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At least 163 records · Page 9Linked to original sources
PubMed1992

Anti-fibronectin antibodies that modify heparin binding and cell adhesion: evidence for a new cell binding site in the heparin binding region.

A panel of monoclonal antibodies (mAbs) to bovine fibronectin (FN) is described which modulates either heparin binding or cell adhesion to FN, or both. A combination of competitive exclusion and binding to proteolytic fragments identified epitopes in the Hep II, Hep III/I and CBF (cell binding fragment) regions of FN. mAb A17, which bound to the CBF region, strongly inhibited the cell adhesion of BHK-21 fibroblasts, primary corneal fibroblasts and endothelial cells, and NM4 mammary adenocarcinoma cells, to FN at mAb concentrations as low as 1 microgram/ml. This mAb was not so effective at inhibiting the adhesion of B16 mouse melanoma cells. Adhesion of B16 cells to FN was more sensitive to inhibition by mAbs binding to Hep II (A2, A9, A32, A35). Of these, A32 and A35 significantly increased the binding of 35S-heparin to FN, whereas A2 and A9 did not affect it. mAbs A2, A9 and A32 showed good binding to HBF, the 40 kDa proteolytic fragment of human FN which contains both Hep II and IIICS (type III connecting segment). These mAbs inhibited B16 cell adhesion to the HBF (heparin binding fragment) by 30-50%, the greatest inhibition being shown by mAb A32. Two synthetic peptides from the HBF, CS1 (peptide 1) from the IIICS region and peptide I from the Hep II region, also inhibited B16 cell adhesion to HBF by approximately 70 and 30%, respectively. These results suggest that maximal cell adhesion to the HBF involves both CS1 and Hep II. The inhibitory effects of the two peptides were linearly additive in combination, whereas the inhibitory mAbs A2, A9 and A32 showed synergistic additive effects with each of the peptides. This points to the existence of an additional important cell binding site in Hep II, other than peptide I. Recent independent evidence for an additional cell binding site in Hep II supports this view. Melanoma cellular receptor(s) for the Hep II region may be cell surface proteoglycans but do not appear to bind to areas of Hep II with high affinity for soluble heparin, as the latter was not an inhibitor of B16 cell adhesion to the HBF. The increased effectiveness of A32 in inhibiting cell adhesion, compared to A2 and A9, may be due to conformational effects which increase the binding of soluble heparin, but reduce affinity for the cellular receptor. These results are discussed in context with other reports in the literature.

Amino Acid Sequence↗
PubMed2005

Reduced tumor necrosis factor signaling in primary human fibroblasts containing a tumor necrosis factor receptor superfamily 1A mutant.

OBJECTIVE: Tumor necrosis factor receptor-associated periodic syndrome (TRAPS) is an autoinflammatory syndrome associated with mutations in the gene that encodes tumor necrosis factor receptor superfamily 1A (TNFRSF1A). The purpose of this study was to describe a novel TNFRSF1A mutation (C43S) in a patient with TRAPS and to examine the effects of this TNFRSF1A mutation on tumor necrosis factor alpha (TNFalpha)-induced signaling in a patient-derived primary dermal fibroblast line. METHODS: TNFRSF1A shedding from neutrophils was measured by flow cytometry and enzyme-linked immunosorbent assay (ELISA). Primary dermal fibroblast lines were established from the patient with the C43S TRAPS mutation and from healthy volunteers. Activation of NF-kappaB and activator protein 1 (AP-1) was evaluated by electrophoretic mobility shift assays. Cytokine production was measured by ELISA. Cell viability was measured by alamar blue assay. Apoptosis was measured by caspase 3 assay in the fibroblasts and by annexin V assay in peripheral blood mononuclear cells. RESULTS: Activation-induced shedding of the TNFRSF1A from neutrophils was not altered by the C43S TRAPS mutation. TNFalpha-induced activation of NF-kappaB and AP-1 was decreased in the primary dermal fibroblasts with the C43S TNFRSF1A mutation. Nevertheless, the C43S TRAPS fibroblasts were capable of producing interleukin-6 (IL-6) and IL-8 in response to TNFalpha. However, TNFalpha-induced cell death and apoptosis were significantly decreased in the samples from the patient with the C43S TRAPS mutation. CONCLUSION: The C43S TNFRSF1A mutation results in decreased TNFalpha-induced nuclear signaling and apoptosis. Our data suggest a new hypothesis, in that the C43S TRAPS mutation may cause the inflammatory phenotype by increasing resistance to TNFalpha-induced apoptosis.

Apoptosis↗
PubMed1982

A comparison of the cytogenetic response to asbestos and glass fibre in Chinese hamster and human cell lines. Demonstration of growth inhibition in primary human fibroblasts.

Asbestos and fine glass fibre, which induce high levels of chromosome aberrations and polyploidy in Chinese hamster permanent cell lines, were found to cause no increase in chromosome damage or polyploidy in primary human fibroblasts or in human lymphoblastoid lines. In common with permanent cell lines of hamster or human origin, treatment of primary human fibroblasts with higher doses of asbestos or fine glass resulted in almost total growth inhibition, showing that the primary cells are not unaffected by these agents. The reason for lack of evident cytogenetic damage in primary cells may lie in the greater spontaneous karyotype instability of transformed (permanent) cell lines or may be connected with the less efficient DNA repair capacity of Chinese hamster ovary cells.

Animals↗
PubMed2005

Differential effect of BMP4 on NIH/3T3 and C2C12 cells: implications for endochondral bone formation.

UNLABELLED: After intramuscular implantation, BMP4-expressing NIH/3T3 fibroblasts and BMP4-expressing C2C12 myoblasts can promote ectopic cartilage and bone formation. Fibroblasts tend to undergo chondrogenesis, whereas myoblasts primarily undergo osteogenesis. These results suggest that endochondral bone formation may involve different cell types, a finding that could have major implications for the tissue engineering of bone and cartilage. INTRODUCTION: The delivery of BMP4 through cell-based gene therapy can trigger ectopic endochondral bone formation in skeletal muscle. We hypothesized that, when stimulated with or transduced to express BMP4, different types of cells residing within skeletal muscle might participate in different stages of endochondral bone formation. MATERIALS AND METHODS: We compared the responses of a fibroblast cell line (NIH/3T3), a myoblast cell line (C2C12), primary fibroblasts, and primary myoblasts to BMP4 stimulation in vitro. We then transduced the four cell populations to express BMP4 and compared their ability to promote ectopic endochondral bone formation in skeletal muscle. RESULTS: Under the influence of BMP4 in vitro and in vivo, NIH/3T3 cells differentiated toward both chondrogenic and osteogenic lineages, whereas most C2C12 cells underwent primarily osteogenic differentiation. NIH/3T3 cells genetically modified to express BMP4 induced delayed but more robust cartilage formation than did genetically modified C2C12 cells, which promoted rapid ossification. These differences in terms of the timing and amount of cartilage and bone formation persisted even after we introduced a retrovirus encoding dominant negative Runx2 (DNRunx2) into the C2C12 cells, which interferes with the function of Runx2. Superior osteogenic potential was also displayed by the primary myoblasts in vitro and in vivo compared with the primary fibroblasts. The different proliferation abilities and differentiation potentials exhibited by these cells when influenced by BMP4 may at least partially explain the differing roles that BMP4-expressing myogenic cells and BMP4-expressing fibroblastic cells play in endochondral bone formation. CONCLUSIONS: Our findings suggest that the process of endochondral bone formation in skeletal muscle after delivery of BMP4 involves different cell types, including fibroblastic cells, which are more involved in the chondrogenic phases, and myoblastic cells, which are primarily involved in osteogenesis. These findings could have important implications for the development of tissue engineering applications focused on bone and cartilage repair.

Alkaline Phosphatase↗
PubMed1998

Increased activity of cathepsin B in fibroblasts isolated from primary melanoma in comparison to fibroblasts from normal skin.

We determined activity of cathepsin B in early-passage fibroblasts isolated from primary melanoma and in fibroblasts from normal skin. Our results show an up to 5-fold increase in activity of cathepsin B in the tumor-derived fibroblasts in comparison to the fibroblasts from normal skin. We conclude that fibroblasts isolated from melanoma tissue are altered with regard to their specific activity of cathepsin B and preserve this elevated activity in early-passage cell culture. The data support the idea that stromal cells are not passive elements of the peritumoral environment but actively participate in the production of proteolytic enzymes.

Cathepsin B↗
PubMed1997

Receptor-mediated and protein kinase-dependent growth enhancement of primary human fibroblasts by platelet activating factor.

Chronic inflammation is a recognized risk factor for human cancer, but the causal mechanisms are poorly understood. We previously demonstrated that platelet activating factor (PAF) can induce alterations in the in vitro growth properties of primary rat fibroblasts. In the study reported here, exposure of primary human skin fibroblasts to PAF for 1 h in serum-free medium was shown to cause sustained proliferation over 50 d in medium containing low serum and anchorage-independent growth in soft agarose. Both properties could be inhibited by pretreatment with a PAF receptor antagonist, CV3988 (10 microM); a tyrosine-kinase inhibitor, genistein (1 microgram/mL); or a protein kinase C (PKC) inhibitor, staurosporine (50 nM) but not with a cyclooxygenase inhibitor, indomethacin (200 nM-20 microM). PAF had no effect on doubling time, saturation density, or cell viability under normal monolayer growth conditions in complete medium. Treatment with lyso-PAF, an inactive metabolite of PAF, had no effect in either of the assays. Control and PAF-induced cell proliferation in low-serum medium was inhibited by PAF receptor antagonists present during the extended growth period. The presence of PAF receptor mRNA in human skin fibroblasts was demonstrated by reverse transcriptase-polymerase chain reaction. The presence of a functional receptor was indicated by an early (2 min) transient increase in PKC activity and an increase in fos mRNA after PAF treatment. PAF-induced PKC activity was blocked by pretreatment with either staurosporine (50 nM) or CV3988 (1 microM). These results suggest that PAF is a mitogenic factor that contributes to the known increase in risk of malignancy associated with chronic inflammatory conditions.

Animals↗
PubMed1993

Formation of large deletions by illegitimate recombination in the HPRT gene of primary human fibroblasts.

HPRT gene mutants were isolated from untreated and x-irradiated cultures of primary human fibroblasts, and mutants carrying large deletions were identified. The breakpoints of the deletions were mapped by methods based on the polymerase chain reaction, and the deletion junctions of four different mutants were sequenced. Alu repeats were associated with one end of three of these junctions, but in each case repeat sequences were not found at the other end. Sequence features found at the deletion breakpoints included in particular short direct and inverted repeats, which may mispair to promote illegitimate recombination. One mutant had additional bases inserted at the deletion junction; these bases formed a direct repeat with a sequence immediately adjacent to the junction, suggesting a mechanism of templated repair of broken DNA in deletion formation.

Base Sequence↗
PubMed1998

Survival of normal colonic epithelial cells from both rats and humans is prolonged by coculture with rat embryo colonic fibroblasts.

Primary cultures of normal colonic epithelial cells from both humans (HCEC) and rats (RCEC) have been established using coculture with colon fibroblasts isolated from rat term embryos. While no other factors we have analyzed had any effect on the survival of epithelial cells, which is normally 3-4 days, coculture with viable fibroblasts extended this period to at least 2 weeks. The effects depended on early passages and low seeding densities of the fibroblasts and on direct cell-cell contact. We have obtained cultures of epithelial cells expressing keratin, laminin, and uvomorulin, displaying a polygonal, epithelial morphology and forming microvilli. DNA synthesis as measured by BrdU uptake into DNA varied widely between colonies of the same culture depending on cell morphology: flat colonies of RCECs contained 5.7% +/- 0.56% BrdU-positive cells, while the proportion in dense three-dimensional colonies reached 50.3% +/- 2.6%. In HCECs the growth fraction was lower, but showed the same distribution between classes of colonies. In the presence of rat embryonic colon fibroblasts, growth factors exerted survival activity on colonic epithelial cells. Consecutive addition of insulin and epidermal growth factor/fibroblast growth factor (EGF/FGF) increased colony number (15.0 +/- 1.0 and 23.0 +/- 2.0 colonies/well respectively; p < or = 0.05 increased above control) and size (1022 +/- 155 and 1207 +/- 158 cells/colony respectively; p < or = 0.05 increased above control) compared to serum-free control medium and basic MEM without growth factors. BrdU labeling index was not increased, however: EGF/FGF actually decreased BrdU labeling from 33.2% +/- 3.9% in controls to 21.3% +/- 3.8% in the EGF/FGF group (p < or = 0.05) owing to the high proportion of flat colonies consisting of resting cells. The newly established culture model can now be used to investigate growth control mechanisms in colonic mucosa and the effects of toxic and/or tumor-promoting substances on these mechanisms.

Animals↗
PubMed1997

Evaluation of the trophic effect of human placental polydeoxyribonucleotide on human knee skin fibroblasts in primary culture.

A simple assay capable of evaluating the trophic effect of growth factors or active principles on human skin diploid fibroblasts in primary culture has been developed. The results indicate that at physiological concentrations (20-100 micrograms/ml) a human placental polydeoxyribonucleotide (PDRN) preparation enhances the growth of human skin diploid fibroblasts of the knee in primary culture. This effect is consistently reproducible in the case of patients over 60 years of age, and may explain previously reported data on the successful clinical applications of human placental preparations, suggesting a selective benefit of PDRN in wound healing when compared to other treatments.

Cell Line↗
PubMed2001

Direct evidence for a bystander effect of ionizing radiation in primary human fibroblasts.

Bystander responses underlie some of the current efforts to develop gene therapy approaches for cancer treatment. Similarly, they may have a role in strategies to treat tumours with targeted radioisotopes. In this study we show direct evidence for the production of a radiation-induced bystander response in primary human fibroblasts. We utilize a novel approach of using a charged-particle microbeam, which allows individual cells within a population to be selected and targeted with counted charged particles. Individual primary human fibroblasts within a population of 600-800 cells were targeted with between 1 and 15 helium ions (effectively, alpha-particles). The charged particles were delivered through the centre of the nucleus with an accuracy of +/- 2 micrometer and a detection and counting efficiency of greater than 99%. When scored 3 days later, even though only a single cell had been targeted, typically an additional 80-100 damaged cells were observed in the surviving population of about 5000 cells. The yield of damaged cells was independent of the number of charged particles delivered to the targeted cell. Similar results of a 2-3-fold increase in the background level of damage present in the population were observed whether 1 or 4 cells were targeted within the dish. Also, when 200 cells within one quadrant of the dish were exposed to radiation, there was a 2-3-fold increase in the damage level in an unexposed quadrant of the dish. This effect was independent of the presence of serum in the culture medium and was only observed when a cell was targeted, but not when only the medium was exposed, confirming that a cell-mediated response is involved.

Alpha Particles↗
PubMed1978

Macrophage-mediated in vitro sensitization of lymphocytes. II. The detection of neo-antigens on transformed lymphocytes and passages of normal fibroblasts.

Unprimed lymphocytes were sensitized in vitro by incubating them with syngeneic macrophages that had been fed with viral or cellular antigens. The sensitized lymphocytes were tested for their cytotoxic activity against virus-infected and noninfected fibroblasts. The antigenic preparations used for priming the macrophages were either tumor cell-free extracts or supernatants from virus productive cells. Cell-free extracts from the productive RadLV-induced lymphoma cells or the nonproductive radiation-induced lymphoma cells were immunogenic when presented to lymphocytes by macrophages. In contrast, cell-free extracts from normal thymocytes were much less immunogenic, suggesting that the presence of viral associated antigens (VAA) can selectively be detected on lymphoma cells by this assay. Fibroblastic cell lines but not primary fibroblasts were also susceptible to the cytotoxic lymphocytes induced by RadLV-fed macrophages. Primary fibroblasts became susceptible to the sensitized lymphocytes either after infection with the corresponding virus, or if not infected, after several passages in vitro, suggesting that neo-antigens cross-reacting with viral antigens appear during sub-culturing of fibroblasts in vitro. This system makes it possible to detect VAA either as immunogens when presented to lymphocytes by macrophages, or as targets for cytotoxic lymphocytes.

Animals↗
PubMed1984

The nature and function of granulopoietic microenvironments.

A significant volume of reviewed literature in combination with observations presented here suggest that fibroblastic reticulum cells of the hematopoietic stroma may play a decisive role in the regulation of granulopoiesis. The histology of both bone marrow and spleen demonstrates an association between granulopoiesis and fibroblastic reticulum cells. Using in vitro cultured primary fibroblastic cells from murine bone marrow (BM) as antigenic source, we have prepared a hybridoma cell line secreting a monoclonal antibody (mAb) to fibroblastic reticular cells of the nonlymphoid domains in the spleen and BM. The mAb (ER-HR1) was demonstrated to exclusively detect primary fibroblastic cells cultured from spleen and bone marrow and in situ reticular cells with characteristic dendritic morphology and a dispersed distribution pattern. These reticular cells had extensive ramifications in the hemopoietic parenchyma. With the exception of some sparse fibroblastic cells in the dermis of the skin, the mAb did not react with any other cell type or extracellular substance elsewhere in the mouse, nor did it cross-react with human hemopoietic tissues. Support for a role of fibroblastic reticular cells in the regulation of granulopoiesis was obtained from in vitro studies. Primary BM fibroblasts produced granulocyte-macrophage colony stimulating activity (CSA). In contrast, we have been unable to detect CSA production by BM macrophages. Rather, we have found that BM macrophages deplete exogenous CSA from their culture medium. It is concluded that CSA may be produced by fibroblastic cells in adherent BM cultures and that the low CSA levels detected in their medium is due to depletion of fibroblastic cell-derived CSA by macrophages. We conclude that fibroblastic reticulum cells of the hemopoietic stroma carry unique antigenic determinants and together with macrophages, endothelial cells, and lymphocytes contribute to a complex network of cellular interactions providing an integrated microenvironmental control of granulopoiesis.

Animals↗
PubMed1984

Cell membrane polarity in primary human fibroblasts.

The topologies of bindings of cationized CF and native ferritin (NF) on plasma membranes of primary human fibroblasts were examined by transmission electronmicroscopy. Both ligands bound mainly to apical surfaces and less abundantly to lateral regions. At basal localization they were observed only at cell-to-cell connections. The polarity of negatively charged binding sites was not altered by glutaraldehyde fixation. The definite polarity of plasma membranes of cultured cells express alternating surface areas charged negatively or positively.

Binding Sites↗
PubMed2001

Contraction-dependent apoptosis of normal dermal fibroblasts.

The mechanisms underlying the contraction-dependent apoptosis of primary fibroblasts are of prime importance in understanding anchorage-dependent survival/apoptosis of dermal fibroblasts. As integrins are essential extracellular matrix receptors in fibroblasts, their role in anchorage-dependent apoptosis/survival of fibroblasts was analyzed. Primary human fibroblasts displayed a marked reduction of apoptosis in mechanically relaxed collagen matrices in the presence of adhesion-blocking antibodies against alpha1beta1 or alpha2beta1. Anti-alphavbeta3 antibodies had a considerably weaker effect. In additional experiments RD cells, which lack alpha2 integrin, displayed no apoptosis in mechanically relaxed collagen matrices. Their susceptibility to apoptosis was restored after transfection with functional alpha2 integrin, and it could be blocked again by adhesion-blocking antibodies against alpha2beta1 integrin. Therefore we conclude that apoptosis of human primary fibroblasts in contractile collagen matrices is - at least in part - inhibited by adhesion-blocking anti-integrin antibodies, suggesting that the mode of apoptosis in this case is different from anoikis. Further, apoptosis in a mechanically relaxed collagen matrix could be abrogated by depolymerization of F-actin using cytochalasin D and also by disturbing actin-myosin interaction using 2,3-butanedione monoxime, indicating a possible dependence of apoptosis on mechanical forces and/or cell shape.

Antibodies↗
PubMed1994

Three-dimensional contact with type I collagen mediates tyrosine phosphorylation in primary human fibroblasts.

By applying Western blot analysis using anti-phosphotyrosine antibodies, primary human dermal fibroblasts were examined after having been cultured on type I collagen-coated surfaces or in free-floating type I collagen gels. In both systems cells showed enhanced tyrosine phosphorylation of a M(r) 120,000 protein (pp120) and of a M(r) 42,000 protein (pp42). Phosphorylation was apparent 6 h at the latest after initiation of the culture and was only slightly induced on polylysine or on plastic. In contrast to pp42, pp120 was rapidly dephosphorylated in cells suspended by trypsinization or released from collagen gels by collagenase treatment, but regained phosphorylation in cells cultured in/on type I collagen. Two human sarcoma cell lines (HT-1080 and RD) exhibited identical tyrosine phosphorylation of pp120 but not of pp42. pp120 is identical with pp125FAK, a novel tyrosine kinase localized in focal adhesions, as proved by immunological cross-reactivity with anti-pp125FAK antibodies. Our results suggest that tyrosine phosphorylation is involved in signal transduction triggered by two- and three-dimensional type I collagen-fibroblast contact.

Adult↗
PubMed2006

Effect of meso-dihydroguaiaretic acid from Machilus thunbergii Sieb et Zucc on MMP-1 expression in heat shock-induced cultured primary human fibroblasts.

Ethanol and aqueous extracts of Machilus thunbergii Sieb et Zucc (Lauraceae) used traditionally for the treatment of a variety of diseases were screened in vitro for MMP-1 inhibitory actions. Meso-dihydroguaiaretic acid (MDGA) from the stem bark of M. thunbergii showed a significant inhibition of matrix metalloproteinase (MMP)-1 in primary human fibroblasts by heat shock-induced. This study investigated the effect of MDGA isolated from M. thunbergii on heat shock-induced premature skin aging. MDGA reduced the expression of MMP-1 at the protein level in a dose-dependent manner in heat shock-induced cultured primary human fibroblasts. Taken together, these results show that MDGA can prevent the harmful effects of heat (and/or IR) that lead to skin aging.

Cell Proliferation↗
PubMed1993

Expression of recombinant human methylmalonyl-CoA mutase: in primary mut fibroblasts and Saccharomyces cerevisiae.

Methylmalonyl-CoA mutase is an adenosylcobalamin-dependent enzyme which catalyzes isomerization of methylmalonyl-CoA to succinyl-CoA. Previous reports have described cloning and sequencing of a cDNA for human methylmalonyl-CoA mutase. This clone does not express an active apoenzyme after gene transfer into primary MCM-deficient fibroblasts and contains several sequences which differ from the consensus sequence of other cDNA clones. We describe reconstruction of a functional MCM cDNA and expression of recombinant enzyme activity in primary fibroblasts and Saccharomyces cerevisiae. This consensus human MCM cDNA is capable of complementing the inherited defect in mut MMA and overexpressing an enzyme in yeast with kinetic properties indistinguishable from the enzyme in murine or human tissues.

Acyl Coenzyme A↗
PubMed1993

High-resolution cytogenetic analysis of X-ray induced mutations of the HPRT gene of primary human fibroblasts.

We report cytogenetic analysis of X-ray induced mutants of the HPRT gene isolated from a primary human fibroblast line. The mutants were shown previously by molecular analysis to have total or partial HPRT gene deletions. Detailed analysis of the chromosomal region containing HPRT (the cytogenetic band Xq26) identified Xq26 aberrations in five of five total HPRT gene deletions but failed to detect any changes in two of two partial gene deletions. Four microdeletions were verified and quantified by a method termed the band ratio, which compares the distance between bands encompassing Xq26 and an adjacent X-chromosome region in elongated chromosomes. These measurements were supported by the presence or absence of the sub-band Xq26.2. One total HPRT gene deletion was also associated with an X;11 translocation involving band Xq26. The data strengthen earlier findings on the tolerance of the Xq26 region to large genetic changes (> 1 Mb), but also indicate that some mutations are more complex than simple deletion of DNA sequence.

Cells, Cultured↗