Tenascin-C inhibits beta1 integrin-dependent T lymphocyte adhesion to fibronectin through the binding of its fnIII 1-5 repeats to fibronectin.

The extracellular matrix consists of different proteins interacting to form a meshwork-like structure. T lymphocyte adhesion to individual matrix proteins is mainly regulated at the adhesion receptor level, but it is conceivable that the composition of the matrix itself may affect T lymphocyte adhesion to individual proteins. We have addressed the latter point by studying the effect of the matrix protein tenascin-C (TN-C) on T lymphocyte adhesion to fibronectin. Here we report that TN-C inhibits adhesion of T lymphocytes and MOLT-4 lymphoma cells to fibronectin. We demonstrate that a TN-C fragment consisting of fibronectin type III repeats 1-5 (TNfnIII 1-5) but not TNfnIII A-D and TNfnIII 6-8 inhibited alpha5beta1 and alpha4beta1 integrin-mediated T lymphocyte and MOLT-4 adhesion to fibronectin. At concentrations that did not inhibit adhesion, TNfnIII 1-5 still prevented MOLT-4 cells from spreading on fibronectin. Preincubation and co-immobilization of TNfnIII 1-5 with fibronectin was more effective in inhibiting MOLT-4 adhesion to fibronectin than soluble TNfnIII 1-5 present during the adhesion test. Using an enzyme-linked immunosorbent assay we could demonstrate binding of TNfnIII 1-5 to fibronectin and fibronectin fragments. Taken together, these data demonstrate that the TNfnIII 1-5 domain is implicated in the inhibition of T lymphocyte adhesion to fibronectin caused by TN-C, and indicate that this effect involves the binding of TN-C repeats TNfnIII 1-5 to fibronectin.     

Progestin stimulates the biosynthesis of fibronectin and accumulation of fibronectin mRNA in human endometrial stromal cells.

Fibronectin is a major component of decidual basement membrane. In the present study, we have investigated the effect of progestin on the synthesis and secretion of fibronectin in human endometrial stromal cells. Stromal cells were isolated during the menstrual cycle and cultured in RPMI-1640 with 2% fetal calf serum supplemented with progesterone or medroxyprogesterone acetate (MPA) in a long-term culture system. Indirect immunofluorescent staining showed that fibronectin was uniformly distributed in the intracellular and extracellular regions of stromal cells treated with MPA for 14 days. The biosynthesis and secretion of this protein and the accumulation of cellular fibronectin mRNA were studied after various culture periods. Cells were pulse-labelled with [35S]methionine to determine the amount of newly synthesized fibronectin secreted into the culture medium. A monoclonal antibody (Mab) identified human fibronectin on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), showing a predominant band (Mr 230-250 kDa) which migrated with authentic fibronectin run in parallel. In six endometrial specimens, the amount of radioactivity incorporated as [35S]fibronectin was increased by progestin. Maximal stimulation occurred after 6 days treatment with MPA. Culture beyond 16 days reduced the rate of synthesis and secretion to 40% of the maximum. The effect of progestin was dose dependent with 0.02, 0.2 and 1 microM progesterone, producing 2.0, 3.8 and 11-fold increases respectively, over the control. Medroxyprogesterone acetate was more effective than progesterone, the maximal response (10-fold increase) being achieved at 0.02 microM MPA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bcl-2反义核酸增强HL-60、K562细胞对柔红霉素敏感性的研究

目的:研究bcl-2反义寡核苷酸作用后,白血病细胞株HL60、K562细胞对柔红霉素(DNR)敏感性的影响。方法:MTT法测HL60、K562细胞中DNR的半数抑制率(IC50);免疫荧光标记观测细胞Bcl-2蛋白水平;用Hoechest33258和碘化丙锭双染色法及流式细胞仪检测细胞凋亡。结果:靶向bcl-2mRNA蛋白编码区反义寡核苷酸(AS-ODN1)和靶向翻译起始区的反义寡核苷酸(AS-ODN2)分别与DNR联合作用于HL60细胞后DNR的IC50值分别为0.124±0.011、0.149±0.012,分别与不加寡核苷酸组DNR的IC50值(0.173±0.021)或无义寡核苷酸联用DNR的IC50值(0.180±0.023)相比有显著差异,P＜0.05。AS-ODN1和AS-ODN2分别与DNR联合作用于K562细胞后DNR的IC50值分别为0.078±0.007、0.079±0.008,分别与不加寡核苷酸组DNR的IC50值(0.106±0.011)或无义寡核苷酸联用DNR的IC50值(0.107±0.012)相比有显著差异,P＜0.05。AS-ODN1和AS-ODN2分别与DNR同时作用于HL60或K562细胞后抑制Bcl-2蛋白表达及诱导细胞凋亡率分别与单用DNR或无义寡核苷酸联用DNR相比有显著差异,P＜0.05。与AS-ODN2比较,AS-ODN1提高HL60细胞对DNR的敏感性作用要强些(P＜0.05)。结论:靶向bcl-2mRNA蛋白编码区反义寡核苷酸和靶向翻译起始区的反义寡核苷酸能增强HL60和K562细胞对DNR的敏感性。     

人参皂苷Rh2调节白血病细胞增殖分化的作用

目的:观察人参皂苷Rh2对白血病细胞HL60增殖分化的影响.方法:免疫印迹法及流式细胞分析法分别检测未处理组HL60细胞及药物处理组HL60细胞的核增殖抗原(PCNA)及成熟粒细胞表面抗原分子CD15的表达水平.结果:药物处理组的HL60细胞体积增大,形状不规则;与未处理组HL60细胞相比,药物处理组的HL60细胞增殖...     

钙调素拮抗剂EBB逆转HL60／Har细胞对抗肿瘤药的抗性

本文采用细胞增殖抑制、流式细胞术等方法研究钙调素拮抗剂０－４－乙氧基－丁基－小檗胺对ＨＬ６０／Ｈａｒ细胞多药抗性的逆转，发现２ｍｍｏｌ／ＬＥＢＢ与阿霉素联合用药可使细胞的ＩＣ５０值由阿霉素单独用药时的２．１６ｍｇ／Ｌ降低至０．２８ｍｇ／Ｌ。     

Nano-TiO2 particles impair adhesion of airway epithelial cells to fibronectin

Titanium dioxide engineered nanoparticles (nano-TiO₂) are widely used in the manufacturing of a number of products. Due to their size (<100 nm), when inhaled they may be deposited in the distal lung regions and damage Clara cells. We investigated the mechanisms by which short-term (1-h) incubation of human airway Clara-like (H441) cells to nano-TiO₂ (6 nm in diameter) alters the ability of H441 cells to adhere to extracellular matrix. Our results show that 1 h post-incubation, there was a 3-fold increase of extracellular H₂O₂, increased intracellular oxidative stress as demonstrated by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) oxidation, and a 5-fold increase of phosphor-ERK1/2 as measured by Western blotting. These changes were accompanied by a 25% decrease of H441 adherence to fibronectin (p < 0.05 compared to vehicle incubated H441 cells). Pretreatment with the ERK1/2 inhibitor U0126 for 3 h, partially prevented this effect. In conclusion, short-term exposure of H441 cells to nano-TiO₂ appears to reduce adherence to fibronectin due to oxidative stress and activation of ERK1/2.     

Requirement of MEF2D in the induced differentiation of HL60 promyeloid cells

The regulatory role of MEF2 (myocyte enhancer binding factor 2) proteins in nonmuscle tissues has not been well characterized. We examined the expression of MEF2 family members, namely, MEF2A, -B, -C, and -D, in the differentiation of HL60 promyeloid cells and observed the remarkable increase in the expressions of MEF2A and MEF2D proteins during the differentiation process into monocytes. To examine the role of MEF2, we expressed a dominant-negative form of MEF2D, without its transactivation domain, in HL60 cells. When the HL60 cell line expressing the mutant MEF2D was induced to differentiate by VitD₃ treatment, cell surface expression of CD14 and the ability to reduce NBT, which are important characteristics of differentiated monocytes, were significantly decreased compared with control HL60 cells. These results show that MEF2D is required in the differentiation process along the monocyte/macrophage lineage,     

Livin反义寡核苷酸对人HL60细胞增殖和凋亡的影响

目的 探讨Livin mRNA反义寡核苷酸（ASODN）对人白血病细胞（HL60）增殖及凋亡的影响。方法 用免疫组织化学检测Livin蛋白表达,设计合成特异性的Livin硫代磷酸ASODN及其对照错义寡核苷酸（MSODN）,脂质体转染HL60细胞。用四甲基偶氮唑盐光吸收法(MTT)检测细胞增殖,反转录-聚合酶链反应（RT-PCR）检测Livin mRNA的表达,原位末端标记技术（TUNEL）、电镜检测细胞凋亡率和形态学改变。结果Livin ASODN在终浓度为600nmol／L作用HL60细胞48h时,能明显地抑制其增殖,降低Livin mRNA的表达,HL60细胞在形态学上出现明显的凋亡改变,细胞凋亡率显著增加 (P<0.01)。结论Livin ASODN可有效抑制HL60细胞的增殖,下调Livin基因表达,并促进HL60细胞凋亡,在诱导肿瘤细胞凋亡、抑制增殖中发挥重要作用。     

A rough morphology of the adsorbed fibronectin layer favors adhesion of neuronal cells

A range of substrates made of polystyrene (PS) and poly(methyl methacrylate)-poly(methacrylic acid) copolymer (PMMA-PMAA) containing 98 and 80% PMMA (PA98, PA80) and presenting a homogeneous or a patterned surface were used to study fibronectin adsorption and neuronal cell behavior. Fibronectin adsorption showed weak differences regarding the adsorbed amount (evaluated by XPS), but large differences in adsorbed layer morphology as observed by AFM. A fine granular morphology, with dimensions up to 8 nm height and 50-150 nm width, was observed on top of a thin adsorbed layer in the case of PS, PA98, and of a surface made of nanoscale inclusions of the latter in PS. In contrast, the layer adsorbed on PA80, which carries more ionizable groups, showed a higher roughness on the PA80 zones with differences in height up to 30 nm and characteristic lateral dimensions of 400 nm. On substrates of the former category, the cells formed large clusters, revealing poor interactions with the substrate. On PA80, the cells formed large networks with only a few small clusters. The adsorbed layer roughness, resulting from aggregation of fibronectin upon adsorption and from the substrate surface chemical composition, is responsible for neuronal cell spreading and growth. Its effect is not prevented by the presence of inclusions (< 30% of the surface) responsible for smoother areas of adsorbed fibronectin and for protrusions below 40 nm.     

Decrease in cAMP levels modulates adhesion to fibronectin and immunostimulatory ability of human dendritic cells

The aim of the present study was to analyze the early events elicited by tumor necrosis factor alpha (TNF-alpha) on monocyte-derived dendritic cells (moDC) adhesion to fibronectin (FN) and the involvement of cAMP in the signal transduction mechanism. The intracellular concentration of cAMP and moDC adhesion to FN decreased after TNF-alpha treatment. An inverted dose-dependency for TNF-alpha effect was observed for adhesion and cAMP levels. The presence of a phosphodiesterase (PDE) inhibitor (IBMX) and cAMP analogs (8Br-cAMP, Db-cAMP) reversed the observed TNF-alpha effects. The role of cAMP was analyzed further by examining the cAMP levels in nonadhered and adhered, TNF-alpha-treated moDC. Nonadhered moDC showed lower cAMP levels compared with adhered moDC. Furthermore, nonadhered moDC showed higher IL-12 content and allostimulatory ability compared with adhered moDC. The higher allostimulatory capacity was abolished in the presence of cAMP analogs and a PDE inhibitor. These results suggest that cAMP levels correlate with TNF-alpha-induced changes of moDC adhesion and allostimulatory capacity.     

Microtiter plate immunoassay for the evaluation of platelet adhesion to fibronectin

Investigations of platelet adhesion to adhesive proteins have been pursued to understand the basic mechanisms of hemostasis and thrombosis. Most assays used to determine platelet adhesion under stasis conditions rely on radiolabeled platelets. We describe a new microtiter immunoassay to study platelet adhesion to adhesive proteins under statis conditions. Direct comparison of platelet adhesion to fibronectin using a standard platelet adhesion assay based on ⁵¹Cr-labeled platelets and the new immunoassay showed that the optical density values obtained with the immunoassay are directly proportional to the number of platelets bound. The choice of platelet suspension buffer appears crucial for the design of such experiments, because the adhesion of resting platelets to fibronectin was significantly higher when platelets were suspended in Tyrode's buffer rather than Tris buffer. Platelet adhesion to fibronectin is increased in response to thrombin stimulation. This increase can be inhibited by synthetic RGD peptides. The thrombin-induced increase of platelet adhesion to fibronectin could be detected with antibodies against actin and glycoprotein IIb-IIIa, but not against the α-granule constituent platelet factor 4 (PF4). This assay is very versatile, because it avoids the use of radioactivity, and allows the parallel processing of a large number of samples. In addition, the parallel use of antibodies against different platelet antigens allows the screening for platelet activation events associated with the measured platelet adhesion.     

硫酸软骨素对HL60细胞增殖的影响及化疗增敏作用

目的观察硫酸软骨素(ChS)对HL60细胞增殖的影响,探讨其对肿瘤生长的作用及其在恶性肿瘤化疗中的应用价值。方法应用细胞计数法进行细胞计数;用MTT法测定细胞存活率;用酶联免疫检测仪测定各组细胞的酸性磷酸酶活性。结果硫酸软骨素<50mg/L时显著促进HL60细胞增殖(P<0·01),但>50mg/L时无影响;加入阿霉素(ADR)后,ChS组的细胞生存率均有不同程度的降低,ChS浓度>25mg/L时明显降低(P<0·01);加入ChS后各组细胞的酸性磷酸酶活性明显增强,75mg/L时最为显著。结论硫酸软骨素在适当浓度时对HL60细胞的增殖有促进作用;ChS可增强ADR对HL60细胞的杀伤作用。     

Influence of polypeptide molecular and side chain lengths and of side chain steric location on the kinetics of basic polypeptide-induced sensitization of primate cells to transfection.

Kinetics of sensitization of chimpanzee cell sheets to transfection by poliovirus RNA was determined for 5 basic polypeptides. With basic olypeptide hydrobromide at 100 microng/ml, initial sensitization rate was faster for poly-L-ornithine of average molecular weight (AMW) 15500 than of AMW 105000, and much faster for poly-L-lysine of AMW 1700 than of AMW 140000. Desensitization phases were observed with the 2 shorter polypeptides. Sensitization was much faster and sensitivity maxima were considerably higher for the polyornithines than for the polylysines. Poly-D-lysine of AMW 160000 sensitized cells slightly faster than poly-L-lysine of AMW 140000, but gave about the same sensitivity maximum. Analysis of the slow cell sensitization by poly-L-lysine of AMW 140000 revealed 2 steps: a fast step 1 (attachment) and a slow step 2 (processing).     

Presence of sialic acids in bronchioloalveolar cells and identification and quantification of N-acetylneuraminic and N-glycolylneuraminic acids in the lung

The lung, in air-breathing vertebrates, is a tree-like structure composed of branching tubes ending in alveoli and lined by diverse and highly specialized epithelial cells.A dense array of complex and diverse glycoconjugates is present on essentially all animal cell surfaces. Sialic acids are widely allocated at the outermost ends of glycan chains, attached to membrane proteins and lipids below. Due to their abundance and their terminal position in glycans, sialic acids are implicated in many physiological and pathological functions. Although the composition of lung epithelial cell-surface glycans has been studied over the years, it is not yet completely understood. In the present work, we aimed to histochemically localize N-acetylneuraminic acid (Neu5Ac)>N-glycolylneuraminic acid (Neu5Gc) residues on rat bronchioloalveolar epithelial cell surfaces using light microscopy (LM) methods. In lung membranes isolated from adult rat lung homogenates, we also separated, identified and quantified Neu5Ac and Neu5Gc by means of high-performance liquid chromatography (HPLC), and systematically described the optimized HPLC methods used. Sialic acid residues were localized on the surface coat of bronchioloalveolar cells, and the mean quantification of Neu5Ac and Neu5Gc in the adult rat lung homogenates was 12,26 and 2,73 μg/mg prot., respectively, revealing a manifest preponderance of Neu5Ac. A coefficient of variation (CV) of 4,98% and 4,40%, respectively was obtained and an optimal dispersion variability expressed by the SD and the CV was also reported, confirming the efficiency of the methodology.To the best of our knowledge, our group was the first to identify, separate and quantify sialic acids in purified lung membranes.The presence of these residues contributes to a strong anionic shield and may provide an hydrating and protective barrier as well as a repulsive structure that is crucial to lung physiology.     

The effect of histamine on the oxidative burst of HL60 cells before and after exposure to reactive oxygen species

During an inflammation neutrophils are stimulated to produce reactive oxygen species (ROS). These ROS induce the release of histamine from mast cells, which are also present at the inflammation site. In this study dibutyryl cAMP differentiated HL60 cells are used as a model for human neutrophils. The effect of histamine on formyl-methionyl-leucyl-phenylalanine (fmlp) stimulated cells is examined. Except for histamine also an accumulation of ROS takes place at the inflammation site and we investigated if ROS can influence the response of the stimulated HL60 cells. It is found that 10^–3 M histamine can inhibit the fmlp induced superoxide anion radical production. This occurs partly via an H₂ receptor because H₂ antagonists like famotidine, mifentidine and ranitidine could partially antagonize this effect of histamine. When HL60 cells are exposed to hydrogen peroxide or hypochlorous acid (20 min), an increased fmlp response is found while the inhibiting effect of histamine remains unchanged.     

Comparative analysis of gene expression between articular cartilage-derived cells to assess suitability of fibronectin adhesion assay to enrich chondroprogenitors

BackgroundEnhanced chondrogenesis and reduction in hypertrophy are essential pre-requisites for cell-based therapy in regenerative research for cartilage loss. Chondroprogenitors, isolated by fibronectin adhesion assay (FAA), have shown promising results in various preclinical studies due to their inherent characteristics. However, the need for monolayer culture and the effect of expansion on cell phenotype render differentiation between chondroprogenitors and chondrocytes (native cartilage cells) difficult. This is further complicated due to reported de-differentiation of chondrocytes in culture. Thus, the aim of our study was to harvest cells from articular cartilage and compare their gene expression to cells demonstrating adherence and non-adherence to fibronectin.MethodFresh-cells (FC) were isolated from human osteoarthritic knee joints(n = 3) and subjected to FAA. Cells unbound to fibronectin (20 min after plating) were termed as FAA − ve. Attached cells were further cultured for five population doublings and designated FAA + ve. RNA from all three cell groups was assessed for SOX-9, ACAN, COL2A1, COL1A1, RUNX2 and COL10A1.ResultsAll three groups exhibited moderate to high expression of markers of chondrogenesis and marker of chondrocyte hypertrophy. FAA + ve group exhibited significantly lower levels of hypertrophy markers: RUNX2 (vs FC and FAA − ve, P = 0.018) and COL10A1(vs FAA − ve, P = 0.005).ConclusionsOur results demonstrated that fibronectin effectively isolated cells distinct from mature chondrocytes in terms of reduced hypertrophic tendency. This is noteworthy as cells isolated by FAA, retaining their inherent progenitor phenotype, with upregulation of chondrogenic markers may be used successfully for cartilage repair in future translational work.     

Dexamethasone affects cytokine-mediated adhesion of HL-60 human promyelocytic leukemia cells to cultured dermal microvascular endothelial cells

Leukocyte endothelial adhesion (LEA) is the prelude to a complex cascade of reactions following an immunological challenge. Recently, LEA has been implicated in the molecular basis of several dermatological disorders. While the role of proinflammatory cytokines, such as interleukin-1 beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), in LEA has been investigated using nondermal models, limited data exist regarding their effects on LEA in dermal models. This study shows that cotreatment of cultured human dermal endothelial cells (CADMEC) with IL-1beta and TNF-alpha resulted in a marked increase in the adherence of human promyelocytic leukemia (HL-60) cells to CADMEC and an increase in expression of intercellular adhesion molecule-1 and E-selectin. Pretreatment of CADMEC with dexamethasone, a long-lasting glucocorticoid, resulted in a decrease in both HL-60 cell adhesion to CADMEC and adhesion molecule expression. Taken together, these data demonstrate that LEA may play a role in inflammatory skin conditions and in the mechanisms underlying the potential use of glucocorticoids as a treatment option.     

The effect of chitosan on the migration of neutrophil-like HL60 cells, mediated by IL-8

Research interest in chitosan stems in part from the demonstrated wound healing properties. The benefits of chitosan as a therapeutic agent appear to be paradoxical because chitosan also elicits neutrophil infiltration indicative of an inflammatory response. While the affinity between chitosan and neutrophils has been well documented, the underlying mechanism is unclear. To our knowledge, no studies have investigated the consequences of chitosan-neutrophil interaction to explain neutrophil migration. To that end, transwell migration assays to chitosan of varying extent of acetylation were conducted using a differentiated model cell line (HL60-PMN) in order to assess the effect of chitosan chemistry and the resultant physical properties such as charge and hydrophobicity on neutrophil migration. As chitosan N-acetylation increased, neutrophil migration increased and chitosan became less positively charged and more hydrophobic. Moreover, HL60-PMN cells secreted the potent neutrophil chemokine IL-8, also known as CXCL8, when exposed to chitosan and IL-8 levels increased with N-acetylation, and migration was inhibited by anti-IL-8 antibodies. Collectively these results suggest that chitosan-neutrophil interaction is encouraged by material properties, results in IL-8 secretion, and causes migration of neutrophils to chitosan. The implication is that the wound healing properties of chitosan may be enhanced through the attenuation of overabundant neutrophils, and thus the inflammatory response, simply by changing chitosan N-acetylation.     

双歧杆菌脂磷壁酸对HL-60细胞端粒酶活性的影响

目的 探讨双歧杆菌表面分子脂磷壁酸（ＬＴＡ）对体外培养的ＨＬ－６０白血病细胞端粒酶活性的影响。方法 采用ＰＣＲ－ＥＬＩＤＡ法检测经ＬＴＡ处理前后的ＨＬ－６０白血病细胞株端粒酶活性的改变。结果 经ＬＴＡ处理后,ＨＬ－６０白血病细胞的生长受到抑制,端粒酶活性明显降低。结论 双歧杆菌ＬＴＡ对ＨＬ－６０白血病细胞具有生长抑制作用,其抗肿瘤作用的机理可能与抑制肿瘤细胞的端粒酶活性有关。