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PubMed2003

Age-dependent variations of human and rat colon myofibroblasts in culture: Influence on their functional interactions with colon cancer cells.

Epithelial-mesenchymal interactions play a pivotal role in colon cancer invasion and metastasis. We aimed at elucidating the impact of long-term cultivation on the phenotypic and functional characteristics of primary fibroblasts and their interaction with the human colon adenocarcinoma cell line LoVoC5. We used fibroblasts from human colon tumor tissue, normal human colon mucosa, rat normal colon and 2 rat colon-derived myofibroblast cell lines, MIC316 and MG. The following parameters were studied: cell shape and size, growth curve, intermediate filament expression and extracellular matrix synthesis. Coculture models with or without cell contacts were used to test the effects on LoVoC5 cell proliferation, spreading and adhesion. Irrespective of their origin, fibroblastic cells in primary cultures presented marked phenotypic and functional changes with time. Before passage 5, they presented as large, slow-growing cells expressing vimentin and alpha-smooth muscle actin; synthesizing laminin-1, fibronectin and collagens I and IV; and inducing LoVoC5 proliferation, spreading and adhesion. After passage 15, they presented as small, fast-growing cells inconstantly expressing alpha-smooth muscle actin and synthesizing mainly type I collagen. In coculture with or without cell contacts, they inhibited LoVoC5 proliferation and allowed only limited cell spreading and adhesion. Myofibroblastic cell lines presented as large, fast-growing cells expressing vimentin and alpha-smooth muscle actin and synthesizing mainly type I collagen. They had no significant effects on LoVoC5 proliferation, spreading and adhesion. Our results underline the importance of age-dependent variations in colon mesenchymal cells in culture and for the in vitro study of epithelial-mesenchymal interactions in colon cancer.

Actins↗
PubMedDate not supplied

Junctional Adhesion Molecules (JAMs) are differentially expressed in fibroblasts and co-localize with ZO-1 to adherens-like junctions.

Junctional Adhesion Molecules (JAMs) are components and regulators of the well-characterized epithelial and endothelial tight junction. Since the molecular components of native fibroblast adherens-like junctions remain poorly described we determined JAM expression profiles in fibroblasts. We found JAM-C on human dermal, lung, and corneal primary fibroblast cultures. Within murine lines, JAM-A was found in L-cells, JAM-C in 3T3 L1 cells, and both JAM-A and JAM-C were co-expressed in NIH 3T3 fibroblasts. In primary dermal fibroblasts, JAM-C concentrated at zipper-like junctions that formed between apposing cells. Dual immunostaining showed JAM-C co-localization with the ZO-1 intracellular scaffolding molecule at cell contacts that ranged from 7 microm to over 25 microm in length. JAM-C also labeled similar zipper-like junctions detected with N-Cadherin and Cadherin-11 antibodies. We conclude that endogenous JAM-C is an integral component of the dermal fibroblast adherens-like junction, and our data extend the expression and potential function of JAMs into mesenchymal tissues.

3T3-L1 Cells↗
PubMed2003

The Moloney murine leukemia virus repressor binding site represses expression in murine and human hematopoietic stem cells.

The Moloney murine leukemia virus (MLV) repressor binding site (RBS) is a major determinant of restricted expression of MLV in undifferentiated mouse embryonic stem (ES) cells and mouse embryonal carcinoma (EC) lines. We show here that the RBS repressed expression when placed outside of its normal MLV genome context in a self-inactivating (SIN) lentiviral vector. In the lentiviral vector genome context, the RBS repressed expression of a modified MLV long terminal repeat (MNDU3) promoter, a simian virus 40 promoter, and three cellular promoters: ubiquitin C, mPGK, and hEF-1a. In addition to repressing expression in undifferentiated ES and EC cell lines, we show that the RBS substantially repressed expression in primary mouse embryonic fibroblasts, primary mouse bone marrow stromal cells, whole mouse bone marrow and its differentiated progeny after bone marrow transplant, and several mouse hematopoietic cell lines. Using an electrophoretic mobility shift assay, we show that binding factor A, the trans-acting factor proposed to convey repression by its interaction with the RBS, is present in the nuclear extracts of all mouse cells we analyzed where expression was repressed by the RBS. In addition, we show that the RBS partially repressed expression in the human hematopoietic cell line DU.528 and primary human CD34(+) CD38(-) hematopoietic cells isolated from umbilical cord blood. These findings suggest that retroviral vectors carrying the RBS are subjected to high rates of repression in murine and human cells and that MLV vectors with primer binding site substitutions that remove the RBS may yield more-effective gene expression.

Animals↗
PubMed1994

Lipopolysaccharide is a frequent contaminant of plasmid DNA preparations and can be toxic to primary human cells in the presence of adenovirus.

Endotoxin (lipopolysaccharide, LPS) is commonly found as a contaminant in plasmid DNA preparations. We demonstrate here that the quantities of LPS typically contaminating DNA preparations can generate a toxicity to primary cells (primary human skin fibroblasts, primary human melanoma cells) in the presence of entry-competent adenovirus particles. Toxicity can be observed with as little as 100 ng/ml free LPS or 100 pg/ml LPS when the LPS is assembled into polylysine/adenovirus complexes. Simple and effective methods of removing the contaminating LPS using either a polymyxin B resin or Triton X-114 extraction are described. Treatment of DNA samples to remove LPS eliminates the toxicity to primary cells.

Adenoviridae↗
PubMed2005

IGF-I differentially regulates IGF-binding protein expression in primary mammary fibroblasts and epithelial cells.

Elucidating how mitogens facilitate epithelial/stromal interactions is critical given that mitogens regulate mammary gland development and function. IGF-I is a potent mammary cell mitogen that is locally produced in the mammary gland. Since IGF-binding proteins (IGFBPs) regulate IGF-I bioavailability, we characterized the cell-type-specific production of IGFBP in primary bovine mammary epithelial (BME) and fibroblast (BMF) cells. Cells were treated with IGF-I and mRNA levels were analyzed via quantitative real-time (qRT)-PCR and Northern blot analysis. Media conditioned by cells treated with IGF-I for 48 h were analyzed via ligand blotting with 125I-labeled IGF-I and -II and immunoblotting with specific IGFBP antibodies. A reciprocal regulation of IGFBP-3 and -5 by IGF-I was observed between the two cell types. IGF-I induced large dose-dependent increases in IGFBP-3 mRNA and protein levels in BME cells, while IGFBP-5 protein was barely detectable and mRNA levels were detectable only by qRT-PCR. In BMFs, IGF-I induced large increases in IGFBP-5 mRNA and protein while IGFBP-3 mRNA was only slightly increased by IGF-I treatment and the protein was difficult to detect. IGFBP-6 mRNA was detected by Northern blot analysis in both cell types but was not regulated by IGF-I. In BME cells, IGFBP-6 protein levels were readily detectable under basal conditions and were increased by IGF-I. Interestingly, IGFBP-6 protein could not be detected in media conditioned by BMFs. IGFBP-4 mRNA was readily seen by Northern blot analysis in BMFs, however qRT-PCR was required to detect IGFBP-4 mRNA in BME cells. IGF-I increased IGFBP-4 mRNA levels by 2-fold in both cell types. IGFBP-4 protein was only detectable in media conditioned by BME cells when stimulated by IGF-I. In contrast, IGFBP-4 was present in media conditioned by untreated BMFs but was not consistently increased by IGF-I treatment. This was explained by the finding that IGF-I stimulated proteolysis of IGFBP-4, as evidenced by the appearance of two immuno-responsive fragments of 18 and 14 kDa. This proteolysis was specific to IGFBP-4, and was not observed in BME cells. We confirmed the protease to be pregnancy-associated plasma protein A (PAPP-A) by immunoblotting with an antibody against human PAPP-A/proMBP (pro form of eosinophil major basic protein) complex. In vitro immuno-neutralization experiments showed that blocking PAPP-A prevented the ability of IGF-I to stimulate IGFBP-4 proteolysis. IGFBP-2 mRNA and protein levels were observed under basal conditions in both cell types, with no significant regulation by IGF-I. The analysis of cell-type-specific regulation of the IGF system in both primary mammary epithelial cells and stromal cells will assist in the characterization of the mechanisms behind the role of the IGF system in normal mammary physiology and ultimately breast cancer.

Animals↗
PubMed1997

Transgenic mice overexpressing ornithine and S-adenosylmethionine decarboxylases maintain a physiological polyamine homoeostasis in their tissues.

Recent work has shown that transgenic mice overexpressing human ornithine decarboxylase display no marked changes in the tissue concentrations of spermidine or spermine in spite of a dramatic increase in putrescine levels. In the tissues of transgenic mice carrying the human spermidine synthase gene and in those of hybrid mice overexpressing both ornithine decarboxylase and spermidine synthase, spermidine and spermine levels remain within normal limits. To test whether the amount of the propylamine group donor, decarboxylated S-adenosylmethionine, limits the conversion of putrescine into the higher polyamines, we have produced transgenic mouse lines harbouring the rat S-adenosylmethionine decarboxylase gene in their genome. However, neither these mice nor the hybrid mice overexpressing both ornithine decarboxylase and S-adenosylmethionine decarboxylase displayed significant changes in their spermidine and spermine tissue levels. To study the mechanism by which cells maintain the constancy of the polyamine concentrations, we have determined the metabolic flux of polyamines in transgenic primary fibroblasts using pulse labelling. The results indicate that the polyamine flow is faster in transgenic primary fibroblasts than in non-transgenic fibroblasts and that the intracellular homoeostasis of higher polyamines is maintained at least partly by the acetylation of spermidine and spermine and their secretion into the medium.

Adenosylmethionine Decarboxylase↗
PubMed1999

Effect of polydeoxyribonucleotides on human fibroblasts in primary culture.

The effects of a mixture of oligo- and polydeoxyribonucleotides (PDRN) on the growth and protein secretion of cultured human skin fibroblasts were investigated. Both intact and DNAase-digested PDRN stimulated cell proliferation to a similar extent. When cultured fibroblasts were incubated with radioactive amino acids in the presence of intact or digested PDRN the incorporation of the tracer into secreted proteins increased significantly. This stimulation appears to be specific for certain protein components, including fibronectin. These results are interpreted assuming that PDRN and the nucleotides and nucleosides resulting from its degradation, can act as signal transducers or, alternatively, can be internalized and utilized to provide purine and pyrimidine rings for the salvage pathways.

Cell Division↗
PubMed1991

Reactive astrocytes are substrates for the growth of adult CNS axons in the presence of elevated levels of nerve growth factor.

To assess the necessary parameters for the growth of axons within the adult rat CNS, we have used intracerebral grafts of primary fibroblasts genetically engineered to express nerve growth factor (NGF). Following the implantation of NGF-producing primary fibroblasts within the striatum, cholinergic axons arising from the nucleus basalis grow toward and penetrate these grafts between 1 and 8 weeks. Grafts of noninfected control cells do not elicit axon sprouting at any time. Unmyelinated axons grow into grafts of NGF-producing cells only on reactive astrocytic processes, which contribute to a surrounding glial border. From our data concerning axon growth within the adult rat CNS, we conclude that reactive astrocytes can act as conducive substrates for growing axons; and only in the presence of elevated levels of NGF will permissive substrates (e.g., astrocytes) support axon growth by NGF-sensitive neurons.

Animals↗
PubMedDate not supplied

Mutagenesis of the v-mht/mil oncogene in avian carcinoma virus MH2.

Avian carcinoma retrovirus MH2 induces leukemia and solid tumors in chickens and transforms fibroblasts and macrophages in vitro. The genome of MH2 consists of two oncogenes, v-mht/mil and v-myc. Most of the transforming activity of MH2 is attributed to the v-myc oncogene. In contrast, the v-mht/mil oncogene alone does not induce a fully transformed phenotype of avian primary fibroblasts in vitro. It was shown previously that v-mht/mil is the avian homology of the v-raf oncogene in murine sarcoma retrovirus 3611. Because the v-raf oncogene transforms murine fibroblasts very efficiently, the present study tested the hypothesis that an extra segment in the 5' end of v-mht/mil relative to v-raf suppressed the fibroblast-transforming activity of v-mht/mil. By introducing an in-frame deletion of 195 nucleotides into the 5' end of v-mht/mil, the results demonstrate that in the presence of an inactive v-myc oncogene, the 5'-deleted v-mht/mil oncogene fails to transform chicken embryo fibroblasts. Therefore, it is likely that avian primary fibroblasts lack a cellular component that serves as a critical substrate/target for v-mht/mil-induced cellular transformation.

Animals↗
PubMed1999

Transplants of fibroblasts genetically modified to express BDNF promote regeneration of adult rat rubrospinal axons and recovery of forelimb function.

Adult mammalian CNS neurons do not normally regenerate their severed axons. This failure has been attributed to scar tissue and inhibitory molecules at the injury site that block the regenerating axons, a lack of trophic support for the axotomized neurons, and intrinsic neuronal changes that follow axotomy, including cell atrophy and death. We studied whether transplants of fibroblasts genetically engineered to produce brain-derived neurotrophic factor (BDNF) would promote rubrospinal tract (RST) regeneration in adult rats. Primary fibroblasts were modified by retroviral-mediated transfer of a DNA construct encoding the human BDNF gene, an internal ribosomal entry site, and a fusion gene of lacZ and neomycin resistance genes. The modified fibroblasts produce biologically active BDNF in vitro. These cells were grafted into a partial cervical hemisection cavity that completely interrupted one RST. One and two months after lesion and transplantation, RST regeneration was demonstrated with retrograde and anterograde tracing techniques. Retrograde tracing with fluorogold showed that approximately 7% of RST neurons regenerated axons at least three to four segments caudal to the transplants. Anterograde tracing with biotinylated dextran amine revealed that the RST axons regenerated through and around the transplants, grew for long distances within white matter caudal to the transplant, and terminated in spinal cord gray matter regions that are the normal targets of RST axons. Transplants of unmodified primary fibroblasts or Gelfoam alone did not elicit regeneration. Behavioral tests demonstrated that recipients of BDNF-producing fibroblasts showed significant recovery of forelimb usage, which was abolished by a second lesion that transected the regenerated axons.

Animals↗
PubMed1990

Ornithine and S-adenosylmethionine decarboxylase activities and polyamine contents in developing muscle tissues and primary cultures of normal and polymyopathic hamsters.

Polyamine (putrescine, spermidine, and spermine) contents and ornithine (ODC) and S-adenosylmethionine (SAMDC) decarboxylase activities have been assessed in an age-dependent manner, in normal and polymyopathic (dystrophic) hamster skeletal muscle, heart, and tongue extract and in primary tongue myoblast and skin fibroblast cultures. At 2 weeks of age, polyamine contents were significantly elevated in all of the dystrophic hamster tissues studied when compared with their age-matched controls. The degree of this elevation decreased with the age of the animals, generally, to a level where no significant difference in polyamine contents could be noted between normal and dystrophic hamster tissues. ODC and SAMDC activities in whole tissue extracts were consistently highest in 2-week-old muscle extracts and also declined with age. However, no significant changes in ODC or SAMDC activities were evident in any of the dystrophic muscle tissues studied when compared with their age-matched controls. Polyamine contents in dystrophic hamster myoblast and fibroblast primary cultures were also during proliferation (1 and 2 days after the initial seeding) compared with cultures prepared from normal hamsters. ODC and SAMDC activities in primary myoblast and fibroblast cultures clearly reflected the rate of cell proliferation, with highest activities found in subconfluent cell cultures. However, in general, no significant dystrophic-related abnormality in ODC or SAMDC activity was evident in proliferating myoblast or fibroblast cultures. These results suggest that the elevated polyamine contents of dystrophic hamster tissues and primary cultures may be due to a deficiency in polyamine catabolism or transport.

Adenosylmethionine Decarboxylase↗
PubMed1986

Immunoglobulin M to the membrane of uninfected fibroblasts in primary human cytomegalovirus infections.

Thirty-two patients with primary human cytomegalovirus (HCMV) infection were examined for presence of antibody to the membrane (Me) of uninfected human embryonic lung fibroblasts (HELF) using an anti-complement immunofluorescence (ACIF) test. Patients were divided into three groups according to age: less than or equal to 12 months (11 cases), 2-6 years (8 cases), greater than or equal to 13 years (13 cases). Convalescent-phase sera from 13 out of 13 of the older and 6 out of 8 of the middle-aged group gave positive fluorescence staining of the membrane of living uninfected HELF monolayers, whereas only one out of 11 younger patients showed antibody reactivity to HELF Me. ACIF performed on IgM and IgG fractions of randomly selected Me antibody-positive sera, showed a reactivity to HELF Me restricted to antibody of the IgM class (Me IgM). HELF Me IgM were first detected during the second week after the onset of symptoms of primary HCMV infection and disappeared usually within 4-6 months. Me IgM were not detected in sera from patients with HCMV infections other than primary (i.e. recurrent or congenital), or with other herpesvirus infections and were not detected in sera from patients with severe immunological diseases. HELF Me IgM were shown not to be related to rheumatoid factor and their reactivity was shown to be restricted to HELF cells, for it was not observed in any of the other cell types tested. The determination of HELF Me IgM antibody appears to be a new, practical and useful serological tool for the diagnosis of primary infections in patients over 2 years.

Cell Line↗
PubMed1998

Radiosensitivity of normal fibroblasts from breast cancer patients assessed by the micronucleus and colony assays.

PURPOSE: To investigate whether radiation-induced micronucleus formation as expressed by the cytochalasin-blocked Mn-assay correlates with cellular radiosensitivity measured by a colony assay in primary fibroblast cultures from cancer patients. MATERIALS AND METHODS: Studies were made on skin fibroblasts from 36 breast cancer patients. The micronucleus assay was performed using treatment with cytochalasin-B to create binucleate cells. Response was scored in terms of the percentage of binucleate cells with micronuclei, also as the number of micronuclei per binucleate cell. The data were related to previously published results of cell survival measurements on these cell lines. RESULTS: Neither endpoint for micronucleus formation showed a correlation with radiosensitivity by the colony assay. The fraction of fibroblasts that reach mitosis without micronuclei also failed to correlate with cell survival. CONCLUSIONS: Among these primary fibroblast cell lines radiation-induced micronucleus formation was not associated with radiosensitivity as measured by a colony assay.

Adult↗
PubMedDate not supplied

Cytogenetic methodologies for gene mapping and comparative analyses in mammalian cell culture systems.

Presented here are the detailed methods employed in our laboratory for gene mapping and cytogenetic analyses in human beings, in the domestic cat, and in other mammalian species. Induced in the procedures are: 1) establishment of primary fibroblast and lymphoid cell cultures; 2) heterologous cell fusion for production of rapidly proliferating cell hybrids; 3) cellular transformation of primary fibroblasts using an oncogenic retrovirus; 4) cell synchronization for high-resolution banding of promethaphase chromosomes; 5) chromosome-banding procedures, including G-banding, alkaline G-11, and Q-banding; and 6) in situ hybridization of radiolabeled molecular clones to metaphase chromosomes for regional gene localization.

Animals↗
PubMed2000

Trehalose expression confers desiccation tolerance on human cells.

Many organisms that withstand desiccation express the disaccharide trehalose. We have now expressed the otsA and otsB genes of Escherichia coli, which encode trehalose biosynthetic enzymes, in human primary fibroblasts using a recombinant adenovirus vector. Infected cells produced increased amounts of trehalose with increasing multiplicity of infection (MOI). Human primary fibroblasts expressing trehalose could be maintained in the dry state for up to five days. Fourier transform infrared spectroscopy indicated that dry, but viable, human cells contained no detectable water. This study shows that mammalian cells can be engineered to retain viability in the absence of water.

Bacterial Proteins↗
PubMed2000

Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture--primary culture cells markedly differ from fourth-passage cells.

To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies. This method yielded highly enriched SFB (85% prolyl-4-hydroxylase+/74% Thy-1/CD90+ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1beta. This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture.

Arthritis, Rheumatoid↗
PubMed1991

Rat primary embryo fibroblast cells suppress transformation by the E6 and E7 genes of human papillomavirus type 16 in somatic hybrid cells.

The E6 and E7 genes of human papillomavirus type 16 (HPV-16) transform established lines of rat cells but not rat cells in primary culture irrespective of the expression of the two genes. The reason for this difference between the susceptibilities of cell lines and primary cells was examined by using hybrid cells obtained by somatic cell fusion of rat cell lines transformed by the E6 and E7 genes of HPV-16 and freshly isolated rat embryo fibroblast cells. In these hybrid cells, transformed phenotypes, including colony formation in soft agar, saturation density, and tumorigenicity in nude mice, were suppressed, whereas hybrid cells between E6/E7-transformed cell lines and normal rat cell lines retained these transformed phenotypes. RNA analysis showed that the E6 and E7 genes were transcribed in both types of hybrid cells. These results suggest that primary rat cells possess intracellular functions that cause posttranscriptional suppression of induction of the transformed phenotypes by the E6 and E7 genes of HPV-16.

Animals↗