Inhibition of purine phosphoribosyltransferases of Ehrlich ascites-tumour cells by 6-mercaptopurine

1. The formation of adenosine 5′-phosphate, guanosine 5′-phosphate and inosine 5′-phosphate from [8-¹⁴C]adenine, [8-¹⁴C]guanine and [8-¹⁴C]hypoxanthine respectively in the presence of 5-phosphoribosyl pyrophosphate and an extract from Ehrlich ascites-tumour cells was assayed by a method involving liquid-scintillation counting of the radioactive nucleotides on diethylaminoethylcellulose paper. The results obtained with guanine were confirmed by a spectrophotometric assay which was also used to assay the conversion of 6-mercaptopurine and 5-phosphoribosyl pyrophosphate into 6-thioinosine 5′-phosphate in the presence of 6-mercaptopurine phosphoribosyltransferase from these cells. 2. At pH 7·8 and 25° the Michaelis constants for adenine, guanine and hypoxanthine were 0·9 μm, 2·9 μm and 11·0 μm in the assay with radioactive purines; the Michaelis constant for guanine in the spectrophotometric assay was 2·6 μm. At pH 7·9 the Michaelis constant for 6-mercaptopurine was 10·9 μm. 3. 25 μm-6-Mercaptopurine did not inhibit adenine phosphoribosyltransferase. 6-Mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 4·7 μm) and hypoxanthine phosphoribosyltransferase (K_i 8·3 μm). Hypoxanthine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 3·4 μm). 4. Differences in kinetic parameters and in the distribution of phosphoribosyltransferase activities after electrophoresis in starch gel indicate that different enzymes are involved in the conversion of adenine, guanine and hypoxanthine into their nucleotides. 5. From the low values of K_i for 6-mercaptopurine, and from published evidence that ascites-tumour cells require supplies of purines from the host tissues, it is likely that inhibition of hypoxanthine and guanine phosphoribosyltransferases by free 6-mercaptopurine is involved in the biological activity of this drug.     

Metabolic properties of an azaguanine-resistant variant of Chinese hamster ovary cells (azarts) with normal levels of hypoxanthine-guanine phosphoribosyltransferase activity

Azarts Chinese hamster ovary cells were 20 to 50 times more resistant to 8-azaguanine and 50 to 10 times more resistant to both 6-thioguanine and 6-mercaptopurine than wild-type cells. Resistance correlated with a failure of azarts cells to incorporate 8-azaguanine into the nucleotide pool and into nucleic acids. The uptake of hypoxanthine and guanine, on the other hand, was about the same in both types of cells and the hypoxanthine-guanine phosphoribosyltransferase of the azarts cells as measured in cell lysates was unaltered both in concentration and kinetic properties with hypoxanthine as well as 8-azaguanine as substrate. Plasma membrane permeability to 8-azaguanine and the regulation of intracellular pH were also not altered in azarts cells and there was no significant degradation of 8-azaguanine or azaguanine nucleotides. We conclude therefore that in azarts cells the phosphoribosylation of 8-azaguanine per se is specifically blocked but that this effect is abolished upon cell lysis.     

Facilitated transport of uracil and 5-fluorouracil,and permeation of orotic acid into cultured mammalian cells

The mode of permeation of uracil, 5–fluorouracil, and orotic acid into cells has been investigated in four established cell lines (Novikoff rat hepatoma, P388 mouse leukemia, mouse L, and Chinese hamster ovary cells) in attempts to assess the rate-determining step(s) in their incorporation into the nucleotide pool and nucleic acids. Uracil and 5–fluorouracil shared a saturable transport system (K_m = 5 to 15 mM) capable of rapid equilibration of these substrates across the cell membrane (t_1/2 at 25 in first-order range of concentration = 25 to 58 sec). Thus it seems unlikely that transport is limiting the incorporation hypoxanthine. Only the non-ionized form of fluorouracil was a substrate for the transporter; exclusion of charged pyrimidines may explain why orotate was not a substrate at physiological pH. Orotate permeated the cell membrane much more slowly (t_1/2 = 2890 to 6930 sec); its permeation was apparently non-mediated and rate-determining in the conversion of extracellular orotate to intracellular nucleotides.     

Isolation and characterization of mutants of mesophilic methanogenic bacteria resistant to analogues of DNA bases and nucleosides

Spontaneous mutants of mesophilic Methanobacterium, Methanobrevibacter and Methanosarcina species resistant to 6-mercaptopurine, 5-fluorouracil, 8-azaguanine, 6-azauracil or 5-fluorodeoxyuridine were isolated. Low level resistant mutants were unstable but highly resistant strains (resistance factor greater than 10-fold) were stable and showed growth characteristics comparable to the parent. Wild type strains showed linear uptake of hypoxanthine and uracil into cells, but guanine uptake was only detected in Methanosarcina mazei. 6-Mercaptopurine-resistant clones of Methanobacterium and Methanobrevibacter species and 8-azaguanine-resistant clones of Methanosarcina mazei showed reduced uptake of hypoxanthine and guanine respectively, but no evidence for altered permeability of 5-fluoro-and 6-azauracil-resistant strains to uracil was obtained. Double resistant mutants of Methanobacterium sp. strain FR-2 were characterised. Although these generally exhibited reduced specific growth rates, several were selected which showed similar growth to the parent.Abbreviations DSM Deutsche Sammlung von Mikroorganismen, Federal Republic of Germany - MJC minimum inhibitory concentration - cfu colony forming unit - MP 6-mercaptopurine - FU 5-fluorouracil - FDU 5-fluorodeoxyuridine - AG 8-azaguanine - AU 6-azauracil - DA l-deazaadenosine     

Purine and pyrimidine transport by cultured Novikoff cells. Specificities and mechanism of transport and relationship to phosphoribosylation.

Adenine, guanine, and hypoxanthine were rapidly incorporated into the acid-soluble nucleotide pool and nucleic acids by wild type Novikoff cells. Incorporation followed normal Michaelis-Menten kinetics, but the following evidence indicates that specific transport processes precede the phosphoribosyltransferase reactions and are the rate-limiting step in purine incorporation by whole cells. Cells of an azaguanine-resistant subline of Novikoff cells which lacked hypoxanthine-guanine phosphoribosyltransferase activity and failed to incorporate guanine or hypoxanthine into the nucleotide pool, exhibited uptake of guanine and hypoxanthine by a saturable process. Similarly, wild type cells which had been preincubated in a glucose-free basal medium containing KCN and iodoacetate transported guanine and hypoxanthine normally, although a conversion of these purines to nucleotides did not occur in these cells. The mutant and KCN-iodoacetate treated wild type cells also exhibited countertransport of guanine and hypoxanthine when preloaded with various purines, uracil, and pyrimidine nucleosides. The cells also possess a saturable transport system for uracil although they lack phosphoribosyltransferase activity for uracil. In the absence of phosphoribosylation, none of the substrates was accumulated against a concentration gradient. Thus transport is by facilitated diffusion (nonconcentrative transport). Furthermore, the apparent Km values for purine uptake by untreated wild type and azaguanine-resistant cells were higher and the apparent Vmax values were lower than those for the corresponding phosphoribosyltransferases...     

Inhibition by 6-mercaptopurine of purine phosphoribosyltransferases from Ehrlich ascites-tumour cells that are resistant to this drug

1. A strain of Ehrlich ascites-tumour cells that showed little inhibition of growth in the presence of 6-mercaptopurine accumulated less than 5% as much 6-thioinosine 5′-phosphate in vivo, in the presence of 6-mercaptopurine, as did the sensitive strain from which it was derived. 2. Specific activities of the phosphoribosyltransferases that convert adenine, guanine, hypoxanthine and 6-mercaptopurine into AMP, GMP, IMP and 6-thioinosine 5′-phosphate were similar in extracts of the resistant and the sensitive cells. 3. As found previously with sensitive cells, 6-mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase from the resistant cells and does not inhibit the adenine phosphoribosyltransferase from these cells. Michaelis constants and inhibitor constants of the purine phosphoribosyltransferases from resistant cells did not differ significantly from those measured with the corresponding enzymes from sensitive cells. 4. Resistance to 6-mercaptopurine in this case is probably not due to qualitative or quantitative changes in these transferases.     

Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. I. Kinetics of incorporation of nucleosides into nucleotide pools and pool sizes during growth cycle

Results from kinetic studies on the incorporation of ³H-5-uridine and ³H-8-adenosine into the acid-soluble nucleotide poor and nucleic acids by Novikoff hepatoma cells (subline N1S1-67) in suspension culture indicate that the uridine transport reaction is saturated at about 100 μM and that for adenosine at about 10 μM nucleoside in the medium, and that above 100 μM simple diffusion becomes the predominant mode of entry of both nucleosides into the cell. The Km of the transport reactions is approximately 1.3 × 10^?5 M for uridine and 6 × 10^?6 M for adenosine. The incorporation of these nucleosides into both the nucleotide pool and into nucleic acids seems to be limited by the rate of entry of the nucleic acid synthesis from the rate of incorporation of nucleosides. Other complicating factors are a change with time of labeling in the relative proporation of nucleoside incorporated into DNA and into the individual nucleotides of RNA, the splitting of uridine to uracil by th ecells, the deamination of adenosine kto inosine and the subsequent cleavage of inosine to hypoxanthine. Various lines of evidence are presented which indicate that the overall nucleotide pools of the cells are very small under normal growth conditions. During growth in the presence of 200 μM uridine or adenosine, however, the cells continue to convert the nucleosides into intracellular nucleotides much more rapidly than required for nucleic acid synthesis. This results in an accumulation of free uridine and adenosine nucleotides in the cells, the maximum amounts of which are at least equivalent to the amount of these nucleotides in total cellular RNA.     

Metabolism of 6-Mercaptopurine by Resistant Escherichia coli Cells

Coggin, Joseph H. (University of Chicago, Chicago, Ill.), Muriel Loosemore, and William R. Martin. Metabolism of 6-mercaptopurine by resistant Escherichia coli cells. J. Bacteriol. 92:446-454. 1966.-6-Mercaptopurine (MP) utilization as a source of purine in MP-sensitive and -resistant cultures of Escherichia coli was investigated. The label of MP-8-C(14) appeared in adenine and guanine of ribonucleic acid and deoxyribonucleic acid in sensitive and resistant cultures. Studies using MP-S(35) further demonstrated that the MP moiety was degraded, as shown by a rapid decrease in radioactivity from cells upon exposure to MP for 20 min. Enzymatic analysis showed that MP was converted to 6-mercaptopurine ribonucleotide (MPRP) by extracts derived from both sensitive and resistant cells. Resistant cell preparations, however, degraded MPRP to inosine monophosphate (IMP) rapidly when compared with analogue degradation by sensitive cells. Inosineguanosine-5'-phosphate pyrophosphorylase from resistant cells did not catalyze the synthesis of IMP from hypoxanthine when the cells were cultured in the presence of MP, but these enzyme preparations actively converted guanine to guanosine monophosphate (GMP). Pyrophosphorylase derived from resistant cells cultured in medium without MP catalyzed the conversion of hypoxanthine to IMP and also guanine to GMP. These observations suggest that inosine-guanosine-5'-phosphate pyrophosphorylase is composed of two distinct enzymes. The mode of resistance to MP in E. coli is related to an enhancement of the enzymatic degradation of MPRP to the pivotal purine intermediate, IMP.     

Toxicity of 6-thioguanine and 8-azaguanine to non-dividing liver cell cultures

8-azaguanine and 6-thioguanine were both toxic to non-dividing liver cells in primary cultures. In addition, these agents were toxic to an established line of liver-derived epithelial cells brought to growth arrest by serum deprivation. These observations demonstrate that the toxicity of 8-azaguanine and 6-thioguanine can occur at least in part through mechanisms that do not involve effects on DNA synthesis or incorporation of the analogs into DNA.Abbreviations AG 8-azaguanine - ARL adult rat liver epithelial cell line - HGPRT hypoxanthine-guanine phosphoribosyl transferase - WME Williams Medium E     

Intracellular conversions of deoxyribonucleosides by Novikoff rat hepatoma cells and effects of hydroxyurea

The incorporation of ³H-labeled deoxyadenosine and deoxyguanosine into nucleic acids by cultured Novikoff rat hepatoma cells is about 80% into RNA and 20% into DNA. The pathways of incorporation have been elucidated in studies with whole cells and cell-free extracts. Deoxyadenosine is very rapidly deaminated to deoxyinosine. Most of the deoxyinosine formed by whole cells is transported out of the cells and accumulates in the medium. A portion of the deoxyinosine, and deoxyguanosine are phosphorolyzed by purine nucleoside phosphorylase to hypoxanthine and guanine, respectively. The latter are subsequently converted by hypoxanthine-guanine phosphoribosyl transferase to IMP and GMP, respectively. Incorporation of the purine deoxyribonucleosides into DNA is mainly via this pathway and the subsequent reduction of ADP and GDP by ribonucleoside reductase, although a small proportion of the deoxyadenosine and deoxyguanosine taken up by the cells seems to be directly phosphorylated to dAMP and dGMP, respectively. Deoxyguanosine is incorporated only into guanine residues of RNA and DNA. Deoxyadenosine is also mainly incorporated into guanine residues of RNA and DNA, although the radioactivity of deoxyadenosine in the acid-soluble pool is almost exclusively associated with ATP. A similar labeling pattern is observed with labeled deoxyinosine, inosine or hypoxanthine. The pyrimidine deoxyribonucleosides, on the other hand, are specific precursors for their respective bases in DNA. Hydroxyurea inhibits the incorporation of all deoxyribonucleosides into DNA. Results from pulse-chase experiments indicate that the inhibition of DNA synthesis is prevented by the presence of high concentrations of deoxyadenosine plus deoxyguanosine in the medium. Either purine deoxyribonucleoside alone or deoxycytidine, hypoxanthine or inosine alone or in combination with deoxyadenosine or deoxyguanosine are ineffective. The results are consistent with the conclusion that the inhibition of DNA synthesis is due to a depletion of the dATP and dGTP pools as a result of the hydroxyurea treatment. On the other hand, hydroxyurea causes an increased incorporation of thymidine and deoxycytidine into the dTTP and dCTP pools, respectively. Evidence is presented to indicate that this effect of hydroxyurea is due to an increased synthesis of dTTP and dCTP rather than to an inhibition of their turnover.     

Functional Identification of the Hypoxanthine/Guanine Transporters YjcD and YgfQ and the Adenine Transporters PurP and YicO of Escherichia coli K-12

The evolutionarily broad family nucleobase-cation symporter-2 (NCS2) encompasses transporters that are conserved in binding site architecture but diverse in substrate selectivity. Putative purine transporters of this family fall into one of two homology clusters: COG2233, represented by well studied xanthine and/or uric acid permeases, and COG2252, consisting of transporters for adenine, guanine, and/or hypoxanthine that remain unknown with respect to structure-function relationships. We analyzed the COG2252 genes of Escherichia coli K-12 with homology modeling, functional overexpression, and mutagenesis and showed that they encode high affinity permeases for the uptake of adenine (PurP and YicO) or guanine and hypoxanthine (YjcD and YgfQ). The two pairs of paralogs differ clearly in their substrate and ligand preferences. Of 25 putative inhibitors tested, PurP and YicO recognize with low micromolar affinity N⁶-benzoyladenine, 2,6-diaminopurine, and purine, whereas YjcD and YgfQ recognize 1-methylguanine, 8-azaguanine, 6-thioguanine, and 6-mercaptopurine and do not recognize any of the PurP ligands. Furthermore, the permeases PurP and YjcD were subjected to site-directed mutagenesis at highly conserved sites of transmembrane segments 1, 3, 8, 9, and 10, which have been studied also in COG2233 homologs. Residues irreplaceable for uptake activity or crucial for substrate selectivity were found at positions occupied by similar role amino acids in the Escherichia coli xanthine- and uric acid-transporting homologs (XanQ and UacT, respectively) and predicted to be at or around the binding site. Our results support the contention that the distantly related transporters of COG2233 and COG2252 use topologically similar side chain determinants to dictate their function and the distinct purine selectivity profiles.     

Enhanced uptake of drugs in liposomes: Use of labile vitamin B12 complexes of 6-mercaptopurine and 8-azaguanine

Vitamin B₁₂a complexes of 8-azaguanine, 8-AZG-B₁₂, and of 6-mercaptopurine, 6-MP-B₁₂, have been isolated. Upon standing, these complexes decompose to reform vitamin B₁₂a and 8-azaguanine, 8-AZG, or 6-mercaptopurine, 6-MP. Extents of entrapments of 8-AZG-B₁₂ and 6-MP-B₁₂ in negatively charged dipalmitoyl-D, L-α-phosphatidylcholine liposomes are substantially greater than those of 8-AZG or 6-MP. Each single compartment phospholipid vesicle incorporates 11, 5, 49 and 79 molecules of 8-AZG, 6-MP, 8-AZG-B₁₂ and 6-MP-B₁₂, respectively. Rates of decompositions of 8-AZG-B₁₂ and 6-MP-B₁₂ are considerably slower in liposomes than in water. Potentials of liposome entrapped 8-AZG-B₁₂ and 6-MP-B₁₂ in cancer chemotherapy are discussed.     

Incorporation of Exogenous Purines and Pyrimidines by Methanococcus voltae and Isolation of Analog-Resistant Mutants

Methanococcus voltae incorporated exogenous adenine, guanine, hypoxanthine, and uracil, but not thymine. Growth of M. voltae was also sensitive to purine and pyrimidine analogs. Of the 20 analogs tested, 12 were inhibitory at 1 mg/ml. The most effective inhibitors were purine analogs with endocyclic substitutions. Nucleoside analogs and analogs with exocyclic substitutions or additions were less effective. Four purine analogs, 8-aza-2,6-diaminopurine, 8-azaguanine, 8-azahypoxanthine, and 6-mercaptopurine and one pyrimidine analog, 6-azauracil, were especially toxic. The MICs were 20, 0.5, 2.0, 80, and 10 μg/ml, respectively. Spontaneous resistance mutants were isolated for these five analogs. The MICs for these mutants were 20.5, 8.2, >65, >41, and 20.5 mg/ml, respectively. These concentrations far exceeded the solubilities of the analogs and represented an increase in resistance of at least three orders of magnitude. In addition to demonstrating cross resistance to several of the analogs, four of these mutants lost the ability to incorporate exogenous bases. These appeared to be mutations in the salvage pathways for purines and pyrimidines. In contrast, the mutant resistant to 6-mercaptopurine was not defective in purine uptake. Instead, it degraded 6-mercaptopurine. In the presence or absence of high concentrations of the analogs, the growth rates of the resistant mutants were no less than one-half of the growth rate of the wild type in the absence of the analog. The high level of resistance and rapid growth are very desirable properties for the application of the mutants in genetic experiments.     

Toxoplasma gondii: Purine synthesis and salvage in mutant host cells and parasites

Toxoplasma gondii, growing exponentially in heavily infected mutant Chinese hamster ovary cells that had a defined defect in purine biosynthesis, did not incorporate [U-¹⁴C]glucose or [¹⁴C]formate into the guanine or adenine of nucleic acids. Intracellular parasites therefore must be incapable of synthesizing purines and depend on their host cells for them. Extracellular parasites, which are capable of limited DNA and RNA synthesis, efficiently incorporated adenosine nucleotides, adenosine, inosine, and hypoxanthine into their nucleic acids; adenosine 5′-monophosphate was the best utilized precursor. Extracellular parasites incubated with ATP labeled with ³H in the purine base and ³²P in the α-phosphate incorporated the purine ring 50-fold more efficiently than they did the α-phosphate. Thus, ATP is largely degraded to adenosine before it can be used by T. gondii for nucleic acid synthesis. Two pathways for the conversion of adenosine to nucleotides appear to exist, one involving adenosine kinase, the other hypoxanthine—guanine phosphoribosyl transferase. In adenosine kinase-less mutant parasites, the efficiency of incorporation of ATP or adenosine was reduced by 75%, which indicates the adenosine kinase pathway was predominant. Extracellular parasites incorporated ATP into both the adenine and the guanine of their nucleic acids, so ATP from the host cell could supply the entire purine requirement of T. gondii. However, ATP generated by oxidative phosphorylation in the host cell is not essential for parasites because they grew normally in a cell mutant that was deficient in aerobic respiration and almost completely dependent upon glycolysis.     

Chemical and Morphological Studies of Bacterial Spore Formation : III. The Effect of 8-Azaguanine on Spore and Parasporal Protein Formation in Bacillus cereus var. Alesti

The purine analogue, 8-azaguanine, was added to cultures of the parasporal crystal-forming organism Bacillus cereus var. alesti at different times during growth and synchronous sporulation. The effect of its incorporation has been studied with particular reference to cell growth, nucleic acid composition, cytology, and the synthesis of the spore and crystal protein. Additions of the analogue during any stage of growth prevented further cell proliferation and all spore and crystal formation. Since both nucleic acids continued to be formed, cells of an increased size developed, containing large masses of chromatin in the form of condensed balls or axial cords. Lipid-containing inclusions also appeared following these additions and were usually aggregated at the centre or poles of the cells. The analogue could be isolated as the ribonucleotide from both the acid soluble and RNA fractions of these inhibited cells. Additions of the analogue following commencement of sporulation did not prevent either spore or crystal formation or affect the nucleic acid content of the sporulating cells. However, as before, the 8-azaguanine was incorporated into both the acid soluble and RNA of the cells, but not into these fractions of the spores ultimately formed. The implications of these findings are discussed in relation to crystal protein synthesis.     

Effects of benzimidazole on the purine and pyrimidine metabolism of yeast

Yeast cells inhibited by benzimidazole accumulate hypoxanthine with an associated efflux of xanthine. Unlike control cells, inhibited cells contain no detectable free UMP and CMP. Benzimidazole decreases uptake of [8-¹⁴C]-hypoxanthine into the intracellular pool of hypoxanthine and xanthine but causes radioactive xanthine to accumulate in the medium. In inhibited cultures there is a threefold increase in incorporation of [8-¹⁴C]hypoxanthine into the total (intracellular plus extracellular) xanthine. Uptake of [8-¹⁴C]hypoxanthine into free nucleotides and into bound adenine and guanine was inhibited by 70%. Uptake of [U-¹⁴C]glycine into IMP, AMP, GMP, DNA and RNA was also substantially decreased. Incorporation of [2-¹⁴C]uracil into the intracellular uracil pool was inhibited by 30% and into free uridine and cytidine by over 90%. Benzimidazole inhibited incorporation of [8-³H]IMP into AMP and GMP, and decreased substantially the activity of glutamine-amidophosphoribosyltransferase (EC 2.4.2.14). Yeast cultures were shown to N-ribotylate benzimidazole. Results are consistent with benzimidazole inhibiting yeast growth by competing for P-rib-PP and so depriving other ribotylation processes such as the ‘salvage’ pathways and de novo synthesis of purines and pyrimidines.     

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.     

Hypoxanthine transport in mammalian cells: Cell type-specific differences in sensitivity to inhibition by dipyridamole and uridine

Summary We have measured by rapid kinetic techniques the zero-trans influx of hypoxanthine in various cell lines and its sensitivity to inhibition by uridine, dipyridamole, nitrobenzylthioinosine and nitrobenzylthiopurine. The results and those reported earlier divided the cells into two distinct groups. In mouse P388, L1210 and L929 cells uridine and hypoxanthine had little effect on the transport of each other, supporting the view that nucleosides and hypoxanthine are transported by different carriers. In these cells, hypoxanthine transport was also uniquely resistant to inhibition by dipyridamole (IC₅₀ (50% inhibition dose) >30M). In Novikoff and HTC rat hepatoma, Chinese hamster ovary and Ehrlich ascites tumor cells, on the other hand, hypoxanthine and uridine inhibited the transport of each other about 50% at a concentration corresponding to the Michaelis-Menten constant of their transport, and hypoxanthine transport was strongly inhibited by dipyridamole (IC₅₀=100 to 400nM). Although these results are compatible with the view that nucleosides and hypoxanthine are transported by a common carrier in these cells, this conclusion is not supported by the finding that uridine transport is strongly inhibited in some of these cell lines, as in first group of cells, by nitrobenzylthioinsine, whereas hypoxanthine transport is highly resistant in all cell lines tested. In contrast, the transport of both substrates is highly resistant to inhibition by nitrobenzylthiopurine. The Michaelis-Menten constants for uridine transport are about the same in all cell lines. The Michaelis-Menten constants for hypoxanthine transport are similar to those for uridine transport in some cell lines, but are much higher in others. This difference is unrelated to the sensitivity of uridine and hypoxanthine transport to inhibition by each other or dipyridamole.     

Cellular reactions of O6-methylguanine, a product of some alkylating carcinogens

Cultures of a purine-requiring mutant of Chinese hamster ovary cells (CHO-104b), randomly bred hamster embryo cells, or Escherichia coli B_s−1 were treated with non-toxic doses of ³H-labelled O⁶-methylguanine. DNA and RNA were isolated and subjected to enzymic digestion to nucleosides at pH8. The products of digestion were analysed by ion-exchange chromatography on columns of Dowex 50 (NH₄⁺ form) at pH8.9. No ³H-labelled O⁶-methylguanosine was detected in nucleic acid digests. ³H-labelled O⁶-methylguanine was O-demethylated yielding [³H]guanine in CHO-104b cells. Radioactivity in nucleic acid digests was associated with thymidine, guanosine, deoxyguanosine and an unidentified early-eluting product. Reports of similar unidentified products from nucleic acids labelled with various agents are discussed.     

Cytochalasin B. VI. Competitive inhibition of nucleoside transport by cultured Novikoff rat hepatoma cells

Cytochalasin B competitively inhibits the transport of uridine and thymidine by Novikoff rat hepatoma cells growing in suspension culture with apparent K_i''s of 2 and 6 µM, respectively, but has no effect on the intracellular phosphorylation of the nucleosides. Choline transport is not affected by cytochalasin B. Results from pulse-chase experiments indicate that cytochalasin B has no direct effect on the synthesis of RNA, DNA, or uridine diphosphate-sugars. The inhibition of uridine and thymidine incorporation into nucleic acids by cytochalasin B is solely the consequence of the inhibition of nucleoside transport.