Nucleotide metabolism and nucleic acid synthesis in mouse thymocytes.

Nucleotide pool sizes, DNA polymerizing enzymes, and DNA synthesis were studied in mouse thymocytes over a 24-hr period of culture in Marbrook chambers. The initial rate of cell division was high with an average mitotic index of 1.6%/hr for 12 hr, followed by a decline to 0.14%/hr at 24 hr. The decline in DNA synthesis was not closely correlated with the activities of DNA-dependent DNA polymerases-α or -β or with the activity of terminal deoxynucleotidyl transferase. The amounts of several ribo- and deoxyribonucleotides per thymocyte were measured using high performance liquid chromatography and DNA polymerase. Pool sizes of ATP, GTP, dATP, and dTTP were less than 10% of pool sizes commonly observed in mammalian cells. The rates of DNA and RNA synthesis in thymocytes may be critically affected by minor changes in the availability of nucleotides. Cultures of thymocytes serve as useful experimental systems for investigation of nucleotide and nucleic acid metabolism during lymphoid differentiation.     

Cyclic AMP dependent regulation of mitosis in human lymphoid cells

Intracellular levels of cyclic AMP (cAMP), cAMP-dependent phosphodiesterase activity, and adenylate cyclase activity are examined in an established line of human lymphoid cells synchronized by either excess thymidine or by colcemid treatment. cAMP levels and adenylate cyclase activities during the two G periods are high when compared with the values in M. cAMP-dependent phosphodiesterase activity, which is low during early G 2, is shown to increase during G 2 and reach a maximum activity during M. Agents such as dibutyryl cAMP, 1-methyl-3-isobutyl xanthine, noradrenaline, and isopropyl noradrenaline, which increase the levels of intracellular cAMP were examined to determine their effects on mitosis and on DNA synthesis. In thymidine-synchronized cells the onset of mitosis is prevented by increasing or maintaining high levels of cAMP during G 2. The specificity of inhibition of DNA synthesis or mitosis by dibutyryl cAMP is a function of the time, during the cell cycle, when the analogue is added. The elevation of cAMP by methyl xanthine results in a more general inhibition of nucleic acid synthesis and mitosis. Although both catecholamine hormones inhibit mitosis, isopropylnoradrenaline also inhibits DNA synthesis while noradrenaline treatment does not result in such inhibition.     

Comparative induction of unscheduled DNA synthesis by physical and chemical agents in non-proliferating primary cultures of rat hepatocytes

The incorporation of [³H]thymidine into DNA due to unscheduled DNA synthesis (UDS) induced by N-OH-2-acetylaminofluorene (N-OH-AAF), aflatoxin B₁ (AFB₁), ethyl methanesulfonate (EMS) and ultra-violet light was quantitated by autoradiography and by scintillation spectrometry on acid precipitable macromolecules or DNA insolated by isopycnic banding in cesium chloride (CsCl). Dose-dependent increases in UDS due to N-OH-AAF and AFB₁ treatment were found. Only 2-fold increases at the highest dose levels were found, however, when incorporated [³H]thymidine was quantitated by scintillation spectrometry. Seven, 11, and 25-fold increases in UDS induced by AFB₁, N-OH-AAF and ultra-violet light, respectively, were found when incorporated [³H]thymidine was quantitated by autoradiography, indicating a high sensitivity for detecting ‘long patch’ repair by this technique. Scintillation spectrometry was completely ineffective in detecting EMS-induced UDS, whereas autoradiography demonstrated a small, but significant induction in [³H]thymidine incorporation at high dose levels. The non-proliferative nature of the primary hepatocyte prohibits the uniform radioactive prelabeling of DNA, necessary in other techniques, for the detection of ‘short patch’ repair induced by compounds such as EMS. Therefore, the sensitivity of the primary cultured rat hepatocyte in conjunction with UDS for detecting DNA damage caused by mutagens and carcinogens which induce ‘short patch’ repair may be limited to the autoradiographic analysis of the unscheduled incorporation of [³H]thymidine.     

The relation between the increase in reduced nicotinamide nucleotides and the initiation of DNA synthesis in sea urchin eggs

Previous work has suggested that the activation of the sea urchin egg at fertilization is the result of a transient increase in intracellular free calcium and an increase in intracellular pH. We have investigated the absence of nuclear activation in incompletely activated eggs and have found a correlation between nuclear activation and the levels of total reduced nicotinamide nucleotides (NAD[P]H). Eggs activated with ammonia show a similar correlation: besides its action as a weak base in raising intracelluar pH (which we conclude is insufficient to stimulate or maintain nuclear activation as judged by nuclear envelope breakdown or DNA synthesis), ammonia increases NAD(P)H. This increase is associated with the stimulation of 6-³H-thymidine incorporation into egg DNA. Removing ammonia decreases NAD(P)H, and tritiated thymidine incorporation ceases. We conclude that a critical level of NAD(P)H is essential to nuclear activation and that the increase of NAD(P)H at fertilization must be included with the increase in calcium and pH as a causal agent in development.     

Involvement of proteolytic acivity in early events in lymphocyte transformation by phytohemagglutinin

The stimulation by phytohemagglutinin (PHA) of DNA synthesis in cultured blood lymphocytes of guinea pig was markedly inhibited by addition of leupeptin, a well-characterized, powerful protease inhibitor of tripeptide nature. About 30 to 40 per cent inhibition was observed at 40 μg/ml of leupeptin when leupeptin was added 30 min prior to or together with PHA. Per cent inhibition by the appropriate amount of leupeptin was proportional to the amount of PHA added in the range of 0.6 to 3.0 μg of PHA at which the per cent inhibition reached maximum. This inhibitory effect of leupeptin on PHA stimulation was abolished when the lymphocytes were preincubated with PHA for more than 10 min before addition of leupeptin or preincubated with leupeptin for more than 60 min prior to PHA addition.     

Biosynthesis of cyclopiazonic acids in Penicillium cyclopium: The isolation of dimethylallylpyrophosphate: Cyclo-acetoacetyltryptophanyl dimethylallyltransferase

During the production of β-cyclopiazonic acid (βCA) by Penicillium cyclopium, the following points are noted: (a) Dimethylallylpyrophosphate (DMAPP), which is incorporated into α-cyclopiazonic acid (αCA) in vivo, stimulates overall CA synthesis, whereas tryptophan, although incorporated into αCA, inhibits overall CA synthesis. (b) A previously suggested substrate, γ,γ-dimethylallyltryptophan, is not a precursor of αCA. (c) The accumulation of cyclo-acetoacetyl-l-tryptophanyl (cAATrp) is described in both the culture medium and mycelium with increasing growth. (d) A cell-free extract of mycelium will catalyze the conversion of exogenous cAATrp and exogenous DMAPP into βCA in 1:1 stoichiometry, the βCA being bound to a protein.     

Changes in polyamine metabolism in WI38 cells stimulated to proliferate.

Quiescent confluent monolayers of WI38 human diploid fibroblasts were stimulated to proliferate by replacement of the exhausted medium with fresh medium containing 10% fetal calf serum. The cellular content of the polyamines, putrescine, spermidine, and spermine was studied at various intervals after the nutritional change. The putrescine content increased during the pre-replicative phase of the cell cycle, whereas the content of spermidine and spermine did not increase until after the initiation of DNA synthesis. By varying the composition of the stimulating medium it was possible to alter the percentage of cells that were stimulated to proliferate. Measurement of the cellular polyamine content and ³H-thymidine (³H-TdR) incorporation into DNA at the time of the maximal rate of DNA synthesis showed that the magnitude of putrescine accumulation depended on the percentage of cells that were stimulated to proliferate. These results indicate that there may be a connection between polyamine synthesis and subsequent DNA replication.     

Aphidicolin does not inhibit the repair synthesis of mitotic chromosomes

In order to determine the possible role of α-polymerase in repair synthesis, we measured the unscheduled DNA synthesis stimulated by UV-irradiation in mitotic HeLa cells in the presence of Aphidicolin, a specific inhibitor of this enzyme. The results do not show any inhibition of the unscheduled DNA synthesis,indicating that the function of α-polymerase is not essential for repair synthesis of mitotic cells.     

Mitochondrial DNA synthesis in cell cycle mutants of saccharomyces cerevisiae

Mitochondrial DNA replication was examined in mutants for seven different Saccharomyces cerevisiae genes which are essential for nuclear DNA replication. In cdc8 and cdc21, mutants defective in continued replication during the S phase of the cell cycle, mitochondrial DNA replication ceases at the nonpermissive temperature. Replication is temperature sensitive even when these mutants are arrested in the G1 phase of the cell cycle with α factor, a condition where mitochondrial DNA replication continues for the equivalent of several generations at the permissive temperature. Therefore the cessation of replication results from a defect in mitochondrial replication per se, rather than from an indirect consequence of cells being blocked in a phase of the cell cycle where mitochondrial DNA is not normally synthesized. Since the temperature-sensitive mutations are recessive, the products of genes cdc8 and cdc21 must be required for both nuclear and mitochondrial DNA replication. In contrast to cdc8 and cdc21, mitochondrial DNA replication continues for a long time at the nonpermissive temperature in five other cell division cycle mutants in which nuclear DNA synthesis ceases within one cell cycle: cdc4, cdc7, and cdc28, which are defective in the initiation of nuclear DNA synthesis, and cdc14 and cdc23, which are defective in nuclear division. The products of these genes, therefore, are apparently not required for the initiation of mitochondrial DNA replication.     

The effect of cyclic nucleotides on purine biosynthesis and the induction of PRPP synthetase during lymphocyte activation

The activation of lymphocytes has been used to study the regulation of mammalian gene expression. Concanavalin A (Con A) added to mouse spleen lymphocytes in serum-free medium leads to an increase in the rate of DNA synthesis as great as 1000 fold, commencing 20 hr after its addition. Prior to 20 hr, the rate of purine synthesis increases 10–100 fold as measured by accumulation of the purine intermediate, formyl glycineamide ribonucleotide (FGAR). Addition of dibutyryl cyclic GMP to the lymphocyte suspensions results in a 10 fold increase in the rate of DNA synthesis in the absence of Con A and enhances both purine synthesis and DNA synthesis in its presence. The activity of phosphoribosyl pyrophosphate synthetase (PRPP synthetase), an enzyme central to purine and pyrimidine biosynthesis, is increased 2–10 fold during the activation. The increase begins to appear 8 hr after Con A addition and requires concomitant protein synthesis. The induced PRPP synthetase activity is stimulated by the presence of cyclic GMP in the enzyme assay. Addition of dibutyryl cyclic AMP to Con A-stimulated lymphocytes inhibits FGAR production, the stimulation of DNA synthesis, and the appearance of cyclic GMP-sensitive PRPP synthetase. These studies suggest that cyclic nucleotides play a significant role in the molecular mechanism of lymphocyte activation, the regulation of purine biosynthesis, and of eucaryotic genetic expression.     

Age-related alterations in plasma membrane glycoprotein content and scheduled or unscheduled DNA synthesis.

WI-38 cells of various ages and SV40-transformed WI-38 cells were examined for differences in plasma membrane composition of glycoproteins and DNA synthesis. Sialic acid per milligram of protein content of the membranes of WI-38 cells decreased with passage of time in culture. Other glycoprotein fractions and alkaline phosphatase activity disappeared in the WI-38 cells with passage of time in culture (Phase III). Studies of DNA repair correlated with changes observed in the plasma membrane glycoprotein content of WI-38 cells over a passage of time in culture were also reported. Both the extent and rate of ultraviolet-induced unscheduled DNA synthesis remained relatively constant during the passage of the WI-38 cells until late phase III. At that time the extent of unscheduled DNA synthesis was measurably reduced. The number of cells in a population of phase III cells able to perform semiconservative DNA synthesis diminished with age in culture but not to an extent capable of explaining the observed changes seen in membrane composition of semiconservative DNA synthesis during passage of the cells in culture. Cells with an extended lifespan SV40-transformed WI-38 (VA 13.2 RA) cells, did not vary in membrane composition, semiconservative DNA synthesis, or unscheduled DNA synthesis over 200 serial subpassages of the cells in culture.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. II. Inhibition by inhibitors of protein synthesis.

The effect of inhibitors of protein synthesis on the killing of tumor cells by in vitro activated macrophages was determined. Cytotoxicity was inhibited by concentrations of puromycin, pactamycin, and actinomycin D that almost completely inhibited protein synthesis by guinea pig macrophages, but not by concentrations of drug that inhibited protein synthesis by only ± 50%. Cytotoxicity was inhibited when the effector macrophages were pretreated with the metabolic inhibitors, but not when the drugs were added 30 to 60 min after the initiation of the reaction. Pretreatment with puromycin or pactamycin also markedly inhibited the binding of tumor cells by mediator activated macrophages. These results are consistent with the hypothesis that one possible mechanism by which inhibitors of protein synthesis inhibit macrophage mediated cytotoxicity is by inhibiting close contact between effector and target cells. The finding that pretreatment of activated macrophages with trypsin also inhibits tumor cell killing suggests that protein synthesis may be necessary to maintain an adequate number of “recognition structures” on the macrophage membrane, structures that mediate the initial contact between the activated macrophage and the target tumor.     

14 C-acetate incorporation and nucleic acid synthesis in human lymphocytes stimulated by PHA and tuberculin in vitro

Studies on the timing of incorporation of labeled acetate in relationship to other cellular events in phytohemagglutinin (PHA)-treated lymphocytes have suggested that acetylation of nuclear histones may constitute an important regulatory mechanism for gene activation. In the present investigation, it was shown that PHA stimulation of lymphocytes from a tuberculin-positive patient caused an early increased incorporation of ¹⁴C-acetate prior to RNA and DNA synthesis. Lymphocytes from the same patient, however, repeatedly showed no increased incorporation of ¹⁴C-acetate following exposure to the sensitizing antigen, tuberculin (PPD), even though RNA and DNA synthesis were markedly stimulated. These results suggest that regulatory mechanisms of DNA template activity other than acetylation may be operative in sensitized lymphocytes responding to specific antigen. One possible explanation for the differences in ¹⁴C-acetate incorporation is that the increased uptake of acetate exhibited by PHA-treated cells is an effect related to nonspecific membrane changes caused by the PHA. If this is the case, then template regulation in PHA and antigen-stimulated lymphocytes may be achieved via similar but yet to be defined mechanisms.     

SCE evaluations in human lymphocytes after G0 exposure to mitomycin C Lack of expression of MMC-induced SCEs in cells that have undergone greater than two in vitro divisions

We conducted a series of experiments designed to determine whether DNA damage induced in G₀ lymphocytes by mitomycin C (MMC) would be expressed as sister-chromatid exchanges during the second and third post-treatment cell cycles. Lymphocytes from normal donors were exposed to MMC for 2 h prior to culture in the presence of phytohemagglutinin. MMC-treated and control cells were subsequently exposed to bromodeoxyuridine (BrdUrd) for the entire culture period (i.e. 48 h or 72 h) or for the terminal 24 h of 72-h cultures. We observed a 3–4-fold increase in SCEs in MII metaphases from lymphocytes treated with MMC and cultured in the presence of BrdUrd for the entire culture period. In contrast, in replicate cultures of MMC-treated lymphocytes that were exposed to BrdUrd for the terminal 24 h only, the SCE frequency in uniformly harlequinized metaphases was not significantly different from that observed in control cultures. We interpret these data as providing evidence that MMC-induced lesions (or alterations) in the DNA of G₀ lymphocytes are probably expressed as SCEs during the first period of mitogeninduced DNA synthesis, and that these lesions do not persist and give rise to SCEs in subsequent cell divisions.     

Effects of indomethacin on intestinal secretion, prostaglandin E and cyclic AMP: Evidence against a role for prostaglandins in cholera toxin-induced secretion

The effects of indomethacin on intestine mucosal cAMP, intestinal fluid secretion, and mucosal and fluid PGE were studied in rabbits in vivo following challenge with cholera toxin. Indomethacin had no effect on cholera toxin-induced fluid secretion or cAMP accumulation. Inhibition of PGE synthesis was achieved by the administration of two but not one injection of indomethacin. These studies provide evidence against a role for PGE in mediating cholera toxin-induced secretion and point out the need to measure prostaglandin levels when using prostaglandin synthetase inhibitors in vivo.     

Ornithine decarboxylase and polyamines in the resumption of cycling by diluted and reoxygenated mammalian cells

Plateau-phase V79 Chinese hamster cells induced to reenter the proliferative cell cycle from G1/G0 by dilution in fresh medium showed an early increase in ornithine decarboxylase (ODC) followed by increases in some polyamines and in DNA synthesis. In contrast, cells accumulated in G1/G0 by growth in extreme hypoxia for 60 h and induced to recycle by reoxygenation did not respond with early increases in either ODC or polyamines.     

Inhibition of DNA synthesis in permeabilized L cells by novobiocin

Novobiocin was equipotent in inhibiting DNA and RNA synthesis in cultured mouse L cells. It also suppressed in vitro DNA and RNA synthesis in permeabilized L cells and nuclei; 50 percent inhibition of DNA and RNA synthesis was obtained by 1 mM and 20 mM novobiocin, respectively. ATP antagonized the effect of novobiocin. Nalidixic acid had a weak inhibitory effect on in vitro DNA synthesis; 10 mM nalidixic acid showed 60 percent inhibition. ATP did not antagonize nalidixic acid. The inhibitory effect of novobiocin exceeded that of aphidicolin. These findings suggest a participation of a gyrase- and/or type II topoisomerase-like enzyme in the DNA replication machinery in L cells.     

Transient lysozymuria during Sarcoma I tumor rejection

The present studies on the in vivo regulation of lysozyme synthesis by cytotoxic macrophages infiltrating Sarcoma I (Sa I) tumor ascites of C57BL/KS mice are an extension of previous observations that the elevation of serum and urinary lysozyme levels might reflect the degree of macrophage-mediated host resistance. Cytotoxic macrophages of host origin are required in the natural rejection of tumors by C57BL/Ks mice challenged with Sa I. The amounts of lysozyme in the serum and urine of the affected mice are quantitated during Sa I growth and rejection. The enzyme levels are correlated with the number of macrophages in the tumor and with the capacity for lysozyme synthesis measured in vitro on monolayers of macrophages isolated from the ascites. The results indicate that the elevation of serum and urinary lysozyme activity is a reflection of both the increased number of cytotoxic macrophages in the host and the qualitative change in the functional state of the cells. These parameters alone cannot account for the extent of the in vivo release of lysozyme in the serum and urine of the affected animals. High levels of lysozyme are present in the extracellular fluids of the mice only when the macrophages are actively participating in host defense. We conclude that in vivo control mechanisms which depend on the presence of viable tumor cells are responsible for the regulation of lysozyme release by cytotoxic macrophages. However, the high levels of lysozyme do not always indicate complete rejection, as indicated by a preliminary observation in which extensive lysozymuria was followed by tumor relapse.     

The effect of arachidonic acid and prostanoids on collagen dissolution in human uterine cervix in vitro

Explants of human non-pregnant cervix produce collagenolytic enxymes which degrade collagen over a 10 day period in culture. This is significantly enhanced by the presence of very low concentrations of arachidonic acid (10⁻¹⁶−10⁻¹¹M). Prostaglandin E₂, F_2α and 6-keto-F_1α were synthesised in declining amounts over the 10 day period and synthesis was not increased by adding arachidonic acid (10−11M). Meclofenamic acid (10⁻⁶M) and indomethacin (10⁻⁵M), but not tranylcypromine (10⁻⁵) suppressed prostaglandin synthesis yet all reduced collagen dissolution. Mepacrine (phospholipase A₂ inhibitor) also suppressed collagen dissolution. Remodelling of the structure of the cervix matrix may, in part, depend upon arachidonic acid or one of its cyclo-oxygenase or lipoxygenase derived products.     

Estradiol and tamoxifen stimulation of lapine articular chondrocyte prostaglandin synthesis

The effect of estradiol and tamoxifen on prostaglandin (PG) synthesis by rabbit articular chondrocytes in secondary monolayer cultures was investigated. Radioimmunoassay for PGE₂, PGF_2α, 6-oxo-PGF_1α and thromboxane B₂ was performed on media from cultures containing estradiol and tamoxifen (10⁻¹²M-10⁻⁷-M). Radiometric thin-layer chromatography was also carried out. The time course of estradiol/tamoxifen effect on chondrocyte PG synthesis was evaluated and its relationship to cell density in culture examined. Estradiol stimulated the synthesis of PGs by chondrocytes. Stimulation was noted at picomolar concentrations of estradiol without further stimulation at markedly higher concentrations. In time studies, after a lag, the effect of estradiol was present fully by 5 hrs, remained steady for 24 hrs and then declined by 48 hrs. Estradiol stimulation of PG synthesis was dependent upon chondrocyte culture plating density. Tamoxifen stimulated chondrocyte PG synthesis to relatively lower levels than estradiol. The characteristics of estradiol/tamoxifen stimulation of chondrocyte PG synthesis suggest a mechanism involving estradiol cytoplasmic receptors.