Inhibition of fibroblast growth by polyanions; effects of dextran sulfate and lignin derivatives.

When macrophages prepared from rat bone marrow were cultured for more than one week, contaminating fibroblasts grew and formed large colonies which were observable with the naked eye after Giemsa staining. The addition of dextran sulfate or water-soluble lignin derivatives (acetyl or sulfonyl) to the culture medium almost completely inhibited the growth of fibroblasts at 10 micrograms/ml. These polyanions also inhibited the growth of chick embryo fibroblasts in a dose-dependent manner: at 5 micrograms/ml the inhibitory effect was 20-30% of the control and at 100 micrograms/ml it was 50-70%. In addition, these polyanions inhibited the mitosis of hepatocytes, although their inhibitory effects on the growth of hepatocytes were smaller than those observed on the growth of fibroblasts. On the other hand, these polyanions did not affect the growth of human urinary bladder carcinoma cells (HUB-4 and HUB-15). In conclusion, dextran sulfate and lignin derivatives may be useful in eliminating fibroblasts from certain cells.     

Effect of microinjected amine and diamine oxidases on the ultrastructure of eukaryotic cultured cells

Diamine oxide and serum amine oxidase, which catalyse the oxidation of diamines and polyamines, respectively, were trapped within reconstituted Sendai virus envelopes. These loaded envelopes were incubated with cultured normal chick fibroblasts or with fibroblasts transformed by Rous sarcoma viruses. The binding of the reconstituted envelopes to the cultured cells was confirmed by scanning electron microscopy. It has been shown that the reconstituted envelopes (1-3 microns diameter) were attached to the eukaryotic cells. No significant changes in the morphology of the normal chick embryo fibroblasts were noted upon treatment with enzyme-loaded envelopes. On the other hand, chick embryo fibroblasts transformed by Rous sarcoma virus were affected by the microinjected amine oxidases. Scanning electron microscopy demonstrated the formation of holes in the microinjected cells. Similar morphological changes were also observed when diamine oxidase was microinjected into cultured glioma cells. These holes may be the result of the ejection of the nucleus. These findings are in line with the observed effect of the injected amine oxidases on macromolecular synthesis in normal and transformed chick embryo fibroblasts.     

Inhibition of chick embryo hepatic uroporphyrinogen decarboxylase by components of xenobiotic-treated chick embryo hepatocytes in culture. II.

A variety of xenobiotics, viz., 3,3',4,4'-tetrachlorobiphenyl (TCBP), sodium phenobarbital (PB), 3,5-diethoxycarbonyl-2, 4,6-trimethylpyridine (OX-DDC), and nifedipine, cause a decrease in uroporphyrinogen decarboxylase (UROG-D) activity, accompanied by uroporphyrin accumulation, in chick embryo hepatocytes in culture. In this study the activity of 17-day-old chick embryo hepatic UROG-D was determined by measuring the conversion of pentacarboxylporphyrinogen I to coproporphyrinogen I, and it was shown that a UROG-D inhibitor, previously reported to accumulate in TCBP-treated and PB-treated chick embryo hepatocytes in culture, also accumulates in OX-DDC-treated and nifedipine-treated chick embryo hepatocytes in culture. It was concluded that the accumulation of a UROG-D inhibitor provides an explanation for the UROG-D inhibition observed in this culture system with xenobiotics that cause uroporphyrin accumulation. Studies of the UROG-D inhibitory fraction isolated from the 10,000 x g, 40,000 x g, and 100,000 x g supernatant fractions of cultured chick embryo hepatocyte homogenate led to the conclusion that the UROG-D inhibitor is derived from a soluble component of the homogenate.     

Tryptophan 2,3-dioxygenase in chick embryo hepatocytes: studies in ovo and in culture

Tryptophan dioxygenase is a hemoprotein in its active form, which has a relatively low affinity for heme. From previous studies in rats, the ratio of holoenzyme/total enzyme activity of tryptophan dioxygenase has been proposed to reflect the size of a "free" heme pool in hepatocytes. Chick embryo hepatocytes in ovo and in culture are other systems that have proven useful for study of hepatic heme metabolism and its control. Heretofore, there have been few studies of tryptophan dioxygenase activity in chick embryo hepatocytes. As part of studies on hepatic heme metabolism, using two different assays, we have measured tryptophan dioxygenase activity and percentage of heme saturation of the enzyme in chick embryo livers cells in ovo and in culture. One method of assay relies on endogenous formamidase to generate the final product, kynurenine, which is measured directly, whereas the other method uses a chemical hydrolysis step to form kynurenine which is further diazotized prior to measurement. The latter method is shown to be preferable for studies with chick embryo hepatocytes. In addition, we show that (i) tryptophan dioxygenase activity is present and can be increased by tryptophan and phenobarbital-like drugs in chick embryo hepatocytes in ovo; (ii) total enzyme activity falls markedly in cultured hepatocytes despite the presence of high concentrations of glucocorticoids in the culture medium; and (iii) under all conditions studied thus far in the cultures, the enzyme is nearly saturated with heme. Results are discussed in relation to regulation of heme metabolism in chick embryo hepatocytes.     

The intracellular significance of pH, its dynamics and possible role in regulating cell functions]

The intracellular and intralysosomal pH were studied on living monolayer cell cultures SPEV (pig embryo kidney), CHC (Chinese hamster's fibroblasts), NGUK-1 (neurinoma of Gasser's gland of rat), and also on primary cell cultures of rat's hepatocytes and chick embryo fibroblasts. In cytoplasm and lysosomes of these cells were found out pH changes during cells cycle and cell cultivation. There was also showed a heterogeneity of different parts of cells and lysosomes, and circahoral pH oscillations were registered. So there was found a spatial and temporal pH mosaity of living cells. The probable role of intracellular pH changes via regulator of cell vital activity is discussed.     

Study of storage iron in cultured chick embryo fibroblasts and rat glioma cells, using M?ssbauer spectroscopy

57Fe M?ssbauer spectra of normal and Rous sarcoma virus-infected cultured chick embryo fibroblasts and rat glioma cells have been measured between 0.08 and 318 K. Ferritin-like iron and bacterio-ferritin-like iron have been found in these cells, in various relative amounts, indicating a close relationship between the two storage materials. The bacterio-ferritin-like iron was found to be predominantly membrane-bound. Above 260 K very wide lines were observed in the M?ssbauer spectra, yielding an effective viscosity of about 1 poise in the normal chick embryo fibroblasts and about 0.5 poise in the virus-infected chick embryo fibroblasts.     

Study of lysosomal enzymes in chick embryo fibroblasts infected with microorganisms of family Halprowiaceae (Chlamydiaceae)

It is shown that infection of chick embryo fibroblasts with agents of paratrachoma and meningopneumonia Halprowiaceae (Chlamydiaceae) causes a sharp decrease of the activities of lysosomal enzymes, e.g. acidic alpha-glucosidase, beta-glucuronidase, beta-galactosidase, alpha-mannosidase, acid phosphatase, etc. The activity of cytosol enzymes (neutral alpha-glucosidase, amylo-1,6-glucosidase) does not change, however. A decrease in the activities of lysosomal enzymes in infected fibroblasts occurs some time later after inoculation and is due to a release of lysosomal enzymes from the fibroblasts into the culture medium, without loss of cell integrity. No changes in the activity of lysosomal enzymes in fibroblasts and culture medium is observed in the case of inoculation of cells with a killed agents, as well as after contact of cells with a suspension of normal chick embryo yolk sacs. The release of lysosomal enzymes from halprowiae-infected chick embryo fibroblasts probably occurs by the exocytosis.     

Study of storage iron in cultured chick embryo fibroblasts and rat glioma cells,using Mössbauer spectroscopy

⁵⁷Fe Mössbauer spectra of normal and Rous sarcoma virus-infected cultured chick embryo fibroblasts and rat glioma cells have been measured between 0.08 and 318 K. Ferritin-like iron and bacterio-ferritin-like iron have been found in these cells, in various relative amounts, indicating a close relationship between the two storage materials. The bacterio-ferritin-like iron was found to be predominantly membrane-bound. Above 260 K very wide lines were observed in the Mössbauer spectra, yielding an effective viscosity of about 1 poise in the normal chick embryo fibroblasts and about 0.5 poise in the virus-infected chick embryo fibroblasts.     

Metallothionein mRNA stability in chicken and mouse cells

Northern blot analysis revealed that metallothionein (MT) mRNAs accumulate after inhibition of protein synthesis with cycloheximide (CHX) in primary cultures of chick embryo hepatocytes and fibroblasts, as well as in an established mouse hepatoma cell line. Inhibition of RNA synthesis with actinomycin D (AMD) led to rapid loss of MT mRNAs in these cells, whereas CHX dramatically retarded the rate of MT mRNA decay (t1/2 greater than 24 h). These results suggest that CHX causes MT mRNA accumulation primarily by increasing stability of MT mRNA. Thus, changes in MT mRNA turn-over rates may play an important role in regulating the accumulation of MT mRNA. The half-lives of MT mRNAs in chicken and mouse cells were determined by oligodeoxyribonucleotide excess solution hybridization with RNA samples extracted after different periods of exposure to AMD. The half-life of chicken MT (cMT) mRNA in uninduced chicken embryo hepatocytes was 3.6 h. Induction of cMT mRNA by pretreatment of these cells with zinc (Zn) prior to exposure to AMD, did not alter the half-life of cMT mRNA significantly. In contrast, cadmium (Cd) induction led to a 2.5-fold increase in the stability of this mRNA. In uninduced chicken embryo fibroblasts, cMT mRNA levels were too low to allow accurate determination of half-life using the methods employed here. However, the half-life of this mRNA in Zn-induced chicken embryo fibroblasts was 6.2 h, whereas it was 9.3 h in Cd-induced cells. Thus, the turn-over rate of cMT mRNA after Cd-induction is very similar in chick embryo fibroblasts and hepatocytes. These data suggest that the accumulation of MT mRNA in chicken cells may reflect, in part, metal-specific effects on MT mRNA stability. The half-lives of mouse MT-I and MT-II (mMT-I and mMT-II) mRNAs in uninduced BNL hepatoma cells were identical (9.2 h), and were not effectively altered after induction by metals (Zn, Cd) or interleukin-1 beta (IL-1 beta). However, mMT mRNAs in pachytene spermatocytes and round spermatids, freshly isolated from the adult testes, were 2.2- to 4.5-fold more stable than in hepatoma cells. These results suggest that cell-type specific accumulation of mMT mRNAs may be regulated, in part, by mRNA stability.     

Immunological evidence for phenobarbital-stimulated synthesis of cytochrome P-450 in primary cultures of chick embryo hepatocytes

The induction of cytochrome P-450 by phenobarbital was studied in primary cultures of chick embryo hepatocytes. The rate of the de novo synthesis of the induced form of cytochrome P-450 was measured directly and specificially, using form-specific anti-cytochrome antibodies that quantitatively immunoprecipitated this form from the radiolabeled hepatocytes. Additionally, the steady-state levels of the cytochrome were estimated spectrophotometrically and electrophoretically. In the presence of phenobarbital the synthesis of cytochrome P-450_PB by cultured hepatocytes was markedly accelerated. Furthermore, the same cytochrome P-450_PB form was induced by phenobarbital in vivo in chicken liver and in the cultured chick embryo hepatocytes. Their identity was judged from immunological and electrophoretic properties of these induced cytochromes. Immunological cross-reactivity was also detected between the cytochrome P-450_PB forms from chick embryo hepatocytes and from adult rat liver. The immunological cross-reactivity observed between the phenobarbital-induced cytochrome P-450 forms from different species was not observed between the different cytochrome forms with the same liver (Thomas, P.E., Reik, L.M., Ryan, D.E. and Levin, W. (1981) J. Biol. Chem. 256, 1044–1052). Implications as to the evolutionary origin of the different cytochrome forms are discussed.     

Phospholipid turnover in hamster and chick embryo fibroblasts

The stability of the glycerol backbone of phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl serine was measured in growing and non-growing hamster and chick embryo fibroblasts. Major differences were found for the rates of degradation of the individual glycerophospholipids in both hamster and chick embryo fibroblasts: considerable degradation of phosphatidyl choline was detected over a 24 h period while at the same time no degradation of the glycerol backbone of phosphatidyl ethanolamine and phosphatidyl serine was observed. The patterns of stability of these glycerophospholipids were similar in growing and non-growing cells.     

The mechanism of the antiviral activity of an antiviral factor of a non-interferon nature]

Antiviral factor (AF) of protein nature has been isolated from chick embryo fibroblasts infected with Venezuelan equine encephalitis virus. The suppression of virus reproduction has been observed both in homologous and heterologous cell cultures when the preparation was introduced immediately after the adsorption of the virus after pretreatment of the cell monolayer. The study has demonstrated that the antiviral effect of AF is not linked with its IFN-alpha and TNF-alpha activity. Analysis of the results obtained in this study and earlier data contained in literature suggests that infected chick embryo fibroblasts release original cytokine of non-interferon nature with antiviral activity.     

Alteration of insulin binding and cytoskeletal organization in cultured fibroblasts by tertiary amine local anesthetics

Tertiary amine local anesthetics cause a time- and dose-dependent, reversible increase in insulin binding sites in cultured chick embryo fibroblasts. Incubation of fibroblasts with 0.2 mM dibucaine for 3 h at 37°C results in a twofold to threefold increase in insulin binding, with an increase in average number of binding sites (K_a = 3.0 × 10⁷M^?1) from 9 × 10³ to 29 × 10³ per cell. Trypsin or ethylenegly coltetraacetic acid (EGTA) alone increases insulin binding twofold to threefold, but fails to further increase ¹²⁵I-insulin binding in cells pretreated with dibucaine. Transformation of chick embryo fibroblasts with Rous sarcoma virus causes a threefold to fivefold increase in insulin binding, which is not further increased by incubation with dibucaine. As demonstrated by transmission electron microscopy, dibucaine and trypsin also induce changes in the cytoskeleton of chick embryo fibroblasts, characterized by disorganization and disappearance of microfilament and microtubule bundles. These alterations are accompanied by gross morphologic changes, including rounding of cells and appearance of numerous ruffles and blebs on the cell surface. These observations are consistent with the hypothesis that expression of surface receptors in cultured chick embryo fibroblasts is related to the organization and disorganization of cytoskeletal structures.     

Regulation of deoxyglucose uptake by adrenocorticotropic hormone in cultured neurons

The influence of ACTH and some of its N-terminal related peptides was investigated on the uptake of (3H)-2-deoxy-D-glucose in pure cultures of neurons from chick embryo cerebral hemispheres. ACTH influences deoxyglucose uptake in a time and dose-dependent fashion. The stimulation of deoxyglucose uptake is observed after a delay of 6-8 h and requires active protein synthesis. ACTH does not affect deoxyglucose in non-neuronal cells (astroglial cells, hepatocytes, myoblasts, fibroblasts). The effect of various peptide hormones, neuropeptides and growth factors, active in the central nervous system or other tissues, has also been examined. None of these were able to stimulate deoxyglucose uptake, suggesting that the regulation of hexose uptake in neurons is specific for the ACTH-related peptides.     

Cell growth and thymidine incorporation changes induced by Dolichos lectin in embryo fibroblasts at various stages of differentiation.

We report here the effect of Dolichos lectin on chick embryo fibroblasts from embryos between 6th and 16th day of development. There is evidence that Dolichos lectin decreases cell number and proportion of cells incorporating tritium labelled thymidine in case of chick embryo fibroblasts of 6th, 8th and 10th day of development. Dolichos lectin stimulated the proliferation of 16-day old embryo cells. No effect was noticed on 12-day embryo cells at different concentrations of Dolichos lectin used. This lectin is specifically inhibited by N-acetyl-D-galactosamine and anti-Dolichos lectin serum. The difference in response by cells during different stages of embryonic development could perhaps be explained as some regulatory changes occurring on the cell surface.     

Modulating effects of beta-naphthoflavone on the induction of SCEs by model compounds with emphasis on benzo[a]pyrene

The inducing capability of the synthetic flavonol beta-naphthoflavone (beta-NF) on cytochrome P-450 content was studied in primary chick embryo hepatocytes. In addition, the modulating effects of pretreatment with beta-NF on the induction of sister-chromatid exchanges (SCEs) in V79 cells by mutagens from different chemical classes were investigated in a co-cultivation system consisting of primary chick embryo hepatocytes and V79 Chinese hamster cells. Finally, the effects of pretreatment on benzo[a]pyrene (B(a)P) metabolism were studied in more detail. Pretreatment of cultured primary chick embryo hepatocytes with beta-NF resulted in a large increase in cytochrome P-450 content (a 2.8-fold increase after 31 h). Pretreatment with beta-NF had no effect on the level of SCEs induced by N-nitroso-dimethylamine (NDMA) and 2-aminoanthracene (2AA). Pretreatment with beta-NF resulted in a decrease in B(a)P-induced SCEs. This inhibitory potential was positively related to the beta-NF dose. However, there was an inverse relationship between the inhibitory action of beta-NF and the dose of B(a)P, at higher doses less inhibition was observed. When beta-NF was applied simultaneously with B(a)P the percentage of decrease was about the same as for pretreatment. Pretreatment with beta-NF followed by simultaneous application of beta-NF and B(a)P did not result in larger effects. In addition, subcellular fractions were prepared from chick embryos pretreated with beta-NF in ovo. The use of the S9 fraction resulted in a large decrease (80%) in the induction of SCEs in V79 cells by B(a)P whereas the use of the microsomal fraction resulted in a 70% increase in SCE induction compared with non-pretreated microsomes. Pretreatment with beta-NF in ovo gave rise to a large increase in aryl hydrocarbon hydroxylase (AHH) activity in the hepatic microsomal fraction. Increases were observed in the formation of all B(a)P metabolites. In particular the formation of the proximate carcinogenic and mutagenic metabolite B(a)P-7,8 dihydrodiol was increased 7-fold. The data strongly suggest that the inhibitory effects of pretreatment of cultured primary chick embryo hepatocytes with beta-NF cannot be ascribed to its inducing capabilities but instead seem to be due to the formation of an intracellular pool of beta-NF which acts as a competitive inhibitor for B(a)P metabolism.     

Biosynthesis in vitro of mono- and di-sialosylgangliosides from gangliotetraosylceramide by cultured cell lines and young rat brain. Structure of the products, and activity and specificity of sialosyltransferase

Incubations in vitro of GA1, labeled with 3H in the terminal D-galactopyranosyl group, with nonradioactive CMP-NeuNAc in the presence of homogenates of C21 rat brain glial cells, NIE mouse neuroblastoma cells, 3T3 mouse fibroblasts, SV 40-transformed 3T3 cells, chick embryo fibroblasts, Rous sarcoma virus-transformed chick embryo fibroblasts, and 9-day old rat brain resulted in all cases in the formation in high yield of GM1b, in which the neuraminidase-labile NeuNAc group is linked at O-3 of the terminal D-galactosyl residue, as shown by permethylation studies. No trace of the naturally occurring neuraminidase-stable GM1a was detected in any case. In addition, with NIE cells, and normal and RSV-transformed chick embryo fibroblasts, a disialosylganglioside (GD1) differing from GD1a and GD1b, and bearing only one substituent at O-3 of the terminal D-galactopyranosyl residue was formed. It was also biosynthesized from GM1b and CMP-NeuNAc by NIE and chick embryo cells but not by C21 cells, or rat brain. However, C21 cells and rat brain were capable of synthesizing GD1a from GM1a. Periodate oxidation degraded both NeuNAc groups in GD1 to a 7-carbon fragm:nt, indicating lack of substitution at O-8. GM1b could not be detected as a natural product in rat brain.     

Effect of 5''-deoxy-5''-isobutylthioadenosine on putrescine uptake and polyamine biosynthesis by chick embryo fibroblasts.

The effect of 5'-deoxy-5'-S-isobutylthioadenosine (SIBA) on polyamine biosynthesis has been studied by using cultured chick embryo fibroblasts. It has been shown that the drug inhibits the uptake of [14C]putrescine and its conversion into labelled spermidine or spermine. The inhibitory effect is reversed by removing the inhibitor after exposing the cells to the drug for 24 h. SIBA also caused a significant decrease in cellular spermine levels and an accumulation of putrescine. These changes are reversed by removing the inhibitor. SIBA had the same effect on chick embryo fibroblasts transformed by Rous sarcoma virus; a decrease in cellular spermine levels in SIBA-treated cells was observed. In all the experiments SIBA caused a reduction in the spermine/putrescine and spermidine/putrescine ratios. It is suggested that SIBA is not only an inhibitor of transmethylation but also interferes with polyamine biosynthesis, probably by blocking aminopropyltransferase.     

Evidence for a dual mechanism of chick embryo fibroblast adhesion on fibronectin and laminin substrata

Eight-day-old chick embryo fibroblasts were shown to adhere specifically to fibronectin and laminin substrata. Moreover, the Scatchard analysis reveals 540,000 binding sites per cell for the fibronectin with a dissociation constant (Kd) of 1.35 microM and 5,500 binding sites per cell for laminin with a Kd of 1.5 nM. Furthermore, cell-fibronectin interactions are mediated by plasma membrane proteins of high molecular weight (HMW) (150K and 125K) insensitive to trypsin treatment and low molecular weight (LMW) proteins (95K, 80K, 65K and 45K) sensitive to trypsin treatment. Adhesion of 8-day-old chick embryo fibroblasts on laminin is mediated by plasma membrane proteins highly sensitive to trypsin treatment. Regarding the paucity of laminin-binding sites, the identification of laminin receptor could not be achieved. Nevertheless, this study provides quantitative and qualitative evidences for different mechanisms of 8-day-old chick embryo fibroblasts on laminin and fibronectin.     

Antiviral factor production by infected chick embryo fibroblasts]

The secretion of antiviral factor (AF) by infected cell cultures was examined. Activity of AF depended on the cell culture used. AF produced by infected chick embryo fibroblasts had maximal activity. No activity was registered in BHK-21 cells, whereas human embryo fibroblasts and cell line Vero produced a low level of activity. Actinomycin D and cycloheximide prevented the production of AF. The results indicate that VEE virus-infected chick embryo fibroblasts produce AF which may be attributed to nonspecific factors of cell defense.