell number and OB proliferation in the presence of low or high concentrations of serum. However, the HMW/nuclear isoforms preferentially mediate augmented OB proliferation. We conclude that differential expression of FGF‐2 proteins isoforms is important in modulating OB function. © 2003 Wiley‐Liss, Inc.
LPS induces pulmonary intravascular macrophages producing inflammatory mediators via activating NF‐κB
Authors: Chen Zheng‐Tang , Li Sheng‐Liang , Cai En‐Qi , Wu Wei‐Ling , Jin Jing‐Sheng , Zhu Bo 
Published: July 2003

AbstractPulmonary intravascular macrophages (PIMs) are often responsible for the clearance of blood‐borne pathogens, including endotoxin, lipopolysaccharide of Gram‐negative bacteria. It is well accepted that PIMs play a pivotal role in the pathogenesis of endotoxin‐induced acute lung injury. However, the mechanisms by which PIMs are involved in the lipopolysaccharide‐induced inflammatory responses remain unclear. Through the present study the following results were found: (1) When challenged with lipopolysaccharide (10 μg/ml), PIMs underwent marked cellular enlargement, intercellular adhesion plaques became longer, and some particulates were enwrapped in the pseudopods. (2) Lipopolysaccharide could up‐regulate the expression of some inflammatory mediators in PIMs, including TNF‐α, IL‐1β, IL‐6, IL‐8, and COX‐2, and these up‐regulated expression of inflammatory mediators correlated with NF‐κB activation. (3) Dexamethasone as well as acetylsalicylic acid reduced the expression of TNF‐α in lipopolysaccharide‐challenged PIMs, and the decreased expression of TNF‐α was also consistent with decreased NF‐κB activation. Our results suggest that NF‐κB activation in PIMs followed by phagocytizing lipopolysaccharide resulted in the up‐regulation of TNF‐α, IL‐1β, IL‐6, IL‐8, and COX‐2, which could be alleviated by dexamethasone. © 2003 Wiley‐Liss, Inc.
Non‐random features of loop‐size chromatin fragmentation
Authors: Szilágyi Ildikó , Varga Tamás , Székvölgyi Lóránt , Hegedüs Éva , Goda Katalin , Kaczur Viktória , Bacsó Zsolt , Nakayama Yuji , Pósafi János , Pongor Sándor , Szabó Gábor 
Published: July 2003

AbstractUpon isolation of DNA from normal eukaryotic cells by standard methods involving extensive proteolytic treatment, a rather homogeneous population of loop‐size, double‐stranded DNA fragments is regularly obtained. These DNA molecules can be efficiently end‐labeled by the DNA polymerase I Klenow fragment, as well as by a 3′‐ to ‐5′‐exonuclease‐free Klenow enzyme, but not by terminal transferase (TdT) unless the ends have been filled up by Klenow, suggesting that dominantly 5′ protruding termini are generated upon fragmentation. The filled‐up termini were used for cloning the distal parts of the ∼50 kb fragments. BLAST analysis of the sequence of several clones allowed us to determine the sequence of the non‐cloned side of the breakpoints. Comparison of 25, 600 bp‐long breakpoint sequences demonstrated prevalence of repetitive elements. Consensus motives characteristic of the breakpoint sequences have been identified. Several sequences exhibit peculiar computed conformational characteristics, with sharp transition or center of symmetry located exactly at the breakpoint. Our data collectively suggest that chromatin fragmentation involves nucleolytic cleavages at fragile/hypersensitive sites delimiting loop‐size fragments in a non‐random manner. Interestingly, the sequence characteristics of the breakpoints are reminiscent of certain breakpoint cluster regions frequently subject to gene rearrangements. © 2003 Wiley‐Liss, Inc.
Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection
Authors: Baddoo Melody , Hill Katy , Wilkinson Robin , Gaupp Dina , Hughes Catherine , Kopen Gene C. , Phinney Donald G. 
Published: July 2003

AbstractMesenchymal stem cells (MSCs) are typically enriched from bone marrow via isolation of the plastic adherent, fibroblastoid cell fraction. However, plastic adherent cultures elaborated from murine bone marrow are an admixture of fibroblastoid and hematopoietic cell types. Here we report a reliable method based on immunodepletion to fractionate fibroblastoid cells from hematopoietic cells within plastic adherent murine marrow cultures. The immunodepleted cells expressed the antigens Sca‐1, CD29, CD44, CD81, CD106, and the stem cell marker nucleostemin (NST) but not CD11b, CD31, CD34, CD45, CD48, CD90, CD117, CD135, or the transcription factor Oct‐4. They were also capable of differentiating into adipocytes, chondrocytes, and osteoblasts in vitro as well as osteoblasts/osteocytes in vivo. Therefore, immunodepletion yields a cell population devoid of hematopoietic and endothelial cells that is phenotypically and functionally equivalent to MSCs. The immunodepleted cells exhibited a population doubling time of approximately 5–7 days in culture. Poor growth was due to the dramatic down regulation of many genes involved in cell proliferation and cell cycle progression as a result of immunodepletion. Exposure of immunodepleted cells to fibroblast growth factor 2 (FGF2) but not insulin‐like growth factor (IGF), murine stem cell factor, or leukemia inhibitory factor (LIF) significantly increased their growth rate. Moreover, 82% of the transcripts down regulated by immunodepletion remain unaltered in the presence of FGF2. Exposure to the later also reversibly inhibited the ability of the immunodepleted cells to differentiate into adipocytes, chondrocytes, and osteoblasts in vitro. Therefore, FGF2 appears to function as a mitogen and self‐maintenance factor for murine MSCs enriched from bone marrow by negative selection. © 2003 Wiley‐Liss, Inc.
Jun N‐terminal kinase pathway enhances signaling of monocytic differentiation of human leukemia cells induced by 1,25‐dihydroxyvitamin D3
Authors: Wang Qing , Wang Xuening , Studzinski George P. 
Published: July 2003

AbstractRecent studies revealed that the MEK/ERK module of the mitogen‐activated protein kinase (MAPK) signaling cascades is up‐regulated in the early stages of 1α,25‐dihydroxyvitamin D3 (1,25D3)‐induced monocytic differentiation of human leukemia cells HL60. In the present study, we investigated whether another MAPK module, the JNK pathway, also participates in this form of differentiation. We found that the dependence on the concentration of the inducer, the vitamin‐hormone 1,25D3, in two types of human leukemia cells, HL60 and U937, and the kinetics of monocytic differentiation in HL60 cells, parallel the degree of the activation of the JNK pathway. A blockade of JNK signaling by a stable expression of dominant negative (dn) JNK1 mutant in U937 cells resulted in reduced c‐jun phosphorylation, and the differentiation of these cells was markedly decreased. Similarly, inhibition of JNK1 and JNK2 activities by the selective inhibitor SP600125 led to both dose‐dependent reduction of c‐jun and ATF‐2 phosphorylation, and of the differentiation of HL60 cells. In addition, we found that JNK activity is essential for the AP‐1 DNA binding induced by 1,25D3 in HL60 and U937 cells. The results indicate that in cultured human leukemia cells, the JNK pathway participates in the induction of monocytic differentiation by 1,25D3, probably by activating the AP‐1 transcription factor. © 2003 Wiley‐Liss, Inc.
Increased expression of Mcl‐1 is responsible for the blockage of TRAIL‐induced apoptosis mediated by EGF/ErbB1 signaling pathway
Authors: Henson Elizabeth S. , Gibson Erika M. , Villanueva Jacylyn , Bristow Nicolle A. , Haney Neil , Gibson Spencer B. 
Published: July 2003

AbstractEpidermal growth factor (EGF) protects against death receptor induced apoptosis in epithelial cells. Herein, we demonstrate that EGF protection against tumor necrosis factor related apoptosis‐inducing ligand (TRAIL) induced apoptosis is mediated by increased expression of the Bcl‐2 family member myeloid cell leukemia 1 (Mcl‐1). EGF increased the mRNA and protein levels of Mcl‐1. Furthermore, expression of ErbB1 alone or in combination with ErbB2 in NIH3T3 cells up‐regulates Mcl‐1 following EGF treatment. In addition, up‐regulation of Mcl‐1 by EGF is mediated through AKT and NFκB activation since kinase inactive AKT and ΔIκB effectively blocks this up‐regulation. NFκB was also critical for the ability of EGF to prevent TRAIL induced apoptosis as a dominant negative IκB (ΔIκB) blocked NFκB activation, and relieved EGF protection against TRAIL mediated mitochondrial cytochrome‐c release and apoptosis. Finally, anti‐sense oligonucleotides directed against Mcl‐1 effectively reduced the protein levels of Mcl‐1 and blocked EGF protection against TRAIL induced mitochondrial cytochrome‐c release and apoptosis. Taken together, EGF signaling leads to increased Mcl‐1 expression that is required for blockage of TRAIL induced apoptosis. © 2003 Wiley‐Liss, Inc.
Development of a dual‐luciferase fusion gene as a sensitive marker for site‐directed DNA repair strategies
Authors: Bennett Michael , Schaack Jerome 
Published: July 2003

AbstractBackgroundSeveral novel techniques have been developed recently for the site‐specific repair of DNA as an approach to gene therapy. Correction efficiencies as high as 40% have been reported, well within the range of therapeutic impact for a number of genetic diseases. Unfortunately, many of the model systems in which these methods have been employed typically target genes that are not well suited for analyzing the various techniques.MethodsTo address this, we have constructed and characterized a dual‐luciferase fusion gene as a sensitive marker for optimizing repair strategies. The genes encoding two distinct luciferase proteins were fused so that expression of one luciferase necessitated expression of the other. Engineering a stop codon in the downstream luciferase gene created an ideal tool to study the efficiency of various site‐directed DNA repair techniques as one luciferase can act as an internal control while the other is targeted for correction.ResultsFusing two luciferase genes resulted in a single protein that produces two bioluminescent activities in a constant ratio. The utility of this system as a target for site‐directed DNA repair research was demonstrated using two of the recently developed gene repair techniques, small fragment homologous replacement and oligonucleotide‐mediated repair, to mediate correction and by the ability to detect repair efficiencies of less than 5 × 10−6 (<1 event in 200 000).ConclusionsThe ability to rapidly and accurately quantify the amount of correction using the dual‐luciferase fusion system will allow the comparison and evaluation of the many factors involved in successful gene repair and lead to the optimization of these techniques, both in cell culture and in whole animals. Copyright © 2003 John Wiley & Sons, Ltd.
Dendritic cells engineered to express the Flt3 ligand stimulate type I immune response, and induce enhanced cytoxic T and natural killer cell cytotoxicities and antitumor immunity
Authors: Liu Yongqing , Huang Hui , Chen Zhuang , Zong Li , Xiang Jim 
Published: July 2003

AbstractBackgroundTumor antigen presentation by dendritic cells (DCs) to T cells in lymphoid organs is crucial for induction of antitumor immune responses. Fms‐like tyrosine kinase 3 ligand (Flt3L) is a regulator of hematopoietic cell development.MethodsTo investigate the potential effect of Flt3L transgene expression on DC‐based cancer vaccines, we constructed a recombinant adenovirus AdVFlt3L expressing Flt3L, transfected DCs with AdVFlt3L, and investigated the efficacy of antitumor immunity by vaccination of DCFlt3L engineered to express Flt3L transgene.ResultsOur data demonstrated that AdVFlt3L transfection up‐regulated the expression of cytokine IL‐1β and chemokines MIP‐1α, MIP‐1β, IP‐10, MCP‐1 and MIP‐2, and stimulated DCFlt3L cell proliferation in vitro and migration toward regional lymph nodes in vivo. Our data also demonstrated that vaccination of Mut1‐pulsed DCFlt3L cells was able to stimulate (i) a type 1 immune response comprising CD4+ Th1 and CD8+ Tc1 activation and (ii) around 2‐ and 3‐fold enhanced tumor‐specific cytotoxic T lymphocyte (CTL) and non‐specific NK responses (p < 0.05) than vaccination with similarly pulsed control virus‐transfected and untransfected DCs, respectively. More importantly, vaccination of Mut1‐pulsed DCFlt3L cells induced enhanced antitumor immunity in vivo, even against poorly immunogenic 3LL tumor cells. Vaccinations of Mut1‐pulsed DCs, DCpLpA and DCFlt3L all protected mice from challenge of low dose (0.5 × 105) tumor cells. However, only vaccination of the last one was able to protect 63% (6/8) mice from challenge of high dose (3 × 105) 3LL tumor cells (p < 0.01).ConclusionsDCs engineered to secrete Flt3L may offer a new strategy in DC‐based cancer vaccines. Copyright © 2003 John Wiley & Sons, Ltd.