The inhibition of nucleic acid synthesis by mycophenolic acid

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 [(14)C]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 [(14)C]thymidine into DNA in suspensions of Landschütz and Yoshida ascites cells in vitro. 6. Mycophenolic acid inhibits the conversion of [(14)C]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 [(14)C]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 K(i) values of between 3.03x10(-8) and 4.5x10(-8)m. 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.     

The improbability of prebiotic nucleic acid synthesis

Many accounts of the origin of life assume that the spontaneous synthesis of a self-replicating nucleic acid could take place readily. Serious chemical obstacles exist, however, which make such an event extremely improbable.Prebiotic syntheses of adenine from HCN, of D, L-ribose from adenosine, and of adenosine from adenine and D-ribose have in fact been demonstrated. However these procedures use pure starting materials, afford poor yields, and are run under conditions which are not compatible with one another.Any nucleic acid components which are formed on the primitive earth would tend to hydrolyze by a number of pathways. Their polymerization would be inhibited by the presence of vast numbers of related substances which would react preferentially with them.It appears likely that nucleic acids were not formed by prebiotic routes, but are later products of evolution.     

The improbability of prebiotic nucleic acid synthesis

Many accounts of the origin of life assume that the spontaneous synthesis of a self-replicating nucleic acid could take place readily. Serious chemical obstacles exist, however, which make such an event extremely improbable. Prebiotic syntheses of adenine from HCN, of D,L-ribose from adenosine, and of adenosine from adenine and D-ribose have in fact been demonstrated. However these procedures use pure starting materials, afford poor yields, and are run under conditions which are not compatible with one another. Any nucleic acid components which were formed on the primitive earth would tend to hydrolyze by a number of pathways. Their polymerization would be inhibited by the presence of vast numbers of related substances which would react preferentially with them. It appears likely that nucleic acids were not formed by prebiotic routes, but are later products of evolution.     

Inhibition of nucleic acid synthesis by maleic hydrazide

Maleic hydrazide (MH), which causes chromosome breakage, inhibitionof cell division and retardation of plant growth, inhibits nucleicacid synthesis in corn and pea seedling roots. DNA synthesisin corn roots is affected sooner than RNA synthesis; the lagtimes for inhibition are 4 hr and 8–12 hr respectively.MH inhibits nucleic acid synthesis in the root apices most rapidly,while it acts on the subapical portions only after a much longerdelay and sometimes not at all. Likewise, certain fractionsof RNA synthesis are inhibited preferentially (ribosomal RNA),and others are relatively unaffected (transfer RNA). Proteinsynthesis is not affected during the early stages of MH treatment;however, it too may be reduced after a long exposure. Since0.2% colchicine does not inhibit DNA synthesis in corn rootswithin 24 hr, it seems unlikely that MH inhibits DNA synthesisindirectly through an effect on cell division. Although MH mayalso interfere with solute uptake, there is evidence that itis fairly selective in its action, i.e. it does not inhibitrespiration or cell expansion in corn roots. (Received February 22, 1972; )     

Amino acid-directed nucleic acid synthesis

Summary The fact that proteins contain onlya-amino acids and that protein structure is determined by 3 5 linked ribonucleotides is postulated to be the result of the copolymerization of these molecules in the prebiotic environment. Ribonucleotides therefore represent partial degradation products and proteins represent a side reaction developing from copolymerization. The basic structural unit of copolymerization is a nucleotide substituted with an amino acid at the 2 position. Characteristics of modern amino and ribonucleic acid structure are all consistent with and necessary for this hypothesis. The characteristics and individual base assignments of the code also provide strong support for origin from the postulated copolymers. All characteristics of the code can be accounted for by this single hypothesis.An Established Investigator of the American Heart Association     

Modulation of transferrin receptor expression by inhibitors of nucleic acid synthesis

We investigated the effects of the iron chelator desferrioxamine on the expression of transferrin receptors (TfR) by CCRF-CEM human T-cell leukaemia and B16 mouse melanoma cells growing in tissue culture. Desferrioxamine (DFOA) enhanced TfR expression when added in the dose range of 10(-5)-10(-4) to CCRF-CEM cells, but was toxic to these cells, the lower concentrations producing a slowing of cell growth with a build up in S-phase, while higher concentrations caused cell death with a block at the G1/S-phase interface. These toxic effects are compatible with its previously reported inhibition of the non-haem iron containing (M2) subunit of ribonucleotide reductase. In marked contrast, DFOA caused the growth of B16 melanoma cells to arrest in G1, without loss of cloning efficiency, and resulted in a fall in TfR expression to approximately 50% of control values. These results suggested that the effects of DFOA on TfR expression were linked to DNA synthesis rather than to a more generalised inhibition of iron-dependent cellular processes. It was subsequently found that inhibition of the M2 subunit of ribonucleotide reductase in CCRF-CEM cells with 5 X 10(-5) M hydroxyurea, which is not an iron chelator, also enhanced TfR expression, as did thymidine and cytosine arabinoside, which have different enzyme targets. By measuring cellular DNA and RNA content simultaneously it was shown that all of these agents caused unbalanced growth, i.e., inhibited DNA synthesis more than RNA synthesis. In contrast, 6-thioguanine was more inhibitory to RNA synthesis, and treatment with this drug caused a fall in TfR expression. Thus, although CCRF-CEM cells treated with DFOA show enhanced TfR expression, similar effects are also seen with other inhibitors of DNA synthesis, provided that RNA synthesis is allowed to continue. These results provide further evidence that the regulation of TfR expression by proliferating cells is specifically linked to DNA synthesis rather than to the iron requirements of other cellular processes.     

Primary antibody response studied by using inhibitors of nucleic acid synthesis

Inhibitors of nucleic acid synthesis were tested for the effect on the primary antibody response. Antibody formation by adult spleen cells mixed with antigenin vitro and transferred to newborn rabbits was completely suppressed by 6-mercaptopurine administered in the initial phase of antibody induction. The extent of inhibition decreased proportionately to the time interval between cell transfer and treatment with 6-MP. Different sensitivity of immunologically competent cells towards 6-MP at various periods of differentiation and interference of 6-MP with synthesis of specific ribonucleic acid is suggested. Actinomycin D did not show any inhibitory effect on the primary antibody responsein vivo which may be due either to the toxicity of the drug or to some other mechanism of synthesis of immunoglobulins than is known for bacterial proteins. Mitomycin C was also ineffective in suppressing the inductive phase of antibody formation. From results with 6-MP and mitomycin C it is concluded that DNA synthesis and mitotic division are not essential steps in the induction of antibodies.     

Phosphatidic acid synthesis in bacteria

Membrane phospholipid synthesis is a vital facet of bacterial physiology. Although the spectrum of phospholipid headgroup structures produced by bacteria is large, the key precursor to all of these molecules is phosphatidic acid (PtdOH). Glycerol-3-phosphate derived from the glycolysis via glycerol-phosphate synthase is the universal source for the glycerol backbone of PtdOH. There are two distinct families of enzymes responsible for the acylation of the 1-position of glycerol-3-phosphate. The PlsB acyltransferase was discovered in Escherichia coli, and homologs are present in many eukaryotes. This protein family primarily uses acyl–acyl carrier protein (ACP) endproducts of fatty acid synthesis as acyl donors, but may also use acyl-CoA derived from exogenous fatty acids. The second protein family, PlsY, is more widely distributed in bacteria and utilizes the unique acyl donor, acyl-phosphate, which is produced from acyl-ACP by the enzyme PlsX. The acylation of the 2-position is carried out by members of the PlsC protein family. All PlsCs use acyl-ACP as the acyl donor, although the PlsCs of the γ-proteobacteria also may use acyl-CoA. Phospholipid headgroups are precursors in the biosynthesis of other membrane-associated molecules and the diacylglycerol product of these reactions is converted to PtdOH by one of two distinct families of lipid kinases. The central importance of the de novo and recycling pathways to PtdOH in cell physiology suggest that these enzymes are suitable targets for the development of antibacterial therapeutics in Gram-positive pathogens. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.