Neonatal Foreskin Fibroblasts Research Articles

Use of An in Vitro Skin Model for Determining Epidermal and Dermal Contributions to Irritant Responses

AbstractWe have used an in vitro skin model to study the responses of epidermal and dermal cells to irritants. This model consists of neonatal foreskin fibroblasts grown into a three-dimensional dermal structure on a nylon mesh. The dermal structure is seeded with keratinocytes and grown in calcium-containing medium at the air-liquid interface until a fully differentiated epidermis was formed. Phorbol-myristate acetate (PMA), a skin irritant, was incubated with this model. The PMA-conditioned culture medium was assayed for interleukin 1 alpha (IL-1 alpha) and tissue-type plasminogen activator (t-PA) by enzyme-linked immunosorbent assay (ELISA). Gelatin-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels were used to measure production of gelatinases. Exposure of the skin model to PMA resulted in the release of IL-1 alpha, t-PA, and gelatinases. This increase can be detected prior to a decrease in cell viability as measured by reduction of the mitochondrial dye MTT. Separat...

Electron microscopy of keratinocytes cultured on a collagen substrate used as an allograft for the treatment of chronic wounds

Grafts of cultured allogenic or autogenic keratlnocytes have proven to be an effective treatment of chronic wounds and burns. This study utilized a collagen substrate for keratinocyte and fibroblast attachment. The substrate provided mechanical stability and augmented graft manipulation onto the wound bed. Graft integrity was confirmed by light and transmission electron microscopy.Bovine Type I dermal collagen sheets (100 μm thick) were crosslinked with 254 nm UV light (13.5 Joules/cm2) to improve mechanical properties and reduce degradation. A single cell suspension of third passage neonatal foreskin fibroblasts were plated onto the collagen. Five days later, a single cell suspension of first passage neonatal foreskin keratinocytes were plated on the opposite side of the collagen. The grafts were cultured for one month.The grafts were fixed in phosphate buffered 4% formaldehyde/1% glutaraldehyde for 24 hours. Graft pieces were then washed in 0.13 M phosphate buffer, post-fixed in 1% osmium tetroxide, dehydrated, and embedded in Polybed 812.

Spontaneous production of growth factors for human lymphocytes from a human papillomavirus type 18-contained foreskin fibroblast cell line.

An immortalized fibroblast cell line, designated as CCFS-1/KMC, derived from human neonatal foreskin fibroblasts, contained human papillomavirus (HPV) type 18 DNA. Since this newly established cell line could spontaneously secrete activating factors for normal human blood lymphocytes, the synthesis and release of potent inflammatory cytokines from this cell line were checked. To determine the presence of cytokines in the supernatant collected from the cell line, tests by a cytokine-specific ELISA and a mitogenesis bioassay were done. The cell line could spontaneously produce several immunoreactive cytokines, such as tumor necrosis factor (TNF), interleukin 1 (IL-1) and interleukin 6 (IL-6). It also could stimulate the mitosis of human blood lymphocytes and Raji lymphoblast cell line. These cytokines were present in the same fraction of isoelectric points (pI) from 5.4 to 5.6. This study suggests that non-immune bystander cells may exert immunomodulatory effect on the immune cells via the production of potent inflammatory cytokines during viral infection.

HNF transfection with chondrosarcoma DNA results in the development of a sarcoma cell surface-associated epitope

High molecular weight DNA, which was isolated from a chondrosarcoma cell line, was transfected into human neonatal foreskin fibroblasts and NIH/3T3 cells. Both types of transfected cells expressed anchorage-independent growth in soft agar and produced tumors in nude mice. The tumors which developed in nude mice following injection of transfected human fibroblasts grew to ∼0.8 cm in diameter in four weeks. The tumors which developed from transfected NIH/3T3 cells grew to>2.0 cm in diameter in 6 weeks. After growth in soft agar the transfected human fibroblasts expressed a cell surface sarcoma-associated epitope recognized by the monoclonal antibody 345.134S. In addition to the transfected human fibroblasts, the original human chondrosarcoma tumor, the chondrosarcoma cell line derived from the tumor, and the nude mouse tumor which developed from transfected human fibroblasts all exhibited positive reactivity with the monoclonal antibody 345.134S. Transfected NIH/3T3 cells that exhibited anchorage-independent growth and tumorigenicity did not exhibit detectable reactivity with the monoclonal antibody. These results suggest that the expression of the tumor-associated cell surface antigen appears to be an early event correlated with transformation of the transfected human cells but not directly related to the tumorigenic potential of the DNA. The transfected cells which expressed anchorage independent growth exhibited the sarcoma cell surface antigen prior to attaining the potential for tumorigenicity.

Expression and Regulation of mRNA Coding for Acidic and Basic Fibroblast Growth Factor and Transforming Growth Factorα in Cells Derived from Human Skin

We investigated the regulation of mRNAs coding for acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), and transforming growth factor-alpha (TGF alpha) in cultures of human neonatal foreskin fibroblasts, keratinocytes, and melanocytes. Each cell type was propagated in an optimized serum-free medium. In rapidly growing fibroblasts, the addition of fetal bovine serum caused a modest induction of aFGF message within 2 h in conjunction with a concomitant elevation of bFGF transcripts. In these same cells, TGF alpha mRNA could not be detected in any experimental condition. In contrast, keratinocytes rapidly growing in the presence of epidermal growth factor (EGF) contained transcripts for TGF alpha that increased substantially when these cells were treated with serum. This observation suggests that factors present in serum can elevate the levels of TGF alpha mRNA beyond the levels already present in keratinocyte cultures growing in the presence of EGF. These same keratinocyte cultures had low to undetectable levels of bFGF or aFGF message, and the levels of these mRNAs were not affected by serum treatment. Treatment of keratinocytes proliferating in the presence of EGF with TGF beta for 48 h caused expression of bFGF mRNA in four of six independent cell strains. TGF beta-enhanced expression of bFGF mRNA occurred as early as 12-24 h after TGF beta exposure. TGF beta did not enhance the expression of mRNA for aFGF or TGF alpha in keratinocytes. Melanocytes failed to express detectable levels of mRNA coding for any of these growth factors in the presence of absence of TGF beta or serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytotoxicity, anchorage‐independent growth, and DNA adduct formation in human neonatal fibroblasts by 1, 2, 3, 4‐tetrahydro‐7, 12‐dimethylbenz(a)‐anthracene (TH‐DMBA), its six aryl fluoro regioisomers, and an exo methylene tautomer

1,2,3,4-Tetrahydro-7,12-dimethylbenz(a)anthracene (TH-DMBA), its six possible fluorosubstituted regioisomers, and the C-7 exo methylene tautomer of the 11F derivative have been investigated for their cytotoxicity and for their ability to induce anchorage-independent growth and to form adducts in human neonatal foreskin fibroblasts. All compounds tested exhibited a low level of cytotoxicity, determined as percent cloning efficiency, up to a final concentration of 30 micrograms/ml. Except for 5F-TH-DMBA and the C-7 exo methylene tautomer, all compounds induced anchorage-independent growth of neonatal foreskin fibroblasts in soft agar at all concentrations tested (1, 3, 10 and 30 micrograms/ml). The C-7 exo methylene tautomer induced anchorage-independent growth only at a concentration of 10 micrograms/ml. Among the compounds tested the 6F derivative was the most effective compound at 1 microgram/ml. The D-ring fluoro isomers induced anchorage-independent growth at a frequency comparable to TH-DMBA itself, with the 11F derivative being the least effective of the four D-ring regioisomers. All compounds except 5F-TH-DMBA formed detectable adducts with cellular DNA as determined by 32P postlabeling procedures, when the cells were treated at 1 microgram/ml. Two adducts were detected in cells treated with TH-DMBA and four adducts were detected in DNA obtained from cells treated with 6F-TH-DMBA. The level of bonding for the D-ring fluoro isomers was quantitatively less and sometimes qualitatively different than that for TH-DMBA. For the D-ring compounds, the ability to induce anchorage-independent growth frequency correlated with the total quantity of adduct formed. The C-7 exo methylene tautomer formed a single adduct and the level of bonding was less than one adduct per 10(9) nucleotides. Analysis of these results led to the proposal that the planar anthracene ring structure (rings B, C, and D) of TH-DMBA and possibly oxidative metabolism at benzylic carbon 4 of the A-ring are important to DNA bonding and initiation of induction of anchorage-independent growth.

Cultured endothelial cells produce a platelet-derived growth factor-like protein.

The platelet-derived growth factor (PDGF) binds specifically to high-affinity receptors on the surface of bovine aortic smooth muscle cells and 3T3 cells. Conditioned medium from cultured bovine aortic endothelial cells (EC) prevents PDGF binding to these receptors in a dose-dependent manner at 4 degrees C. The (125)I-labeled PDGF that is displaced by the conditioned medium shows no increase in trichloroacetic acid solubility or decrease in binding capability to fresh cells. The competitor activity was identified as a protein by ammonium sulfate precipitability and sensitivity to trypsin. The competitor protein also is found in the serum-free conditioned media from porcine aortic EC and human umbilical vein EC but not in media from bovine aortic smooth muscle cells, human neonatal foreskin fibroblasts, or the interleukin-producing thyoma cell line EL-4. The competitor protein, like PDGF, has no effect on the specific 4 degrees C binding of either (125)I-labeled insulin to 3T3 cells or (125)I-labeled epidermal growth factor to human epidermoid A431 cells. Saturation curves of PDGF binding to smooth muscle cells that had been preincubated in the presence and absence of competitor indicate that the concentration for half-maximal binding of (125)I-labeled PDGF to its receptor ( approximately 30 pM) is unchanged by the competitor, whereas the apparent number of available receptor sites or maximal level of binding is greatly diminished. The competitor activity produced by cultured human umbilical vein EC is completely inhibited by antiserum against pure human PDGF, whereas the same PDGF antiserum only partially inhibits the mitogenic activity of the conditioned media. In addition, approximately 7-fold more crude endothelium-derived growth factor is required for half-maximal inhibition of (125)I-labeled PDGF binding as is required for half-maximal stimulation of DNA synthesis. These results suggest that EC secrete a PDGF-like protein that is biochemically distinct from the majority of EC-derived mitogenic activity.

Effect of Hydrocortisone on the Proliferation of Cultured Skin Fibroblasts from Normal Individuals and Individuals at High Risk of Colon Cancer

In the present study we have compared the mitogenic effects of hydrocortisone (HC), both in the cloning efficiency (CE) assay and in semisolid agar, on skin fibroblasts (SF) obtained from individuals with hereditary adenomatosis of the colon and rectum (ACR) and their age-matched controls. The maximum stimulation of both cell types in the CE assay occured at an HC concentration of 500 ng/ml, but normal adult SF (NSF) were more sensitive to the mitogenic effects of HC than were ACR SF. The stimulation of cell proliferation in agar by HC (range 0–20 µg/ml) was apparently maximal at about 5 µg/ml of HC. However, NSF proliferated in agar containing HC considerably better, proportionately, than did ACR cells, whereas no growth of normal cells in agar was seen in the absence of HC. Normal neonatal foreskin fibroblasts (FOS) and colon adenocarcinoma cells (HT-29), in that order, were increasingly more refractory to the proliferative effects of HC in both the CE assay and in semi-solid agar. Charcoal-treated serum (from which steroids were largely removed) supported to a varying degree the growth of colon adenocarcinoma cells, FOS and ACR SF, but not of NSF cells. On the other hand, addition of HC to charcoal-treated serum primarily stimulated the proliferation of NSF cells. The reduced sensitivity of ACR SF to HC may be closely linked to mechanisms associated with initiation and promotion for this form of cancer.

Multiparametric evaluation of the toxic responses of normal human cells treated in vitro with different classes of environmental toxicants.

Eight compounds representing three classes of chemicals were evaluated for their toxic effects on normal neonatal human foreskin fibroblasts in vitro. A battery of toxicity assays was employed to measure the effects of the chemicals on cell viability, DNA synthesis, protein synthesis, DNA repair synthesis, cell ultrastructure, membrane-bound and soluble cytoplasmic proteins, and the activities of six enzymes: beta-glucuronidase, acid phosphatase, gamma-glutamyl transpeptidase, alkaline phosphatase, 5'mononucleotidase, and calcium-magnesium activated (Na+,K+)-dependent ATPase. The compounds evaluated included two antibiotics, each with a metabolic derivative-sulfamethazine (SMZ) and acetylsulfamethazine (ASZ), and carbadox (CBX) and desoxycarbadox (DCX); two anthelmintics-haloxon (HAL) and sansalid (SAN); and a steroid with a metabolic derivative, 17 alpha-estradiol (17-AE) and 17 alpha-estradiol-17-beta-D-glucoside (AE-G). Compounds with similar biological functions often elicited different patterns of response in the normal fibroblasts. For example, the two anthelmintics, HAL and SAN, were similar to each other in that they induced 50% relative cloning efficiencies (EC50) at approximately the same concentrations (HAL = 52 microgram/ml, SAN = 58 microgram/ml), and neither inhibited protein synthesis. They differed, however, in their effects of DNA synthesis. SAN did not inhibit DAN synthesis, while HAL was a profound inhibitor of DNA synthesis (98% inhibition after 4 h at 100 microgram/ml). Because the various toxicants elicited such a variety of response patterns as measured by a multiplicity of parameters, we conclude that similarities in survival responses of cells to closely related toxicants may arise frequently through toxic action at different sites within the cells.

Growth of human foreskin fibroblasts in a serum-free, defined medium without platelet-derived growth factor.

The hormones which support growth, in vitro, of normal, neonatal human foreskin fibroblasts were determined. Whereas thrombin and hydrocortisone were major growth stimulants, platelet-derived growth factor was not. Human foreskin fibroblasts grew in a serum-free, biochemically defined medium consisting of epidermal growth factor (100 ng/ml), insulin (100 ng/ml), transferrin 10 micrograms/ml), thrombin (1 microgram/ml), ascorbic acid (10 micrograms/ml), and hydrocortisone (5 x 10(-5) M) in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12, supplemented with ovalbumin (1 mg/ml) and trace elements. The growth achieved was comparable to that achieved with 5% fetal bovine serum. Neither platelet-derived growth factor, fibroblast growth factor, nor somatomedin activity increased proliferation. This serum-free medium, designated Defined Medium F, provides a biochemically defined system for growth and limited subcultivation of human foreskin fibroblasts in vitro.

Trophoblast transferrin and transferrin receptors in the host--parasite relationship of human pregnancy.

Transferrin and specific transferrin receptors are demonstrated on the microvillous surface of syncytiotrophoblast in human immature and term placentae by immuno histological techniques with the use of light and electron microscopy. That the distribution of transferrin is limited to the materno-foetal interface supports the hypothesis that binding of maternal transferrin to trophoblast receptors is involved in the process of iron transport to the foetus. Parallel studies with baboon placentae demonstrate the presence of trophoblast receptors which bind both baboon and human transferrin, thereby putting forward an experimental model which might be used to test the biological significance of placental transferrin receptors in primates. In addition, investigation of a large number of human cell lines shows that many transformed cells, but no normal cells (such as blood lymphocytes) or cells from primary culture (such as neonatal foreskin fibroblasts), possess the ability to bind transferrin to their membranes. These findings suggest that transferrin receptors may play important biological roles in addition to that of iron transport from mother to foetus. One such role could be the limitation of iron in intervillous spaces, thus depriving iron-requiring microorganisms of iron, hence serving as a non-specific factor of resistance for placentae. Another role for foetal transferrin receptors on trophoblasts could be to bind maternal transferrin at the materno-foetal interface, thus frustrating maternal immunosurveillance. This is similar to a mechahism used by schistosomes in the host-parasite relation where host proteins are bound by the parasite to escape immunological recognition. The presence of transferrin receptors on transformed cells suggests that this mechanism might also be employed by tumour cells. Finally, in view of previous studies which show that transferrin is required by stimulated lymphocytes to pass from the G1 to the S phase of cellular replication, it is proposed that trophoblast transferrin receptors could limit the amount of transferrin in intervillous spaces and thus impede the proliferation and possible cytotoxicity of maternal activated lymphocytes at the materno-foetal interface.

Localization of the Gene AVG for the Antiviral Expression of Immune and Classical Interferon to the Distal Portion of the Long Arm of Chromosome 21

The responses of normal fibroblasts and of fibroblasts trisomic and monosomic for chromosome 21 to exogenously administered virus-induced (classical) and phytohemagglutinin-induced (immune) human interferon were determined. The virus-induced interferon was obtained from leukocytes treated with Sendai virus and from neonatal foreskin fibroblasts treated with Newcastle disease virus. With both classical and immune interferons, the mean response of the trisomic cell lines was three times that of the normal cells, whereas that of the monosomic lines was half or less that of the normal cells, Furthermore, a line trisomic for only the distal half of the long arm of chromosome 21 (q21 leads to qter) also demonstrated increased sensitivity to virus- and phytohemagglutinin-induced interferons, a fact that indicated that the gene responsible for the antiviral effect of interferon, AVG, is located on this part of chromosome 21. Responses to the two categories of interferon (virus-induced and phytohemagglutinin-induced) of individual cell lines of different degrees of sensitivity were strongly correlated (r=0.79). It is concluded, therefore, that despite their physical and antigenic differences, the antiviral expressions of both classical and immune interferons are ultimately mediated by the same genetic locus, AVG.