Differential proliferative response of cultured fetal and regenerating hepatocytes to growth factors and hormones

Upon epidermal growth factor (EGF) stimulation, fetal (20 days of gestation) and regenerating (44-48 h after partial hepatectomy) rat hepatocytes, isolated and cultured under identical conditions, increased DNA synthesis and entered into S-phase and mitosis, measured as [3H]thymidine incorporation and DNA content per nucleus in a flow cytometer, respectively. Fetal hepatocytes consisted of a homogeneous population of diploid (2C) cells. Two different populations of cells were present in regenerating liver, diploid (2C) and tetraploid (4C) cells, that responded to EGF. Glucagon or norepinephrine did not affect EGF stimulation of DNA synthesis in fetal liver cells, but they potentiated EGF response in regenerating hepatocyte cultures. Glucocorticoid hormones (dexamethasone) inhibited DNA synthesis in fetal hepatocyte cultures, an effect potentiated by the presence of glucagon or norepinephrine. In contrast, in regenerating hepatocytes, dexamethasone increased EGF-induced proliferation. EGF-dependent DNA synthesis was inhibited by TGF-beta in both fetal and regenerating cultured hepatocytes. TGF-beta action was partially suppressed by norepinephrine in regenerating hepatocytes, but was without effect in fetal hepatocyte cultures, whereas a synergistic action between TGF-beta and dexamethasone inhibiting growth in fetal but not in regenerating hepatocytes was found. Taken together, these results may suggest that there are significant differences between fetal and regenerating hepatocyte growth in their response to various hormones.     

Experimental evaluation of conversion factors for the [h]thymidine incorporation assay of bacterial secondary productivity

The relationship between bacterial growth and incorporation of [methyl-H]thymidine in oligotrophic lake water cultures was investigated. Prescreening, dilution, and addition of organic and inorganic nutrients were treatments used to prevent bacterivory and stimulate bacterial growth. Growth in unmanipulated samples was estimated through separate measurements of grazing losses. Both bacterial number and biovolume growth responses were measured, and incorporation of [H]thymidine in both total macromolecules and nucleic acids was assayed. The treatments had significant effects on conversion factors used to relate thymidine incorporation to bacterial growth. Cell number-based factors ranged from 1.1 x 10 to 38 x 10 cells mol of total thymidine incorporation and varied with treatment up to 10-fold for the same initial bacterial assemblage. In contrast, cell biovolume-based conversion factors were similar for two treatment groups across a 16-fold range of [H]thymidine incorporation rates: 5.54 x 10 mum mol of total thymidine incorporation and 15.2 x 10 mum mol of nucleic acid incorporation. Much of the variation in cell number-based conversion factors was related to changes in apparent mean cell volume of produced bacteria. Phosphorus addition stimulated [H]thymidine incorporation more than it increased bacterial growth, which resulted in low conversion factors.     

H]thymidine incorporation to estimate growth rates of anaerobic bacterial strains

The incorporation of [H]thymidine by axenic cultures of anaerobic bacteria was investigated as a means to measure growth. The three fermentative strains and one of the methanogenic strains tested incorporated [H]thymidine, whereas the sulfate-reducing bacterium and two of the methanogenic bacteria were unable to incorporate [H]thymidine during growth. It is concluded that the [H]thymidine incorporation method underestimates bacterial growth in anaerobic environments.     

Inhibition of Macrophage DNA Synthesis by Immunomodulators

Oil-induced guinea pig peritoneal exudate macrophages were found to incorporate actively [³H]thymidine without any tissue fluids such as conditioned medium, lymphokines or inflammatory tissue exudates. The [³H]thymidine incorporation was markedly suppressed by macrophage stimulants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS), while glucosamine incorporation was simultaneously increased by these stimulants. The degree of suppression of thymidine incorporation depended on the cell density, the concentrations of the stimulants, and sera or culture media used. The exposure of macrophages to MDP for 30 min was sufficient to cause significant suppression.     

Thymidine incorporation into lake water bacterioplankton and pure cultures of chemolithotrophic (CO, H2) and methanotrophic bacteria

Abstract Incorporation of [³H]methyl thymidine into bacterial DNA was measured using samples of bacterioplankton from Lake Constance and pure cultures of CO, H₂ and CH₄-oxidizing bacteria. Thymidine was incorporated by Pseudomonas carboxydovorans, Paracoccus denitrificans, Methylosinus trichosporium, Methylomonas agile , and by various chemolithotropic or methylotrophic isolates from Lake Constance. Thymidine incorporation by bacterial cultures was stimulated by increasing concentrations of CO or H₂. Increased CH₄ concentrations stimulated thymidine incorporation by Ms. trichosporium only if the cells had been starved. In contrast to bacterial cultures, thymidine incorporation by bacterioplankton samples was not stimulated by increasing     

Antagonism between interleukin 3 and erythropoietin in mice with azidothymidine-induced anemia and in bone marrow endothelial cells

Azidothymidine (AZT)-induced anemia in mice can be reversed by the administration of IGF-IL-3 (fusion protein of insulin-like growth factor II (IGF II) and interleukin 3). Although interleukin 3 (IL-3) and erythropoietin (EPO) are known to act synergistically on hematopoietic cell proliferation in vitro, injection of IGF-IL-3 and EPO in AZT-treated mice resulted in a reduction of red cells and an increase of plasma EPO levels as compared to animals treated with IGF-IL-3 or EPO alone. We tested the hypothesis that the antagonistic effect of IL-3 and EPO on erythroid cells may be mediated by endothelial cells. Bovine liver erythroid cells were cultured on monolayers of human bone marrow endothelial cells previously treated with EPO and IGF-IL-3. There was a significant reduction of thymidine incorporation into both erythroid and endothelial cells in cultures pre-treated with IGF-IL-3 and EPO. Endothelial cell culture supernatants separated by ultrafiltration and ultracentrifugation from cells treated with EPO and IL-3 significantly reduced thymidine incorporation into erythroid cells as compared to identical fractions obtained from the media of cells cultured with EPO alone. These results suggest that endothelial cells treated simultaneously with EPO and IL-3 have a negative effect on erythroid cell production.     

3H]thymidine incorporation into whole liver as an alternative to [3H]thymidine incorporation into DNA as a parameter of cell proliferation in regenerating liver tissue in rats

OBJECTIVE: To monitor liver regeneration following partial hepatectomy, liver cell proliferation can be measured by assaying in vivo [3H]thymidine incorporation into liver cell DNA. We hypothesized that [3H]thymidine incorporation into whole liver tissue parallels [3H]thymidine incorporation into liver cell DNA, both in high proliferating and low proliferating liver. STUDY DESIGN: Liver cell proliferation in rats after partial hepatectomy or a sham operation was studied by measuring incorporation of [3H]thymidine into various fractions of liver tissue on days 1, 2, 3, 4 and 10 after surgery. RESULTS: [3H]thymidine incorporation into whole liver tissue and in the protein fraction correlated well with DNA-specific [3H]thymidine incorporation into regenerating (r > .80, P < .0001) and nonregenerating liver (r > .69, P < .005). [3H]thymidine incorporation into DNA was < 5% of the total amount of administered [3H]thymidine in both sham-operated and hepatectomized rats. Significant differences in [3H]thymidine incorporation into partially hepatectomized livers as compared to sham-operated rat livers were found on days 1 and 2 (whole liver tissue and protein fraction) or day 1 (DNA) after surgery. CONCLUSION: [3H]thymidine incorporation into whole liver tissue is a simple technique that can be used for the study of liver cell proliferation after partial hepatectomy in rats.     

Synthesis of DNA in the liver and testes of cadmium-tested partially hepatectomized rats.

Cadmium affects the induction of thymidine and thymidylate kinases in regenerating rat liver. EDTA administered simultaneously with cadmium reverses its inhibitory action on enzyme synthesis, and prevents the depression of thymidine incorporation into DNA observed in cadmium-treated animals. Zinc does not abolish the inhibitory action of cadmium on the synthesis of DNA in regenerating liver, and the incorporation of thymidine into DNA in the testes was inhibited more by intraperitoneal injection of cadmium plus zinc than by injection of cadmium alone. Inhibition of thymidine incorporation into DNA in the liver and testes was proportional to the amount of cadmium administered up to about 2 mg CdCl2/kg body weight, but surprisingly, higher doses of cadmium caused less inhibition.     

Soluble age-related factors from skeletal muscle which influence muscle development

Successful regeneration of damaged striated muscle in adult mice is dependent on the regeneration of newly differentiated myofibers from proliferating satellite cells and inhibition of scar tissue formation by fibroblasts. As with most tissues, the ability of skeletal muscle to regenerate decreases in older animals. In this study, we have analysed soluble extracts from intact and regenerating skeletal muscle from mice of different ages for their ability to affect avian myogenesis in tissue culture. We were interested in determining whether an age-dependent difference could be detected with this tissue culture bioassay system. Total cell proliferation in the cultures, measured by [3H]thymidine incorporation was increased equally by muscle extracts from both young and older mice but the resulting cell populations differed in proportion of cell types. The ratio of myoblasts to fibroblasts was significantly greater in cultures exposed to extracts from younger mouse muscle as compared with cultures exposed to extracts from older animals. This age-related activity was found to reside in a low molecular weight (MW) (greater than 12 kD) component of the extract. This fraction had dissimilar effects on myoblasts and fibroblasts. Relative to saline controls, myoblast proliferation was increased and fibroblast proliferation decreased. The low MW fraction from younger mouse muscle extracts stimulated myogenic cell proliferation and myotube formation to a greater extent than the similar fraction prepared from older mouse muscle. Conversely, younger mouse muscle fractions had significantly greater inhibitory activity against fibroblast proliferation than did older mouse muscle fractions.     

Modulation of EL-4 mouse lymphoma cell proliferation by macrophages and tumor related factors

It is well known that macrophages play an important role in the control of tumor growth. This control may be the result of a direct action of macrophages or mediated by several biologically active products or factors elaborated by these and other cell populations. Our studies on the proliferation of a murine T-cell lymphoma (EL-4) showed that the treatment of the ascitic fluid (from the peritoneum of EL-4 bearing mice) with carbonyl iron resulted in a depletion of phagocytes concomitant with a significant increase of [3H] thymidine uptake by EL-4 cells. Further, the growth of EL-4 cells cultured in semisolid agar was significantly inhibited by an underlayer of large quantities of macrophages both from normal and EL-4 bearing mice as well as when cultured in the presence of PGE2. The underlayer of tumor macrophages P388 D1 resulted in an increase of the EL-4 cell growth. Also, conditioned media obtained from in vitro liquid cultures of EL-4 cells and L 1210 cells (B-lymphoma) produced a remarkable inhibition of the in vitro cloning capacity and [3H] thymidine uptake by EL-4 cells. These data support the hypothesis that different factors from normal and hemopoietic tumor cells may control the tumor growth and point out that self-produced factors may modulate the proliferation of tumor cells.     

Effect of different factors on proliferation of antler cells, cultured in vitro

Antlers as a potential model for bone growth and development have become an object of rising interest. To elucidate processes explaining how antler growth is regulated, in vitro cultures have been established. However, until now, there has been no standard method to cultivate antler cells and in vitro results are often opposite to those reported in vivo. In addition, many factors which are often not taken into account under in vitro conditions may play an important role in the development of antler cells. In this study we investigated the effects of the antler growth stage, the male individuality, passaged versus primary cultures and the effect of foetal calf serum concentrations on proliferative potential of mixed antler cell cultures in vitro, derived from regenerating antlers of red deer males (Cervus elaphus). The proliferation potential of antler cells was measured by incorporation of (3)H thymidine. Our results demonstrate that there is no significant effect of the antler growth stage, whereas male individuality and all other examined factors significantly affected antler cell proliferation. Furthermore, our results suggest that primary cultures may better represent in vivo conditions and processes occurring in regenerating antlers. In conclusion, before all main factors affecting antler cell proliferation in vitro will be satisfactorily investigated, results of in vitro studies focused on hormonal regulation of antler growth should be taken with extreme caution.     

[3H]Thymidine Incorporation To Estimate Growth Rates of Anaerobic Bacterial Strains

The incorporation of [³H]thymidine by axenic cultures of anaerobic bacteria was investigated as a means to measure growth. The three fermentative strains and one of the methanogenic strains tested incorporated [³H]thymidine, whereas the sulfate-reducing bacterium and two of the methanogenic bacteria were unable to incorporate [³H]thymidine during growth. It is concluded that the [³H]thymidine incorporation method underestimates bacterial growth in anaerobic environments.     

Isolation of a growth and mitosis inhibitory peptide from mouse liver

The isolation of a liver peptide that inhibits the growth, mitosis rate and thymidine incorporation in regenerating liver is described. The peptide has the structure Pyroglu-gln-gly-ser-asn, and the deamidated forms are also active. The peptide probably belongs to a class of growth inhibitors with a high degree of tissue specificity. Two such peptides have previously been isolated from the epidermis (Reichelt et al. 1987) and from colonic tissue (Skraastad et al. 1987).     

Isolation of a growth and mitosis inhibitory peptide from mouse liver

The isolation of a liver peptide that inhibits the growth, mitosis rate and thymidine incorporation in regenerating liver is described. The peptide has the structure Pyroglu-gln-gly-ser-asn, and the deamidated forms are also active. The peptide probably belongs to a class of growth inhibitors with a high degree of tissue specificity. Two such peptides have previously been isolated from the epidermis (Reichelt et al. 1987) and from colonic tissue (Skraastad et al. 1987).     

Effects of fetal versus postnatal sera upon adipose tissue stromal-vascular cells in primary culture

Summary This experiment was conducted to determine if serum factors are responsible for differences in cellularity of prenatal and postnatal pig adipose tissue as determined by in vitro measurement of cellular proliferation and enzyme-histochemical metabolic development. Cellular proliferation of stromal-vascular cells derived from rat inguinal adipose tissue was measured by [³H]-thymidine incorporation. Coverslip cultures were used for analysis of histochemical differentiation. Cells were incubated in media containing 10% fetal bovine, fetal pig, mature pig, or various combinations of these sera. Fetal bovine serum promoted more [³H]-thymidine incorporation than fetal or postnatal pig sera. Fetal pig sera also stimulated more [³H]-thymidine incorporation than mature pig sera. Sera from adult pigs promoted differentiation and lipid filling of adipocytes. Fetal pig sera stimulated histochemical expression of enzymes, but did not induce lipid filling. Fetal bovine serum produced histochemically undifferentiated cells. Addition of fetal bovine serum to media containing mature pig sera reduced lipid accumulation and histochemical reactivity of cells. This effect of fetal serum was thus due to specific inhibition of lipid deposition and not substrate restriction. These experiments demonstrated that serum factors have a major influence on morphological development of fetal and postnatal adipose tissue.     

Calculation of cell production from [h]thymidine incorporation with freshwater bacteria

The conversion factor for the calculation of bacterial production from rates of [H]thymidine incorporation was examined with diluted batch cultures of freshwater bacteria. Natural bacterial assemblages were grown in aged, normal, and enriched media at 10 to 20 degrees C. The generation time during 101 growth cycles covered a range from 4 to >200 h. The average conversion factor was 2.15 x 10 cells mol of thymidine incorporated into the trichloroacetic acid (TCA) precipitate (standard error = 0.29 x 10; n = 54), when the generation time exceeded 20 h. At generation times of <20 h, the average conversion factor was 11.8 x 10 cells mol of thymidine incorporated into TCA precipitate (standard error = 1.72 x 10; n = 47). The amount of radioactivity in purified DNA increased with decreasing generation time and increasing conversion factor (calculated from the TCA precipitate), corresponding to a decrease in the percentage in protein. The conversion factors calculated from purified DNA or from the TCA precipitate gave the same variability. Conversion factors did not change significantly with the medium, but were significantly higher at 20 degrees C than at 15 and 10 degrees C. A detailed examination of the [H]thymidine concentrations that were needed to achieve maximum labeling in DNA was carried out 6 times during a complete growth cycle. During periods with low generation times and high conversion factors, 15 nM [H]thymidine was enough for the maximum labeling of the TCA precipitate. This suggests that incorporation of [H]thymidine into DNA is probably limited by uptake during periods with generation times of <20 h and that freshwater bacterioplankton cell production sometimes is underestimated when a conversion factor of 2.15 x 10 cells mol of thymidine incorporated is used.     

Bacterial Growth in Mixed Cultures on Dissolved Organic Carbon from Humic and Clear Waters

Interactions between bacterial assemblages and dissolved organic carbon (DOC) from different sources were investigated. Mixed batch cultures were set up with water from a humic and a clear-water lake by a 1:20 dilution of the bacterial assemblage (1.0 μm of prefiltered lake water) with natural medium (sterile filtered lake water) in all four possible combinations of the two waters and their bacterial assemblages. Bacterial numbers and biomass, DOC, thymidine incorporation, ATP, and uptake of glucose and phenol were followed in these cultures. Growth curves and exponential growth rates were similar in all cultures, regardless of inoculum or medium. However, bacterial biomass produced was double in cultures based on water from the humic lake. The fraction of DOC consumed by heterotrophic bacteria during growth was in the same range, 15 to 22% of the total DOC pool, in all cultures. Bacterial growth efficiency, calculated from bacterial biomass produced and DOC consumed, was in the order of 20%. Glucose uptake reached a peak during exponential growth in all cultures. Phenol uptake was insignificant in the cultures based on the clear-water medium, but occurred in humic medium cultures after exponential growth. The similarity in the carbon budgets of all cultures indicated that the source of the bacterial assemblage did not have a significant effect on the overall carbon flux. However, fluxes of specific organic compounds differed, as reflected by glucose and phenol uptake, depending on the nature of the DOC and the bacterial assemblage.     

Estimates of bacterial growth from changes in uptake rates and biomass.

Rates of nucleic acid synthesis have been used to examine microbiol growth in natural waters. These rates are calculated from the incorporation of [3H]adenine and [3H]thymidine for RNA and DNA syntheses, respectively. Several additional biochemical parameters must be measured or taken from the literature to estimate growth rates from the incorporation of the tritiated compounds. We propose a simple method of estimating a conversion factor which obviates measuring these biochemical parameters. The change in bacterial abundance and incorporation rates of [3H]thymidine was measured in samples from three environments. The incorporation of exogenous [3H]thymidine was closely coupled with growth and cell division as estimated from the increase in bacterial biomass. Analysis of the changes in incorporation rates and initial bacterial abundance yielded a conversion factor for calculating bacterial production rates from incorporation rates. Furthermore, the growth rate of only those bacteria incorporating the compound can be estimated. The data analysis and experimental design can be used to estimate the proportion of nondividing cells and to examine changes in cell volumes.     

Quantitative protein sequencing using mass spectrometry: thermally induced formation of thiohydantoin amino acid derivatives from N-methyl- and N-phenylthiourea amino acids and peptides in the mass spectrometer

A simple technique is described for continuous labeling of mammalian DNA with radioactive precursors adsorbed onto activated charcoal and administered by a single intraperitoneal injection. It is shown that adsorbed labeled thymidine is available in the organism for incorporation into DNA for at least 50 hr.The same technique can be used to substitute up to 20% of the thymidine with 5-bromodeoxyuridine in regenerating rat liver.     

Long-term primary culture of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver

Summary Long-term primary cultures of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver have been established. Nearly homogenous (>97%) populations of hepatocytes were placed into primary culture and remained viable and proliferative for at least 70 d. In addition to hepatocytes, proliferative biliary cells persisted in the cultures for at least 30 d. Finally, a third type of epithelial cell, which we have termed a “spindle cell,” consistently appeared and proliferated to confluence in these cultures. The confluent cultures of spindle cells were successfully subcultured and passaged. The initial behavior, growth, and optimization of serum and media requirements for these cells is described. All three cell types proliferated as measured by thymidine incorporation, autoradiography, proliferating cellular nuclear antigen analysis, and propidium iodine staining. Further efforts to characterize the cells included western blotting and immunohistochemical staining with antibodies to cytokeratins previously reported in fish liver. From these data, it appears that all three cell populations are epithelial in nature. Furthermore, significant changes in actin organization, often indicative of transformation or pluripotent cells, were observed with increased time in primary culture.