Thymidine Metabolism and the Measurement of the Rate of DNA Synthesis in Carrot Suspension Cultures: EVIDENCE FOR A DEGRADATION PATHWAY FOR THYMIDINE

The kinetics of [³H]thymidine incorporation into the DNA of carrot suspension cultures were investigated. At a thymidine concentration of 0.1 micromolar, incorporation into DNA is not quantitative but ceases after only 14% of the thymidine has been incorporated. Thymidine incorporation into DNA is resumed following addition of a second aliquot of thymidine, which is consistent with substrate depletion. In vivo tracer experiments indicate that this may be due to a catabolic route for converting thymidine to β-aminoisobutyric acid. Bearing these observations in mind, conditions for determining the rate of DNA synthesis using [³H]thymidine incorporation have been investigated. It is concluded that by increasing the thymidine concentration to 10 micromolar the assay period may be increased, by reducing the influence of the degradative pathway, and that cell density and incubation time are critical factors in establishing a valid measure of the rate of DNA synthesis using this method.     

Cyclic GMP and cyclic GMP-dependent protein kinase in brown adipose tissue of developing rats.

Human cells (normal and xeroderma pigmentosum variant) irradiated with ultraviolet light and pulse-labelled with [3H]thymidine underwent transient decline and recovery of molecular weights of newly synthesized DNA and rates of [3H]thymidine incorporation. The ability to synthesize normal-sized DNA recovered more rapidly in both cell types than thymidine incorporation. During recovery cells steadily increased in their ability to replicate normal-sized DNA on damaged templates. The molecular weight versus time curves fitted exponential functions with similar rate constants in normal and heterozygous xeroderma pigmentosum cells, but with a slower rate in two xeroderma pigmentosum variant cell lines. Caffeine added during the post-irradiation period eliminated the recovery of molecular weights in xeroderma pigmentosum variant but not in normal cells. The recovery of the ability to synthesize normal-sized DNA represents a combination of a number of cellular regulatory processes, some of which are constitutive, and one of which is altered in the xeroderma pigmentosum variant such that recovery becomes slow and caffeine sensitive.     

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.     

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.     

DNA Biosynthesis in Chloroplasts: II. DEVELOPMENTAL VARIATION IN TTP INCORPORATION INTO DNA BY PLASTID EXTRACTS

Incorporation of [³H]TTP into DNA by pea chloroplast extractswas highly dependent on the age of the tissue from which plastidswere prepared. Catalytic activity was greatest in samples from6- to 9-d-old plants; preparations from more mature tissueswere much less effective. Moreover, activity was 3 to 10 timesgreater in younger tissues regardless of whether chlorophyll,protein or plastid number was used as the index of comparison.Enzymes from the first (oldest), second, third, and fourth (youngest)leaves of the same plants were also studied. Again, activitywas 4 to 10 times greater in samples from the youngest tissues.When plastid extracts from older leaves were mixed with thosefrom younger tissues, they did not reduce synthesis. Thus, thedecline in activity does not appear to be due to the productionof an inhibitor during plant development. One explanation forthese data is that enzymes of ctDNA replication, such as DNApolymerase, vary in activity during leaf development; thereforechanges in enzyme levels may be an important factor in controllingchloroplast DNA replication during development. We have alsoexamined the incorporation of [³H]TTP into DNA by isolated intactpea chloroplasts; in general, labelled TTP was less readilyincorporated into chloroplast DNA than was [³H]thymidine. Key words: Chloroplast DNA replication, chloroplast DNA polymerase     

The synthesis of polydeoxyribonucleotide by cell-free extracts of embryonic mouse tissue

The conditions under which soluble extracts prepared from mouse embryos incorporate [(3)H]thymidine 5'-triphosphate into polydeoxyribonucleotide have been studied. In common with similar preparations from other mammalian tissues, mouse-embryo DNA nucleotidyltransferase requires the four complementary deoxyribonucleoside 5'-triphosphates, primer DNA and a bivalent cation for activity. Unlike other mammalian DNA nucleotidyltransferases, the rate and extent of the incorporation of [(3)H]thymidine 5'-triphosphate is much greater with Mn(2+) than with Mg(2+) and, with either Mg(2+) or Mn(2+), maximum activity occurs at pH6.4. The difference between Mg(2+) and Mn(2+) varies markedly with pH, reaching a maximum of six- to eight-fold at pH6.4.     

Rapid Initiation of Thymidine Incorporation into Deoxyribonucleic Acid in Vegetative Tobacco Stem Segments Treated with Indole-3-acetic Acid

The short term effect of 11.4 mum indoleacetic acid on the incorporation of (methyl-(3)H)thymidine into DNA in vegetative tobacco (Nicotiana tabacum cv. Wis. 38) stem segments has been investigated. In segments that are defoliated, inverted, and kept in the dark for 7 hours, indoleacetic acid very rapidly (about 60 minutes) and strikingly initiates thymidine incorporation into DNA. The time required before enough indoleacetic acid (2.8 mum) to enhance thymidine incorporation moves into a segment has been found to be about 35 minutes. The initiation response time for segment tissue that already contains 2.8 mum indoleacetic acid should be no more than about 25 minutes. The rate of labeled thymidine incorporation into DNA is affected by physiological treatments of segments. Moving segments from the light into the dark or defoliating segments or inverting defoliated segments decreases the rate of thymidine incorporation. For segments given all three treatments, indoleacetic acid restores the rate of thymidine incorporation as compared to controls. Darkness, or defoliation or inversion of segments, therefore, may decrease thymidine incorporation into DNA by effecting reduced auxin levels in stem segments.     

Biochemical aspects of cardiac muscle differentiation. Possible control of deoxyribonucleic acid synthesis and cell differentiation by adrenergic innervation and cyclic adenosine 3':5'-monophosphate.

A single injection of either isoproternol or N6, O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) results in an inhibition in the rate of [3H]thymidine incorporation into DNA of differentiating cardiac muscle of the neonatal rat. This inhibition is not due to substantially altered cellular uptake or catabolism of [3H]thymidine. Inhibition of [3H]thymidine incorporation by isoproterenol or dibutyryl cyclic AMP is potentiated by theophylline. Maximal inhibition (95%) is observed 24 h after administration of isoproterenol, and the rate of incorporation returns to a value 80% of control by 72 h. Norepinephrine also inhibits [3H]thymidine incorporation whereas cyclic GMP, N2, 02-Dibutyryl guanosine 3':5'-monophosphate (dibutyryl cyclic GMP), and phenylephrine have little effect. Equilibrium sedimentation analysis of cardiac muscle DNA in neutral and alkaline cesium chloride gradients using bromodeoxyuridine as a density label indicate that isoproterenol and dibutyryl cyclic AMP inhibit [3H]thymidine incorporation into DNA that is replicating semiconservatively. Administration of isoproterenol or dibutyryl cyclic AMP to neonatal rats inhibits by approximately 60% the incorporation of [3H]thymidine into DNA of tissue slices of cardiac muscle prepared 16 h later. [3H]Thymidine incorporation into DNA of tissue slices is into chains that were growing in vivo. This incorporation is linear for at least 4 h of incubation and is inhibited by isoproterenol and dibutyryl cyclic AMP. Inhibition is not due to altered cellular uptake of [3H]thymidine nor is it due to a cytotoxic action. Several other compounds which elevate intracellular levels of cyclic AMP (epinephrine, norepinephrine, glucagon, and prostaglandin E1) also inhibit [3H]thymidine incorporation into DNA or cardiac muscle tissue slices. Cyclic GMP, dibutyryl cyclic GMP, sodium butyrate, and phenylephrine have little effect. Isoproterenol administered together with theophylline to neonatal rats signficantly stimulates the in corporation of [3H]phenylalanine into total cardiac muscle protein and into myosin. This enhanced incorporation may be due in part to an increase in the cellular uptake of [3H]phenylalanine. DNA synthesis decreases progressively in differentiating cardiac muscle of the rat during postnatal development and essentially ceases by the middle of the third week (Claycomb, W. C. (1975) J. Biol. Chem. 250, 3229-3235). In reviewing the literature it was found that this decline in synthetic activity correlates temporally with a progressive increase in tissue concentrations of norepinephrine and cyclic AMP and with the anatomical and physiological development of the adrenergic nerves in this tissue. Because of these facts and data presented in this report it is proposed that cell proliferation and cell differentiation in cardiac muscle may be controlled by adrenergic innervation with norepinephrine and cyclic AMP serving as chemical mediators.     

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.     

The effect of microwave radiation on the cell genome

Cultured V79 Chinese hamster cells were exposed to continuous radiation, frequency 7.7 GHz, power density 30 mW/cm2 for 15, 30, and 60 min. The parameters investigated were the incorporation of [3H]thymidine and the frequency of chromosome aberrations. Data obtained by 2 methods (the incorporation of [3H]thymidine into DNA and autoradiography) showed that the inhibition of [3H]thymidine incorporation took place by complete prevention of DNA from entering into the S phase. The normal rate of incorporation of [3H]thymidine was recovered within 1 generation cycle of V79 cells. Mutagenic tests performed concurrently showed that even DNA macromolecules were involved in the process. In comparison with the control samples there was a higher frequency of specific chromosome lesions in cells that had been irradiated. Results discussed in this study suggest that microwave radiation causes changes in the synthesis as well as in the structure of DNA molecules.     

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.     

Relationship between release of LH and incorporation of tritiated thymidine in the anterior pituitary gland of the castrated female rat

Castration of female rats during diestrus increases the concentration of circulating LH from days 3 and the incorporation of 3H thymidine into pituitary DNA from day 5. Both effects are completely abolished by the administration of dihydrotestosterone. Although the i.v. injection of LHRH markedly enhances the concentration of LH in serum, it does not modify the incorporation of 3H thymidine into pituitary DNA. Castration might produce a maximal stimulation in 3H thymidine incorporation and a further stimulation of LH release with LHRH is unable to enhance the incorporation of the radioactive precursor. The results suggest a relationship between LH secretion and DNA synthesis in the pituitary gland of the rat.     

Effect of estradiol on the early incorporation of (3H) thymidine into uterine DNA on immature mouse.

The incorporation of [3H]thymidine into uterine DNA was markedly depressed within 10 to 30 minutes after intraperitoneal administration of 17beta-estradiol to immature mouse. Maximum inhibition occurred about 6 hours after the hormone was administered. Uterine DNA content and the amount of [3H]thymidine incorporated into the acid-soluble fraction was not affected during the period of hormone-induced inhibition. Moreover, the in vitro incorporation of [3H]thymidine by isolated estradiol-treated mouse uterus was blocked. In contrast to the uterus, 17beta-estradiol did not influence the incorporation of thymidine into mouse liver DNA. Evidence is presented to show that the incorporation of thymidine into uterine DNA was blocked initially by 17beta-estradiol.     

IN VIVO SPECIFIC LABELING OF CHLAMYDOMONAS CHLOROPLAST DNA

When Chlamydomonas reinhardi is supplied with (methyl-³H)-thymidine, radioactivity is incorporated specifically into chloroplast DNA Chromatographic analysis of the products of enzymatic hydrolysis of the DNA reveals that only thymidine monophosphate has been labeled. Use of thymidine-6-³H yields an identical result. If thymidine-³H monophosphate is supplied, a small amount of radioactivity is incorporated into both nuclear and chloroplast DNA in proportion to the abundance of these DNA components. These observations are consistent with earlier suggestions that algae lack cytoplasmic thymidine kinase, but that the enzyme is present within their chloroplasts.     

Meiotic DNA synthesis during mouse spermatogenesis

The incorporation of radioactivity into various cells in the sequence of spermatogenesis was measured by preparing highly purified spermatozoan nuclei from the cauda epididymidis of mice at daily intervals after injection of (3H)thymidine. The stages of differentiation of these sperm at the time of thymidine administration were calculated from the kinetics of spermatogenesis. The procedure for purification of sperm nuclei included sonication, mechanical shearing, and treatment with trypsin, DNase, Triton X-100, 2M NaC1, and sodium dodecyl sulfate. DNA was isolated from these nuclei by treatment with dithiothreitol and pronase, followed by phenol extraction and ethanol precipitation. The levels of radioactivity in the epididymal sperm head preparations were low (less than 13 dpm/mouse) for 27 days after injection, and then rose dramatically to over 4 times 104 dpm/mouse. Further experiments demonstrated that the 11 dpm of 3H radioactivity contained in sperm heads at 21 or 26 days after injection of (3H)TdR was significantly above background and contamination levels from other cells or other sources. Most of the radioactivity was in the sperm DNA and represented incorporation of tritium from (3H)TdR into the nuclear DNA of meiotic cells at 0.002 percent of the rate of incorporation into S-phase cells. Little, if any, (3H)TdR was incorporation into the DNA of spermatids. The levels of DNA synthesis during the meiotic prophase in the mouse appear to be much lower than those reported for other organisms.     

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.     

Thymidine metabolism and DNA synthesis in Newcastle disease virus-infected cells.

The inhibition of thymidine incorporation into DNA in Newcastle disease virus-infected cells has been studied. At 6 h after infection of L-929 cells at high multiplicity, transport of exogenous thymidine across the cell membrane was inhibited. The kinetics of this inhibition, decreased Vmax with no change in Km, suggest that there are fewer sites available for transport in infected cells. The conversion of thymidine to dTTP was not inhibited. Equilibrium of exogenous thymidine with the acid-soluble pool occurred more slowly and at a lower level of radioactivity than in uninfected cells, and there was a reduction in the rate of incorporation of exogenous thymidine into DNA. The reduction of incorporation into the pool and into DNA was proportionate. The size of total cellular dTTP pools was changed very little in infected cells. DNA synthesized in infected cells in the presence of [3H]BrdUrd had reduced incorporation of tritium but similar buoyant density to that from uninfected cells. The results show that Newcastle disease virus inhibits DNA synthesis directly and, in addition, decreases thymidine transport. Together these account for the overall decrease in thymidine incorporation into DNA of infected cells.     

Variation of thymidine incorporation patterns in the alternating vegetative and sexual life cycles of Chlamydomonas reinhardtii

The cellular distribution of thymidine kinase activities in Chlamydomonas reinhardtii, as manifested by the in vivo incorporation of exogenous thymidine and 5-bromodeoxyuridine into different DNA species, appeared to be organelle specific and varied with different developmental stages in the life cycle of this organism. During vegetative growth and gametogenic differentiation, thymidine and 5-bromodeoxyuridine were shown to be selectively incorporated into chloroplast but not nuclear DNA. On the other hand, during zygotic germination in which meiosis occurs and the ensuing vegetative divisions of meiotic products, thymidine as well as 5-bromodeoxyuridine were incorporated into both nuclear and chloroplast DNA. These results suggest that, in addition to the thymidine kinase activity that is constantly present in the chloroplast, a cytoplasmic thymidine kinase is derepressed only during the sexual reproductive cycle of C. reinhardtii.     

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.     

Inhibition of cell division by interferons. The relationship between changes in utilization of thymidine for DNA synthesis and control of proliferation in Daudi cells.

Inhibition of the proliferation of Daudi cells by exposure to human lymphoblastoid interferons is associated with an early and marked decrease in the incorporation into DNA of exogenous [3H]thymidine when cells are incubated with trace amounts of this precursor. In contrast, incorporation of exogenous deoxyadenosine into DNA is unchanged under the same conditions. Interferon treatment results in a lowering of thymidine kinase activity, an effect which may be largely responsible for the inhibition of incorporation of labelled thymidine into DNA. At higher concentrations of exogenous thymidine, which minimize the contribution of intracellular sources to the dTTP pool, the inhibition of thymidine incorporation is abolished. Under conditions in which exogenous thymidine is rigorously excluded from the medium or, conversely, in which cells are entirely dependent on exogenous thymidine for growth, the magnitude of the inhibition of cell proliferation by interferons is the same as under normal culture conditions. We conclude that, even though cell growth is impaired, the rate of DNA synthesis is not grossly inhibited up to 48 h after commencement of interferon treatment. Furthermore, changes in neither the utilization of exogenous thymidine nor the synthesis of nucleotides de novo are responsible for the effect on cell proliferation.