Cultured Rodent Cells Research Articles

Metabolic activation of 3-methylcholanthrene and benzo(a)pyrene to mutagens in the L5178Y/TK assay by cultured embryonic rodent cells.

AbstractEarly passage cultured rodent cells obtained from mouse, rat, or Syrian hamster embryos were cocultivated with L5178Y/TK+/‐ cells in the presence of benzo(a)pyrene (B(a)P) or 3‐methylcholanthrene (3MCA) for 6 hr to determine if active metabolic intermediates could be detected in this cell‐mediated assay through the induction of TK+/‐ mutants. Neither chemical was directly active as a mutagen when tested with L5178Y/TK+/‐ cells alone. As a mutagen, 3MCA was slightly active in the presence of mouse or rat embryo cells and clearly active when tested with hamster embryo cells. B(a)P was active only in the presence of hamster embryo cells when tested at concentrations that would be mutagenic in the presence of rodent liver microsomal preparations. These results indicate the utility of L5178Y/TK+/‐ plus hamster embryo cell combinations in a cell‐mediated mutation assay for the detection of these two polycyclic hydrocarbon carcinogens.

Similarity of teleocidin B and phorbol ester tumour promoters in effects on membrane receptors.

The tumour promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and structurally related phorbol esters effect changes in avian and mammalian cell cultures that mimic transformation by oncogenic viruses or chemical carcinogens and the inhibition or induction of various types of differentiation (for review see refs 1--3). Unlike initiating carcinogens, which seem to act by binding covalently to cellular DNA, the primary site of action of the phorbol ester tumour promoters seems to be the cell membrane; indeed, specific high-affinity saturable receptors for phorbol esters have been identified in cell membranes. The recently discovered class of tumour promoters, the teleocidins, are as potent as TPA in the induction of ornithine decarboxylase in mouse skin, the inhibition of differentiation of Friend erythroleukaemia cells, the induction of HL-60 cell adhesion and the promotion of tumours on mouse skin. As the teleocidins are structurally unrelated to the phorbol esters, we set out to determine their effects on cell membranes and receptors. We found that in rodent cell cultures, teleocidin B and dihydroteleocidin B have effects similar to those of TPA and that, at nanomolar concentrations, teleocidin inhibits the binding of phorbol esters to cell-surface receptors, which suggests that the action of both classes of compounds may be mediated by the same or a similar receptor system.

Enhancement of the primary antibody response by 2-mercaptoethanol is mediated by its action on glutathione in the serum.

2-Mercaptoethanol (2-ME) is widely used in rodent lymphoid cell cultures as an enhancer of multiple cellular functions. We have confirmed that the action of 2-ME must be on a serum component(s), rather than a direct action on the cells. The serum component(s) is contained within the dialyzable fraction of fetal calf serum (FCS) since: (a) dialysis of FCS diminished the ability of FCS to support an antibody response even in the presence of 2-ME; and (b) FCS dialysate, pulsed with 2-ME, restored the ability of dialyzed FCS to support an antibody response. Diminution of the reduced glutathione content of FCS by heating reduced the capacity of FCS to support an antibody response, whereas addition of 2-ME-pulsed glutathione restored the supportive capacity of heated FCS. Conversely, oxidized glutathione inhibited the antibody response in the absence of 2-ME, but that inhibition was not seen in the presence of 2-ME. We have concluded that reduced glutathione is an essential component in FCS in order for 2-ME to produce its enhancing effect. The most plausible explanation for the enhancement of antibody responses, in vitro, by 2-ME, is the concomitant reversal of the inhibitory effect of oxidized glutathione and the increased availability of reduced glutathione which can scavenge oxygen-derived radicals, thus protecting macrophages and lymphocytes from the deleterious effects of oxygen-derived free radicals.

Uptake of minute virus of mice into cultured rodent cells.

The uptake of minute virus of mice into cells in tissue culture was examined biochemically and by electron microscopy. Cell-virus complexes were formed at 4 degrees C, and uptake of virus was followed after the cells were shifted to 37 degrees C. The infectious particles appeared to enter cells at 37 degrees C by a two-step process. The first and rapid phase was measured by the resistance of cell-bound virus to elution by EDTA. The bulk of the bound virus particles became refractory to elution with EDTA within 30 min of incubation at 37 degrees C. The infectious particles became resistant to EDTA elution at the same rate. The second, slower phase of the uptake process was measured by the resistance of infectious particles to neutralization by antiserum. This process was complete within 2 h of incubation at 37 degrees C. During this 2-h period, labeled viral DNA became progressively associated with the nuclear fraction of disrupted cells. The uptake of infectious virus could occur during the G1 phase of the cell cycle and was not an S phase-specific event. The uptake process was not the cause of the S phase dependence of minute virus of mice replication. In electron micrographs, virus absorbed to any area of the cell surface appeared to be taken into the cell by pinocytosis.

Membrane effects of tumor promoters: Stimulation of sugar uptake in mammalian cell cultures

Phorbol esters stimulate 2-deoxy-D-glucose (DG) uptake in rodent and human cell cultures. The potent tumor promoting agent, 12-0-tetradecanoyl phorbol-13 acetate (TPA), induces a 12-fold stimulation in confluent 3T3 cells and 2.5-fold stimulation in HeLa cells. When a series of macrocyclic deterpenes are assayed, their relative potencies in stimulating DG uptake in 3T3 cells correlate with other known biologic effects of these compounds. On a molar basis, TPA is a much more potent stimulator of DG transport than insulin or epidermal growth factor. In HeLa cells, the ED50 value of the TPA effect is 0.2 nM. The increase in DG uptake occurs immediately after the addition of TPA, reaches a maximum at 90 minutes, persists for at least three hours after removal of TPA from the medium, and is temperature dependent. The stimulation is not inhibited by cycloheximide or actinomycin D. As in control cells, DG uptake in TPA treated cells is inhibited by p-hydroxymercuribenzoate, phyloridzin, cytochalasin B, and dexamethasone. Although the precise mechanism is not known, evidence is presented that the TPA stimulation of DG uptake is due to enhanced transport of the sugar rather than to effects on intracellular metabolism. The enhanced transport may be secondary to a more generalized change in membrane structure.

Enhancement of aryl hydrocarbon hydroxylase activity in cultured rodent cells by x-irradiation

X-irradiation (500 rads) was found to enhance the aryl hydrocarbon hydroxylase (AHH) activity of three cell lines. Radiation followed by induction with benz (a) anthracene (5–15 μg/ml) produced a synergistic effect on AHH. These effects were highly significant and were observed most dramatically with a hamster tumor cell line, A(Tl)Cl-3,a nd to a lesser extent in secondary hamsters embryo cells and mouse C3H/10T12 CL8 cells.

Studies on Simian Virus 40 Excision from Cellular Chromosomes

Rodent cells in culture which do not support SV40 replication can be stably transformed by the virus. The resulting cells, which invariably harbor integrated viral DNA sequences (Sambrook et al. 1968; Hirai et al. 1971; Gelb et al. 1971), often can be manipulated to become infectious centers of viral production by fusion with permissive simian cells (Gerber 1966; Watkins and Dulbecco 1967; Koprowski et al. 1967). The source of the progeny viral DNA molecules is the integrated copy (see Discussion). This is remarkable in view of the fact that previous work from our laboratory and others has shown that integrated SV40 DNA can be attached to chromosomal DNA at various positions on the viral genome and also that multiple sites in cellular DNA are available for this viral insertion (Botchan et al. 1976; Ketner and Kelly 1976; Kucherlapati et al. 1978).

Immunological identification of two adenovirus 2-induced early proteins possibly involved in cell transformation.

HUMAN adenovirus 2(Ad2), a DNA tumour virus that replicates in the nucleus of permissive human cells1,2, transforms non-permissive or semipermissive cultured rodent cells, but does not induce tumours in newborn hamsters (oncogenic group C). The constant functioning of protein product(s) from Ad2 ‘transforming gene(s)’ is probably responsible for the transformed phenotype, although this has not been proved. Identification and functional characterisation of ‘transforming protein(s)’ are important for the understanding of cell transformation and growth control. We describe here immunoprecipitation studies made with antisera against presumptive Ad2 ‘transforming protein(s)’, that identify two candidate transforming proteins of 53,000 (53k) and 15kdaltons.

Transformation of rat and mouse embryo cells by a new class of carcinogenic compounds isolated from particles in city air.

AbstractThe customary benzene extraction of airborne particulate matter collected in Los Angeles has previously been shown to yield carcinogenic material. The residue from the benzene extraction contains substances soluble in methanol, some of which are organic. This methanol extract and a number of its component fractions have been tested in rodent cell culture systems developed as assay methods for screening carcinogens. In one system a high‐passage Fischer rat embryo cell line was used. In the other a low‐passage cell line derived from NIH Swiss albino mouse embryos and infected with AKR leukemia virus was used. In both systems the methanol extract showed cell transformation activity approaching that of 3‐methylcholanthrene. In the mouse cell system the activities of several fractions were also examined. The major inorganic component, ammonium nitrate, showed no activity. Neither the neutrals alone nor the recombined non‐neutral fractions separately showed any activity, although they were active together. The basic materials separated by ion exchange were also active. The methanol extract combined with the conventional benzene extract showed much lower activity than either extract alone.

Studies on the xanthine oxidase activity of mammalian cells

Xanthine oxidase in man is confined to but a few tissues and is absent from cultured cell strains. In rodents, however, the enzyme is more widely distributed among the tissues and can be demonstrated in most cell lines. Rodents possess the enzyme uricase and are therefore able to carry purine catabolism one step further than man. Preliminary results suggest that uricase is restricted to but a few rodent tissues and is absent from cultured rodent cells. Hence it may be that in each species only the final enzyme of purine catabolism is tissue restricted. In other experiments, mammalian cells were grown in the presence of compounds known to induce xanthine oxidase in a eukaryotic fungus (Aspergillus nidulans). These compounds did not induce the enzyme in mammalian cells.

Agglutination of normal and rous sarcoma virus-transformed chick embryo cells by concanavalin A and wheat germ agglutinin.

RODENT cells in culture transformed by oncogenic DNA viruses have surface sites that on normal cells are usually present in latent form only1,2. This difference in surface properties can be detected by plant glycoproteins such as wheat germ agglutinin (WGA) and concanavalin A (Con A), which agglutinate only transformed cells, because they have certain carbohydrate moieties on their neoplastic surfaces1–4. According to some investigators, normal and neoplastic cells that have been freshly isolated also exhibit this marked difference3,5; according to others6,7, there is no such distinction. We have looked for such differences in cells transformed by RNA tumour viruses and in several types of normal and naturally occurring malignant cells and their normal counterparts.

In Vitro Transformation of Rodent Cells by K-Region Derivatives of Polycyclic Hydrocarbons

The K-region epoxides and cis-dihydrodiols derived from benz(a)anthracene and from dibenz(a,h)-anthracene have been found to be more active in the production of malignant transformation in hamster embryo cells than the hydrocarbons or the corresponding K-region phenols. The K-region epoxides derived from benz(a)-anthracene and from 3-methylcholanthrene were also active in transforming a clone of ventral prostate cells from the C3H mouse that was not readily transformed by the parent hydrocarbons. The phenols were the most toxic compounds tested but did not transform cells; this confirms that toxicity and transformation are not directly related events. The results obtained support the view that metabolism of polycyclic hydrocarbons precedes toxicity and transformation in rodent cells in culture.

Comparative Studies on the Interaction of Benzo[<italic>a</italic>]pyrene With Cells Derived From Poikilothermic and Homeothermic Vertebrates. I. Metabolism of Benzo[<italic>a</italic>]pyrene<xref ref-type="fn" rid="FN2">2</xref>

The metabolism of benzo[a]pyrene (BP) to water-soluble derivatives was measured in cell cultures derived from poikilothermic and homeothermic vertebrates. Tritiated BP was added to the cultures and, after 24 hours of incubation, the medium was extracted with chloroform:methanol:water. The amount of BP metabolized per cell (relative efficiency of metabolism) was calculated from the 3H-BP equivalents in the aqueous phase of the extraction mixture. Many poikilothermic vertebrate cell cultures, including those derived from fish, toad, lizard, and turtle, were able to metabolize BP. In some instances, metabolism was as efficient as in rodent cell cultures. Cell multiplication was inhibited by BP, only in those cultures in which the metabolism of BP was highly efficient. Conversely, cytotoxicity was not observed in cell cultures with little or no ability to metabolize the carcinogen.

Measles virus growth in canine renal cell cultures.

Introduction The successful isolation and characterization of measles virus in cell cultures of human and simian renal tissue by Enders and Peebles 1 in 1954 was the first sound basis for intensive efforts to develop a practical measles vaccine. An immediate problem, however, was the need for a source of cells capable of supporting adequate growth of the virus, yet suitable for large-scale production of vaccine. In recent years, there have been many reports of the growth of measles virus in various cell culture systems. Thus, the virus has been grown in human amnion 2 as well as in rodent renal cell cultures. 3 Milovanovic et al. 4 succeeded in the adaptation of measles virus to the developing chick embryo and Katz et al., 5 by further adaptation, successfully propagated measles in chick embryo tissue cultures. Frankel et al. 6 described the use of canine renal cell cultures for the