Electrochemical immunoassay on expression of integrin β1 on tumor cells and drug-resistant tumor cells

An electrochemical indirect competitive immunoassay protocol as a promising cytosensing strategy was developed to detect integrin β1 expression on human breast cancer MCF-7 cells and adriamycin-resistant human breast cancer MCF-7 (MCF-7/ADR) cells and quantify the cell number. Integrin α5β1 was adsorbed on the gold-nanoparticle modified glassy carbon electrode to bind integrin β1 monoclonal antibody (anti-CD29 mAb). A sandwich structure was then formed using nanocomposites which consisted of horseradish peroxidase (HRP) labeled anti-antibody and gold nanoparticles. HRP bound on the electrode surface could cause an amperometric response of the hydroquinone-H(2)O(2) system. The assembly of the sandwich structure was inhibited by tumor cells to give decreased enzyme-catalytic signals due to the capture of anti-CD29 mAb by integrin β1 on cell membranes. Under optimal conditions the relative current change (S) was proportional to the cell concentration from 1.6×10(3) to 2.0×10(6)cellsmL(-1) with a detection limit of 700cellsmL(-1). Integrin β1 expression in MCF-7/ADR cells was found to be significantly higher than that in MCF-7 cells, indicating the increased adhesion ability of MCF-7/ADR cells.     

Effects of daintain/AIF-1 on β cell dysfunction in INS-1 cells

We investigated the effects of daintain/AIF-1, a novel inflammatory cytokine, on INS-1β cells. Cells incubated with daintain/AIF-1 showed decreased cell viability and glucose-stimulated insulin secretion, as well as upregulated apoptosis and NO production. These deleterious effects of daintain/AIF-1 indicate that daintain/AIF-1 plays important roles in the dysfunction of pancreatic β cells in type-1 diabetes.     

Effects of integrin α6β1 on migration of hepatocellular carcinoma cells

In this study, we applied specific blocking antibodies for integrin α6 or β1 subunit, and evaluated the in vitro effects of integrins α6β1 on the adhesion, chemotaxis and migration of hepatocellular carcinoma (HCC) cell line SMMC-7721 to type IV collagen. The adhesion force and cell migration, as measured by a micropipette aspiration system and Boyden chamber assay respectively, was dramatically reduced when either integrin subunits was blocked. The chemotaxis, as determined using a dual-micropipette system, was only affected by the antibody against β1 subunit. This study suggests that integrin α6β1 is an important cell surface receptor that mediates the adhesion of SMMC-7721 to type IV collagen. But the α6 subunit has minimal effect on pseudopod formation in response to type IV collagen. Therefore, the integrin α6β1-mediated cell migration is, at least in part, through the regulation on the cell adhesion step.     

Production of interferon-β by NB1-RGB cells cultured on peptide-lipid membranes

Cell growth and production of interferon-β (IFN-β) were investigated for normal human skin fibroblast cells (NB1-RGB) cultured on membranes prepared from peptide-lipids containing the arginine-glycine-aspartic acid [Arg-Gly-Asp] (RGD), tyrosine-isoleucine-glycine-serine-arginine [Tyr-Ile-Gly-Ser-Arg] (YIGSR) and arginine-glutamic acid-aspartic acid-valine [Arg-Glu-Asp-Val] (REDV) peptides. Cell density was found to be approximately the same on various peptide-lipid membranes, whereas production of IFN-β depended significantly on the peptide-lipid membranes on which NB1-RGB cells were cultured. The highest production of IFN-β was observed for NB1-RGB cells on REDV-lipid membranes prepared by a casting method (REDV-cast membranes) after 24 hr of cultivation. Specific binding between REDV of REDV-cast membranes and the receptor on the NB1-RGB cells may have caused the specific cell response for the production of IFN-β. This revised version was published online in July 2006 with corrections to the Cover Date.     

Trehalose uptake and dehydration effects on the cryoprotection of CHO–K1 cells expressing TRET1

Chinese hamster ovary cells (CHO–K1 cells) in which the trehalose transporter (TRET1) is expressed can have greater cryoprotection than ordinary CHO–K1 cells. This study examines the uptake characteristics of trehalose into cells via TRET1 and determines the influence of intracellular trehalose on the freeze–thaw viabilities. In our experiments, the intracellular trehalose concentration is controlled by the extracellular trehalose concentration and the immersion time in a freezing solution. In this freezing solution, both kinds of CHO–K1 cells are independently dispersed with various amount of trehalose, and then put into the CO₂ incubator for 0–6 h. After a set immersion time, the cell-suspended sample is cooled to 193 K, stored for 1 week, then quickly thawed at 310 K and its viability measured. The uptake amount of intracellular trehalose is measured before freezing. We find an upper limit for the uptake amount of trehalose when the extracellular trehalose concentration is about 400 mM, at which the freeze–thaw viability is the highest. When the extracellular trehalose concentration exceeds 400 mM, shorter immersion times are needed to obtain the maximum freeze–thaw viability. Also, longer immersion weakens the cells. Our analyses indicate that when the extracellular trehalose-concentration is less than 400 mM, the trehalose uptake occurs more slowly with less dehydration, resulting in less stress on the cell. When the extracellular trehalose concentration exceeds the saturation level, the cell is stressed by the excess dehydration due to the remaining osmotic pressure, with apoptosis occurring before freezing.     

A new 'spin' on neural stem cells?

The existence of neural stem cells in the adult brain was essentially denied until the last decade. Within the past ten years, considerable progress has been made in examining the fundamental properties of neural stem cells. Most recently there has been much interest in the identification and precise location of the adult neural stem cells in vivo. Studies examining the localization of neural stem cells are controversial and suggest two distinct locations within the adult brain: the ependymal layer lining the ventricles, and the subependymal layer immediately adjacent to the ependyma.     

Effects of rhDecorin on TGF-β1 induced human hepatic stellate cells LX-2 activation

Decorin is a small leucine-rich extracellular matrix proteoglycan composed of a core protein with a single glycosaminoglycan (GAG) chain near the N-terminus and N-glycosylated at three potential sites. Decorin is involved in the regulation of formation and organization of collagen fibrils, modulation of the activity of growth factors such as transforming growth factor β (TGF-β), and exerts other effects on cell proliferation and behavior. Increasing evidences show that decorin plays an important role in fibrogenesis by regulating TGF-β, a key stimulator of fibrosis, and by directly modulating the degradation of extracellular matrix (ECM) from activated hepatic stellate cells (HSCs). In this study, the core protein of human decorin was cloned and expressed in Escherichia coli. The purified recombinant human decorin (rhDecorin) significantly inhibited the proliferation of LX-2 cells, a human HSC cell line, stimulated by TGF-β1. RT-PCR result showed that the expression of metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were reduced by rhDecorin in LX-2 cells stimulated by TGF-β1. Furthermore, the protein expression of smooth muscle-α-actin (α-SMA), collagen type III and phosphorylated Smad2 (p-Smad2) was significantly decreased in the presence of rhDecorin. rhDecorin also reduced fibrillogenesis of collagen type I in a dose-dependent manner. Gene expression profiles of LX-2 cells stimulated by TGF-β1 in the presence and the absence of rhDecorin were obtained by using cDNA microarray technique and differentially expressed genes were identified to provide further insight into the molecular action mechanism of decorin on LX-2 cells.     

Increase of β2-integrin on adhesion of THP-1 cells to collagen vitrigel membrane

When human monocyte-derived leukemia (THP-1) cells, which are floating cells, are stimulated with lipid peroxides, or Streptococcus suis, these cells adhere to a plastic plate or endothelial cells. However, it is unclear whether or not non-stimulated THP-1 cells adhere to collagen vitrigel membrane (CVM). In this study, firstly, we investigated the rate of adhesion of THP-1 cells to CVM. When THP-1 cells were not stimulated, the rate of adhesion to CVM was high. Then, to identify adhesion molecules involved in adhesion of THP-1 cells to CVM, expressions of various cell adhesion molecules on the surface of THP-1 cells adhering to CVM were measured. β-actin, β-catenin, and β1-integrin expressions did not change in non-stimulated THP-1 cells cultured on CVM compared with those in cells cultured in a flask, but β2-integrin expression markedly increased.     

Real-time detection of β1 integrin expression on MG-63 cells using electrochemical impedance spectroscopy

Beta 1 integrin is a membrane protein responsible for attachment and migration of osteosarcoma cells. In this study, expression of β1 integrin on MG-63 cells, a human osteogenic sarcoma cell line, was monitored using electrochemical impedance spectroscopy (EIS). ITO-based biochips were developed using a semiconductor technique. Differences in electric resistance (ΔR) were measured continuously when cells binding with anti-β1 integrin antibody coagulated with nano-scale gold particles. The results of the EIS system were compared with traditional immunofluorescence staining. We found that sample chambers with higher cell densities had larger ΔR values. When the cell densities increased from 5 × 10(4) cells/ml to 5 × 10(5) cells/ml, the ΔR value dose-dependently increased from 14 Ω to 37 Ω. In addition, a highly linear relationship (correlation coefficient, 0.921) was found between the ΔR values and the corresponding fluorescence intensities (p<0.05). These results suggest that electrochemical impedance spectroscopy can be a useful tool for evaluating β1 integrin expression on cell membranes.     

Transforming growth factor-β1 modulates the effect of 1α,25-dihydroxyvitamin D3 on leukemic cells

Summary The human leukemic cells HL-60, U937, KG-1 and THP-1 incubated with transforming growth factor-β1 (TGF-β1) were studied by examining cell surface antigens and macrophage-specific activities. The addition of 0.5 ng/ml (20 pM) of TGF-β1 with 1α,25-dihydroxyvitamin D₃ [1α, 25(OH)₂D₃] induced more Leu-M3 (CD14)-positive cells (approximately 80%) than 5×10⁻⁸ M 1α,25(OH)₂D₃ alone did (30 to 50%), although original HL-60 cells did not express any Leu-M3 antigen at all. Tumor necrosis factor-α (TNF-α) with TGF-β1 and 1α,25(OH)₂D₃ was found to potentiate the expression of these surface antigens. Furthermore, the phagocytic activity was also induced strongly. The expression of CR3 (CD11b) antigen was also increased, and all Leu-M3-positive cells were found CR3-positive when HL-60, U937, and THP-1 cells were treated with these stimulants. In contrast, CR3 but not Leu-M3 was induced in KG-1 cells after the same treatment. This may indicate that the responsiveness of leukemic cells to TGF-β1 and 1α,25(OH)₂D₃ might vary depending on a differentiation stage of the target cells. Furthermore, K562 cells originated from a more undifferentiated precursor, were not able to respond to these two inducers. These results suggested that some of TGF-β superfamily proteins might represent potent modulators in hematopoiesis, especially in the development of monocytes-macrophages or their precursors.     

Influence of α-interferon therapy on blood lymphoid cells

Summary The influence of natural -interferon (-IFN) therapy (3×10⁶ units i.m. daily) on blood lymphoid cells was studied in 20 patients with gynecological neoplasias (7 patients with condylomata accuminata and 13 patients with ovarian carcinoma). There was a statistically significant increase in the intracellular levels of 2'–5'oligoadenylate synthese 1 day after the first injection of IFN and with few exceptions this activity remained increased during 3 months of treatment. In most of the patients, the capacity of blood lymphoid cells to produce IgA, IgG, and IgM following stimulation with pokeweed mitogen was decreased 1 day after the first injection of IFN and with few exceptions it remained low during 6 months of IFN therapy. In most patients there was a decrease in the capacity of lymphoid cells to act as stimulator or responder cells in a mixed lymphocyte culture during IFN therapy. The -IFN therapy had no major influence on the response of lymphoid cells to mitogens. We conclude, that neither this nor our previous studies on the influence of IFN therapy on immunological functions have given support to the hypothesis that the antitumor action of IFN is mediated by the immune system.