TTP Pools Research Articles

Differential modulation of 1-β- d-arabinofuranosylcytosine metabolism by hydroxyurea in human leukemic cell lines

The ability of hydroxyurea (HU) to modulate 1-β- d-arabinofuranosylcytosine (Ara-C) metabolism was investigated in human leukemic cell lines. Exposure of HL-60 cells to 1 mM HU enhanced the accumulation of Ara-CTP up to 2.5-fold, whereas HU did not have significant effects on Ara-C metabolism in CEM cells. In addition, two adenine nucleosides, deoxyadenosine (dAdo) and 9-β- d-arabinofuranosyladenine (Ara-A), which are known to be activated by deoxycytidine (dCyd) kinase as Ara-C, were more effectively phosphorylated after the addition of HU only in HL-60 cells. However, the changes of intracellular dCTP and TTP pools induced by HU, i.e. decrease in dCTP and increase in TTP, were the same in both cell lines. Finally, dCyd production under normal culture conditions was at least 3- to 4-fold higher in HL-60 cells and was inhibited significantly by HU administration. These results suggest that the modulation of Ara-C metabolism by HU occurs at the level of dCyd kinase through the regulation of de novo dCyd generation.

DNA nucleotide excision-repair synthesis is dependent of pertubations of deoxynucleoside triphosphate pool size

We have investigated the effects of fluctuations in deoxynucleoside triphosphate (dNTP) pool size on DNA repair and, conversely, the effect of DNA repair on dNTP pool size. In confluent normal human skin fibroblasts, dNTP pool size was quantitated by the formation of [ 3H]TTP from [ 3H]thymidine; DNA repair was examined by repair replication in cultures irradiated with UV light. As defined by HPLC analysis, the [ 3H]TTP pool was formed within 30 min of the addition of [ 3H]thymidine and remained relatively constant for the next 6 h. Addition of 2–10 mM hydroxyurea (HU) caused a gradual 2–4-fold increase in the [ 3H]TTP pool as HU inhibited DNA synthesis but not TTP production. No difference was seen between the [ 3H]TTP pool size in cells exposed to 20 M/m 2 and unrradiated controls, although DNA-repair synthesis was readily quantitated in the former. This result was observed even though the repair replication protocol caused an 8–10-fold reduction in the size of the [ 3H]TTP pool relative to the initial studies. In the UV excision-repair studies the precense of hydroxyurea did not alter the specific activity of [ 3H] thymidine 5'-monophospahte incorporated into parental DNA due to repaier replication. These results suggest that fluctuations in the deoxynucleoside triphosphate pools do not limit the extent of excision-repair sythesis in human cells and demonstrate that DNA nucleotide excision-repair synthesis does not significantly diminish the size of the [ 3H]TTP pool.

Deoxynucleoside triphosphate pool of mouse FM3A cell lines unaffected by mutagen treatment

The size of deoxynucleoside triphosphate pool in cultured mouse FM3A cells and mutator mutants isolated from this cell line was determined by high-pressure liquid chromatography after treatment of the cells with ultraviolet light, N-methyl-N′-nitro-N-nitrosoguanidine or mitomycin C. The results showed that, in all the FM3A cell clones, no large increase in the dATP or TTP pool was induced after treatment, while in some cases 40–50% decrease in dCTP pool was observed. It is concluded that the induction of large increase in dNTP pool is not the general effect of the mutagens.

Dipyridamole inhibits reversion by thymidine of methotrexate effect and increases drug uptake in Sarcoma 180 cells.

The combined effect of methotrexate (MTX) with dipyridamole, an inhibitor of nucleoside transport, was studied in ascitic Sarcoma 180 cells. It was determined that 10 microM MTX inhibits by greater than 90% deoxy[3H]uridine incorporation into DNA and that this MTX concentration inhibits DNA synthesis as revealed by deoxy[3H]cytidine but not [3H]thymidine incorporation into DNA. Exogenous thymidine (greater than or equal to 1 microM) in the cell culture medium enhances DNA synthesis in nontreated cells and fully restores it in MTX-treated cells, whereas hypoxanthine has no appreciable effect on DNA synthesis. Dipyridamole inhibits deoxy[3H]cytidine and [3H]thymidine uptake by these cells (IC50 = 0.2 and 3 microM, respectively) and blocks the increase in TTP pool produced by 1 microM thymidine in MTX-treated cells (23.1 +/- 4.7 pmol per 1 X 10(6) cells vs. 80.4 +/- 18.9 pmol per 1 X 10(6) cells). Dipyridamole at 10 microM enhances [3H]MTX accumulation by Sarcoma 180 cells and diminishes the efflux of the drug in previously loaded cells. It is suggested that the combination of inhibitors of the de novo pathway for pyrimidine biosynthesis, such as MTX, with inhibitors of the salvage pathway, such as dipyridamole, may increase the cytotoxic activity of MTX alone.

Kinetic behaviour and allosteric regulation of human deoxycytidylate deaminase derived from leukemic cells.

Deoxycytidylate deaminase has been highly purified (1232-fold) from human leukemia CCRF-CEM cells. The native molecular weight of the enzyme is 108 000 and subunit molecular weight 50 500, suggesting that the native enzyme exists as a dimer. The enzyme exhibits a sigmoidal initial velocity vs substrate concentration curve and is regulated by allosteric effectors, dCTP and TTP. The curve relating substrate concentration to initial velocity was changed from a sigmoidal shape to a hyperbolic one by the activator dCTP, while the inhibitor TTP increased the sigmoidicity of the curve. The molecular weight of deoxycytidylate deaminase was unchanged in the presence of allosteric effectors, indicating that aggregation-disaggregation is not the basis of regulation. Deoxycytidylate deaminase exhibited the greatest affinity for the substrate dCMP, with lesser affinity for ara-CMP, and least affinity for CMP. Ara-CMP was an effective substrate in the presence of dCTP concentrations exceeding 4 microM. These data indicate that human neoplastic cell deoxycytidylate deaminase is a highly regulated allosteric enzyme, which is likely to have a significant influence on cellular dUMP, dCTP and TTP pools. These findings further suggest, that the enzyme through its influence on dUMP levels is likely to modulate the biochemical effects of pyrimidine antimetabolites active against the thymidylate synthetase reaction and in the presence of elevated dCTP pools will promote deamination of ara-CMP to the inactive ara-UMP.

Mechanism of 2-aminopurine mutagenesis in mouse T-lymphosarcoma cells.

We investigated the mechanism of action of 2-aminopurine (Apur) in eucaryotic cells. By analogy with studies in procaryotic systems, the base analog is presumed to incorporate into DNA predominantly opposite T where, upon subsequent DNA replication, it can mispair with C, inducing an A:T leads to G:C transition. This model predicts that Apur-induced mutagenesis will be enhanced by factors that favor formation of Apur-C mispairs, e.g., high levels of dCTP or low levels of TTP. We describe the use of a mutant T-lymphosarcoma cell line, AraC-6-1, which has an abnormally high dCTP pool and a low TTP pool, to test this prediction. AraC-6-1 cells were three- to fivefold more mutable by Apur than their parental cell line, NSU-1. This enhanced mutability by Apur could not be explained by altered incorporation of 3H-labeled Apur, by generally impaired ability to repair DNA damage, or by a direct effect of Apur on the endogenous deoxynucleotide pools. The addition of 10 microM thymidine to the growth medium of AraC-6-1 cells lowered their high dCTP pool (two- to threefold), raised the TTP pool (two- to threefold), and abolished their enhanced mutability by Apur. Further manipulation to produce an abnormally high TTP/dCTP ratio suppressed Apur-induced mutagenesis (8- to 10-fold) in both AraC-6-1 and NSU-1 cells. These observations support the hypothesis that Apur induces A:T leads to G:C transitions in mammalian cells by a mispairing mechanism.

Mechanism of 2-aminopurine mutagenesis in mouse T-lymphosarcoma cells.

We investigated the mechanism of action of 2-aminopurine (Apur) in eucaryotic cells. By analogy with studies in procaryotic systems, the base analog is presumed to incorporate into DNA predominantly opposite T where, upon subsequent DNA replication, it can mispair with C, inducing an A:T leads to G:C transition. This model predicts that Apur-induced mutagenesis will be enhanced by factors that favor formation of Apur-C mispairs, e.g., high levels of dCTP or low levels of TTP. We describe the use of a mutant T-lymphosarcoma cell line, AraC-6-1, which has an abnormally high dCTP pool and a low TTP pool, to test this prediction. AraC-6-1 cells were three- to fivefold more mutable by Apur than their parental cell line, NSU-1. This enhanced mutability by Apur could not be explained by altered incorporation of 3H-labeled Apur, by generally impaired ability to repair DNA damage, or by a direct effect of Apur on the endogenous deoxynucleotide pools. The addition of 10 microM thymidine to the growth medium of AraC-6-1 cells lowered their high dCTP pool (two- to threefold), raised the TTP pool (two- to threefold), and abolished their enhanced mutability by Apur. Further manipulation to produce an abnormally high TTP/dCTP ratio suppressed Apur-induced mutagenesis (8- to 10-fold) in both AraC-6-1 and NSU-1 cells. These observations support the hypothesis that Apur induces A:T leads to G:C transitions in mammalian cells by a mispairing mechanism.

Early effects of phytohemagglutinin on induction of DNA polymerase, thymidine kinase, deoxyribonucleoside triphosphate pools and DNA synthesis in human lymphocytes.

In phytohemagglutinin stimulated human lymphocytes the time relationship was determined between induction of the parameters mentioned. The results indicate that the induction occurred in a specific sequence. Thus, a simultaneous increase in the activity of DNA polymerase and thymidinekinase occurred after 15 h of incubation with Phytohemagglutinin. Furthermore, this enhancement occurred 2 h before the expansion of the TTP and dCTP pools and 4 h before the expansion of the dATP and dGTP pools. The rate of [3H] deoxyguanosine incorporation into DNA increased simultaneously with the expansion of the TTP and dCTP pools.

THIAMINE PHOSPHATE METABOLISM AND POSSIBLE COENZYME‐INDEPENDENT FUNCTIONS OF THIAMINE IN BRAIN1

Abstract—The effect of depolarization of rat brain cortex slices on the relative distribution of thiamine among its various phosphate esters and on the efflux of thiamine was studied as a probe of possible coenzyme‐independent neurophysiological functions of thiamine. Electrical pulses for 30 min increased lactate production but did not affect the levels of thiamine esters. Depolarization with 41 mM‐potassium decreased thiamine diphosphate by only 3 percent (P= 0.05). Thiamine triphosphate levels (TTP) were unaffected by depolarization but doubled during incubation for 1 h in which time efflux of 40 percent of the total thiamine from the slices as unesterified thiamine occurred. Depolarization by potassium released a small but highly variable portion of the thiamine content of superfused cortex slices above the basal rate of efflux. The basal efflux was partially sodium dependent. Thiamine efflux was unaffected by acetylcholine, ouabain, or tetrodotoxin, compounds previously reported to increase thiamine efflux. The incorporation of 32P1 into the endogenous thiamine phosphates of cortex slices was studied. Incorporation into thiamine diphosphate reached only 20 percent of the specific activity of its precursor, ATP, after 2h of incubation while the incorporation into TTP approached equilibrium with ATP in 15‐30 min indicating that the TTP pool was the most rapidly turning over of the thiamine phosphates. The data suggest that only a small portion of the TDP pool undergoes rapid turnover and serves as a precursor for TTP. The rapid turnover of TTP phosphoryl groups is consistent with specific functions for this compound related to its potential for phosphorylation reactions. An analog of TTP with the β, γ oxygen bridge replaced by a methylene group decreased TDP levels and increased thiamine when incubated with cortex slices, but did not effect thiamine monophosphate or triphosphate levels indicating inhibition of thiamine pyrophosphokinase.

Utilization of thymine, thymidine and TMP by Neisseria meningitidis. 2. Lack of enzymes for specific incorporation of exogenous thymine, thymidine and TMP into DNA.

Activities corresponding to thymidine phosphorylase and nucleoside deoxyribosyl transferase could not be found in Neisseria meningitidis. 14C‐2‐thymine was bound to or taken up by whole cells, but this did not result in labelling of the TMP, TDP or TTP pools. No significant thymidine kinase activity could be induced in N. meningitidis. 14C‐2‐thymidine which was bound to or taken up by whole cells resulted in faint labelling of the TMP, TDP and TTP pools after prolonged incubation, but no radioactive thymine appeared. The N. meningitidis extracts had activities corresponding to TMP and TDP kinases, but whole cells which were exposed to TMP‐methyl‐3H did not seem to take up significant amounts of this compound. The findings indicate that N. meningitidis lacks the enzyme systems that are known to mediate a specific incorporation of thymine or thymidine into DNA.

DNA Polymerase, Triphosphates and Deoxyribonuclease in a Systemof Synchronized L Cells

In a system of synchronously dividing mouse fibroblasts (L cells), the activity of several enzymes was determined in relation to initiation of DNA synthesis. Synchrony of DNA synthesis and cell division was obtained by selective mechanical detachment of cells in mitosis, thereby avoiding the use of blocking agents. The activity of DNa polymerase under assay condition spermitting terminal or replicative activity of DNA polymerase under assay conditions permitting terminal or replicative activity to occur shows characterstic fluctuations throughout the life cycle of the cell: whereas the terminal enzyme activity exhibits a peak at about 12 h, the replicative form of theenzyme has its maximum 16 h after mitosis. DNA synthesis starts at about 9 h and reaches its peak at 16 h after mitosis. The two enzyme fractions examined (particular and supernatant) prefer native DNA as primer. The supernatant enzyme exhibits exclusively terminal activity with heat‐denatured DNA as primer. The degradation of TTP by TTPase activity of the particular enzymes fraction in the S‐phase (phase of DNA synthesis) may contribute to a preservation of the TTP pool, thus acting as an additional regulatory emchanism. DNase activity nearly constant throughout the mitotic cycle.