Cytosine arabinoside transport and metabolism in acute leukemias and T cell lymphoblastic lymphoma.

Cytosine arabinoside (araC) has proven efficacy in acute myeloid leukemia (AML), but its place in the treatment of acute lymphoblastic leukemia (ALL) and T lymphoblastic lymphoma is uncertain. The therapeutic potential of araC has been assessed in patients with AML, ALL, and T lymphoblastic lymphoma by measuring the conversion of araC to its active metabolite, the 5'-triphosphate of araC (araCTP), in purified blasts from patients as well as in normal polymorphs and lymphocytes. In all leukemias, araCTP was the major intracellular metabolite of araC. The highest araCTP formation was in blasts from T lymphoblastic lymphoma, which formed threefold more nucleotide than myeloblasts, and in turn myeloblasts formed twofold more araCTP than lymphoblasts from ALL. The mean araCTP formation in myeloblasts was sixfold greater than polymorphs, but in contrast, lymphoblasts and lymphocytes formed low and similar amounts of this nucleotide. Reasons for the sixfold range in araCTP accumulation in the various leukemic blasts were studied. The mean size of myeloblasts was 35-70% larger than lymphoblasts when compared on the basis of protein or intracellular water content, but T lymphoblastic lymphoma blasts and lymphoblasts were the same size. Activities of deoxycytidine kinase, deoxycytidylate deaminase, and pyrimidine nucleoside monophosphate kinase were not different between any of the leukemic cell types. The number of nucleoside transport sites on blasts was estimated by measuring the equilibrium binding of [3H]nitrobenzylthioinosine (NBMPR), which binds with high affinity to the transporter. Scatchard analysis yielded mean values of 27,500 sites/cell for T lymphoblastic lymphoma blasts, 10,000 sites/cell for myeloblasts, and 2,300 sites/cell for lymphoblasts. Our previous work has shown that araC influx correlates with the maximum number of 3H-NBMPR binding sites in leukemic and normal white cells. A strong correlation was observed between the number of nucleoside transport sites per leukemic blast cell and the accumulation of intracellular araCTP from extracellular araC at 1 microM. Membrane transport of araC at the low concentrations (approximately 1 microM), which are achieved therapeutically, is a major rate-limiting step in its conversion to araCTP by leukemic blast cells. Myeloblasts form more araCTP than lymphoblasts because of both higher nucleoside transport capacity and larger cell size. The highest nucleoside transport capacity and largest conversion of araC to araCTP is in T lymphoblastic lymphoma, which suggests that araC may be effective in the treatment of this disease.

Effect of thymidine on the sensitivity of cultured mouse tumor cells to 1-beta-D-arabinofuranosylcytosine.

Assays of thirteen cell lines, derived from mouse lymphomas, myelomas, myeloid tumors, and a mastocytoma, for sensitivity to growth inhibition by 1-beta-D-arabinofuranosylcytosine (ara-C) revealed a spectrum between the most and least sensitive which differed 100-fold from each other. An inverse correlation between sensitivity and cellular deoxycytidine 5'-triphosphate (dCTP) content was found, and this suggested that sensitivity of cells might be increased if the dCTP content was lowered during cell exposure to ara-C. Previous work has shown that thymidine treatment of cells lowers their dCTP content, and the effect of thymidine on the sensitivity of six of the cell lines to ara-C was therefore measured. Concentrations of thymidine below those inhibitory to cell growth by themselves caused an increase in ara-C sensitivity by up to 3-fold in 4 cell lines in which thymidine causes a depression in dCTP content but not in 2 myeloid lines in which the dCTP content was found not to be depressed by the same thymidine treatment. The results confirm an important role for dCTP in determining cellular sensitivity to ara-C. The finding that the sensitivity of 2 lymphoma cell lines to ara-C could be increased by concentrations of thymidine in the region of 10 micrometer, which are attainable clinically in humans, suggests that a combination of ara-C with thymidine might be useful in the treatment of some human tumors.

Degradation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate in human leukemic myeloblasts and lymphoblasts.

The intracellular half-life for retention of the active triphosphate metabolite 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (araCTP) of 1-beta-D-arabinofuranosylcytosine was measured in vitro in blast cells from patients with acute myeloblastic leukemia, acute lymphoblastic leukemia, and T-cell lymphoblastic lymphoma. araCTP accumulation from 1 microM 1-beta-D-arabinofuranosylcytosine in leukemic blast cells was closely correlated with the nucleoside transport capacity as measured by equilibrium binding of [3H]nitrobenzylthioinosine. The half-life of araCTP retention was related to araCTP accumulation only when the level of araCTP was expressed as a percentage of total intracellular 1-beta-D-arabinofuranosylcytosine metabolites. Accumulation of 1-beta-D-arabinofuranosyluracil 5'-monophosphate was inversely related to the half-life of araCTP retention and directly related to dCMP deaminase activity in cell free extracts. No conversion of 1-beta-D-arabinofuranosyluracil to 1-beta-D-arabinofuranosyluracil 5'-monophosphate was detectable in intact cells. The end product of araCTP degradation was 1-beta-D-arabinofuranosyluracil and it is proposed that conversion of 1-beta-D-arabinofuranosylcytosine 5'-monophosphate to 1-beta-D-arabinofuranosyluracil 5'-monophosphate is a step in the degradative pathway of araCTP. However, it is the cells' nucleoside transport capacity which primarily determines the level of intracellular araCTP accumulation.

Regulation of aspartate carbamoyltransferase of Escherichia coli by the interrelationship of magnesium and nucleotides.

Purified aspartate carbamoyltransferase from Escherichia coli K12 (carbamoylphosphate: L-aspartate carbamyltransferase, EC 2.1.3.2) shows greater activity with nucleotide effectors as the magnesium nucleotide complex than with similar amounts of the sodium nucleotide. Regulation of aspartate carbamoyltransferase activity in vivo may occur by changes in the total concentration of regulatory nucleotides or, under conditions of magnesium-limited growth, by variation of the saturation of the nucleotides with magnesium.

Effects of hydroxyurea on DNA synthesis in mouse L-cells.

The effect of the anti-metabolite hydroxyurea on DNA synthesis in mouse L-cells has been examined. It was shown previously that when DNA synthesis was diminished to very low levels by treatment with the drug there was preferential incorporation of added [3H]dThd into low molecular weight fragments (Martin, R.F., Radford, I. And Pardee, M. (1977) Biochem. Biophys. Res. Commun. 74, 9-15). On the basis of several criteria it is concluded here that these fragments are a product of semi-conservative nuclear DNA replication. The preferential labelling of DNA fragments, but not their size, is shown to be dependent on the hydroxyurea concentration used. These DNA fragments are also shown, by comparison with normal DNA replication intermediates, to comprise a heterogeneous population of 'larger-than-normal' fragments. Different models to account for these findings are considered and it is concluded that the results are compatible with a loss of coordination of DNA synthesis following drug treatment.

Inhibition of DNA synthesis and cell death.

The association between DNA synthesis inhibition and cell death in mouse L-cells was investigated using the drug hydroxyurea. This drug produces a preferential labelling of low molecular weight DNA and dose-response studies revealed a correlation between this effect and cytoxicity. Investigation of the reassociation kinetics of DNA labelled during hydroxyurea inhibition showed an over-replication of middle repetitive sequences, but the concentration dependence of this effect was quite different to that of cytotoxicity.

Deoxyribonucleoside triphosphate pools and differential thymidine sensitivities of cultured mouse lymphoma and myeloma cells.

The effects of various concentrations of thymidine on DNA synthesis and deoxyribonucleoside triphosphate contents of a highly thymidine-sensitive cultured mouse lymphoma cell line (WEHI-7) and a relatively resistant mouse myeloma cell line (HPC-108) have been studied by 32P-labelling techniques. DNA synthesis in the myeloma cells was inhibited by thymidine at concentrations of 10(-3) M or greater, while DNA synthesis in the lymphoma cells was inhibited by concentrations 30-fold lower, consistent with the 25-fold difference between the two cell lines in sensitivity to growth inhibition by thymidine. Thymidine caused marked elevation of the dTTP and dGTP pools, slight elevation or no change in the dATP pool and a marked decrease in the dCTP pool in cells of both lines. The greater resistance of HPC-108 cells to thymidine inhibition was related to the finding that they normally contained a much higher concentration of dCTP than did the WEHI-7 cells. Pool size measurements on thymidine-treated (10(-4) M) cells of an additional seven sensitive lymphoma and six relatively resistant myeloma cell lines indicated that in all 15 lines studied, with one exception, a critical concentration of dCTP of about 32 nmol per ml of cell volume was required for the maintenance of normal rates of DNA synthesis. The dCTP content found normally in the lymphoma cells was only a little above this concentration. Amongst the myeloma lines, three contained similarly low levels of dCTP, but were more resistant to thymidine inhibition probably because of their inefficient production of dTTP from thymidine. Cells of the other four myeloma lines (including HPC-108) normally contained much higher dCTP concentrations. The mechanism of thymidine action was explained by reference to the known allosteric properties of ribonucleotide reductase.

Isolation and characterization of Mycoplasma mycoides subsp. mycoides mutants deficient in nucleoside monophosphate transport.

To select mutants lacking dAMP uptake, log-phase cells of Mycoplasma mycoides subsp. mycoides Y were incubated with high-specific-activity [32P]dAMP and then stored several weeks at -20 degrees C to allow 32P decay before plating out. Mutants were screened for lack of labeling by [32P]dAMP. Two mutants were studied further by uptake and growth experiments with other nucleotides.

Preparation and FIGE separation of infrequent restriction fragments from Mycoplasma mycoides DNA.

Use of procedures for obtaining satisfactory preparation and digestion of intact DNA of Mycoplasma mycoides subsp. mycoides Y in agarose blocks is reported. The use of inverted field agarose gel electrophoresis (FIGE) for separation of the small number of fragments derived from the genome by several restriction endonuclease digestions is shown. An effect that fragments containing replication forks remain in the well during FIGE, distorting the representative yield of restriction fragments on the gels, is overcome by incubating cells with chloramphenicol for 1 1/2 h before harvest to allow rounds of replication to go to completion without new initiations of DNA synthesis.

A physical map of the genome of Mycoplasma mycoides subspecies mycoides Y with some functional loci.

A physical map is presented for the 1200 kb genome of Mycoplasma mycoides subsp. mycoides Y, locating 32 cleavage sites for 8 restriction endonucleases. The large restriction fragments involved were separated and sized by pulsed-field agarose gel electrophoresis. Their locations on the map were determined by probing Southern blots of digests with individual fragments isolated from other digests and by correlating the products of double and triple digestions. Loci for 2 ribosomal RNA operons and 2 tRNA operons have been determined by probing with cloned genes and the broad regions of the replication origin and terminus have also been outlined by in vivo labelling studies.

Location of sites of transposon Tn916 insertion in the Mycoplasma mycoides genome.

The genome of Mycoplasma mycoides subsp. mycoides GC1176-2 was cleaved into six large fragments by the endonuclease KpnI which also cleaved the transposon Tn916 once. This has allowed genomic mapping of insertion sites for 50 transformants of GC1176-2 containing Tn916. Almost all of the mapped sites were clearly separate. The transformants provide a bank of genomes each with a KpnI site at a different position to facilitate mapping of gene loci.

Enzymes of pyrimidine metabolism in Mycoplasma mycoides subsp. mycoides.

The major pathways of ribonucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides have been proposed from studies on its use of radioactive purines and pyrimidines. To interpret more fully the observed pattern of pyrimidine usage, cell extracts of this organism have been assayed for several enzymes associated with the salvage synthesis of pyrimidine nucleotides. M. mycoides possessed uracil phosphoribosyltransferase, uridine phosphorylase, uridine (cytidine) kinase, uridine 5'-monophosphate kinase, and cytidine 5'-triphosphate synthetase. No activity for phosphorolysis of cytidine was detected, and no in vitro conditions were found to give measurable deamination of cytidine. Of the two potential pathways for incorporation of uridine, our data suggest that this precursor would largely undergo initial phosphorolysis to uracil and ribose-1-phosphate. Conversely, cytidine is phosphorylated directly to cytidine 5'-monophosphate in its major utilization, although conversion of cytidine to uracil, uridine, and uridine nucleotide has been observed in vivo, at least when uracil is provided in the growth medium. Measurements of intracellular nucleotide contents and their changes on additions of pyrimidine precursors have allowed suggestions as to the operation of regulatory mechanisms on pyrimidine nucleotide biosynthesis in M. mycoides in vivo. With uracil alone or uracil plus uridine as precursors of pyrimidine ribonucleotides, the regulation of uracil phosphoribosyltransferase and cytidine 5'-triphosphate synthetase is probably most important in determining the rate of pyrimidine nucleotide synthesis. When cytidine supplements uracil in the growth medium, control of cytidine kinase activity would also be important in this regard.

Isolation and restriction endonuclease analysis of a mycoplasma plasmid.

A 1.7-kb plasmid was isolated from a large colony type strain of Mycoplasma mycoides subsp. mycoides. It was cloned into the Escherichia coli vector M13mp19, and DNA from the resultant recombinant was used to construct a restriction map of the plasmid. Because mycoplasmas show deviation from normal coding patterns, the availability of a mycoplasma plasmid could be useful in the development of a cloning vector for them.

Response of the pyrimidine pathway of Escherichia coli K 12 to exogenous adenine and uracil.

The effect of exogenous adenine or uracil upon the de novo pathway for synthesis of pyrimidine nucleotides in Escherichia coli K12 was investigated. Parameters studied were levels of the enzymes carbamoyl phosphate synthase (EC 2.7.2.9), aspartate carbamoyltransferase (EC 2.1.3.2) and orotate phosphoribosyltransferase (EC 2.4.2.10) and the intermediates carbamoyl phosphate, aspartate and orotate, together with the contributions of exogenous uracil and aspartate to intracellular pyrimidine nucleotide. Taken with earlier data [Bagnara, A.S. & Finch, L. R. (1974) Eur. J. Biochem- 41, 421--430] on contents of UTP, CTP and 5-phosphoribosyl 1-diphosphate in cultures of this strain after the addition of adenine or uracil, the results obtained provide new insights into the regulatory mechanisms operating on the pathway in vivo. These insights enable evaluation of the contributions of such factors as limitation for a substrate, feed-back allosteric control by end products and enzyme repression/depression mechanisms. The evidence presented indicates that depressed levels of orotate phosphoribosyltransferase in E. coli K12 result in the wasteful ultilization of asparatate for excess synthesis of pyrimidine nucleotide precursors during balanced growth of the strain in minimal medium. Exogenous adenine increases the excessive accumulation of these precursors by lowering the intracellular content of 5-phosphoribosyl 1-diphosphate (Bagnara and Finch, 1974). This causes a decrease in the conversion of orotate to orotidine 5'-monophosphate, thus lowering the utilization or orotate and its precursors for synthesis of pyrimidine nucleotides. Further, since the contents of these nucleotide end products are thereby decreased (Bagnara nad Finch, 1974), theri feed-back on the early steps in the pathway is diminished and the production of the precursors is increased. It is postulated that growth of E. coli K12 under these conditions is limited by a compound that is metabolically related to precursors to aspartate.

Novel arrangement of rRNA genes in Mycoplasma gallisepticum: separation of the 16S gene of one set from the 23S and 5S genes.

Large restriction fragments from the DNA of Mycoplasma gallisepticum S6 and PG31, which were prepared by digestion with BglI, BssHII, SmaI, or XhoI and which were separated by pulsed-field electrophoresis, were hybridized with probes containing most, or different parts, of an rRNA operon of Mycoplasma capricolum. The results showed that the genomes contained three widely separated rRNA loci. One locus contained genes for all three rRNA species and another contained 23S and probably 5S rRNA genes, whereas the third appeared to have only a 16S rRNA gene.

Pathways of nucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides.

By measuring the specific activity of nucleotides isolated from ribonucleic acid after the incorporation of (14)C-labeled precursors under various conditions of growth, we have defined the major pathways of ribonucleotide synthesis in Mycoplasma mycoides subsp. mycoides. M. mycoides did not possess pathways for the de novo synthesis of nucleotides but was capable of interconversion of nucleotides. Thus, uracil provided the requirement for both pyrimidine ribonucleotides. Thymine is also required, suggesting that the methylation step is unavailable. No use was made of cytosine. Uridine was rapidly degraded to uracil. Cytidine competed effectively with uracil to provide most of the cytidine nucleotide and also provided an appreciable proportion of uridine nucleotide. In keeping with these results, there was a slow deamination of cytidine to uridine with further degradation to uracil in cultures of M. mycoides. Guanine was capable of meeting the full requirement of the organism for purine nucleotide, presumably by conversion of guanosine 5'-monophosphate to adenosine 5'-monophosphate via the intermediate inosine 5'-monophosphate. When available with guanine, adenine effectively gave a complete provision of adenine nucleotide, whereas hypoxanthine gave a partial provision. Neither adenine nor hypoxanthine was able to act as a precursor for the synthesis of guanine nucleotide. Exogenous guanosine, inosine, and adenosine underwent rapid cleavage to the corresponding bases and so show a pattern of utilization similar to that of the latter.

Adenine phosphoribosyltransferase in Mycoplasma mycoides and Escherichia coli.

Some kinetic properties of the adenine phosphoribosyltransferases from Escherichia coli and Mycoplasma mycoides have been studied. For the E. coli enzyme, Michaelis constants for adenine and 5-phosphoribosyl-1-pyrophosphate (PRPP) are 1.3 and 10 mum, respectively. Adenosine monophosphate, the most effective nucleotide inhibitor, inhibits competitively with respect to PRPP, the inhibition constant being 26 mum. The M. mycoides enzyme has more complex kinetics. The response to increasing PRPP concentration is sigmoidal, the degree of sigmoidality depending on both the concentration of adenine and the pH. At low PRPP levels, high concentrations of adenine are inhibitory. Guanosine monophosphate is the most effective inhibitor, being inhibitory at all pH values, but other nucleotides have been found to activate at pH 7 and inhibit at pH 8. The elution profile of the M. mycoides enzyme from Sephadex suggests an association of enzyme subunits in the presence of PRPP. This is consistent with the observed kinetics if the associated form has increased stability and activity.

Uptake and utilization of deoxynucleoside 5'-monophosphates by Mycoplasma mycoides subsp. mycoides.

Mycoplasma mycoides subsp. mycoides has been shown to possess an unusual capacity for the uptake and utilization of exogenous deoxyribonucleoside 5'-monophosphates intact without prior dephosphorylation. In this study, it was found that once inside the cell, deoxyribonucleoside 5'-monophosphates were rapidly phosphorylated to the triphosphate level and incorporated into DNA. Catabolism of deoxyribonucleoside 5'-monophosphates was also observed. Competition studies indicated that a single uptake system with a higher affinity for deoxyribonucleotides mediates the uptake of nucleoside 5'-monophosphates.

Genomic maps of some strains within the Mycoplasma mycoides cluster.

Genomic restriction maps for the small colony (SC) strains (PG1, KH3J, Gladysdale, and V5) of Mycoplasma mycoides subsp. mycoides (the agent of contagious bovine pleuropneumonia) and for Mycoplasma strain PG50 (classified as bovine serogroup 7), with respective sizes of 1,280, 1,280, 1,260, 1,230, and 1,040 kbp, were compared with the map (1,200 kbp) for a large colony strain (Y goat) of M. mycoides subsp. mycoides. The number and order of all mapped restriction sites were fully conserved in the SC genomes, as were the approximate positions of mapped loci. A number of these restriction sites in the Y genome and some, but fewer, in the PG50 genome appeared to be conserved. The SC and large colony strains shared conservation in the relative positions of the mapped loci, except for rpoC.

Enzymes of purine metabolism in Mycoplasma mycoides subsp. mycoides.

The major pathways of ribonucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides were proposed previously from studies of its usage of radioactive purines and pyrimidines. To interpret more fully the pattern of purine usage, we have assayed cell-free extracts of this organism for several enzymes associated with the salvage synthesis of purine nucleotides. M. mycoides possessed phosphoribosyltransferases for adenine, guanine, and hypoxanthine, purine nucleoside phosphorylase, GMP reductase, GMP kinase, adenylosuccinate synthetase, and adenylosuccinate lyase. Purine nucleoside kinase and adenosine deaminase were not detected. Examination of kinetic properties and regulation of some of the above enzymes revealed differences between M. mycoides and Escherichia coli. Most notable of these were the greater susceptibility of the enzymes from M. mycoides to inhibition by nucleotides and the more widespread involvement of GMP as an inhibitor. Observations on enzyme activities in vitro allow an adequate explanation of the capacity of guanine to provide M. mycoides with its full requirement for purine nucleotides.