Effects of serum and serum-derived factors on the growth and production of α-fetoprotein and albumin by human hepatoma cell lines

A serum-free culture system of human hepatoma cell lines (HuH-6 and HuH-7) was used to investigate the activity of bovine serum (BS) and of serum-derived factors on the growth and production of -fetoprotein (AFP) and albumin. At higher concentrations, dialyzed BS was inhibitory to the growth of HuH-6 and caused reduction of the level of AFP production by the cells. AFP and albumin levels in HuH-6 and HuH-7 were reduced or unchanged by fetuin, bovine serum albumin (BSA) and transferin (TF), although no cytotoxicity was shown by any of them. Commercial preparations of platelet-derived growth factor exhibited cytotoxicity to HuH-6 and HuH-7 and induced a decrease of AFP and albumin levels in a dose-dependent manner. Transforming growth factor (TGF-) exhibited no cytotoxicity to HuH-6. AFP levels in HuH-6 were unchanged with 1000 pg/ml TGF-, but albumin levels were decreased. TGF-7 at a concentration of 1000 pg/ml was cytotoxic to HuH-7 and AFP levels were a little increased. Albumin levels, however, were unchanged. Following exposure to cycloheximide, AFP and albumin levels in HuH-6 were inhibited.Abbreviations AFP -fetoprotein - BS bovine serum - BSA bovine serum albumin - EDTA ethylenediaminetetraaceticacid - ELISA enzyme-linked immunosorbent assay - HBSS Hank's balanced salt solution - PBS phosphate buffered saline - PDGF platelet-derived growth factor - TF transferrin - TGF-\ transforming growth factor beta     

Transforming growth factor beta 1 differentially regulates alpha-fetoprotein and albumin in HuH-7 human hepatoma cells.

Transforming growth factor beta 1 (TGF-beta 1) is known to inhibit hepatocyte growth in vitro and in vivo. In this study, we analyzed the effect of TGF-beta 1 on alpha-fetoprotein (AFP) and albumin gene expression in HuH-7 human hepatoma cells. TGF-beta 1 inhibited cell growth in a dose dependent manner. The cellular secretion rate of AFP but not albumin was suppressed significantly by TGF-beta 1. TGF-beta 1 caused a significant reduction in the level of AFP mRNA. In contrast, the levels of albumin mRNA or beta-actin mRNA were not changed by TGF-beta 1. In transient transfection experiments, TGF-beta 1 resulted in selective repression of AFP promoter activity. These results suggest that TGF-beta 1 is one of the key factors involved in the differential regulation of the AFP gene and the albumin gene.     

Elevated serum α-fetoprotein level of nude mice bearing hepatoma cells by treatment with monoclonal antibodies to α-fetoprotein

This study was carried out to clarify the reason for elevation of serum α-fetoprotein (AFP) level of nude mice bearing hepatoma cells after treatment with monoclonal antibodies (MoAbs) to AFP. MoAbs to AFP showed no effect on the cumulative amounts of AFP secreted from human hepatoma cell line, HuH-7, in vitro. However, the treatment of nude mice bearing HuH-7N cells (HuH-7 xenograft) with MoAbs to AFP led to elevation of the serum AFP level in spite of the fact that the growth curve of HuH-7N cells was similar to that for PBS treatment. This apparent elevation of the serum AFP level is thought to be due to the slow elimination of AFP-MoAb immune complexes with little lattice structure from circulation, but not the enhancement of AFP secretion of HuH-7N cells. Thus, when using a MoAb alone or MoAb-drug conjugate, the serum AFP level should only be cautiously used as a tumor marker for evaluating the targeting immunotherapy.     

Molecular mapping of functional domains of the leukocyte receptor for endothelium, LAM-1

The human lymphocyte homing receptor LAM-1, like its murine counterpart MEL-14, functions as a mammalian lectin, and mediates the binding of leukocytes to specialized high endothelial cells in lymphoid organs (HEV). LAM-1 is a member of a new family of cell adhesion molecules, termed selectins or LEC-CAMs, which also includes ELAM-1 and PAD-GEM (GMP-140/CD62). To localize the regions of LAM-1 that are involved in cell adhesion, we developed chimeric selectins, in which various domains of PAD-GEM were substituted into LAM-1, and used these chimeric proteins to define the domain requirements for carbohydrate binding, and to localize the regions recognized by several mAb which inhibit the adhesion of lymphocytes to lymph node HEV. The binding of PPME or fucoidin, soluble complex carbohydrates that specifically define the lectin activity of LAM-1 and MEL-14, required only the lectin domain of LAM-1. The LAM1-1, LAM1-3, and LAM1-6 mAb each strongly inhibit the binding of lymphocytes to HEV in the in vitro frozen section assay, and defined three independent epitopes on LAM-1. Blocking of PPME or fucoidin binding by LAM1-3 indicated that this site is identical, or in close proximity, to the carbohydrate binding site, and analysis of the binding of LAM1-3 to chimeric selectins showed that the epitope detected by LAM1-3 is located within the lectin domain. Although the LAM1-6 epitope is also located in the lectin domain, LAM1-6 did not affect the binding of PPME or fucoidin. The LAM1-1 epitope was located in, or required, the EGF domain, and, importantly, binding of LAM1-1 significantly enhanced the binding of both PPME and fucoidin. These results suggest that adhesion mediated by LAM-1 may involve cooperativity between functionally and spatially distinct sites, and support previous data suggesting a role for the EGF domain of LAM-1 in lymphocyte adhesion to HEV.     

Endothelin-1 combined with extracellular matrix proteins promotes the adhesion and chemotaxis of amelanotic melanocytes from human hair follicles in vitro

During the repigmentation of vitiliginous skin, amelanotic melanocytes (AMMCs) migrate from the outer root sheath (ORS) of the hair follicles into depigmented skin. It has been shown that endothelin-1 (ET-1) is an important cytokine in the migration of epidermal melanocytes, produced by keratinocytes particularly after irradiation with ultraviolet B (UVB). To further examine the role of ET-1 on the migration of AMMCs, we investigated the effects of ET-1 to the adhesion and chemotaxis of human AMMCs combined with extracellular matrix proteins (ECMP) and observed the effects on the actin and tubulin cytoskeleton of AMMC by ET-1. Human AMMCs were treated with different concentrations of ET-1 (0.1-100 nM) to observe adhesion on culture dishes coated with fibronectin (FN), laminin (LN) and collagen IV (CIV). In addition, chemotaxis on FN, LN and CIV coated micropore filters, with various concentrations of ET-1 as attractants, was investigated using a Boyden chemotaxis chamber. Cellular microfilaments and microtubules were immunostained with Rhodamine labeled actin and FITC labeled beta-tubulin. The effects of ET-1 on cytoskeleton were observed with laser confocal microscopy. The study demonstrated that ET-1 increases human AMMCs adhesion on FN in a dose-dependent manner, but minor increases are found on the coated surface with LN and CIV. ET-1 also induces chemotaxis of AMMCs on CIV, LN and FN in a dose-dependent manner. The greatest effect was seen with CIV. Minor chemotactic effects were observed with non-coated surfaces. A concentration of >or=10nM ET-1 induced an apparent increase in stress fibers underneath the cell membrane, but no effects were found on tubulin. ET-1 has various effects on the adhesion and chemotaxis of AMMCs on various ECMP, which could be partly due to a modulation and reorganization of the actin cytoskeleton.     

The fibronectin cell attachment sequence Arg-Gly-Asp-Ser promotes focal contact formation during early fibroblast attachment and spreading

Cultured fibroblasts form focal contacts (FCs) associated with actin microfilament bundles (MFBs) during attachment and spreading on serum- or fibronectin (FN)-coated substrates. To determine if the minimum cellular adhesion receptor recognition signal Arg-Gly-Asp-Ser (RGDS) is sufficient to promote FC and MFB formation, rat (NRK), hamster (Nil 8), and mouse (Balb/c 3T3) fibroblasts in serum-free media were plated on substrates derivatized with small synthetic peptides containing RGDS. These cultures were studied with interference reflection microscopy to detect FCs, Normarski optics to identify MFBs, and immunofluorescence microscopy to observe endogenous FN fiber formation. By 1 h, 72-78% of the NRK and Nil 8 cells plated on RGDS-containing peptide had focal contacts without accompanying FN fibers, while these fibroblasts lacked FCs on control peptide. This early FC formation was followed by the appearance of coincident MFBs and colinear FN fibers forming fibronexuses at 4 h. NRK and Nil 8 cultures on substrates coated with native FN or 75,000-D FN-cell binding fragment showed similar kinetics of FC and MFB formation. In contrast, the Balb/c 3T3 mouse fibroblasts plated on Gly-Arg-Gly-Asp-Ser peptide-derivatized substrates, or on coverslips coated with 75,000-D FN cell-binding fragment, were defective in FC formation. These results demonstrate that the apparent binding of substrate-linked RGDS sequences to cell surface adhesion receptors is sufficient to promote early focal contact formation followed by the appearance of fibronexuses in some, but not all, fibroblast lines.     

The Effect of Extracellular Matrix Molecules on the in Vitro Behavior of Bovine Endothelial Cells

Extracellular matrix (ECM) is an important mediator of endothelial functions such as adhesion, spreading, migration, proliferation, and maintenance of differentiated functions. Attachment of cultured cells to tissue culture polystyrene (TCPS) is dependent on vitronectin which adsorbs onto the surface from the serum in the culture medium. Vitronectin (VN) will adsorb efficiently to TCPS even if the latter has been coated with another matrix molecule and blocked with albumin. This means that studies of the interactions of cells with individual coated ECM molecules will be confounded by the presence of adsorbed VN if serum is present in the culture medium. In this study, the adhesion, spreading, growth, and output of endogenous matrix molecules by bovine corneal endothelial (BCE) cells were measured on five different matrix substrates using medium which had been depleted of vitronectin to avoid such confounding effects. The same cell adhesion and spreading maxima were achieved on vitronectin, fibronectin (FN), laminin (LM), and types I and IV collagen (col I, col IV). The coating concentrations required to achieve these maxima, however, differed among the substrates, LM needing considerably higher concentrations than the other substrates for both maximal adhesion and spreading and FN needing higher concentrations for cell spreading. When cells were continuously passaged on each of the five substrates coated at concentrations optimal for cell spreading, no differences in cell proliferation rates or cell morphology were observed. Significant differences, however, were observed in the subcellular output of endogenous matrix molecules (FN, LM, col IV, and thrombospondin) between the different substrates. Col I was a poor substrate for the production of all ECM molecules tested over the 10 passages of the experiment, whereas col IV was a consistently good substrate. LM and FN substrates displayed differential effects on the output of different ECM molecules. VN was unique in that BCE cells at early passage on this substrate produced high levels of endogenous matrix molecules, whereas with continued passage on this substrate, a progressive decline in ECM secretion was observed. These results show that incorporation of individual molecules into the ECM by BCE cells in culture is significantly affected by the nature of the substratum. They further suggest that passage of endothelial cells in media containing serum (which results in coating of VN onto the substrate) may result in a progressive reduction of ECM output.     

Characterization and distribution of extracellular matrix components and receptors in GH3B6 prolactin cells

GH3B6 cells, a rat pituitary tumor cell line, synthesize and secrete large amounts of prolactin (PRL) in vitro. In the present work, we evaluated the capacity of these cells to express extracellular matrix (ECM) components and receptors in vitro. The expression of laminin (LN), fibronectin (FN) and type IV collagen (CIV) was investigated by immunofluorescence assays. In comparison to PRL distribution, where around 50-70% of the cells contained PRL concentrated in the Golgi region, a variable immunolabeling for the three ECM components could be observed in the majority of GH3B6 cells. Importantly, this pattern was not modified when cells were cultured in the presence of 30 nM thyroliberin (TRH). The expression of the ECM receptors: alpha5beta1 (FN receptor), alpha6beta1 (LN receptor) and CD44 (hyaluronic acid receptor) could be demonstrated by cytofluorometric analysis. Using biochemical procedures, we analyzed the synthesis and secretion of glycosaminoglycans (GAGs). The cells synthesized and secreted mainly heparan sulfate (75%) with a minor amount of chondroitin sulfate/dermatan sulfate. In an attempt to evaluate the individual contribution of the ECM components to influence cell morphology and PRL distribution in vitro, GH3B6 cells were cultivated separately on LN, FN and CIV substrates. Under all conditions, it was possible to observe an increase of cell adherence to the substrate, accompanied with changes of cellular morphology, characterized by the appearance of cytoplasmatic processes. However, no changes on PRL distribution could be observed. Our results suggest that endocrine tumor cell lines are involved in synthesis of ECM components and receptors.     

Extracellular matrix components influence the survival of adult cardiac myocytes in vitro

Calcium-tolerant myocytes were isolated from adult rat hearts by collagenase perfusion and plated on various substrates in serum-free medium and their adhesion to various extracellular matrix (ECM) components was determined. The myocytes attached readily to dishes coated with collagen type IV (C-IV), laminin (LN), and to fetal bovine serum (FBS) in a manner dependent on the concentration of the components. Substantially fewer myocytes adhered to dishes coated with fibronectin (FN) or to uncoated plastic dishes. Cells adhered equally well to dishes coated with C-IV, LN and FBS within 1-4 h. However, when examined after 2 weeks in culture it was found that only C-IV and LN could support survival of the attached myocytes, and when cultured on C-IV or LN the myocytes were spread and had formed a dense monolayer. The actin filaments had at this time reorganized linearly along the long axis of the cell and the myocytes contracted spontaneously. Rabbit antibodies were raised against myocyte membranes and their ability to inhibit attachment to ECM components was studied. Purified IgG inhibited attachment to C-IV, while having only a minor effect on attachment to LN. These data are compatible with the presence of a specific cell surface component(s) that interacts with ECM substrates and influences cell shape and possibly thereby influences cellular functions.     

Thrombocyte adhesion and aggregation on surfaces coated with human type-I, -III, -IV and -V collagens

Human collagens of type I, III, IV, and V (CI, CIII, CIV, and CV) can be localized in different anatomic structures of the vessel wall. To investigate the role of vascular collagenous components in mural thrombus formation, the authors studied platelet adhesion to the wells of Falcon culture plates coated with: a) monomeric CI, CIII, CIV, and CV; b) fibrillar CI and CIII, and c) amorphous CIV and CV. On monomeric and amorphous CV, only initial attachment takes place, i.e. platelets bind to the surface without subsequent spreading. Platelet adhesion on monomeric and amorphous CIV proceeds more actively: the total level of adhesion is substantially higher than on CV, with up to 75% of adherent platelets spread out and single unspread platelets from suspension attached to the upper surface of spread platelets. On monomeric and fibrillar CI/CIII, formation of large multi-layer (thrombi-like) aggregates, with spread platelets at the basis, takes place along with processes characteristic for adhesion on CIV/CV. On the contrary, only fibrillar but not monomeric CI and CIII induce platelet aggregation in suspension. The data suggest that the ability of CI and CIII to induce platelet aggregation is fully conditioned by the genetic type of collagen and requires a simultaneous multivalent platelet-collagen interaction, which can be achieved by surface immobilization of collagen or formation of fibrillar structures in suspension.     

Electric field-directed cell motility involves up-regulated expression and asymmetric redistribution of the epidermal growth factor receptors and is enhanced by fibronectin and laminin

Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. This also was serum dependent. Epidermal growth factor (EGF) restored cathodal-directed migration in serum-free medium. Therefore, the hypothesis that EGF is a serum constituent underlying both field-directed migration and enhanced migration on ECM molecules was tested. We used immunofluorescence, flow cytometry, and confocal microscopy and report that 1) EF exposure up-regulated the EGF receptor (EGFR); so also did growing cells on substrates of FN or LAM; and 2) EGFRs and actin accumulated in the cathodal-directed half of CECs, within 10 min in EF. The cathodal asymmetry of EGFR and actin staining was correlated, being most marked at the cell-substrate interface and showing similar patterns of asymmetry at various levels through a cell. At the cell-substrate interface, EGFRs and actin frequently colocalized as interdigitated, punctate spots resembling tank tracks. Cathodal accumulation of EGFR and actin did not occur in the absence of serum but were restored by adding ligand to serum-free medium. Inhibition of MAPK, one second messenger engaged by EGF, significantly reduced EF-directed cell migration. Transforming growth factor beta and fibroblast growth factor also restored cathodal-directed cell migration in serum-free medium. However, longer EF exposure was needed to show clear asymmetric distribution of the receptors for transforming growth factor beta and fibroblast growth factor. We propose that up-regulated expression and redistribution of EGFRs underlie cathodal-directed migration of CECs and directed migration induced by EF on FN and LAM.     

Concurrent changes in sinusoidal expression of laminin and affinity of hepatocytes to laminin during rat liver regeneration.

Distribution of fibronectin, laminin, and collagens type I, III, IV, and V in the lobular regions of regenerating rat liver was studied by indirect immunofluorescence. Little or no laminin was detected in sham-operated controls throughout the experimental period, while it was detected in sinusoids of regenerating liver as early as 6 h after partial hepatectomy (PH). After reaching a maximum at 24 h, it decreased and was barely detectable 6 days after PH. Changes in the other extracellular matrix (ECM) proteins were evident 3 days after PH, but not earlier than 24 h. Hepatocytes isolated from regenerating rat livers were tested in a short term assay for attachment to the substrates coated with the ECM proteins. The attachment of hepatocytes to laminin substrates increased 12 h after PH, reached a maximum at 24 h, and decreased to the control level 6 days after PH, while that of the control remained constant. The attachment to fibronectin substrates was not different between regenerating livers and controls at any time point. The attachment to collagen did not change earlier than 24 h after PH, but increased slightly 3 days after PH. Primary rat hepatocytes cultured on the substrates coated with the ECM proteins were determined for replicative DNA synthesis in response to epidermal growth factor. Both in normal liver and in regenerating liver 24 h after PH, laminin was one of the most effective substrates in supporting the responsiveness of hepatocytes to the growth stimulus. Taken together, these results suggest the importance of hepatocyte-laminin interaction during the early stage of liver regeneration possibly in growth stimulation of hepatocytes and/or maintenance of hepatocyte-specific functions.     

Effects of various factors on the growth and function of a human hepatoblastoma cell line, HuH-6--an approach for serum-free cell culture

The effects on a human hepatoblastoma cell line (HuH-6) of serum and Ca2+ were investigated. A higher concentration of serum reduced the production of alpha-fetoprotein, whereas Ca2+ enhanced that of both albumin and alpha-fetoprotein. In view of these facts, a serum-free medium using RPMI-1640 supplemented with lactalbumin hydrolysate, epidermal growth factor, hydrocortisone, insulin, chrelatoxine, glucagone and selenium was then developed. Collagen type IV was superior to collagen type I, laminin, fibronectin and plastic in supporting the growth of HuH-6 in the serum-free medium. Higher amounts of both albumin and alpha-fetoprotein were secreted in HuH-6 cultured in serum-free medium than in serum-supplemented medium.     

Vitronectin secretion by hepatic and non-hepatic human cancer cells

Summary Thirty-eight human cancer cell lines and subclones derived from 12 different organs were screened for vitronectin secretion in their culture media. By immunoblotting analysis we detected high secretion by three out of five hepatoma cell lines tested but no secretion by the others. In addition, significant secretion was observed in seven non-hepatic cancer cell lines and subclones derived from the cervix, lung, and pancreas. These vitronectin-secreting cells included PLC/PRF/5, HuH-6 #5, HuH-7, HeLa S3, HeLa · P3 #2, #3, #6, #8, A549, and MIAPaCa-2. The results were further confirmed by quantitative analysis using sandwich enzyme-linked immunoassay, and activity analysis of cell attachment promotion on Western blotted filters.     

Regulation of the interleukin-1 beta (IL-1 beta) gene by mycobacterial components and lipopolysaccharide is mediated by two nuclear factor-IL6 motifs.

The cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) are released by mononuclear phagocytes in vitro after stimulation with mycobacteria and are considered to mediate pathophysiologic events, including granuloma formation and systemic symptoms. We demonstrated that the Mycobacterium tuberculosis cell wall component lipoarabinomannan (LAM) is a very potent inducer of IL-1 beta gene expression in human monocytes and investigated the mechanism of this effect. We localized the LAM-, lipopolysaccharide (LPS)-, and TNF-alpha-inducible promoter activity to a -131/+15 (positions -131 to +15) DNA fragment of the IL-1 beta gene by deletion analysis and chloramphenicol acetyltransferase assay. Within this DNA fragment, there were two novel 9-bp motifs (-90/-82 and -40/-32) with high homology to the nuclear factor-IL6 (NF-IL6) binding site. Site-directed mutagenesis demonstrated that the two NF-IL-6 motifs could be independently activated by LAM, LPS, or TNF-alpha and that they acted in an orientation-independent manner. DNA mobility shift assay revealed specific binding of nuclear protein(s) from LAM-, LPS-, or TNF-alpha-stimulated THP-1 cells to the NF-IL6 motifs. We conclude that the two NF-IL6 sites mediate induction of IL-1 beta in response to the stimuli LAM, LPS, and TNF-alpha.     

Human hepatocyte growth factor stimulates the growth of HUH-6 clone 5 human hepatoblastoma cells.

The effects of human hepatocyte growth factor (hHGF), a potent mitogen for rat and human hepatocytes in primary culture, on proliferation of human hepatoma and hepatoblastoma cells were examined. Out of five cell lines; HLE, HuH-6 clone 5, HuH-7, PLC/PRF/5, and Hep G2, only HuH-6 Clone 5 cells were stimulated by recombinant hHGF. Both native and recombinant hHGFs caused dose-dependent increases in cell number and DNA synthesis of cells. This stimulation was strongly inhibited by anti-hHGF monoclonal antibody.