Cultured Mouse Epidermal Cells Research Articles

Tumor promoters inhibit metabolic cooperation in cocultures of epidermal and 3T3 cells

Cultures of mouse epidermal cells ( HEL 37 ) were prelabelled by incubation for 2–3 h with 3H-uridine. The cells were washed and cocultured for 4 h with unlabelled 3T3 cells. Communication between 3T3 and HEL 37 cells, as evidenced by the transfer of label from HEL 37 cells to contacting 3T3 cells, was inhibited by inclusion of the tumor promoting agents, 12-0-tetradecanoylphorbol-13-acetate (10 −7–10 −9 M) and phorbol-12,13-didecanoate (10 −7 M) during coculture. Non-promoting derivatives of these phorbol esters did not inhibit transfer of label.

Binding of epidermal growth factor to primary and permanent cultures of mouse epidermal cells: Inhibition by tumor-promoting phorbol esters

High affinity binding of epidermal growth factor (EGF) was detected with mouse epidermal cells in primary culture and with 2 epidermal cell lines maintained in permanent culture. EGF binding was inhibited by low concentrations of the tumor promoters 12-O-tetradecanoyl phorbol-13-acetate (TPA) and phorbol didecanoate (PDD), but not by the non-promoting phorbol esters 4-O-methyl TPA and 4 alpha-PDD.

A microtechnique for the rapid analysis of macromolecule synthesis in minicultures of mammalian cells.

A new micromethod, called the Stanzen technique, is described for the rapid determination of DNA and protein content as well as the incorporation rates of radioactively labeled precursors into macromolecules in cells growing in replica minicultures on plastic petri dishes. The procedure yielded reproducible results assaying only minimal cell numbers per sample and was applied for studying both primary or early passaged cell cultures (mouse epidermal cells and fibroblasts) and a malignantly transformed epidermal cell line. In four well defined circular areas (called Stanzen) marked on the bottom of tissue-culture plastic petri dishes (by heated stamps), 0.2 to 4 x 10(5) cells per area were plated and grown as four individual cultures in one dish. Both treatment and labeling with radioactive precursors of these Stanzen cultures were performed as with normal petri dishes. After fixation and extraction of the cultures, the singular Stanzen areas (with the cells fixed onto them) were sawed out and transferred into vials for liquid-scintillation counting or determination of DNA and protein. The obtained values of specific activity corresponded well whether the samples compared were derived from the minicultures of the same dish or from several dishes. By modifications of the known colorimetric methods for DNA and protein determination, the sensitivity of these procedures was improved down to values of 1 microgram DNA or 5 micrograms protein per individual culture. These micromodifications yielded the same values as the standard methods whether applied to cell suspensions or to cell cultures. Finally, cell proliferation was not influenced by the growth conditions in the small Stanzen areas and proceeded as in normal dishes or larger areas similarly stamped on the bottom of petri dishes. Since this method proved valuable for biochemical studies using primary cultures of mouse epidermal cells (saving cell material by a factor of 10, test substances and time), it might also be advantageous for other purposes as well where the availability of cells or test substances are limiting factors for large test series.

Mode of action of retinol. Involvement in glycosylation reactions of cultured mouse epidermal cells.

Mode of action of retinol. Involvement in glycosylation reactions of cultured mouse epidermal cells.

Biochemical Evidence for Keratinization by Mouse Epidermal Cells in Culture

More than 70 percent of the urea-extractable proteins from mouse stratum corneum or from differentiated cells of mouse epidermis grown in culture are two proteins of molecular weight 68,000 (keratin I) and 60,000 (kerae are two proteins of molecular weight 68,000 (keratin 1) and 60,000 (keratin 2), which are present in equimolar amounts on polyacrylamide gels. These proteins are the subunits of the keratin filaments, because when isolated from stratum corneum or cells grown in culture they form native-type epidermal keratin filaments in vitro. These observations provide biochemical evidence that epidermal cells grown in culture synthesize the major differentiation products of the epidermis.

Pemphigoid and pemphigus antigens in cultured epidermal cells

Fractions of IgG from sera of patients with pemphigoid and pemphigus added to monolayer cultures of mouse epidermal cells resulted in a sparse distribution of cells. Direct immunofluorescence studies of these monolayers revealed epidermal cell surface antigens reacting with pemphigoid and pemphigus antibodies. We suggest that these antibodies may bind to epidermal cell surface antigenic molecules which are important in maintaining epidermal cell adhesion in culture.

Anti-epidermal-cell-surface pemphigus antibody detaches viable epidermal cells from culture plates by activation of proteinase.

Immunoglobulin from pemphigus patients binds to the surface of mouse epidermal cells in culture. Cells incubated with the pemphigus antibody are easily detached from culture plates whereas cells incubated with serum from normal patients remain on the plate. Pemphigus antibody-mediated cell detachment is blocked by the addition of the proteinase inhibitors soybean trypsin inhibitor and alpha2-macroglobulin to the culture media. Detachable cells are viable, and activation of the complement cascade is not necessary for cell detachment. The anti-cell-surface antibody of pemphigus appears to disrupt adhesion between viable epidermal cells by activation of proteinase.

Retinyl acetate modulation of cell growth kinetics and carcinogen-cellular interaction in mouse epidermal cell cultures

Exposure of mouse epidermal cell cultures to β-retinyl acetate (RA) affects a number of parameters presumed to be important in chemical carcinogenesis. ( 1) RA alters the course of differentiation of the epidermal cells in culture resulting in a reduced rate of cell death which normally follows cellular maturation during the first two weeks in culture. The extended life span of the cultures appeared due to prolonged survival of cells and not to increased growth rate since RA inhibited the rate of cellular proliferation. This inhibition took place only after completion of a full cell cycle in the presence of RA. ( 2) DNA repair in response to physical and chemical agents was quantitatively unaffected in the presence of RA. ( 3) The activity of constitutive aryl hydrocarbon hydroxylase (AHH) was slightly decreased after exposure to RA but the level of enzyme induced by benz[a]anthracene was strongly reduced to 20% of the controls. ( 4) In the presence of RA, binding of 7,12-dimethylbenz[a]anthracene to epidermal cell DNA was markedly decreased. In contrast, binding to cellular protein was significantly increased by the retinoid.

Phorbol esters stimulate DNA synthesis and ornithine decarboxylase activity in mouse epidermal cell cultures.

TUMOUR-PROMOTING agents have been defined by their ability to promote tumour formation on carcinogen-initiated mouse skin. The most potent of these agents are the diesters of phorbol which are the active components of croton oil, the classic promoting substance. Phorbol esters can be chemically modified in a number of positions to form molecules with a wide range of promoter potency1. Such modifications have been useful in determining structure-function relationships2. A good correlation exists between the ability of phorbol esters to promote epidermal tumours and their ability to stimulate epidermal macro-molecular synthesis in vivo3,4. In particular, all tumour promoters stimulate synthesis of epidermal DNA, although not all chemicals which induce hyperplasia are promoters1. Recently O'Brien et al.5 suggested that the degree of induction of ornithine decarboxylase (ODC) activity in mouse skin after application of promoting and non-promoting compounds correlates well with their promoting potency. Evidence from our laboratory indicates that brief exposure to the potent phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), stimulates DNA synthesis and ODC activity in mouse epidermal cell cultures (S.H.Y., T.B., E.P., D. Michael, K. Elgjo and H.H., to be published, and U.L. and S.H.Y., to be published). Using such a culture system, we have examined a series of phorbol esters to test the correlation of promoter potency in vivo with the stimulation of DNA synthesis and ODC activity in vitro.

Mouse epidermal cell cultures: II. Isolation, characterization and cultivation of epidermal cells from perinatal mouse skin

In order to initiate studies on chemical carcinogenesis in an in vitro system analogous to mouse epidermis, primary epidermal cell cultures from perinatal mouse skin were established. A standardized method for the large-scale isolation of epidermal cells from late embryonic or newborn mouse dorsal skin has been developed. The epidermal cells were separated from fibroblasts by two series of discontinuous Ficoll density gradients. Using 80 to 100 animals/experiment, an average yield of 3×10 6 viable epidermal cells/animal was obtained. The viability of the purified cell suspension exceeded 85%, and the plating efficiency, representing the growing cell fraction 24 h after plating, was about 43%. The cultures started as epithelial monolayers without fibroblast contamination. Their epidermal nature and origin was proved immunologically by an in vivo absorbed rabbit anti-mouse epidermis antiserum. The purity of the epidermal cells was quantitatively determined in trypsinized suspensions by the indirect immunofluorescence test yielding more than 95% epidermal antigen-positive cells. About half of the remaining antigen-negative cells could be identified as melanocytes. These highly purified epidermal cells grew in vitro for 2–3 weeks without dermal constituents or diffusible mesenchymal factors. The monolayers differentiated in culture giving rise to keratinizing cell sheets on top of the proliferating basal layer. By morphological, histochemical and physical methods, it could be evidenced that the differentiation processes in vitro are quite similar to keratinization in vivo.

Mouse Epidermal Cell Cultures I. Isolation and Cultivation of Epidermal Cells from Adult Mouse Skin

A modified method was developed for rapid and continuous isolation of viable epidermal cells from adult mouse skin. Using a special type of trypsination apparatus (trypsinator) it was possible to obtain 3–6. 10 6 epidermal cells per 30cm 2 of depilated back skin. Dye exclusion tests indicated a viability of more than 80%. In suspension the isolated epidermal cells incorporated 3 H-thymidine into acid-insoluble material during a 4- to 6-hr period. After plating on plastic culture dishes the cells grew as primary cultures and could be maintained in a proliferative state for 2 months. After 7 days in culture the labeling index reached 17% and the mitotic count 0.67, respectively. The epidermal nature of the cultures was characterized by morphologic features and by detection of a tissue-specific membrane antigen on cultivated cells with a heterologous antiepidermal antiserum. Attempts to increase the low plating efficiency and to maintain the initial proliferation rate were not successful.

Altered glycoprotein synthesis in mouse epidermal cells treated with retinyl acetate in vitro

Retinyl acetate alters glycoprotein synthesis in mouse epidermal cells in culture. Epidermal glycoproteins were enzymatically digested to glycopeptides and separated on DEAE Sephadex A50 columns using different concentrations of LiCl. There was a two-fold increase in incorporation of fucose and glucosamine in the 0.2 M LiCl fraction from cells treated for 3 weeks with 12.5 μg/ml retinyl acetate and 1.25% DMSO as compared with DMSO controls. No changes were noted in other fractions. The glycopeptide from A treated cells isolated on 0.2 M LiCl had a higher molecular weight than glycopeptides from that same fraction eluted by control cells. This isolated newly synthesized glycopeptide from vitamin A treated cells appears to be a single product by rechromatography on DEAE Sephadex A50 and Sephadex G100 columns.

Altered differentiation of mouse epidermal cells treated with retinyl acetate in vitro

Mouse epidermal cells in monolayer culture retain many of the characteristics of differentiating epidermis in vivo. The addition of retinyl acetate dissolved in DMSO to the culture medium alters the direction of differentiation. Desmosome and tonofilament formation are inhibited. There is proliferation of Golgi complexes and endoplasmic reticulum. New or increased production of glycoprotein is suggested by cytochemical procedures. These effects are dose- and vehicle-dependent and require the continued presence of retinyl acetate. At a dose of 12.5 μg/ml of culture medium, retinyl acetate inhibits DNA synthesis. Retinyl acetate or a metabolite remains in the cells for more than 7 days after a single 18 h exposure.