Thymidine Levels Research Articles

Decreased level of thymidine in the serum of obese strain (OS) chickens with spontaneous autoimmune thyroiditis.

We have previously shown that normal chicken serum (NCS) is able to interfere with the IL 2 promoted incorporation of DNA precursors into T lymphoblasts and that serum derived from autoimmunity prone Obese strain (OS) chickens is deficient in this respect. This "defect in non-specific suppression" has been speculated to be one of the causes for T cell hyperreactivity in the OS. In this study we present several lines of evidence that the suppressive effect of normal chicken serum (NCS) on 5-(125Iodo)-2-deoxyuridine (125IUdR) uptake into chicken T blasts is a competition artefact due to cold thymidine (TdR) present in NCS. Inhibition of 125IUdR required the continuous presence of NCS and suppression of 3H-TdR incorporation could be competed for by increasing the dose of the radiolabel. Molecular sieve chromatography followed by reversed phase high performance liquid chromotagraphy revealed the "inhibitory" activity to co-elute with TdR. Moreover, NCS did not suppress protein synthesis by chicken T cells growing with IL 2 and did not affect oxidative metabolism, cell viability, expression of IL 2 receptors, or percentages of cells in the S phase of the cell cycle. In accordance with these data, OS-sera suprisingly contain less TdR than those from normal controls. Experiments involving crosses of the OS with the normal inbred CB strain, revealed that the subnormal serum TdR level of the OS is an autosomally dominant trait which, however, segregates from T cell hyperreactivity. These findings falsify our previous hypothesis that a defect in specific IL 2 antagonists might be involved in T cell hyperfunction of the OS and indicate that NCS is devoid of factors which neutralize IL 2 function.

Phorbol ester inhibition of insulin-stimulated deoxyribonucleic acid synthesis in BC3H-1 myocytes.

Insulin and 12-O-tetradecanoyl phorbol 13-acetate (TPA) each acutely stimulates hexose transport, amino acid uptake, and pyruvate dehydrogenase activity in the BC3H-1 myocyte in a nonadditive fashion, suggesting that the acute effects of insulin and TPA are mediated through a common mechanism of action. Here we have demonstrated that while chronic incubation with insulin stimulated DNA synthesis by 3- to 6-fold, TPA, in contrast, did not stimulate DNA synthesis and, indeed, caused a 70% inhibition of insulin-stimulated DNA synthesis in a dose-dependent fashion. In differentiated myocytes, insulin maximally stimulated hydroxyurea-sensitive [3H]thymidine incorporation into DNA at 200-400 nM with an ED50 of 5-8 nM, suggesting that insulin stimulates DNA synthesis via the insulin receptor rather than through growth factor receptors. Phorbol ester inhibition of insulin-stimulated DNA synthesis was specific for the active tumor-promoting phorbols and the synthetic diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol. Maximal TPA inhibition of insulin-stimulated DNA synthesis was observed at 100 nM with an ID50 of 30 nM TPA, values analogous to those required for TPA stimulation of hexose transport in the myocyte. Chronic incubation with TPA did not inhibit insulin-stimulated protein synthesis, acute K+ flux, K+ accumulation, cytosolic thymidine levels, or insulin binding, indicating that TPA inhibits a specific intracellular event mediating DNA synthesis and suggesting that the acute and chronic effects of insulin in BC3H-1 myocytes are regulated by distinct pathways.

Augmentation of methotrexate cytotoxicity in human colon cancer cells achieved through inhibition of thymidine salvage by dipyridamole

In HCT 116 cells, a human colon cancer cell line, the levels of thymidine (0.6 μM) and hypoxanthine (9 μM) contributed to the tissue culture medium by the fetal bovine serum significantly reduced the growth inhibition and lethality produced by 0.1 μM methotrexate. Dipyridamole, an inhibitor of nucleoside transport, potentiated the growth inhibitory effects of methotrexate when the cells were grown in medium that was changed daily. However, when the medium was supplemented with dialyzed serum, methotrexate cytotoxicity was not increased by dipyridamole. Similarly, in cloning experiments, dipyridamole increased the cell killing produced by methotrexate. The potentiation of methotrexate toxicity produced by dipyridamole was mediated through inhibition of thymidine uptake. The uptake of 1 μM thymidine was inhibited 50% by 0.12 μM dipyridamole but neither hypoxanthine nor guanine uptake was decreased by dipyridamole (5 μM). As a result, the decrease in dTTP pools produced by methotrexate was augmented by dipyridamole. In contrast, dipyridamole did not influence the effect of methotrexate on ribonucleoside triphosphate pools. HCT 116 cells avidly salvaged low concentrations of thymidine, and methotrexate increased this capacity. Conversion of 0.11 μM thymidine to thymidine triphosphate was increased by 55%, from 16.6 to 25.7 pmoles/10 6 cells, following exposure to 1.0 μM methotrexate. Dipyridamole blocked this pool expansion. This study suggests that the salvage of physiological levels of thymidine may diminish the cytotoxic effects of methotrexate on human colon cancer cells. Inhibition of thymidine uptake by dipyridamole may be an effective strategy to increase the cytotoxicity of methotrexate.

Employment of a [<sup>3</sup>H]Thymidine-Incorporation Assay to Distinguish the Effects of Different Friend Erythroleukemia-Inducing Retroviruses on Erythroid Cell Proliferation

Spleen cells taken from mice infected as adults with two different variants of the spleen focus-forming virus (SFFV), SFFVP and SFFVA, as well as spleen cells taken from mice infected as newborns with Friend murine leukemia virus (F-MuLV) were assayed in a proliferation assay in the presence or absence of the erythroid hormone erythropoietin (Epo). Infection of NIH Swiss mice with SFFV resulted in excessive proliferation of erythroid cells that could still differentiate, and spleen cells taken from these mice were able to incorporate high levels of tritiated thymidine ([3H]dThd) in the absence of Epo, even in the presence of antibodies to Epo. In contrast, the level of proliferation of spleen cells from SFFVA-infected mice, but not those from SFFVP-infected mice, could be greatly enhanced by the addition of Epo to the cultures. Infection of newborn mice with F-MuLV resulted in the generation of Friend mink cell focus-inducing virus, which caused excessive proliferation of erythroid cells that appeared to be blocked in differentiation, resulting in severe anemia. Spleen cells from these mice were unable to proliferate in the absence of Epo. However, when increasing doses of Epo were added to the cultures, the cells proliferated at levels equivalent to the levels seen with SFFV. These results indicate that a proliferation assay based on the incorporation of [3H]dThd into spleen cells in response to Epo can be used as a quantitative means of assessing and comparing the effects of erythroleukemia-inducing retroviruses on the proliferation of their target cells.

RBE Determination of Acute In Vivo Effects from Very Low Dose Whole-body Exposure with Different Radiation Qualities by Three In Vitro Assays

Whole body exposure of NMRI mice (0.002-1 Gy) with 137Cs gamma rays or En = 6 MeV cyclotron neutrons increases the thymidine level in the blood serum and decreases the incorporation of 125I-UdR into bone marrow cells. In the very low dose range these effects are related to a time dependent but not dose dependent inhibition of the intracellular enzyme thymidine kinase. In the low dose range, irradiation induces a release of thymidine in the salvage pathway. Dose-effect curves which were obtained 4 h after exposure indicated that in the very low dose range (D(0.02 Gy) there is a fast neutron RBE of about 1 for all assay systems. In the low dose region (0.05 to 1 Gy) the fast neutron RBE increases to 2–3 for inhibition of tracer uptake into DNA of bone marrow cells and the TdR concentration in blood serum.

Excess thymidine induces folate sensitive fragile sites.

Folate sensitive fragile sites on human chromosomes have been found to be inducible in cultured lymphocytes by high levels of thymidine but not by high levels of BrdU. The biochemical interpretation of events leading to fragile site expression has been revised since it is now clear that low levels of either thymidylate or deoxycytidine triphosphate will result in this phenomenon. A model for the DNA at a fragile site, composed of alternating repeating polypurine/polypyrimidine sequences is proposed.

Antiherpes effects and pharmacokinetic properties of 9-(4-hydroxybutyl) guanine and the (R) and (S) enantiomers of 9-(3,4-dihydroxybutyl)guanine.

Three acyclic guanosine analogs with similar structures, the (R) and (S) forms of 9-(3,4-dihydroxybutyl)guanine and 9-(4-hydroxybutyl)guanine, were compared for antiherpes activity in vivo and in vitro. The three guanosine analogs were viral thymidine kinase-dependent inhibitors of virus multiplication. In cell cultures, (S)-9-(3,4-dihydroxybutyl)guanine was the least active of these three drugs against a variety of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) strains. This was also the case for a certain HSV-1 or HSV-2 strain in different cell lines. In cell cultures, (R)-9-(3,4-dihydroxybutyl)guanine and 9-(4-hydroxybutyl)guanine had similar antiherpes activities. However, in vivo in cutaneous HSV-1 infections in guinea pigs treated topically and in systemic HSV-2 infections in mice treated orally or intraperitoneally, only (R)-9-(3,4-dihydroxybutyl)guanine had a therapeutic effect. The extremely short half-life in plasma and the high clearance of 9-(4-hydroxybutyl)guanine as compared with those of (R)-9-(3,4-dihydroxybutyl)guanine probably made 9-(4-hydroxybutyl)guanine inefficacious when given intraperitoneally or orally to mice infected with herpesvirus. On the other hand, no kinetic differences between (R)-9-(3,4-dihydroxybutyl)guanine and 9-(4-hydroxybutyl)guanine were observed in penetration through guinea pig skin ex vivo, and no preferential metabolism of 9-(4-hydroxybutyl)guanine in skin was noted. We deduced that high thymidine levels in guinea pig skin preferentially antagonize the antiviral effect of 9-(4-hydroxybutyl) guanine in cutaneous HSV-1 infections.

The diurnal fluctuations in unincorporated radio-thymidine pool and its relation to the circadian rhythm of thymidine incorporation in mouse pinna skin

The circadian variation in the labelling indices of epidermal basal and sebaceous gland cells in murine pinna is a well documented phenomenon. This, however, observes only the incorporated label and takes no account of any rhythmic changes in the precursor pool. It is reported here that, using a liquid scintillation counting method, there is a diurnal change in the level of unincorporated radio-labelled thymidine, which is apparently not related to the rhythm of thymidine incorporation. The implications of this phenomenon and its relationship to DNA synthesis are discussed.

The effect of age on the accumulation of labile triosephosphate isomerase and thymidine incorporation in pokeweed mitogen stimulated human lymphocytes.

Peripheral lymphocytes from young persons were found to increase the content of labile triosephosphate isomerase (TPI) following pokeweed mitogen stimulation in vitro. The labile form appears to have the same thermal inactivation pattern as a tetradeamidated form of the enzyme previously shown to accumulate in fibroblasts and lymphoblasts. Human lymphocytes from an elderly sample were also subjected to mitogen stimulation under identical conditions. There was a greater accumulation of labile TPI during lymphoblast transformation in the aged sample. Cellular studies revealed that the absolute level of tritiated thymidine incorporated into DNA was significantly decreased with age in both resting and stimulated lymphocytes, but the blastogenic index showed no significant age-dependent changes. The ratio of the accumulation of labile enzyme during mitogen stimulation to the cellular blastogenic index was increased with age. The accumulation of labile forms of this enzyme may relate to the impairment of the energy-supplying glycolytic pathway that occurs in elderly human lymphocytes subjected to mitogenic stress.

Transfer of a mutant viral thymidine kinase gene results in temperature-sensitive mouse cells.

Temperature-sensitive cell lines were obtained by DNA-mediated transfer of the thymidine kinase (TK) gene from a mutant, ts1117, of herpes simplex virus type 1. The cells died at 39 degrees C in selective medium which contained low levels (1 microgram/ml) of thymidine. In this lethal condition, no revertants were detected among 10(8) cells. It was shown by in vitro analysis of the TK activity that the temperature-sensitive cell line contains an enzyme whose activity is temperature sensitive and relatively unaffected by dTTP. The viral enzyme has these properties. The effect of the lethal growth conditions in the cell line was characterized by cell cycle analysis and rescue experiments which involved a shift to the permissive conditions. The successful transfer of the mutant viral TK activity to cells provides an additional selective marker for gene transfer.

Transfer of a mutant viral thymidine kinase gene results in temperature-sensitive mouse cells.

Temperature-sensitive cell lines were obtained by DNA-mediated transfer of the thymidine kinase (TK) gene from a mutant, ts1117, of herpes simplex virus type 1. The cells died at 39 degrees C in selective medium which contained low levels (1 microgram/ml) of thymidine. In this lethal condition, no revertants were detected among 10(8) cells. It was shown by in vitro analysis of the TK activity that the temperature-sensitive cell line contains an enzyme whose activity is temperature sensitive and relatively unaffected by dTTP. The viral enzyme has these properties. The effect of the lethal growth conditions in the cell line was characterized by cell cycle analysis and rescue experiments which involved a shift to the permissive conditions. The successful transfer of the mutant viral TK activity to cells provides an additional selective marker for gene transfer.

Trimethoprim activity in media selective for Campylobacter jejuni.

The activity of trimethoprim (TMP) in two selective media used for isolation of Campylobacter jejuni was evaluated. The two selective media, Campy-BAP and Skirrow medium, contain TMP in addition to other antimicrobial agents. The minimal inhibitory concentrations of TMP in blood agar base (basal agar for Skirrow medium) or brucella agar (basal agar for Campy-BAP) for three sensitive control organisms were compared with those in Mueller-Hinton agar, which contains low levels of thymidine. TMP was inactive in both blood agar base and brucella agar, even when lysed horse blood or thymidine phosphorylase was added. TMP had activity when used in combination with the other antimicrobial agents normally included in Skirrow medium and Campy-BAP, probably indicating synergism between TMP and one or more of the other antimicrobial agents. Sheep blood could be substituted for lysed horse blood in Skirrow medium without compromising the activity of TMP.

Increased levels of unscheduled DNA synthesis in UV-irradiated human fibroblasts pretreated with sodium butyrate.

AbstractPretreatment of growing normal and xeroderma pigmentosum (XP) human fibroblasts with sodium butyrate at concentrations of 5‐20 mM results in increased levels of DNA repair synthesis measured by autoradiography after exposure of the cells to 254 nm UV radiation in the fluence range 0‐25 J/m2. The phenomenon manifests as an increased extent and an increased initial rate of unscheduled DNA synthesis (UDS). This experimental result is not due to an artifact of autoradiography related to cell size. Xeroderma pigmentosum cells from complementation groups A, C, D and E and XP variant cells all exhibit increases in the levels of UV‐induced UDS in response to sodium butyrate proportional to those observed with normal cells. These UDS increases associated with butyrate pretreatment correlate with demonstrable changes in intracellular thymidine pool size and suggest that sodium butyrate enhances uptake of exogenous radiolabeled thymidine during UV‐induced repair synthesis by reducing endogenous levels of thymidine.

Using miniature osmotic infusion pumps to maintain tritiated thymidine exposure to cells in culture.

To provide a constant level of tracer doses of tritiated thymidine to cultured cells during continuous infusion, miniature osmotic infusion pumps were used to provide replacement thymidine. By determining the loss of isotope from the media during nonreplacement, the rate of constant infusion replacement to maintain thymidine levels was calculated. The replacement rates were similar for the three cell lines examined and allowed a standard osmotic pump infusion.

Effect of high-dose thymidine infusions in patients with mycosis fungoides.

The antitumor effect of thymidine has been demonstrated in patients with leukemia and lymphoma. This report summarizes the treatment of three patients with mycosis fungoides, a chronic T-cell lymphoma. Four courses of thymidine (75 g/m2/day) were administered by continuous infusion for 4-7 days. Steady-state serum thymidine levels were in the range of 1-3 mM. Associated toxicities were minimal and consisted of milk headache and anorexia. Myelosuppression was manifested by transient declines in the peripheral leukocyte count. One patient had extensive clearing of diffuse erythematous plaques on the trunk and extremities that persisted for over one month. A second patient had partial clearing of plaques that persisted for two weeks following therapy and a third patient had a minimal response with 25% reduction in lymphadenopathy and noduloulcerative lesions. These responses indicate the effectiveness of thymidine as a single agent in the treatment of mycosis fungoides.

Activity of trifluorothymidine against cytomegalovirus.

Trifluorothymidine (TFT) was tested for antiviral activity against mouse cytomegalovirus (MCMV) and human cytomegalovirus (HCMV) in one-step replication assays. The TFT concentration required to reduce virus yield by 50% (ID50) was 0.22 microM for MCMV and 0.012 microM for HCMV. The antiviral activity of TFT against MCMV was reversed by addition of equimolar thymidine, and no antiviral activity was demonstrable in a host cell line lacking thymidine kinase. Thus, TFT's anti-MCMV activity is dependent on a host cell TK since this herpesvirus lacks thymidine kinase. A continuous subcutaneous infusion of TFT achieving a serum concentration of 1 microM failed to protect mice from lethal MCMV infection, perhaps because serum levels of thymidine were comparable to the drug level. Comparison of the ID50 against HCMV and the ID50 against human bone marrow progenitor cells resulted in an in vitro therapeutic ratio of 108, suggesting that TFT might offer some promise as a clinically useful anti-HCMV agent.

Radioimmunoassays of plasma thymidine, uridine, deoxyuridine, and cytidine/deoxycytidine

Radioimmunoassay techniques have been developed for the assay of thymidine, uridine, deoxyuridine, and deoxycytidine. Plasma levels of the first three nucleosides have been measured, and an upper limit has been determined for the plasma concentration of deoxycytidine. The assays involve displacement of a [ 3H]pyrimidine nucleoside from the appropriate labeled rabbit immunoglobulin. By assaying a mixture of uridine and deoxyuridine in the presence and absence of borax, the concentrations of both nucleosides have been measured. In seven healthy adults, plasma levels of uridine were 21.1 ± 8.4 μ m (mean ± SD) and of deoxyuridine were 0.62 ± 0.39 μ m. In cancer patients, thymidine levels were 7.5 ± 2.7 × 10 −7 m. The upper limit for plasma deoxycytidine levels in six healthy adults was 0.71 ± 0.1 μ m.

Hormonal control of tobacco protoplast nucleic acid metabolism during in vitro culture

Tobacco mesophyll protoplasts cultivated in vitro do not synthesize a measurable quantity of chloroplastic ribosomal RNA, but actively synthesize cytoplasmic ribosomal RNA, polyadenylated RNA, and proteins. These syntheses are essentially independent of the presence of hormones in the culture medium and are thus related to the ageing phenomenon induced by isolation from the plant and in-vitro culture. At all stages of culture and in all culture media, protoplasts incorporate low levels of thymidine into their DNA. However, the incorporation of considerable quantities of thymidine, indicative of the S phase, only takes place after 25-30 h and requires the presence of auxin and cytokinin.

The activities of thymidine metabolising enzymes during the cell cycle of a human lymphocyte cell line LAZ-007 synchronised by centrifugal elutriation

The activities throughout the cell cycle of thymidine kinase (EC 2.7.1.21), dihydrothymine dehydrogenase (EC 1.3.1.2), thymidine phosphorylase (EC 2.4.2.4) and dTMP phosphatase (EC 3.1.3.35) were measured in the Epstein-Barr virally transformed human B lymphocyte line LAZ-007. Cells were synchronised at different stages of the cell cycle using the technique of centrifugal elutriation. The degree of synchrony in each cycle-stage cell population was determined by flow microfluorimetric analysis of DNA content and by measurement of thymidine incorporation into DNA. The activity of the anabolic enzyme thymidine kinase was low in the G 1 phase cells, but increased many-fold during the S and G 2 phases, reaching a maximum after the peak of DNA synthesis, then decreasing in late G 2 + M phase. By contrast, the specific activities of the enzymes involved in thymidine and thymidylate catabolism, dihydrothymine dehydrogenase, thymidine phosphorylase and dTMP phosphatase remained essentially constant throughout the cell cycle, indicating that the fate of thymidine at different stages of the cell cycle is governed primarily by regulation of the level of the anabolic enzyme thymidine kinase and not by regulation of the levels of thymidine catabolising enzymes.

Thymidine resistance in Chinese hamster V79 cells in vitro

Thymidine resistance in V79 Chinese hamster cells has been investigated. Phenotypically stable variant resistant lines occurred at a high frequency, and the mutation rate (2.67 × 10 −3 per cell per generation) to 400 μm/ml thymidine resistance as measured by the standard Luria-Delbrück fluctuation analysis was extremely high. Populations of cells maintained for extended periods in F-10 medium spontaneously increased in resistance, possibly as a result of selective pressures due to the thymidine present in F-10 medium since this change was not observed in Dulbecco's medium. The degree of resistance for a given variant was correlated with the amount of thymidine employed in its selection. Metabolic cooperation, resulting in the suppression of the resistant phenotype, was demonstrated in artificial mixtures of sensitive and resistant clonal lines. Clones isolated in high levels of thymidine possessed lowered uptake of [ 3H]thymidine and the depression in uptake was related to the level of resistance of the particular clone. Although thymidine kinase specific activity levels were slightly depressed in variant cell lines, growth rate and uridine uptake were unaffected. We conclude that thymidine resistance is due to a genetically controlled depression of external thymidine uptake.