Thymidine Triphosphate Incorporation Research Articles

CYCLOSPORINE AND RAPAMYCIN AFFECT PROTEIN KINASE C INDUCTION OF THE INTRACELLULAR ACTIVATION SIGNAL, ACTIVATOR OF DNA REPLICATION

Immunosuppressive agents may initiate their pharmacologic action by disrupting phosphorylation cascades critical to T lymphocyte intracellular signaling after alloantigen recognition. Activator of DNA Replication (ADR), a transduction signal sensitive to CsA as well as rapamycin (RAPA) immunosuppression, seemed a likely candidate for phosphoregulation. This communication reports the development of a cell-free assay wherein CsA and RAPA inhibit ADR induction by protein kinase C (PKC). ADR and PKC are inactive in the cytosol of resting cells. Endogenous PKC activity in quiescent lymphocytes was triggered with the tumor promoter PMA, leading to the appearance of ADR, an event subsequently quantitated by the ability of ADR to trigger [3H]thymidine triphosphate incorporation into isolated nuclei. After PKC induction, ADR activity is increased (890 +/- 120 vs. 3910 +/- 345 cpm, P < 0.001). In the presence of the PKC inhibitor H7, ADR activity fails to increase (100 +/- 50 vs. 3910 +/- 345 cpm, P < 0.001). The high levels of ADR found in PHA-stimulated cells is marginally affected by in situ PKC induction, although the sustained impact of H7 throughout the cell cycle suggests that ADR is constantly being phosphorylated. Titration of CsA or RAPA into the cell-free system inhibited ADR induction in a dose-dependent fashion. The utility of ADR induction as a marker for immunosuppression was investigated by comparing ADR inducibility in resting cells that had been preloaded with CsA or RAPA with the proliferative index of cells cultivated with CsA or RAPA. ADR induction directly correlated with CsA or RAPA proliferative inhibition. The results suggested that ADR is a constituent of the PKC phosphoregulatory cascade essential for cell cycle progression. The correlation between cell-free ADR inducibility and proliferative inhibition by CsA or RAPA suggest that this procedure may be useful for in vitro prediction of allografted patient immunologic response.

Bioaccumulation of tetra-through octachlorinated dibenzo-p-dioxins in two different mixtures and their biochemical effects on enzymes of carbohydrate metabolism in mice

Two different mixtures of various PCDD homologues and congeners originating from octachlorodibenzo-p-dioxin by dechlorination were administered by oral gavage once a week to C57BL/6J (B6) and DBA/2J (D2) mice, respectively, for up to 15 weeks followed by a short recovery period. Each mouse strain was tested with two different doses of these mixtures as well as with 2,3,7,8-TCDD. Neither the clinical symptoms nor the development of body weight varied from those observed in control animals. Liver weight increased in a dose-dependent manner, and significant treatment-specific thymus involution was observed. TCDD and most of the 2,3,7,8-congeners reached steady state concentrations in the liver approximately 10 weeks after starting the administration. Modulations in carbohydrate metabolism were observed in the liver under the influence of all treatments. Investigation of epidermal metabolism revealed signs of hyperproliferation as evidenced by an increase in ornithine decarboxylase activity and of thymidine triphosphate incorporation in B6, but not in D2 mice.

Effects of the Phthalazinone Azelastine on Epidermal Metabolism after Mechanical Skin Irritation

In female NMRI mice, the phthalazinone azelastine was administered orally once daily over 7 days. The drug influenced the epidermal thymidine triphosphate and amino acid incorporation rates at doses between 1 and 5 mg/kg. In control mice, an epidermal hyperproliferation induced by abrasion of superficial epidermal layers was characterized by enhanced prostaglandin and leukotriene concentrations in epidermal homogenate, an increase in thymidine triphosphate and amino acid incorporation and an increase in epidermal thickness. In mice treated with 1 mg/kg azelastine HCl, this epidermal reaction was changed. Compared to controls, the increase in leukotriene concentration was diminished, and that of prostaglandins was enhanced. The incorporation of thymidine triphosphate and of amino acids as well as the epidermal thickness and the ratio cell count/epidermal thickness were increased in irritated skin of azelastine-treated mice. In conclusion, azelastine influences the epidermal metabolism in irritated and unirritated skin. Therefore, a beneficial role of this phthalazinone in the treatment of psoriasis and related skin disorders seems to be possible.

The mouse epidermis as a model in skin pharmacology: influence of age and sex on epidermal metabolic reactions and their circadian rhythms

The influence of age and sex on epidermal metabolism and histology was studied in mice (NMRI). Within a few weeks postpartum, thymidine triphosphate incorporation into DNA and amino acid incorporation into epidermal protein decreased. Differences between tail epidermis and other skin regions (back, abdomen, ear) were found. Sex dependent differences could not be demonstrated. A circadian rhythm of the studied epidermal metabolic processes was shown (artificial 12L:12D light/dark cycle). Maximum incorporation of thymidine triphosphate and of amino acids was found at the end of the light period at 1800 h, while a minimum incorporation was demonstrated at the end of the dark period. Considering described biological variations, the mouse tail assay seems to be suited to study influences of drugs in epidermal metabolism and histology.

Two-stage incorporation of thymidine triphosphate into mammalian DNA as indicated by chromatography on benzoylated DEAE-cellulose

Replicating DNA contains single-stranded regions as indicated by the extent of its recovery in the final fraction, following stepwise elution of sheared preparations from benzoylated DEAE-cellulose using 0.3 M sodium chloride, 1.0 M sodium chloride and caffeine solutions, respectively. In preparations of hepatic DNA, isolated up to 60 min after administration of [ 3H]thymidine to rats subjected earlier to partial hepatectomy, the proportion of radioactivity contained in the caffeine-eluted fraction progressively decreased. This change was attributable to migration of incorporated radioactivity from replicating forms to mature (double-stranded) DNA, the latter being recovered from the column in 1.0 M sodium chloride. In terms of the same chromatograms, the relative size of the 0.3 M sodium chloride-eluted fraction also decreased, this fraction of radioactivity co-eluting from benzoylated DEAE-cellulose with thymidine triphosphate. The precursor—intermediate—product relationship between the three products separated by benzoylated DEAE-cellulose chromatography was confirmed using DNA isolated from cultured mammalian cells. Early eluting ratioactivity co-chromatographed with thymidine triphosphate on thin-layer cellulose, whilst the intermediate status of caffeine-eluted radioactivity was confirmed following pulse-chase labelling procedures. Utilizing stepwise chromatography of such DNA on benzoylated DEAE-cellulose, the effect of three inhibitors could thus be described as affecting either an early or late stage of DNA synthesis. Such an approach offers a simple quantitative method of monitoring influences on DNA synthesis.

DNA synthesis in chromatin preparations from human fibroblasts infected by cytomegalovirus.

Chromatin prepared from (14C)-thymidine pulse labelled cytomegalovirus-infected human fibroblasts 72 hours postinfection exhibited under appropriate conditions endogenous activity of (3H)-thymidine triphosphate incorporation which was relatively salt-resistant and phosphonoacetic acid-sensitive. Isopycnic centrifugation of the doubly labelled DNA in CsCl revealed that cell-free incoporation occurred into viral as well as into host cell DNA. Density labelling experiments with bromodeoxyuridine triphosphate suggested the incoporation into viral DNA to be due to replicative DNA synthesis. Chromatin from infected cells contained, in addition to cellular, viral DNA polymerase activity.

3H]Thymidine triphosphate incorporation into isolated nuclei.

3H]Thymidine triphosphate incorporation into isolated nuclei.

The demonstration of separate DNA polymerase activities in intact isolated rat liver nuclei by means of response to bleomycin and arabinosyl cytosine 5-triphosphate.

Rat hepatic nuclei, isolated and studied in vitro, tend to reflect changes occurring in DNA synthesis in the intact rat (1). Nuclei from normal rat liver synthesize minimal amounts of DNA, while nuclei from a partially hepatectomized rat show a marked increase in thymidine tri-phosphate incorporation. This increase probably represents continuation of elongation of replicating DNA strands begun in vivo (2). Studies of extracts of rat liver nuclei suggest that there are at least two separate DNA polymerase activities: a tightly-bound activity which rises in quantity along with DNA replication following hepatectomy and is inhibited by cytosine arabinoside 5′-triphosphate (ara-CTP), and a loosely-bound activity which rises little or not at all after partial hepatectomy and is insensitive to ara-CTP (3).We have previously reported that Bleomycin causes dose-related and time-related increases of 3H-thymidine triphosphate incorporation into DNA of isolated normal rat liver nuclei (4). This incorporation is presu...

Phleomycin stimulation of thymidine triphosphate incorporation by animal cell nuclei.

Phleomycin stimulation of thymidine triphosphate incorporation by animal cell nuclei.

Effects of bleomycin on PHA-stimulated lymphocytes and isolated hepatic nuclei.

Experiments were carried out in which lymphocytes stimulated by phytohemagglutinin were exposed during 3-day culture to concentrations of bleomycin varying from 0.0 to 300 μg/ml. Inhibition of one hour 3H-thymidine incorporation in these 72-hour cultures increased as the concentration of bleomycin increased from 1.5μg/ml (14.1 % inhibition) to 300μg/ml (96.4% inhibition), and was a linear function when dose and percent control 3H-thymidine uptake were plotted logarithmically. In 3 of 9 experiments, 3H-thymidine uptake at the 1.5μg/ml dose was increased. This increase was not statistically significant, but the possibility of sublethal DNA damage followed by repair was raised. Exposure of resting lymphocytes to bleomycin in the absence of PHA showed no significant increase above baseline thymidine uptake. Addition of bleomycin to cultures at 24 and 48 hours after the addition of PHA produced the same quantitative reduction in uptake of thymidine at 70 hours as the same concentration added at zero time, suggesting that the effect of bleomycin is independent of the initial stimulatory events resulting from the addition of PHA. Evidence of a repair-like process following damage by bleomycin was observed in a different system utilizing isolated hepatic nuclei. A two to five fold increase of thymidine tri-phosphate incorporation was obtained in response to addition of bleomycin in this isolated nuclear system (rat liver nuclei). This process was time and dose related and continued for at least one hour in the presence of bleomycin. These results demonstrate that bleomycin causes dose-dependent reduction of DNA synthesis in PHA-stimulated lymphocytes. Furthermore, they suggest that DNA damage caused by low concentrations of bleomycin may be repaired, and that when damage exceeds the cell's ability to repair, cell death results.

DNA polymerase activity in mammary gland of virgin mice after ovarian hormone treatment.

SUMMARY The effect of ovarian hormones on DNA polymerase activity in the mouse mammary gland was determined. The rate of [3H]thymidine triphosphate incorporation into DNA during incubation in vitro was used as a measure of DNA polymerase activity in the post-microsomal supernatant fraction of the mammary tissue. A negligible amount of DNA polymerase activity was present in the mammary tissue of ovariectomized mice which had no hormone treatment. Daily subcutaneous injections of 1 μg oestradiol-17β plus 1 mg progesterone induced a sixfold increase of DNA polymerase activity in the mammary tissue after two injections. A single intraperitoneal injection of the same dose also induced a similar rise of DNA polymerase activity and the effect was evident within hours after the treatment. The results provide evidence that stimulation of cellular biosynthesis by the growth-promoting ovarian hormones is associated with the activation of a specific enzyme involved in DNA replication.