scholarly journals Compartmentation of guanine nucleotide precursors for DNA synthesis

1986 ◽  
Vol 234 (2) ◽  
pp. 263-269 ◽  
Author(s):  
B T Nguyen ◽  
W Sadée
Keyword(s):  
Dna Synthesis ◽  
Mycophenolic Acid ◽  
Purine Nucleoside Phosphorylase ◽  
Guanine Nucleotide ◽  
Nucleotide Incorporation ◽  
T Lymphoma ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
Inhibition Of Dna Synthesis ◽  
Mutant Cells ◽  
Kinetics Of

We have studied the kinetics of guanine incorporation into DNA in mouse T-lymphoma (S-49) mutant cells [PNPase (purine-nucleoside phosphorylase)- and HGPRTase (hypoxanthine: guanine phosphoribosyltransferase)-deficient] that are incapable of converting dGuo (deoxyguanosine) to Gua (guanine) ribonucleotides. Of the two possible pathways for an exogenous guanine source to reach DNA, firstly: dGuo→dGMP→dGDP→dGTP and secondly: Gua→GMP→GDP→dGDP→dGTP only the second pathway was found to be functional in providing guanine for DNA replication, although deoxyguanosine readily produced toxic cellular dGTP levels via the first pathway. The functional guanine-nucleotide-precursor pools for DNA are rather small; further, the depletion of the small GMP pool, but not that of GDP, GTP and dGTP, correlated well with the inhibition of DNA synthesis by mycophenolic acid, an IMP dehydrogenase inhibitor. These results support the hypothesis that guanine-nucleotide incorporation into DNA is highly compartmentalized and that a small functional guanine-nucleotide pool, e.g., the GMP pool, may serve a crucial role in limiting the availability of DNA precursor substrate.

scholarly journals The inhibition of nucleic acid synthesis by mycophenolic acid

1969 ◽  
Vol 113 (3) ◽  
pp. 515-524 ◽  
Author(s):  
T J Franklin ◽  
Jennifer M. Cook
Keyword(s):  
Dna Synthesis ◽  
Mycophenolic Acid ◽  
Early Stage ◽  
Acid Synthesis ◽  
Purine Nucleotides ◽  
Free System ◽  
L Cells ◽  
A Cell ◽  
Inhibition Of Dna Synthesis

1. Mycophenolic acid, an antibiotic of some antiquity that more recently has been found to have marked activity against a range of tumours in mice and rats, strongly inhibits DNA synthesis in the L strain of fibroblasts in vitro. 2. The extent of the inhibition of DNA synthesis is markedly increased by preincubation of the cells with mycophenolic acid before the addition of [14C]thymidine. 3. The inhibition of DNA synthesis by mycophenolic acid in L cells in vitro is reversed by guanine in a non-competitive manner, but not by hypoxanthine, xanthine or adenine. 4. The reversal of inhibition by guanine can be suppressed by hypoxanthine, 6-mercaptopurine and adenine. 5. Mycophenolic acid does not inhibit the incorporation of [14C]thymidine into DNA in suspensions of Landschütz and Yoshida ascites cells in vitro. 6. Mycophenolic acid inhibits the conversion of [14C]hypoxanthine into cold-acid-soluble and -insoluble guanine nucleotides in Landschütz and Yoshida ascites cells and also in L cells in vitro. There is some increase in the radioactivity of the adenine fraction in the presence of the antibiotic. 7. Mycophenolic acid inhibits the conversion of [14C]hypoxanthine into xanthine and guanine fractions in a cell-free system from Landschütz cells capable of converting hypoxanthine into IMP, XMP and GMP. 8. Preparations of IMP dehydrogenase from Landschütz ascites cells, calf thymus and LS cells are strongly inhibited by mycophenolic acid. The inhibition showed mixed type kinetics with Ki values of between 3·03×10−8 and 4·5×10−8m. 9. Evidence was also obtained for a partial, possibly indirect, inhibition by mycophenolic acid of an early stage of biosynthesis of purine nucleotides as indicated by a decrease in the accumulation of formylglycine amide ribonucleotide induced by the antibiotic azaserine in suspensions of Landschütz and Yoshida ascites cells and L cells in vitro.

scholarly journals Guanine for DNA synthesis. A compulsory route through ribonucleotide reductase

1988 ◽  
Vol 255 (3) ◽  
pp. 1045-1048 ◽  
Author(s):  
D S Duan ◽  
W Sadee
Keyword(s):  
Dna Synthesis ◽  
Cell Line ◽  
Ribonucleotide Reductase ◽  
Purine Nucleoside Phosphorylase ◽  
S Phase ◽  
Alternative Pathways ◽  
Dose Dependent Fashion ◽  
T Lymphoma ◽  
Dose Dependent ◽  
Tracer Experiments

Two alternative pathways for the synthesis of dGTP and its incorporation into DNA were studied: guanine (Gua)→GMP→GDP→dGDP→dGTP→DNA and dG→dGMP→dGDP→dGTP→DNA. To determine the contribution of each pathway to DNA synthesis independently of each other, [14C]Gua and [3H]dG tracer experiments were performed in a double-mutant S-49 mouse T-lymphoma cell line, dGuo-L, with purine nucleoside phosphorylase (EC 2.4.2.1)-deficiency and dGTP-feedback-resistant ribonucleotide reductase (RR, EC 1.17.4.1). In this cell line, dGTP pools can be selectively elevated by exogenous dG without affect RR and DNA synthesis. Although [3H]dG, but not [14C]Gua (up to 200 microM), readily expanded the cellular dGTP pool in a dose-dependent fashion in asynchronous cells, only a small fraction of the Gua flux into DNA was derived from [3H]dG, with the major fraction coming from [14C]Gua. H.p.l.c. analysis of G1- and partially enriched S-phase cells revealed that [3H]dGTP only accumulates in G1- but not in S-phase cells because of a rapid turnover of the dGTP pool during DNA synthesis. These results fail to provide evidence for cellular dGTP compartmentation and suggest that the pathway dG→dGMP→dGDP→dGTP alone has insufficient capacity to maintain DNA synthesis.

scholarly journals Mechanisms of 2′-deoxyguanosine toxicity in mouse T-lymphoma cells with purine nucleoside phosphorylase deficiency and resistance to inhibition of ribonucleotide reductase by dGTP

1990 ◽  
Vol 268 (3) ◽  
pp. 725-731 ◽  
Author(s):  
D S Duan ◽  
T Nagashima ◽  
T Hoshino ◽  
F Waldman ◽  
K Pawlak ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Ribonucleotide Reductase ◽  
Double Mutant ◽  
Purine Nucleoside Phosphorylase ◽  
Feedback Inhibition ◽  
S Phase ◽  
G1 Phase ◽  
Wild Type ◽  
L Cells ◽  
T Lymphoma

Purine nucleoside phosphorylase (PNP; EC 2.4.2.1) deficiency is thought to cause T-lymphocyte depletion by accumulation of dG and dGTP, resulting in feedback inhibition of ribonucleotide reductase (RR; EC 1.17.4.1) and hence DNA synthesis. To test for additional toxic mechanisms of dG, we selected a double mutant of the mouse T-lymphoma S-49 cell line, dGuo-L, which is deficient in PNP and partially resistant to dGTP feedback inhibition of RR. The effects of dG on dGuo-L cells (concn. causing 50% inhibition, IC50 = 150 microM) were compared with those on the wild-type cells (IC50 = 30 microM) and the NSU-1 mutant with PNP deficiency only (IC50 = 15 microM). Fluorescence flow cytometry showed that equitoxic dG concentrations arrested wild-type and NSU-1 cells at the G1-S interface while allowing continued DNA synthesis in the S-phase, whereas the double mutant dGuo-L cells progressed through the cell cycle normally. dGuo-L cells accumulated high levels of dGTP in G1-phase, but not in S-phase cells, because of the utilization of dGTP for DNA synthesis and limited capacity to synthesize dGTP from dG. These results support the hypothesis that dG/dGTP toxicity occurs in the G1-phase or at the G1-S interface. Failure of dG to arrest the double mutant dGuo-L cells at the G1-S interface allows these cells to escape into S-phase, with an accompanying drop in dGTP levels. Thus the partial resistance of dGuo-L cells to dG toxicity may result from their shorter residence time in G1, allowing them to sustain higher dGTP levels. Hence RR inhibition by dGuo may not be the primary toxic mechanism in S-49 cells; rather, it may serve as an accessory event in dG toxicity by keeping the cells in the sensitive phase of the cell cycle. Among the possible targets of dG toxicity is RNA synthesis, which was inhibited at an early stage in dGuo-L cells.

scholarly journals INHIBITION OF DNA SYNTHESIS IN CHICK EMBRYO CULTURES BY DEPRIVATION OF EITHER SERUM OR ZINC

1973 ◽  
Vol 56 (3) ◽  
pp. 777-786 ◽  
Author(s):  
H. Rubin ◽  
T. Koide
Keyword(s):  
Chick Embryo ◽  
Population Density ◽  
Dna Synthesis ◽  
Cell Movement ◽  
Embryo Cultures ◽  
Low Concentrations ◽  
Embryo Cells ◽  
Inhibition Of Dna Synthesis ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

The rate of DNA synthesis in cultures of chick embryo cells is proportional to the concentration of serum added. The concentration of serum required to stimulate DNA synthesis increases with cell population density and with the duration of culture after trypsinization. The increase of the serum requirement with population density is not caused by the depletion of serum constituents. The requirement of cells for external zinc in DNA synthesis also increases with population density and duration of culture. The kinetics of inhibition of DNA synthesis by deprivation of serum or zinc are similar. Serum deprivation, however, inhibits 2-deoxyglucose uptake and cell movement, but zinc deprivation does not. The deprivation of either serum or zinc inhibits RNA synthesis about twofold. Very low concentrations of actinomycin D prevent the resumption of RNA and DNA synthesis upon restoration of serum or zinc to deprived cultures.

scholarly journals Inhibition of DNA synthesis in living cells by microinjection of Gi2 antibodies.

1992 ◽  
Vol 267 (2) ◽  
pp. 691-694 ◽  
Author(s):  
V J LaMorte ◽  
P K Goldsmith ◽  
A M Spiegel ◽  
J L Meinkoth ◽  
J R Feramisco
Keyword(s):  
Dna Synthesis ◽  
Living Cells ◽  
Inhibition Of Dna Synthesis
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