On the mechanism of 5-bromodeoxyuridine induction of prolactin synthesis in rat pituitary tumor cells

GH12C1, a clonal strain of rat pituitary tumor cells in culture (GH cells), does not produce detectable amounts of prolactin. 5-Bromodeoxyuridine (BrdUrd), the thymidine analogue, at sublethal concentrations (3-5 microgram/ml) induces prolactin synthesis in these cells. BrdUrd also induces prolactin synthesis in F1BGH12C1 cells, a BrdUrd resistant (BrdUrdr) substrain isolated from GH12C1 cells. The F1BGH12C1 strain is not drug dependent, but its resistance to BrdUrd is a stable phenotype. The significant features of the induction of prolactin synthesis in the BrdUrdr strain are the increased net synthesis of prolactin and the shortening of the lag period of prolactin induction. As BrdUrd concentration in the growth medium is increased, the rise in prolactin synthesis parallels the increased incorporation of BrdUrd into DNA. Prolactin synthesis is first detected when BrdUrd replaces 20-25% of the thymidine in DNA. BrdUrd can replace up to 75-80% of the thymidine within 2 d of treatment. Partial starvation of these cells under specified growth conditions does not affect the general growth pattern of the cells, general protein synthesis, and thymidine uptake, but does affect DNA synthesis. When cells are cultured under conditions in which DNA synthesis is preferentially inhibited, BrdUrd does not induce prolactin synthesis, suggestive of a DNA-mediated mechanism of action for the drug.     

Induction of sister-chromatid exchanges by the replication of 5-bromouracil-substituted DNA under conditions of nucleotide-pool imbalance

The induction of sister-chromatid exchanges (SCEs) by the replication of 5-bromouracil(BrUra)-containing DNA under conditions of nucleotide-pool imbalance was investigated. A modification of a protocol developed for the induction of mutations under these conditions (E.R. Kaufman, Mol. Cell. Biol., 4, 2449-2454, 1984) was used. To induce SCEs, Chinese hamster ovary cells were grown under non-mutagenic conditions which allowed the uniform incorporation of BrUra into their DNA at specific levels of substitution for thymine residues (25, 50 and 75% BrUra substitution). After 4 and 5 days of growth, the cells, which had incorporated BrUra into their DNA, were washed free of 5-bromodeoxyuridine (BrdUrd) and provided with fresh culture medium supplemented with various concentrations of thymidine (10 microM to 3 mM) and no BrdUrd. The cells were allowed to replicate their BrUra-containing DNA under these conditions, in the absence of BrdUrd, for two rounds of DNA synthesis to achieve sister-chromatid differentiation, and second-division metaphases were scored for SCEs. The results of these studies indicated that the SCEs observed were proportional to the level of BrUra substituted for thymine in the cellular DNA, were induced by increasing concentrations of thymidine in the culture medium during replication of the BrUra-containing DNA, correlated well with the induction of mutations to thioguanine resistance and to ouabain resistance, correlated with increases in the intracellular levels of dTTP and dGTP generated by the high concentrations of thymidine. These findings provide direct evidence for the induction of SCEs by the replication of BrUra-containing DNA and for the importance of the pools of nucleoside triphosphate precursors for DNA replication in these processes. When the effects of 3-aminobenzamide, a potent inhibitor of poly(ADP-?ibose) synthesis, were tested, it was found that 3-aminobenzamide significantly increased SCEs, but it had no effect on mutations induced.     

Replication of DNA containing 5-bromouracil can be mutagenic in Syrian hamster cells.

A new protocol for inducing mutations in mammalian cells in culture by exposure to the thymidine analog 5-bromodeoxyuridine (BrdUrd) was established. This protocol, called "DNA-dependent" mutagenesis, involved the incorporation of BrdUrd into DNA under nonmutagenic conditions and the subsequent replication of the 5-bromouracil (BrUra)-containing DNA under mutagenic conditions but with no BrdUrd present in the culture medium. The mutagenic conditions were induced by allowing BrUra-containing DNA to replicate in the presence of high concentrations of thymidine. This generated high intracellular levels of dTTP and dGTP, causing nucleotide pool imbalance. The mutagenesis induced by this protocol was found to correlate with the level of BrUra substituted for thymine in DNA.     

Effect of 5-fluoro-2'-deoxyuridine (FdUrd) on 5-bromo-2'-deoxyuridine (BrdUrd) incorporation into DNA measured with a monoclonal BrdUrd antibody and by the BrdUrd/Hoechst quenching effect

The purpose of this study was to improve the application of bromodeoxyuridine (BrdUrd) for the flow cytometric analysis of cell kinetics. In order to obtain a quantitative measure of the DNA synthesis rate (or the number of divided cells), BrdUrd should replace thymidine (dThd) completely in the newly synthesized DNA strands. The de novo synthesis of dThd monophosphate competing with BrdUrd incorporation was stopped by fluorodeoxyuridine (FdUrd). Cells of a human leukemic cell line (REH) were exposed to BrdUrd for either 20 min, 8 h, or 24 h. Bromodeoxyuridine incorporation was determined by a monoclonal antibody as well as by the BrdUrd/Hoechst (H) technique. Counterstaining of the DNA was performed with propidium iodide or ethidium bromide. DNA fluorescence was measured in both techniques with a two-parameter flow cytometer, the histograms being analyzed by computer. It was found that FdUrd is required in the BrdUrd/H technique for replacement of dThd at low BrdUrd concentrations and long incubation times. With short incubation periods, as used for detection by the monoclonal anti-BrdUrd antibody, FdUrd increases the incorporated BrdUrd amount when BrdUrd concentrations of 10 microM or less are applied.     

Ataxia telangiectasia cells exhibit the same radiosensitization response by incorporation of BrdUrd or IdUrd as do normal human cells

Normal and ataxia telangiectasia (AT) human cells were exposed to 10(-5) mole/liter bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd). High-pressure liquid chromatography (HPLC) measurements showed that up to 26 and 23% of the thymidine in DNA was substituted by BrdUrd in normal and AT cells, respectively. The incorporation of BrdUrd or IdUrd into DNA resulted in radiosensitization in normal and AT cells. When exposed to equal concentrations of BrdUrd and IdUrd, the BrdUrd caused greater radiosensitization than IdUrd in both normal and AT cells.     

Effect of pyrimidine deoxynucleosides and sodium butyrate on expression of the glycoprotein hormone alpha-subunit and placental alkaline phosphatase in HeLa cells

Production of the glycoprotein hormone common alpha-subunit and placental alkaline phosphatase activity can be modulated in HeLa cells by a variety of deoxynucleosides. Dose response curves for thymidine (Thd), fluorodeoxyuridine (FdUrd), bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd) demonstrate that, in general, alkaline phosphatase was increased by lower concentrations of inducer than was alpha-subunit. The deoxynucleosides were not as effective as sodium butyrate as inducers of either protein. Whereas Thd and the halogenated dUrd derivatives enhanced protein expression, deoxycytidine (dCyd) had negative effects. Induction by deoxynucleosides of both alkaline phosphatase and alpha-subunit was inhibited by dCyd, but induction of alkaline phosphatase by butyrate was more sensitive to dCyd inhibition than was the butyrate-mediated induction of alpha-subunit. These results suggest that the two proteins are not regulated in a coordinate manner. Reversal of alkaline phosphatase induction by dCyd was not observed in cells preincubated with sodium butyrate for 6-24 h before the addition of dCyd, indicating that the deoxynucleoside interferes with an early event in the butyrate-mediated response. Combinations of butyrate with Thd, BrdUrd or IdUrd were synergistic with respect to the induction of HeLa-alpha. It is concluded that incorporation of the deoxynucleosides into DNA may not be required for the synergistic response since 2',5'-dideoxythymidine was an effective as Thd. Cytoplasmic dot hybridizations demonstrate that a primary effect of the various effectors is to increase the steady-state levels of alpha-subunit mRNA. There was a good correlation between alpha-subunit accumulation and corresponding levels of alpha-mRNA, suggesting that regulation occurs at a pretranslational site. Although the mechanism(s) is not understood, these data provide evidence that nucleosides or their derivatives can significantly affect gene expression.     

Simultaneous detection of DNA strand breaks and unscheduled DNA synthesis in mutagen-treated human lymphocytes in the absence of hydroxyurea

Human lymphocytes in the quiescent state were exposed to UVC radiation. After irradiation the cells were allowed to repair for various times in the presence of [3H]thymidine or [3H]deoxycytidine in the culture medium. Hydroxyurea was not used to suppress semiconservative DNA replication in the small number of growing cells. After incubation DNA strand breaks were detected by the DNA-unwinding method and the amount of 3H incorporation in DNA was measured by liquid scintillation counting. The results show that the yield of DNA strand breaks and the amount of unscheduled DNA synthesis (UDS) can be measured from the same lymphocyte sample. A low background 3H incorporation in untreated cells could be achieved even in the absence of hydroxyurea. This requires, however, that 3H incorporation is measured only in the double-stranded DNA and that [3H]dCyd is used instead of [3H]dThd as the labelled deoxynucleoside.     

Mutational and pseudomutational effects of 5-bromodeoxyuridine in human lymphoblasts

We have studied the effects of 5-bromodeoxyuridine (BrdUrd) at two genetic loci in diploid human lymphoblast cells. In thymidine kinase heterozygotes (tk +/-), a 2-h dose of BrdUrd induced a transient, non-heritable resistance to the thymidine analogue, trifluorothymidine (F3TdR). We have called this phenomenon pseudomutation and have shown that affected cells acquire the ability to survive in the presence of F3TdR and then, after degradation of F3TdR in the medium, return to an apparently normal wild-type state. Our data suggest that BrdUrd incorporation into DNA as a thymidine analogue is responsible for the effect, which we interpret as a temporary loss of thymidine kinase activity. This effect is not seen in tk +/+ homozygotes. In contrast, at the hypoxanthine-guanine phosphoribosyl transferase locus in tk +/- heterozygotes, BrdUrd did not induce a permanent, heritable resistance to 6-thioguanine (gene locus mutation). We detected such mutations only in the tk +/+ homozygote and only at external BrdUrd concentrations considerably higher than those which saturate the uptake of BrdUrd into DNA as a thymidine analogue. We postulate that the reduced TK enzyme levels (30%) in the heterozygote prevent the build-up of a sufficiently high intracellular BrdUrd triphosphate pool to promote the misincorporations as deoxycytidine triphosphate which may be responsible for gene locus mutation.     

Inhibition by 2-deoxy-D-ribose of DNA synthesis and growth in Raji cells

When Raji cells were cultured for 3 days in serum-free medium, addition of 2-deoxy-D-ribose at the start of culture inhibited incorporation of [3H]thymidine and cell division. At deoxyribose concentrations between 1 and 5 mM, viability was 80% or greater after 3 days of culture even though 5 mM deoxyribose inhibited thymidine incorporation 95-99%. Inhibition by deoxyribose could be completely reversed if the culture medium was replaced with fresh medium up to 8 hr after the start of culture. The inhibition was specific for deoxyribose since other monosaccharides had no effect. Inhibition of DNA synthesis did not appear to be due to depletion of essential nutrients in the medium since the percentage inhibition of thymidine incorporation by cells cultured either in suboptimal serum-free media or in media supplemented with 0.025-5% human AB serum was similar. When DNA repair synthesis was measured as hydroxyurea-resistant thymidine incorporation, addition of deoxyribose to Raji cultures caused increased thymidine incorporation. These results, together with data from others, suggest that deoxyribose damages DNA.     

5-Bromodeoxyuridine inhibition of differentiation. Kinetics of inhibition and reversal in myoblasts

This paper describes experiments on the kinetics of inhibition of muscle differentiation in vitro in the presence of 5-bromodeoxyuridine (BrdUrd) and the recovery phenomena that occur when such inhibited cells are permitted growth in normal medium. The studies consist of a quantitation of cell fusion in the presence of the analog and during recovery in its absence coupled with simultaneous studies on changes in buoyant density of cellular DNA. We find that if myoblasts are exposed to BrdUrd during the last doubling before cell fusion would normally occur, most cells do not differentiate, but as many as 18% of the cells can fuse in spite of the incorporation of BrdUrd into their nuclei. These nuclei contain approximately the amount of BrdUrd expected for a full round of DNA synthesis. Studies on the rate of recovery of inhibition of cell fusion following one generation in BrdUrd reveal that after one doubling of inhibited cells in the presence of normal medium. fusion reaches about 50% of the control value; after two doublings it reaches 75% of control value; and after 2.5 doublings of reversal, recovery is essentially complete. We find that both the degree of inhibition after approximately one round of BrdUrd incorporation and the rate of cell differentiation after two generations of reversal are consistent with a model which assumes that BrdUrd “sensitivity” resides on single pair of chromosomes and that inhibition occurs in a dominant fashion if approximately 30% or more of the thymidine is replaced by BrdUrd in the readout strand of either chromosome.     

Induction of placental alkaline phosphatase in choriocarcinoma cells by 5-bromo-2'-deoxyuridine.

Growth of choriocarcinoma cells in the presence of 5-bromo-2'-deoxyuridine (BrdUrd) results in a 30- to 40-fold increase in alkaline phosphatase activity. The effects of BrdUrd is specific for phosphatase with an alkaline pH optimum. The induction by BrdUrd is probably not due to the production of an altered enzyme, since the induced enzyme resembles the basal enzyme in thermal denaturation and kinetic properties. Enzyme induction can be prevented by thymidine but not by deoxycytidine or deoxyuridine. The induction of alkaline phosphatase appears to require incorporation of the BrdUrd into cellular DNA. The presence of BrdUrd in the growth medium is not necessary for alkaline phosphatase induction in proliferating cells containing BrdUrd-substituted genomes. However, enzyme induction and maintenance of the induced levels of alkaline phosphatase in nonproliferating cells containing BrdUrd-substituted DNA requires the presence of the analogues in the medium. The induction of alkaline phosphatase by BrdUrd in probably an indirect process.     

The effect of single versus double-strand substitution on halogenated pyrimidine-induced radiosensitization and DNA strand breakage in human tumor cells

To better understand the mechanism underlying halogenated pyrimidine-mediated cytotoxicity and radiosensitization in human tumor cells, a study was undertaken to determine the influence of unifilar (one DNA strand) versus bifilar (both DNA strands) substitution of thymidine by the halogenated bases 5-iodo-2'-deoxyuridine (IdUrd) and 5-bromo-2'-deoxyuridine (BrdUrd) in HT29 human colon cancer cells. Unifilar labeling was obtained by incubating cells with IdUrd or BrdUrd for one doubling time. Cells were incubated for at least three doublings to approximate bifilar substitution. Only IdUrd caused significant cytotoxicity, which correlated with incorporation into DNA. Both BrdUrd and IdUrd were potent radiosensitizers. Radiosensitization was linearly correlated with incorporation of both bases regardless of the number of strands in which thymidine was substituted. In contrast, the relationship between radiosensitization and DNA double-strand breakage was critically dependent in the case of IdUrd, but not for BrdUrd, on whether substitution was unifilar or bifilar. These findings suggest that incorporation is the best predictor of radiation sensitivity, and that the induction of DNA double-strand breaks alone does not account for radiosensitization mediated by halogenated pyrimidines in these human tumor cells.     

The action of 5-amino uracil on log growth and division-synchronized Tetrahymena

The influence of 5-amino uracil (5-AU) was investigated on the cell cycle of log growth and division-synchronized Tetrahymena pyriformis GL. The division index of log growth phase Tetrahymena was suppressed by 50% after 40 min in 8 mM 5-AU. Cells division-synthronized by one heat shock per generation were also treated with 5-AU. Cells treated either prior to the first synchronous division (80 min EH) or up to 25 min prior to the second synchronous division (after 160 min EH) were not delayed in their progress through the cell cycle. Cells treated during the S phase of the first free running cell cycle, however, were delayed 5-30 min from reaching the second synchronous division. The effect of 5-AU on DNA and RNA synthesis was also examined. Incorporation of [3H]thymidine into acid-precipitable material was reduced in the presence of 5-AU; the rate of DNA synthesis was also reduced. The depression in the rate of DNA synthesis was greater at the beginning of S than at the end of S. The size of the thymidine pool (nucleosides + nucleotides) did not change during 5-AU treatment; however, an accumulation of thymidine tri-phosphate and a decrease in the amount of thymidine nucleoside was observed. A suppression of [14C]uridine incorporation resulting from 5-AU treatment was observed throughout the cell cycle. The rate of RNA synthesis as monitored by [14C]uridine incorporation into acid precipitable material was also reduced during 5-AU treatment. No change in either the size or the composition of the pool of uridine (nucleoside + nucleotide) was detected in 5-AU treated cells as compared to controls.     

Effect of pyrimidine deoxynucleosides and sodium butyrate on expression of the glycoprotein hormone α-subunit and placental alkaline phosphatase in HeLa cells

Production of the glycoprotein hormone common α-subunit and placental alkaline phosphatase activity can be modulated in HeLa cells by a variety of deoxynucleosides. Dose response curves for thymidine (Thd), fluorodeoxyuridine (FdUrd), bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd) demonstrate that, in general, alkaline phosphatase was increased by lower concentrations of inducer than was α-subunit. The deoxynucleosides were not as effective as sodium butyrate as inducers of either protein. Whereas Thd and the halogenated dUrd derivatives enhanced protein expression, deoxycytidine (dCyd) had negative effects. Induction by deoxynucleosides of both alkaline phosphatase and α-subunit was inhibited by dCyd, but induction of alkaline phosphatase by butyrate was more sensitive to dCyd inhibition than was the buryrate-mediated induction of α-subunit. These results suggest that the two proteins are not regulated in a coordinate manner. Reversal of alkaline phosphatase induction by dCyd was not observed in cells preincubated with sodium butyrate for 6–24 h before the addition of dCyd, indicating that the deoxynucleoside interferes with an early event in the butyrate-mediated response. Combinations of butyrate with Thd, BrdUrd or IdUrd were synergistic with respect to the induction of HeLa-α. It is concluded that incorporation of the deoxynucleosides into DNA may not be required for the synergistic response since 2′,5′-dideoxythymidine was an effective as Thd. Cytoplasmic dot hybridizations demonstrate that a primary effect of the various effectors is to increase the steady-state levels of α-subunit mRNA. There was a good correlation between α-subunit accumulation and corresponding levels of α-mRNA, suggesting that regulation occurs at a pretranslational site. Although the mechanism(s) is not understood, these data provide evidence that nucleosides or their derivatives can significantly affect gene expression.     

Reversion analysis of mutations induced by 5-bromodeoxyuridine mutagenesis in mammalian cells.

Two protocols have been developed, both of which utilize the thymidine analog 5-bromodeoxyuridine (BrdUrd) to induce mutations in mammalian cells in culture (E. R. Kaufman and R. L. Davidson, Proc. Natl. Acad. Sci. USA 75:4982-4986, 1978; E. R. Kaufman, Mol. Cell. Biol. 4:2449-2454, 1984). The first protocol, termed incorporational (INC) mutagenesis, utilizes high concentrations of BrdUrd in the culture medium to generate a high intracellular ratio of BrdUTP/dCTP. The second protocol, termed replicational (REP) mutagenesis, entails the incorporation of BrdUrd into DNA under nonmutagenic conditions, the removal of all BrdUrd from the culture medium, and the subsequent replication of the bromouracil-containing DNA in the presence of high intracellular levels of dTTP and dGTP. Genetic studies using reversion analysis at the hypoxanthine-guanine phosphoribosyltransferase locus were used to determine whether the mechanisms of these two BrdUrd mutagenesis protocols had enough specificity to be distinguishable by their ability to revert various mutants. The results of these studies indicated that (i) mutants induced by INC mutagenesis were induced to revert only by REP mutagenesis and not by INC mutagenesis, (ii) mutants induced by REP mutagenesis were more efficiently reverted by INC mutagenesis than by REP mutagenesis, and (iii) both spontaneous mutants and mutants induced by the chemical mutagen ethyl methanesulfonate showed a high degree of specificity when tested for reversion by the BrdUrd mutagenesis protocols.     

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.     

Effect of exogenous thymidine on sister-chromatid exchange frequency in Chinese hamster ovary cells with bromodeoxyuridine- and chlorodeoxyuridine-substituted chromosomes

There are conflicting reports on the effect of exogenous thymidine (dThd) on the frequency of sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. Thymidine has been reported either to increase or to have no effect on SCE frequency under similar experimental conditions. To resolve this controversy, we have carried out a series of experiments to examine the effect of dThd on CHO cells cultured with 5-bromodeoxyuridine (BrdUrd). In addition, we have examined the effect of dThd on CHO cells cultured with 5-chlorodeoxyuridine (CldUrd), a much more potent inducer of SCEs than BrdUrd. The addition of 100 microM dThd to the culture medium caused a consistent decrease in the yield of SCEs in cells grown in BrdUrd for two cell cycles. The decrease was even greater when cells were grown in dThd and CldUrd. Analysis of twin and single SCEs indicated that dThd must be present during the first cell cycle to reduce the frequency of SCEs. Because excess dThd is thought to have an effect when DNA replicates on a template substituted with a halogenated nucleoside, dThd at concentrations from 100 microM to 9 mM was added to cultures for the second cell cycle after a first cell cycle in BrdUrd. In this experiment, the presence of dThd increased SCE frequency in a dose-dependent manner. The results suggest that if dThd competes with halogenated nucleosides and thus decreases their incorporation into DNA, SCEs are suppressed in the subsequent cell cycle, whereas if excess dThd creates a dNTP pool imbalance, SCEs can be increased.     

Mutations resistant to bromodeoxyuridine in mouse lymphoma cells selected by repeated exposure to EMS characteristics of phenotypic instability and reversion to HAT resistance by 5-azacytidine

Mutations induced by repeated EMS treatments were investigated by using mouse L5178Y cells. The frequency of TG^r mutations increased linearly with the number of EMS treatments whereas the yield of BrdUrd^r mutations showed a curvilinear dose-response curve. The BrdUrd^r frequency was roughly proportional to the square of the TG^r frequency and the results were compatible with the hypothesis that BrdUrd^r cells were induced by two mutational events within a cell. Most of the BrdUrd^r colonies isolated after 6 EMS treatments, however, were unstable. When BrdUrd^r colonies that had arisen in BrdUrd medium after 2 weeks' incubation were isolated in normal medium, the descendant cells showed a nearly normal level of thymidine incorporation and low plating efficiencies of about 1% in BrdUrd medium. In contrast, after isolation of the same colonies in BrdUrd medium, a low level of thymidine incorporation and high plating efficiencies in BrdUrd medium were observed in the descendant cells.

Reverse selection from BrdUrd^r to HAT^r was accomplished with frequencies of 10⁻⁶−10⁻³ for the descendants grown in BrdUrd medium, and AzaCyd treatment drastically increased the reversion frequency to nearly 10⁻¹. Further re-revertants from HAT^r to BrdUrd^r were also found with frequencies of 10⁻³−10⁻² without treatment. These results indicate that the initial BrdUrd^r cells did not result from inactivation of the thymidine-kinase gene but that the mode of gene expression was altered in some way.     

Reversible permeabilization of lymphocytes destroys the incorporation of deoxythymidine but not of deoxycytidine

The total uptake, phosphorylation and incorporation of thymidine (dThd) and deoxycytidine (dCyd) were compared in intact and reversibly permeabilized human tonsillar lymphocytes. The total uptake of [3H]dThd was lower than that of [5-3H]dCyd, but almost all of [3H]dThd was incorporated into DNA. However, the main part of [5-3H]dCyd taken up by the lymphocytes was found in the pool as phosphorylated nucleoside (55%), and only a smaller part (13%) was incorporated into DNA. Phosphorylated nucleosides were determined by DEAE-cellulose sheets in the ethanol-soluble fraction of the cells. The reversible permeabilization of lymphocytes by Dextran T-150 destroys totally the [3H]dThd incorporation, while [5-3H]dCyd incorporation decreased only to 60% of intact cells. During permeabilization the phosphorylation of both nucleosides increased severalfold. After permeabilization all [3H]dThd was in dTMP form, while [5-3H]dCyd was also found in dCDP (3%) and dCTP (38%) form. In the meanwhile, 22% of thymidine kinase, 63% of deoxycytidine kinase and 98% of DNA polymerase activity were measured in permeabilized cells as compared to intact cells. The results suggest different relationships between the lymphocyte plasma membrane and the salvage pathways of the two pyrimidine nucleosides.     

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.