Effect of 5-Fluoro-2′-Deoxyuridine on [3H]Thymidine Incorporation by Bacterioplankton in the Waters of Southwest Florida

The effect of 5-fluoro-2′-deoxyuridine (FdUrd) on [methyl-³H] thymidine incorporation by bacterioplankton populations in subtropical freshwater, estuarine, and oceanic environments was examined. In estuarine waters, intracellular isotope dilution was inhibited by FdUrd, which enabled us to estimate both intracellular and extracellular isotope dilution. In 2 of 10 cases, extracellular isotope dilution was significant. At low concentrations of [methyl-³H]thymidine or [6-³H]thymidine, FdUrd completely inhibited incorporation of radioactivity into protein and RNA. At high concentrations of [³H]thymidine, however, FdUrd had little effect on labeling patterns. The dihydrofolate reductase inhibitors amethopterin and trimethoprim had no effect on macromolecular labeling patterns. These results suggest that thymidylate synthase is not involved in nonspecific labeling and that FdUrd inhibits nonspecific labeling by blocking some other enzyme involved in thymidine catabolism. In oligotrophic oceanic and freshwater samples, FdUrd did not inhibit intracellular isotope dilution or [³H]thymidine labeling of protein and RNA, but caused some inhibition of [³H]thymidine incorporation into DNA. The ability of FdUrd to inhibit nonspecific macromolecular labeling during [³H]thymidine incorporation was significantly correlated (r = 0.84) with total thymidine incorporation (in picomoles per liter per hour). The results are discussed in terms of applications of FdUrd to routine bacterial production measurements and the general assumptions of [³H]thymidine incorporation.     

Underestimation of DNA synthesis by [h]thymidine incorporation in marine bacteria

A direct comparison of [H]thymidine incorporation with DNA synthesis was made by using an exponentially growing estuarine bacterial isolate and the naturally occurring bacterial populations in a eutrophic subtropical estuary and in oligotrophic offshore waters. Simultaneous measurements of [H]thymidine incorporation into DNA, fluorometrically determined DNA content, and direct counts were made over time. DNA synthesis estimated from thymidine incorporation values was compared with fluorometrically determined changes in DNA content. Even after isotope dilution, nonspecific macromolecular labeling, and efficiency of DNA recovery were accounted for, [H]thymidine incorporation consistently underestimated DNA synthesized by six- to eightfold. These results indicate that although the relationship of [H]thymidine incorporation to DNA synthesis appears consistent, there are significant sources of thymine bases incorporated into DNA which cannot be accounted for by standard [H]thymidine incorporation and isotope dilution assays.     

Underestimation of DNA Synthesis by [3H]Thymidine Incorporation in Marine Bacteria

A direct comparison of [³H]thymidine incorporation with DNA synthesis was made by using an exponentially growing estuarine bacterial isolate and the naturally occurring bacterial populations in a eutrophic subtropical estuary and in oligotrophic offshore waters. Simultaneous measurements of [³H]thymidine incorporation into DNA, fluorometrically determined DNA content, and direct counts were made over time. DNA synthesis estimated from thymidine incorporation values was compared with fluorometrically determined changes in DNA content. Even after isotope dilution, nonspecific macromolecular labeling, and efficiency of DNA recovery were accounted for, [³H]thymidine incorporation consistently underestimated DNA synthesized by six- to eightfold. These results indicate that although the relationship of [³H]thymidine incorporation to DNA synthesis appears consistent, there are significant sources of thymine bases incorporated into DNA which cannot be accounted for by standard [³H]thymidine incorporation and isotope dilution assays.     

Selected Nucleic Acid Precursors in Studies of Aquatic Microbial Ecology

The use of radiolabeled nucleosides and nucleic acid bases to estimate the rates of RNA and DNA synthesis in naturally occurring microbial assemblages requires numerous assumptions, several of which are evaluated herein. Comparative time series analyses of the uptake and incorporation, labeling specificity, and extent of catabolism of [2-³H]adenine, [methyl-³H]thymidine, and [5-³H]uridine were performed with pure bacterial and algal cultures, as well as with environmental samples. [³H]thymidine yielded the most variable results, especially with regard to the extent of nonspecific macromolecular labeling. The pathways of [³H]thymidine and [³H]adenine metabolism were further evaluated by isotope dilution methods and by comparing incorporation patterns of thymidine labeled at different sites of the molecule. The advantages, uncertainties, and limitations of the use of radiolabeled nucleic acid precursors in studies of aquatic microbial ecology are discussed and a prospectus for future studies presented.     

Spatial and Temporal Variations in Bacterial Macromolecule Labeling with [methyl-H]Thymidine in a Hypertrophic Lake

The incorporation of [methyl-H]thymidine into three macromolecular fractions, designated as DNA, RNA, and protein, by bacteria from Hartbeespoort Dam, South Africa, was measured over 1 year by acid-base hydrolysis procedures. Samples were collected at 10 m, which was at least 5 m beneath the euphotic zone. On four occasions, samples were concurrently collected at the surface. Approximately 80% of the label was incorporated into bacterial DNA in surface samples. At 10 m, total incorporation of label into bacterial macromolecules was correlated to bacterial utilization of glucose (r = 0.913, n = 13, P < 0.001). The labeling of DNA, which ranged between 0 and 78% of total macromolecule incorporation, was inversely related to glucose uptake (r = -0.823), total thymidine incorporation (r = -0.737), and euphotic zone algal production (r = -0.732, n = 13, P < 0.005). With decreased DNA labeling, increasing proportions of label were found in the RNA fraction and proteins. Enzymatic digestion followed by chromatographic separation of macromolecule fragments indicated that DNA and proteins were labeled while RNA was not. The RNA fraction may represent labeled lipids or other macromolecules or both. The data demonstrated a close coupling between phytoplankton production and heterotrophic bacterial activity in this hypertrophic lake but also confirmed the need for the routine extraction and purification of DNA during [methyl-H]thymidine studies of aquatic bacterial production.     

Catabolism of Tritiated Thymidine by Aquatic Microbial Communities and Incorporation of Tritium into RNA and Protein

The incorporation of tritiated thymidine by five microbial ecosystems and the distribution of tritium into DNA, RNA, and protein were determined. All microbial assemblages tested exhibited significant labeling of RNA and protein (i.e., nonspecific labeling), as determined by differential acid-base hydrolysis. Nonspecific labeling was greatest in sediment samples, for which ≥95% of the tritium was recovered with the RNA and protein fractions. The percentage of tritium recovered in the DNA fraction ranged from 15 to 38% of the total labeled macromolecules recovered. Nonspecific labeling was independent of both incubation time and thymidine concentration over very wide ranges. Four different RNA hydrolysis reagents (KOH, NaOH, piperidine, and enzymes) solubilized tritium from cold trichloroacetic acid precipitates. High-pressure liquid chromatography separation of piperidine hydrolysates followed by measurement of isolated monophosphates confirmed the labeling of RNA and indicated that tritium was recovered primarily in CMP and AMP residues. We also evaluated the specificity of [2-³H]adenine incorporation into adenylate residues in both RNA and DNA in parallel with the [³H]thymidine experiments and compared the degree of nonspecific labeling by [³H]adenine with that derived from [³H]thymidine. Rapid catabolism of tritiated thymidine was evaluated by determining the disappearance of tritiated thymidine from the incubation medium and the appearance of degradation products by high-pressure liquid chromatography separation of the cell-free medium. Degradation product formation, including that of both volatile and nonvolatile compounds, was much greater than the rate of incorporation of tritium into stable macromolecules. The standard degradation pathway for thymidine coupled with utilization of Krebs cycle intermediates for the biosynthesis of amino acids, purines, and pyrimidines readily accounts for the observed nonspecific labeling in environmental samples.     

Mechanisms of DNA Utilization by Estuarine Microbial Populations

The mechanisms of utilization of DNA by estuarine microbial populations were investigated by competition experiments and DNA uptake studies. Deoxyribonucleoside monophosphates, thymidine, thymine, and RNA all competed with the uptake of radioactivity from [³H]DNA in 4-h incubations. In 15-min incubations, deoxyribonucleoside monophosphates had no effect or stimulated [³H]DNA binding, depending on the concentration. The uptake of radioactivity from [³H]DNA resulted in little accumulation of trichloroacetic acid-soluble intracellular radioactivity and was inhibited by the DNA synthesis inhibitor novobiocin. Molecular fractionation studies indicated that some radioactivity from [³H]DNA appeared in the RNA (10 and 30% at 4 and 24 h, respectively) and protein (approximately 3%) fractions. The ability of estuarine microbial assemblages to transport gene sequences was investigated by plasmid uptake studies, followed by molecular probing. Although plasmid DNA was detected on filters after filtration of plasmid-amended incubations, DNase treatment of filters removed this DNA, indicating that there was little transport of intact gene sequences. These observations led to the following model for DNA utilization by estuarine microbial populations. (i) DNA is rapidly bound to the cell surface and (ii) hydrolyzed by cell-associated and extracellular nonspecific nucleases. (iii) DNA hydrolysis products are transported, and (iv) the products are rapidly salvaged into nucleic acids, with little accumulation into intracellular nucleotide pools.     

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.     

Dynamics of extracellular DNA in the marine environment

The production and turnover of dissolved DNA in subtropical estuarine and oligotrophic oceanic environments were investigated. Actively growing heterotrophic bacterioplankton (i.e., those capable of [3H]thymidine incorporation) were found to produce dissolved DNA, presumably through the processes of death and lysis, grazing by bacteriovores, and excretion. Production of dissolved DNA as determined by [3H]thymidine incorporation was less than or equal to 4% of the ambient dissolved DNA concentration per day. In turnover studies, the addition of [3H]DNA (Escherichia coli chromosomal) to seawater resulted in rapid hydrolysis and uptake or radioactivity by microbial populations. DNA was hydrolyzed by both cell-associated and extracellular nucleases, in both estuarine and offshore environments. Kinetic analysis performed for a eutrophic estuary indicated a turnover time for dissolved DNA as short as 6.5 h. Microautoradiographic studies of bacterial populations in Tampa Bay indicated that filamentous and attached bacteria took up most of the radioactivity from [3H]DNA. Dissolved DNA is therefore a dynamic component of the dissolved organic matter in the marine environment, and bacterioplankton play a key role in the cycling of this material.     

Dynamics of extracellular DNA in the marine environment.

The production and turnover of dissolved DNA in subtropical estuarine and oligotrophic oceanic environments were investigated. Actively growing heterotrophic bacterioplankton (i.e., those capable of [3H]thymidine incorporation) were found to produce dissolved DNA, presumably through the processes of death and lysis, grazing by bacteriovores, and excretion. Production of dissolved DNA as determined by [3H]thymidine incorporation was less than or equal to 4% of the ambient dissolved DNA concentration per day. In turnover studies, the addition of [3H]DNA (Escherichia coli chromosomal) to seawater resulted in rapid hydrolysis and uptake or radioactivity by microbial populations. DNA was hydrolyzed by both cell-associated and extracellular nucleases, in both estuarine and offshore environments. Kinetic analysis performed for a eutrophic estuary indicated a turnover time for dissolved DNA as short as 6.5 h. Microautoradiographic studies of bacterial populations in Tampa Bay indicated that filamentous and attached bacteria took up most of the radioactivity from [3H]DNA. Dissolved DNA is therefore a dynamic component of the dissolved organic matter in the marine environment, and bacterioplankton play a key role in the cycling of this material.     

Inhibition of macrophage DNA synthesis by immunomodulators. II. Characterization of the suppression by muramyl dipeptide or lipopolysaccharide [3H]thymidine incorporation into macrophages

Guinea pig peritoneal exudate macrophages actively incorporated [3H]thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of [3H]thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase alpha and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The [3H]thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of [3H]thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized "cold" thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by [3H]thymidine. These results suggest that the suppression of [3H]thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.     

A correction: inhibitory activity in the conditioned medium of embryonic chick bones is due to thymidine

Earlier studies from this laboratory suggested that embryonic chick bones in organ culture released into the culture medium a specific inhibitor of bone cell proliferation as defined by inhibition of [3H]TdR incorporation into DNA. Dialysis and membrane ultrafiltration experiments suggested that the inhibitory substance (IS) had a molecular weight between 6000 and 14,000. However, subsequent studies on the purification of IS have revealed that the inhibitory activity in bone-conditioned medium is of lower molecular weight and has several properties in common with thymidine (TdR): (1) IS coeluted with [3H]TdR upon gel filtration chromatography on Sephadex G-10. (2) IS bound to charcoal but not to cation or anion exchange resins. (3) Bone-conditioned medium decreased incorporation of [3H]TdR into the free [3H]TdR pool of cells in monolayer culture. (4) Conditioned medium inhibited [3H]TdR incorporation into [3H]thymidine monophosphate in a reaction catalyzed by thymidine kinase. The equivalent concentration of TdR in conditioned medium as estimated by thymidine kinase assay was sufficient to account for the reduction in [3H]TdR incorporation into bone cell DNA. No evidence was found for a specific inhibitor of bone cell proliferation other than TdR. Hence we conclude that the inhibitory effect of IS is due to dilution of [3H]TdR by nonradioactive TdR. Furthermore, media conditioned by several tumor cell lines also contained a low-molecular-weight component which inhibited [3H]TdR incorporation. The results suggest that organ- and cell-conditioned media can contain significant concentrations of TdR which can artifactually inhibit [3H]TdR incorporation in cell proliferation assays.     

Inhibition of cell division by interferons: Changes in the transport and intracellular metabolism of thymidine in human lymphoblastoid (Daudi) cells

The Daudi line of human lymphoblastoid cells shows a high sensitivity towards growth inhibition by human interferons. In cells pretreated with 70 reference units/ml of an interferon preparation for 48 h, the incorporation of exogenous [3H]thymidine into DNA is inhibited by as much as 85%. We are investigating the extent to which this effect reflects a true inhibition of the rate of DNA synthesis or whether it may be caused by changes in the metabolic utilization of exogenous thymidine by the cells. Interferon treatment results in a 30% inhibition of the rate of membrane transport and a 60% decrease in the rate of phosphorylation of [3H]thymidine in vivo. The latter effect is due to a decrease in V of thymidine kinase without any change in the value of Km for this enzyme. In addition to these changes, incorporation of [3H]uridine into DNA, which occurs as a result of the intracellular conversion of this precursor into thymidine nucleotides, is also inhibited by 75%, whereas RNA labelling by [3H]uridine is decreased by only 15% in interferon-treated cells. Thus several different metabolic events associated with thymidine nucleotide metabolism and DNA synthesis in Daudi cells are disrupted by interferon treatment.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of (3H] uridine incorporation into RNA and [3H] leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10-21 M). Insulin stimulated the rate of [3H] thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100-1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [3H] thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of 3H- uridine, [3H] thymidine and [3H] leucine into their respective precursor pools is not responsible for the apparent stimulation of RNA, DNA and protein synthesis.     

Evidence of a role for phosphatidylinositol synthesis in human amnion cell proliferation.

Phosphatidylinositol (PtdIns) is the key precursor of phosphoinositide-derived intracellular mediators. The effects of changing the rate of PtdIns synthesis on mitogenic activity of human amnion-derived WISH cells were investigated. Incubation of the cells with [3H]inositol caused a time- and dose-dependent PtdIns labeling. Exogenous Ca2+ inhibited [3H]inositol incorporation in a dose-dependent fashion; half-maximal inhibition occurred with 0.3-1.0 mM Ca2+. In contrast, removal of cytosolic Ca2+ by ionophore A23187 and 1 mM EGTA induced enhancement of the PtdIns labeling as a function of A23187 concentration, perhaps through release of inhibitory effects of endogenous Ca2+. The A23187-stimulated PtdIns labeling with [3H]inositol was not abolished by additional unlabeled inositol, suggesting that [3H]inositol labeling of PtdIns occurred mainly through de novo synthesis catalyzed by PtdIns synthase (EC 2.7.8.11). In cells with PtdIns synthase activity decreased by exogenous Ca2+, [3H]thymidine incorporation was also inhibited, while A23187 caused dose-dependent enhancement of thymidine incorporation. The changes in PtdIns synthase activity occurred in parallel with changes in mitogenic activity caused by increasing the dose of exogenous Ca2+ or A23187. A similar lowering of mitogenic activity was observed upon suppression of PtdIns synthase by pemirolast potassium (9-methyl-3-1H-tetrazol-5yl-4H-pyrido[1,2-a]pyridin-4-one potassium) via a Ca(2+)-independent mechanism. These data demonstrate that changes in PtdIns synthase activity by some agents acting via different mechanisms are associated with parallel changes in thymidine incorporation, and suggest that PtdIns production is tightly coupled to cell proliferation in human amnion cells.     

Production of dissolved DNA, RNA, and protein by microbial populations in a Florida reservoir.

Production of dissolved macromolecules by ambient autotrophic and heterotrophic microbial populations was measured in a eutrophic Florida reservoir by in situ labeling with various radioactive substrates. When [3H]thymidine was used as the precursor, production of labeled dissolved DNA, RNA, and protein was observed. The rate of production of labeled dissolved macromolecules was 3.1% the rate of cellular incorporation of [3H]thymidine, and the production of dissolved DNA represented 2.3% the rate of cellular DNA incorporation. Microautotrophic populations labeled with NaH[14C]CO3 produced dissolved RNA and protein at rates of 0.24 and 0.11 micrograms of C/liter per h, respectively, or 1.8% the total rate of carbon fixation, with no measurable dissolved DNA production. In an attempt to specifically label phytoplankton DNA, samples were incubated with [3H]adenine or 32Pi in the presence and absence of the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Although DCMU inhibited 14C fixation by approximately 99%, this antimetabolite had only a slight effect on [3H]adenine incorporation and no effect on 32P incorporation into cellular macromolecules. Significant amounts of dissolved DNA were produced in both [3H]adenine and 32Pi incubations, but again DCMU had no effect on the production rates. These results indicate that actively growing populations of both phytoplankton and bacterioplankton produced dissolved RNA and protein, while only active bacterioplankton produced measurable quantities of dissolved DNA. Dead or senescent phytoplankton may have produced dissolved DNA, but would not be measured in the relatively short incubations used. These findings also indicate that [3H]adenine and 32Pi primarily labeled heterotrophic bacterioplankton and not phytoplankton in this environment.     

Production of dissolved DNA, RNA, and protein by microbial populations in a Florida reservoir.

Production of dissolved macromolecules by ambient autotrophic and heterotrophic microbial populations was measured in a eutrophic Florida reservoir by in situ labeling with various radioactive substrates. When [3H]thymidine was used as the precursor, production of labeled dissolved DNA, RNA, and protein was observed. The rate of production of labeled dissolved macromolecules was 3.1% the rate of cellular incorporation of [3H]thymidine, and the production of dissolved DNA represented 2.3% the rate of cellular DNA incorporation. Microautotrophic populations labeled with NaH[14C]CO3 produced dissolved RNA and protein at rates of 0.24 and 0.11 micrograms of C/liter per h, respectively, or 1.8% the total rate of carbon fixation, with no measurable dissolved DNA production. In an attempt to specifically label phytoplankton DNA, samples were incubated with [3H]adenine or 32Pi in the presence and absence of the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Although DCMU inhibited 14C fixation by approximately 99%, this antimetabolite had only a slight effect on [3H]adenine incorporation and no effect on 32P incorporation into cellular macromolecules. Significant amounts of dissolved DNA were produced in both [3H]adenine and 32Pi incubations, but again DCMU had no effect on the production rates. These results indicate that actively growing populations of both phytoplankton and bacterioplankton produced dissolved RNA and protein, while only active bacterioplankton produced measurable quantities of dissolved DNA. Dead or senescent phytoplankton may have produced dissolved DNA, but would not be measured in the relatively short incubations used. These findings also indicate that [3H]adenine and 32Pi primarily labeled heterotrophic bacterioplankton and not phytoplankton in this environment.     

Effects of toxic substances on natural bacterial assemblages determined by means of [h]thymidine incorporation

The effects of 3,5-dichlorophenol, 2,4-dinitrophenol, and potassium dichromate on natural bacterial assemblages were examined by means of [H]thymidine incorporation into trichloroacetic acid-insoluble material. Results from a large number of coastal marine and freshwater samples suggest the following. (i) The effects of the three toxicants included reductions in the bacterial cell number as well as changes in rates of [H]thymidine incorporation and in [H]thymidine incorporation per cell. The concentrations that inhibited [H]thymidine incorporation by 50% ranged from 3 to 11 mg liter for 3,5-dichlorophenol, 6 to 10 mg liter for 2,4-dinitrophenol, and 21 to 123 mg liter for potassium dichromate, with a tendency to higher values in bacterial assemblages from more eutrophic environments. (ii) The effects of 3,5-dichlorophenol and potassium dichromate determined by [H]leucine incorporation into bacterial protein were similar or larger than those obtained from [H]thymidine incorporation. (iii) Two to four hours of exposure to the toxicants was necessary before stable maximum effects were found in [H]thymidine incorporation. (iv) Storage of natural environmental samples should be avoided, since tests with water stored for 1 to 3 days sometimes produced results different from results obtained from in situ tests. (v) The effects of 3,5-dichlorophenol, 2,4-dinitrophenol, and potassium dichromate on natural bacterial assemblages were relatively constant during periods with different growth rates in the assemblages, during various periods of the year, and between samples from freshwater and marine localities. With some precautions, [H]thymidine incorporation can be used as a quick and sensitive method for determining the effects of toxicants on aquatic bacterial assemblages from natural environmental samples.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [³H]uridine incorporation into RNA and [³H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10⁻²¹ M). Insulin stimulated the rate of [³H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [³H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [³H]uridine, [³H]thymidine and [³H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.     

The role of fluorinated pyrimidine analogues in the induction of the in vitro expression of the fragile X chromosome

Summary The modes of action of 5-fluoro-2-deoxyuridine (FdUrd) and 5-fluoro-2-deoxycytidine (FdCyd) were studied in PHA-stimulated lymphocytes from normal volunteer donors and a fragile X patient. In both cell types, FdUrd and FdCyd inhibited cell proliferation at concentrations of 3x10^-8 M. Thymidylate synthetase was identified as the decisive target for the action of both FdUrd and FdCyd, as judged from the following observations: First, addition of thymidine to the culture medium was able to counteract both FdUrd and FdCyd toxicities, whereas addition of dCyd had no observable effect. Second, inhibition of the in situ thymidylate synthetase activity measured as an increase in the level of [³H]-dThd incorporation coincided with the inhibition of cell proliferation. Third, the inhibition of the thymidylate synthetase-dependent incorporation of [³H]-dUrd into newly synthesized DNA coincided with the inhibition of cell proliferation. The effects of FdUrd and FdCyd on the in vitro expression of fragile site Xq27 of fragile X chromosomes was shown to be based on the depletion of the intracellular pool of thymidine-5-monophosphate (dTMP), as fudged from the following observations: First, both the FdUrd- and FdCyd-dependent induction of site Xq27 coincided with the antiproliferative effects of the respective fluoropyrimidines. Second, addition of thymidine (dThd) to the culture medium both prevented the expression of site Xq27 and neutralized the cytotoxicity of FdUrd and of FdCyd. On the basis of these findings, we provide further evidence for the concept that the fragile X site is located in an AT-rich region.