Interferon inhibition of thymidine incorporation into DNA through effects on thymidine transport and uptake

Replenishment of medium after 72 hr of growth of HeLa-S3 cells in dense suspension cultures increased [³H]-thymidine uptake into cells and incorporation into DNA, with the levels reaching a peak ～ 12 hr following medium change; β interferon inhibits the enhanced uptake of [³H]-thymidine and labeling of DNA in a dose-dependent manner. Some reduction in these processes is observed at a concentration as low as 1 u/ml, and ～ 75% inhibition at 640 u/ml. Kinetic analysis has revealed that the rate of labeling of the acid-soluble pool with [³H]-thymidine, measured either at 22°C, or 37°C, is reduced in interferon-treated (640 u/ml, 24 hr) HeLa-S3 cells. At 22°C, the initial rate of thymidine transport at a high (500 μM) thymidine concentration, determined within the first 30 sec of [³H]-thymidine addition was depressed by 44% in interferon-treated HeLa cells. At 37°C, labeled precursors accumulate in acid-soluble material for ～ 8 min after the addition of [³H]-thymidine, after which an apparent equilibrium level is attained. At this temperature, the rate of thymidine uptake and the apparent equilibrium level attained were depressed by 70% in interferon-treated HeLa cells. The reduced incorporation of [³H]-thymidine into DNA in interferon-treated HeLa-S3 cells can be largely explained by interferon inhibition of thymidine transport and phosphorylation.     

RADIOLABELING STUDIES OF LIPIDS AND FATTY ACIDS IN NANNOCHLOROPSIS (EUSTIGMATOPHYCEAE), AN OLEAGINOUS MARINE ALGA1

The synthesis of fatty acids and lipids in Nannochloropsis sp. was investigated by labeling cells in vivo with [¹⁴C]-bicarbonate or [¹⁴C]-acetate. [¹⁴C]-bicarbonate was incorporated to the greatest extent into 16:0, 16:1, and 14:0 fatty acids, which are the predominant fatty acids of triacylglycerols. However, more than half of the [¹⁴C]-acetate was incorporated into longer and more desaturated fatty acids, which are constituents of membrane lipids. [¹⁴C]-acetate was incorporated most strongly into phosphatidylcholine, which rapidly lost label during a 5-h chase period. The label associated with phosphatidylethanolamine also decreased during the chase period, whereas label in other membrane lipids and triacylglycerol increased. The dynamics of labeling, along with information regarding the acyl compositions of various lipids, suggests that 1) the primary products of chloroplast fatty acid synthesis are 14:0, 16:0, and 16:1; 2) C₂₀ fatty acids are formed by an elongation reaction that can utilize externally supplied acetate; 3) phosphatidylcholine is a site for desaturation of C₁₈ fatty acids; and 4) phosphatidylethanolamine may be a site for desaturation of C₂₀ fatty acids.     

Genistein abolishes nucleoside uptake by cardiac fibroblasts

Genistein, a soy isoflavone, is reported to exert significant beneficial action in several human disorders, which has generated immense interest in the mechanisms underlying its effects on diverse cellular processes. It has anti-proliferative action on many cell types, an effect generally attributed to tyrosine kinase inhibition. In this study, genistein was found to cause total inhibition of [³H]-thymidine incorporation into DNA and a modest reduction in [³H]-proline incorporation into protein in primary cultures of cardiac fibroblasts. The decrease in [³H]-thymidine incorporation was not associated with a decrease in cell proliferation but correlated exactly with low intracellular levels of [³H]-thymidine. Genistein dramatically reduced [³H]-thymidine but not [³H]-proline uptake by these cells in which the equilibrative nucleoside transporter may be the major route of nucleoside uptake. The effect was irreversible and was demonstrable in pulmonary fibroblasts as well. The findings suggest that nucleoside uptake mechanisms may be a novel target of genistein action in cardiac fibroblasts and point to serious limitations in using genistein to assess the role of tyrosine kinase in cell proliferation by the standard technique of [³H]-thymidine incorporation.     

Discrimination of two fibroblast progenitor populations in early explant cultures of hamster gingiva

Summary Numerous metabolic studies have demonstrated heterogeneity of fibroblast populations in culture, yet little is known about the structure of fibroblast populations in adult tissues in vivo. To determine if populations of both cycling and non-cycling cells are present in gingiva, hamsters were labelled with [³H]-thymidine to label cycling cells in vivo, and explanted biopsies were subsequently incubated with bromodeoxyuridine to label cycling cells in vitro. Cycling cells were identified by combined immunohistochemistry and radioautography. Fibroblasts were recognized by the presence of vimentin and the absence of keratin as determined by immuno-fluorescence. The largest proportion of cells were double-labelled with [³H]-thymidine and bromodeoxyuridine (43.8%) indicating the presence of actively cycling populations that maintained their proliferative status upon explantation. Cultures also exhibited a second population of cells labelled only with bromodeoxyuridine (38.7%) that did not cycle in vivo, but retained the capacity for proliferation in vitro. However, limiting dilution analysis of single-cell suspensions revealed only a single class of progenitors capable of forming large colonies in vitro. Approximately 1 in 190 plated cells was capable of colony-formation, indicating that, upon explantation, a subset of the cycling cells in vitro exhibits extensive proliferative capacity. There was also a small population of cells unlabeled with either [³H]-thymidine or bromodeoxyuridine (9.4%) that appeared to be terminally differentiated. Different substrates, including glass and thin films of gelatin and collagen, did not significantly alter the fraction of cells labelled with [³H]-thymidine. These data demonstrate the existence of 2 separate progenitor-cell populations with different capacities for proliferation in vivo and in vitro.     

A batch procedure for the assay of cyclic nucleotide phosphodiesterase.

A procedure for the assay of cyclic nucleotide phosphodiesterase is described in which labeled cyclic nucleotide is separated from labeled nucleoside by the batchwise addition of ethanolic slurries of Dowex 2 fluoride. Under the conditions described there is no detectable adsorption of nucleoside by the anion exchanger, which removes more than 95% of the tritium in boiled samples of [8-³H]cAMP or [8-³H]cGMP. Linear time courses and enzyme vs activity relationships are described for 10^?3 and 10^?7m cAMP and 10^?4m cGMP. The method is limited by interference by neutral salts and by the enzymatic conversion of adenosine into inosine.     

The use of 14C-labeled bacteria as a tracer of ingestion and metabolism of bacterial biomass by microbial grazers

A method for radiolabeling marine bacteria with d-[U-¹⁴C] glucose and a radiotracer method for measuring ingestion and metabolism of bacterial biomass by ciliated protozoa and other microzooplankton are presented. Problems associated with using live bacterial tracers, i.e., label retention, label recycling, tracer cell size and morphology, and intracellular distribution of label are evaluated.Bacterioplankton assemblages collected from field samples incorporated and retained label as efficiently as coastal isolates which were selected for glucose incorporation. Under grazing experimental conditions, labeled bacteria retained a high proportion of the label (hourly net loss = 1.2%). Bacterial recycling of released dissolved organic ¹⁴C (DO¹⁴C) was 0–2.2% of total ¹⁴C per h. Addition of labeled assemblages to nearshore water samples did not detectably alter mean cell size or size frequency distribution of the attendant bacterioplankton assemblages.In grazing experiments with cultured ciliates (Euplotes sp. and Uronema sp.), radioassay parameters varied as direct functions of predator and prey concentrations. Microautoradiographic analysis corroborated that ¹⁴C incorporation measured in the radioassay by filtration techniques primarily represented ingested bacterial biomass and that problems associated with attached and adsorbed labeled bacteria were minimized. Grazing experiments conducted with bacteria labeled with [U-¹⁴C]glucose yielded ingestion rates comparable to bacteria labeled with [U-¹⁴C]thymidine and additionally provided respiration and exudation rates.     

3H]-thymidine labelling of DNA triggers apoptosis potentiated by E1A-adenoviral protein

[³H]-thymidine is commonly used to analyze the accumulation of [³H]-labeled chromatin fragments in cells undergoing apoptosis. This study shows that [³H]-thymidine incorporation within DNA is sufficient per se to inhibit growth and to induce apoptosis in canine kidney epithelial cells and porcine aorta endothelial cells. Despite high-level [³H]-thymidine-DNA labeling, rat vascular smooth muscle cells (VSMC) showed only modest inhibition of growth and induction of apoptosis compared to other cell types. Similarly to serum deprivation, apoptosis triggered by [³H]-thymidine labeling was sharply potentiated by VSMC transfection with a functional analogue of c-myc, E1A-adenoviral protein (VSMC-E1A), and was suppressed by stimulation of cAMP signaling with forskolin as well as by and Na/K pump inhibition with ouabain. Both apoptosis induction and growth suppression seen in [³H]-thymidine-treated VSMC-E1A were reduced by the pan-caspase inhibitor z-VAD.fmk. Thus, our results show that the differential efficiency of the apoptotic machinery determines cell type-specific attenuation of growth in cells with [³H]-thymidine-labeled DNA. They also demonstrate that [³H]-thymidine-treated and serum-deprived VSMC employ common intermediates of the apoptotic machinery, including steps that are potentiated by E1A-adenoviral protein and inhibited by activation of cAMP signaling as well as by inversion of the intracellular [Na⁺] _i /[K⁺] _i ratio.     

Ethanol Extraction Requirement for Purification of Protein Labeled with [3H]Leucine in Aquatic Bacterial Production Studies

The trichloroacetic acid (TCA)-insoluble fraction of water column bacteria labeled with [³H]leucine contained an ethanol-soluble fraction accounting for up to 44% of the label. A component of the ethanol-soluble fraction is [³H]leucine. Labeled-protein purification requires an ethanol wash step. Cold TCA can replace hot TCA for precipitation of labeled proteins.     

Requirement for a Microbial Consortium To Completely Oxidize Glucose in Fe(III)-Reducing Sediments

In various sediments in which Fe(III) reduction was the terminal electron-accepting process, [¹⁴C]glucose was fermented to ¹⁴C-fatty acids in a manner similar to that observed in methanogenic sediments. These results are consistent with the hypothesis that, in Fe(III)-reducing sediments, fermentable substrates are oxidized to carbon dioxide by the combined activity of fermentative bacteria and fatty acid-oxidizing, Fe(III)-reducing bacteria.     

Glucose Metabolism in Sediments of a Eutrophic Lake: Tracer Analysis of Uptake and Product Formation

The uptake of glucose and the formation of end products from glucose catabolism have been measured for sediments of eutrophic Wintergreen Lake with a combination of tritiated and ¹⁴C-labeled tracers. Time course analyses of the loss of [³H]glucose from sediments were used to establish rate constants for glucose uptake at natural substrate concentrations. Turnover times from these analyses were about 1 min for littoral and profundal sediments. No seasonal or site differences were noted in turnover times. Time course analyses of [U-¹⁴C]glucose uptake and ¹⁴C-labeled end product formation indicated that glucose mass flow could not be calculated from end product formation since the specific activity of added [¹⁴C]glucose was significantly diluted by pools of intracellular glucose and glucose metabolites. Mass flow could only be accurately estimated by use of rates of uptake from tracer studies. Intermediate fermentation end products included acetate (71%), propionate (15%), lactate (9%), and only minor amounts of butyrates or valerates. Addition of H₂ to sediments resulted in greater production of lactate (28%) and decreased formation of acetate (50%), but did not affect glucose turnover. Depth profiles of glucose uptake indicated that rates of uptake decreased with depth over the 0- to 18-cm interval and that glucose uptake accounted for 30 to 40% of methanogenesis in profundal sediments.     

Utility of the modified ATP III defined metabolic syndrome and severe obesity as predictors of insulin resistance in overweight children and adolescents: a cross-sectional study

Background

Inhibition of vascular smooth muscle cell (vSMC) proliferation by oral anti-hyperglycemic agents may have a role to play in the amelioration of vascular disease in diabetes. Thiazolidinediones (TZDs) inhibit vSMC proliferation but it has been reported that they anomalously stimulate [³H]-thymidine incorporation. We investigated three TZDs, two biguanides and two sulfonylureas for their ability of inhibit vSMC proliferation. People with diabetes obviously have fluctuating blood glucose levels thus we determined the effect of media glucose concentration on the inhibitory activity of TZDs in a vSMC preparation that grew considerably more rapidly under high glucose conditions. We further explored the mechanisms by which TZDs increase [³H]-thymidine incorporation.

Methods

VSMC proliferation was investigated by [³H]-thymidine incorporation into DNA and cell counting. Activation and inhibition of thymidine kinase utilized short term [³H]-thymidine uptake. Cell cycle events were analyzed by FACS.

Results

VSMC cells grown for 3 days in DMEM with 5% fetal calf serum under low (5 mM glucose) and high (25 mM glucose) increased in number by 2.5 and 4.7 fold, respectively. Rosiglitazone and pioglitazone showed modest but statistically significantly greater inhibitory activity under high versus low glucose conditions (P < 0.05 and P < 0.001, respectively). We confirmed an earlier report that troglitazone (at low concentrations) causes enhanced incorporation of [³H]-thymidine into DNA but did not increase cell numbers. Troglitazone inhibited serum mediated thymidine kinase induction in a concentration dependent manner. FACS analysis showed that troglitazone and rosiglitazone but not pioglitazone placed a slightly higher percentage of cells in the S phase of a growing culture. Of the biguanides, metformin had no effect on proliferation assessed as [³H]-thymidine incorporation or cell numbers whereas phenformin was inhibitory in both assays albeit at high concentrations. The sulfonylureas chlorpropamide and gliclazide had no inhibitory effect on vSMC proliferation assessed by either [³H]-thymidine incorporation or cell numbers.

Conclusion

TZDs but not sulfonylureas nor biguanides (except phenformin at high concentrations) show favorable vascular actions assessed as inhibition of vSMC proliferation. The activity of rosiglitazone and pioglitazone is enhanced under high glucose conditions. These data provide further in vitro evidence for the potential efficacy of TZDs in preventing multiple cardiovascular diseases. However, the plethora of potentially beneficial actions of TZDs in cell and animal models have not been reflected in the results of major clinical trials and a greater understanding of these complex drugs is required to delineate their ultimate clinical utility in preventing macrovascular disease in diabetes.     

Phenotype expression of human bone cells cultured on implant substrates

Bone cells derived from the human jaw were cultured on titanium, titanium coated with hydroxyapatite (THA) or with plasma spray (TPS) to study the behaviour of the cells anchored to implant substrates. Bone cells were cultured in MEM with the addition of [³H]-thymidine to evaluate cellular proliferation, and [³H]-glucosamine to evaluate GAG synthesis and accumulation in the extra-cellular matrix (ECM). Moreover, to study the degradation of GAG bone cells were cultured in the presence of NH₄Cl, an amine known to inhibit lysosomal activity. Our results show that TPS is the substrate that favours both cellular proliferation and the accumulation of GAG in the ECM. © 1997 John Wiley & Sons, Ltd.     

Tumor diagnosis by pet: potential of seven tracers examined in five experimental tumors including an artificial metastasis model

The potential of seven tracers for the metabolic imaging of tumors by positron emission tomography was studied using five experimental tumor models. The tracers examined were 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]FDG), 2-deoxy-2-[¹⁸F]fluoro-d-galactose (2-[¹⁸F]FdGal) and 2-deoxy-2-[¹⁸F]fluoro-l-fucose (2-[¹⁸F]FdFuc) for investigating energy metabolism. l-[methyl-¹¹C]Methionine ([¹¹C]Met) and 6-[¹⁸F]fluoro-l-fucose (6-[¹⁸F]FFuc) were used for assessing protein and glycoprotein synthesis, while [³H]thymidine ([³ H]Thd) and 2-deoxy-5′-[¹⁸F]fluorouridine ([¹⁸F]FdUrd) were used to investigate nucleic acid metabolism. The highest mean uptake by the five different tumors was found for [³H]Thd, followed in order by [¹⁸F]FDG, [¹¹C]Met, 2-[¹⁸F]FdGal, [¹⁸F]FdUrd, 2-[¹⁸F]FdFuc and 6-[¹⁸F]FFuc. The tumor-to-tissue uptake ratios indicated that the nucleosides, [¹¹C]Met and 6-[¹⁸F]FFuc were better tracers in the brain region. All the tracers except for the fucose analogs were suitable for the thoracic region, while [¹¹C]Thd and [¹⁸ F]FDG were superior in the abdominal region. In comparison with the primary tumor model of Lewis lung carcinoma (3LL), [³H]Thd uptake in the artificial metastatic 3LL model showed the maximum enhancement, followed by [¹⁸F]FDG, [¹¹C]Met and the other tracers. The [¹⁸F]FDG uptake correlated with the [³H]Thd uptake. [¹⁸F]FdUrd, 6-[¹⁸F]FFuc and 2-[¹⁸F]FdGal could be used for distinguishing different types of tumors. The combined use of these radiotracers can possibly allow the assessment of tumor metabolism, and this indicates the viability of tumors.     

Autoradiographic Studies of [methyl-3H]Thymidine Incorporation in a Cyanobacterium (Microcystis wesenbergii)-Bacterium Association and in Selected Algae and Bacteria

The present investigation showed by means of autoradiography that the cyanobacterium Microcystis wesenbergii did not incorporate [³H]thymidine at nanomolar concentrations, whereas its associated heterotrophic bacteria appearing in the gelatinous cover of the cyanobacterium became labeled. Several other tested cyaobacteria and algae did not incorporate [³H]thymidine.     

Changes of colon epithelium proliferation due to individual aging with cyclin proliferating cell nuclear antigen (PCNA/cyclin) immunostaining compared to [3H]-thymidine radioautography

We report a change in the proliferative activity of mouse colonic epithelium due to development and aging. In order to measure the proliferative activity, colonic epithelium was immunostained for cyclin proliferating cell nuclear antigen (PCNA/cyclin), which appears from the Gl to the S phase of the cell cycle, and compared with labeling obtained by [³H]-thymidine radioautography. Litter mice of six age groups from the fetal period (embryonic day 19), newborn period (postnatal day 1), suckling period (postnatal day 5), weaning period (postnatal dy 21), adult period (2 month old) to the senescent period (11 month old) were examined by immunohistochemistry. The descending colons were fixed in methacarn (method-Carnoy) and embedded in paraffin. Sections were stained for PCNA/cyclin activity using 19A2 monoclonal antibody and the avidin-biotin peroxidase complex (ABC) technique. For radioautography, litter mice of nine age groups using in vivo intraperitoneal administration of [³H]-thymidine. The labeling indices of colonic epithelial cells in the proliferative zone were then analyzed and compared between the two investigative methods. Our results show that the prliferative activity of mice colon was high in the fetal and newborn periods and almost constant from the suckling period to senescence, as demonstrated by both PCNA/cyclin immunohistochemistry and [³H]-thymidine radioautography. The labeling index seen by PCNA/cyclin immunohistochemistry was, however, higher than that seen by [³H]-thymidine radioautography.     

Effects of different vertebrate growth factors on primary cultures of hemocytes from the gastropod mollusc,Haliotis tuberculata

Summary— A useful experimental system from primary cultures of hemocytes from Haliotis tuberculata has been established. Six days after initiation of the culture, the viability of hemocytes remained constant as measured by the MTT assay. In addition, hemocytes showed physiological responses as judged by protein and DNA syntheses in response to treatment with vertebrate growth factors. Porcine insulin and human epidermal growth factor (EGF) stimulated [³H]-leucine and [³H]-thymidine incorporation in hemocytes in a dose-dependent manner. No additive effect of insulin and EGF is observed either for [³H]-leucine or for [³H]-thymidine incorporation. The response of primary cultures of abalone hemocytes to vertebrate growth factors confirms their growth potential in vitro and provides a suitable model for further studies on regulation of the control of cellular processes such as cell growth, differentiation and migration in invertebrate cells.     

Microbial biomass and activity in subsurface sediments from Vejen,Denmark

Subsurface sediment samples were collected from 4 to 31 m below landsurface in glacio-fluvial sediments from the Quaternary period. The samples were described in terms of pH, electrical conductivity, chloride concentration, organic matter content, and grain size distribution. Viable counts of bacteria varied from 0.5 to 1,203 x 103 colony forming units/g dry weight (gdw); total numbers of bacteria acridine orange direct counts (AODC) varied from 1.7 to 147 × 10⁷ cells/gdw; growth rates (incorporation of [³H]-thymidine) varied from 1.4 to 60.7 × 10⁴ cells/(gdw · day); and rate constants for mineralization of ¹⁴C-labelled compounds varied from 0.2 to 2.3 × 10^–3 ml/(dpm · day) for acetate, and from 0 to 2.0 × 10^–3 ml/(dpm · day) for phenol. Sediment texture influenced the total number of bacteria and potential for mineralization; with increasing content of clay and silt and decreasing content of sand, AODC increased and the mineralization rate declined. Intrinsic permeability calculated from grain size correlated positively with mineralization rate for acetate. Statistical correlation analysis showed high correlations between some of the abiotic parameters, but it was not possible to point out a single abiotic parameter that could explain the variation of size and activity of the microbial population. The microbial data obtained in these geologically young sediments were compared to literature data from older sediments, and this comparison showed that age and type of geological formation might be important for the size and activity of the microbial populations.Offprint requests to: H.-J. Albrechtsen.     

Uninterrupted DNA synthesis during S-phase in untransformed diploid hamster fibroblasts

Untransformed Syrian hamster fibroblasts in exponential growth were exposed to a pulse of [³H]-thymidine for 5 min, followed immediately by bromodeoxyuridine, and serial samples were taken up to 16 h. Preparations were autoradiographed and stained for replication banding. No cell with replication bands was found without significant [³H]-thymidine uptake, although the extent of uptake varied between sub-phases of S. Thus there is no indication of a total cessation of synthesis at any period during S-phase.     

Synthesis of nucleic acids and localization of atrial natriuretic peptide in crayfish haemocytes

Three types of cells circulate in the haemolymph of the crayfish Astacus astacus, i.e., agranular haemocytes (HCs I), small-granule haemocytes (HCs II), and large-granule haemocytes (HCs III). Their proliferation, differentiation, and function remain poorly understood. Using light and electron microscopic autoradiography with [³H]-thymidine, we found that only HCs I are capable of DNA synthesis and mitosis whereas HCs II and HCs III are replicatively inactive. To verify whether HCs I are proliferating progenitor cells for granular HCs, we have analyzed autographs of the HC population 1, 2, 7, and 21 days after a single [3H]-thymidine administration. Contrary to our expectations, we have failed to find labeled HCs II and HCs III. These findings have raised doubts as to the capacity of HCs I to differentiate into two other types of HCs. With the use of ³H-uridine autoradiography, it was found that RNA synthesis was the most active in HCs I and 2 and 4 times lower in HCs II and HCs III, respectively. ANP-like immunoreactivity was revealed in large granules of the HCs III by electron microscopic immunocytochemistry. We assume that the presence of ANP in secretory granules extends the possible functions of crayfish HCs and suggests their participation in the regulation of the watersalt balance and immune response.     

A Caution in the Use of Tritiated Substrates for Monoamine Oxidase Assays

Abstract: A monoamine oxidase assay utilizing generally labeled [³H]-serotonin as substrate became nonlinear after only ～5% conversion of initial c.p.m. to product. Subsequent analysis showed that a significant proportion of the tritium label was readily exchangeable into water and that monoamine oxidase activity increased release of label as water. The use of generally labeled substrates for oxidase activities is not recommended.