Deoxycytidine Transport and Metabolism in the Central Nervous System

Abstract: The mechanisms by which deoxycytidine enters and leaves brain, choroid plexus, and CSF were investigated by injecting [³H]deoxycytidine intraarterially, intravenously, and intraventricularly. After intracarotid injection of deoxycytidine (1.0 μM) into rats, deoxycytidine did not pass through the blood-brain barrier at a faster rate than sucrose. [³H]Deoxycytidine, either alone or together with unlabeled deoxycytidine, was infused at a constant rate into conscious adult rabbits. At 130 min, [³H]deoxycytidine readily entered CSF, choroid plexus, and brain. In brain, approx. 60% of the nonvolatile radioactivity was attributable to [³H]deoxycytidine phosphates. The addition of 0.22 mmol/kg unlabeled deoxycytidine to the infusion syringe decreased the phosphorylation of [³H]deoxycytidine in brain by approx. 50%; the addition of 2.2 mmol/kg of unlabeled deoxycytidine to the infusion syringe decreased the relative entry of [³H]deoxycytidine into CSF and brain by approx. 50 and 75%, respectively. Two hours after the intraventricular injection of [³H]deoxycytidine, [³H]deoxycytidine was rapidly cleared from CSF, in part, to brain, where approx. 65% of the [³H]deoxycytidine was converted to [³H]deoxycytidine phosphates. The intraventricular injection of unlabeled deoxycytidine with the [³H]deoxycytidine decreased the phosphorylation of [³H]deoxycytidine in the brain significantly and also decreased the clearance of [³H]deoxycytidine from the CSF. These results were interpreted as showing that the entry of deoxycytidine from blood into CSF occurs by a saturable transport system within the choroid plexus. Once within the CSF, the deoxycytidine can enter brain, undergo phosphorylation to deoxycytidine phosphates, and subsequently be incorporated into DNA.     

Effects of Hydroxyurea and Deoxyadenosine on the Synthesis of Deoxycytidine Phosphate and on the Uptake of Nucleosides in Excised Root Tips of Vicia faba

The effects of hydroxyurea and deoxyadenosine on the synthesis of deoxycytidine phosphate was studied by measuring the incorporation of [¹⁴C]-cytidine into acid soluble deoxycytidine phosphate in root tips of Vicia faba. Hydroxyurea and deoxyadenosine both markedly depressed the incorporation of [¹⁴C]-cytidine. Deoxyadenosine had the additional effect of inhibiting the uptake of [¹⁴C]-cytidine. Furthermore, millimolar concentrations of deoxyadenosine inhibited the uptake of micromolar concentrations of adenosine, thymidine, and deoxycytidine. The incorporation of [¹⁴C]-cytidine into RNA was only slightly affected by hydroxyurea. Deoxyadenosine inhibited the incorporation into RNA to about the same extent as the uptake of [¹⁴C]-cytidine. It is suggested that hydroxyurea reduced the incorporation of radioactive cytidine into deoxycytidine phosphate mainly by interfering with ribonucleotide reduction. The depression of [¹⁴C]-cytidine incorporation into deoxycytidine phosphate in the presence of deoxyadenosine is believed to be the result of an inhibition of both ribonucleotide reduction and [¹⁴C]-cytidine uptake.     

Phosphorylation of Deoxyribonucleosides and DNA Synthesis during Embryogenesis of the Sea Urchin

The phosphorylation of thymidine, deoxycytidine, deoxyadenosine and deoxyguanosine was studied during the embryogenesis of the sea urchin Hemicentrotus pulcherrimus. [³H]Thymidine was taken up, phosphorylated and accumulated mostly as [³H]thymidine triphosphate in the early cleavage stage embryos. As the embryos developed, the formation of [³H]thymidine triphosphate decreased and most of the [³H]thymidine taken up by the blastulae remained be phosphorylated. When [³H]deoxycytidine was added to the cleaving embryos, the resultant labeled pool consisted of almost equal amounts of [³H]deoxycytidine monophosphate and [³H]deoxycytidine triphosphate. The formation of [³H]deoxycytidine monophosphate increased up to 10 hr following fertilization and then decreased, while the formation of [³H]deoxycytidine triphosphate decreased for 10 hr following fertilization and then gradually increased. [³H]Deoxyadenosine was rapidly phosphorylated to monophosphate derivative in the cleavage stage embryos. The formation of [³H]deoxyadenosine triphosphate increased rapidly after cleavage stage with a concomitant decrease of [³H]deoxyadenosine monophosphate. The activity of phosphorylation in [³H]deoxyguanosine to triphosphate derivative increased rapidly reaching a plateau 10 hr after fertilization. At this point, 80 % of the [³H]deoxyguanosine was recovered as [³H]deoxyguanosine triphosphate. Based on the above results, it was concluded that the profile of production of each deoxyribonucleoside triphosphate changed during the embryogenesis of the sea urchin, and the in vivo rate-limiting step of phosphorylation of the individual deoxyribonucleoside was assumed to be different.     

PREPARATION OF BASE-DEUTERATED 2′-DEOXYADENOSINE NUCLEOSIDES AND THEIR SITE-SPECIFIC INCORPORATION INTO DNA

We report a four-step synthesis of 2′-deoxy-2-deuteroadenosine from 2′-deoxyadenosine in 38% overall yield. The more accessible 2′-deoxy-8-deuteroadenosine was also prepared and incorporated into DNA by automated solid phase synthesis (80% deuterium) using N ⁶-benzoyl-2′-deoxy-8-deuteroadenosine-3′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) in combination with acetyl-protected deoxycytidine and phenoxyacetyl-protected purine phosphoramidites.     

Purification of deoxycytidine kinases from two P815 murine neoplasms and their separation from deoxyguanosine kinase

Deoxycytidine kinase, which catalyzes the phosphorylation of deoxycytidine and 1-βd-arabinofuranosylcytosine (Ara-C) at the 5′-position, has been extracted and extensively purified from a murine neoplasm P815, either sensitive (P815) or resistant (P815/ Ara-C) to 1-β-d-arabinofuranosylcytosine. Gel filtration and ion-exchange chromatography were used to accomplish the purification. The purified enzyme exhibited a single band upon disc electrophoresis. During the extraction procedure an enzyme catalyzing the phosphorylation of deoxyguanosine and deoxyadenosine was successfully separated for the first time from deoxycytidine kinase. The K_m values and turnover numbers with deoxycytidine as phosphate acceptor for the kinase from P815 cells sensitive to 1-β-darabinofuranosylcytosine and that from P815 cells resistant to the drug are 9.3 μm, 4.7 × 10⁶/min and 15.4 μm, 8.0 × 10⁴/min, respectively.     

Enzymatic Synthesis of Radioactive 5-Methyl-2′-Deoxycytidine 5′-Monophosphate by Using 32P-Postlabeling

Abstract

5-Methyl-2′-deoxycytidine 5′-[³²P]- and deoxycytidine 5-[32 P]-monophosphates were prepared from corresponding nucleotide homopolymers by using a ³² P-postlabeling procedure. The radioactive monophosphates obtained were well suited for biological and biochemical experiments.     

In vitro and in vivo recombination-related reactions of Escherichia coli recA protein and glucosyl-hydroxymethyl-deoxycytidine DNA

Summary Recombination of T4 phage is not controlled by the host recA gene but by an analogous phage gene, uvsX. We have tested the hypothesis that recA protein is inactive in T4-infected cells because it is unable to catalyze reactions involving single stranded DNA containing glucosyl-hydroxylmethyl-deoxycytidine. We found, however, that with modified and unmodified deoxycytidine containing DNAs, uvsX protein and recA protein catalyze in vitro reactions related to DNA recombination, but in T4-infected cells recA protein fails to promote strand transfer of DNA which contains unmodified deoxycytidine.Abbreviations dC-DNA deoxycytidine containing DNA - dC-T4 T4 phage containing dC-DNA - dHMC-DNA glucosyl-hydroxymethyl-deoxycytidine containing DNA - dsDNA double stranded DNA - gp gene product - ssDNA single stranded DNA     

Specific selection of deoxycytidine kinase mutants with tritiated deoxyadenosine

We have shown previously that a low concentration of tritiated deoxyadenosine, i.e., 1 µCi/ml, selectively kills wild-type S49 murine lymphoma cells. Mutant cells resistant to [³H]deoxyadenosine lacked adenosine kinase completely but retained a significant level of deoxyadenosine phosphorylating activity. To study further the specificity of [³H]deoxyadenosine selection, lymphoma cell clones resistant to 15 µCi/ml [³H]deoxyadenosine have been derived. The resistant line, S49-dA15, is also resistant to high levels of nonradioactive deoxyadenosine and to deoxyguanosine but remains sensitive to thymidine. The thymidine inhibition of the growth of the mutant, in contrast to that of the wild-type cells, cannot be prevented by deoxycytidine. The mutant line lacks deoxycytidine kinase that also phosphorylates deoxyadenosine. In addition, the mutant cells excrete a large amount of deoxycytidine into culture medium, consistent with a failure of salvage of the nucleoside in the absence of an appropriate kinase, i.e., deoxycytidine kinase. In contrast, a deoxycytidine kinase-deficient cell line that was selected with arabinosylcytosine does not excrete deoxycytidine and contains high deoxycytidine deaminase activity. [³H]Deoxyadenosine can be used as a selective agent for specific selection of deoxycytidine kinase-negative mutants.     

Interaction of an Escherichia coli mutator gene with a deoxyribonucleotide effector

Summary Strains of Escherichia coli carrying mutD5 display very high mutation rates (about 10⁴-fold above wild-type) when grown in rich medium, but relatively low mutation rates (about 10- to 50-fold above wild-type) in minimal medium. Thymidine, deoxycytidine, and deoxyuridine are all capable of stimulating mutation when added to minimal medium. Studies with mutants blocked in various steps of thymidine metabolism implicate a phosphorylated thymidine effector which mediates the mutagenic action of the added deoxyribonucleotides. In addition, an unidentified compound or compounds other than thymidine present in rich medium (L-broth) can also increase the mutation rate.     

Deoxycytidine production by metabolically engineered <Emphasis Type="Italic">Corynebacterium ammoniagenes</Emphasis>

Corynebacterium ammoniagenes N424 was metabolically modified to isolate overproducers of deoxycytidine. Inosine auxotrophy (ino) was initially introduced to prevent the flow of PRPP (phosphoribosyl pyrophosphate) into the purine biosynthetic pathway by random mutagenesis using N-methyl-N′-nitro-N-nitrosoguanidine. Following that, mutants possessing hydroxyurea resistance (HU^r) were isolated to increase the activity of ribonucleoside diphosphate reductase, which catalyzes the reduction of ribonucleoside diphosphate to deoxyribonucleoside diphosphate. Then, in order to block the flow of dCTP into the TMP biosynthetic pathway via dUTP, thymine auxotrophy (thy⁻) was introduced into the mutant IH30 with ino⁻ and Hlf. The resulting mutant IM7, possessing the characteristics of ino⁻, HU^r, and thy⁻, was deficient in dCTP deaminase and produced significantly higher amounts of deoxycytidine (81.3 mg/L) compared to its mother strain IH30 (6.2 mg/L). Deoxycytidine productivity was further enhanced by isolating the mutant IU19, which was resistant to 5-fluorouracil, an inhibitor of carbamoyl phosphate synthase. This enzyme catalyzed the synthesis of carbamoyl phosphate from glutamine, HCO₃⁻, and ATP. 5-Fluorouracil also inhibited aspartate trans-carbamoylase, catalyzeing the condensation of carbamoyl phosphate and aspartate. Finally, 5-fluorocytosine resistance (FC^r) was introduced into the mutant strain IU19 to relieve the repression caused by accumulation of pyrimidine nucleosides. The mutant strain IC14-C6 possessing all the five characteristics described above produced 226.3 mg/L of deoxycytidine, which was at least 2,000 fold higher compared to the wild type, and accumulated only a negligible amount of other pyrimidines under shake flask fermentation.     

Inhibition of cell division and induction of chromosome aberrations in cultured Ehrlich ascites tumor cells by deoxyguanosine

Summary Exposure of exponentially growing cultures of Ehrlich ascites tumor cells to 1 or 2×10^-3 M deoxyguanosine resulted in an inhibition of DNA synthesis and cell multiplication. Continued increase in the RNA and protein content of these cultures suggests a state of unbalanced growth. Deoxyguanosine-inhibition is prevented by the presence of deoxycytidine (1×10^-4–2×10^-3 M).Treatment with deoxyguanosine (2×10^-3 M) for about one generation-time (18 hrs) and removal of deoxyguanosine thereafter resulted in chromosome aberrations (breaks and exchange figures) in 30–50% of those mitotic cells which were harvested 5 to 12 hrs after treatment. Chromosome defects were strongly reduced after incubation of cell-cultures in the presence of deoxyguanosine (2×10^-3 M) together with deoxycytidine (5×10^-4 M).The biochemical mechanisms by which deoxyguanosine and other inhibitors of DNA synthesis might produce chromosome damaging effects, are discussed.Supported by grants from Deutsche Forschungsgemeinschaft (Scha 176/1 and Scha 176/2).     

AFFINITY MODIFICATION OF EcoRII DNA METHYLTRANSFERASE BY THE DIALDEHYDE-SUBSTITUTED DNA DUPLEXES: MAPPING THE ENZYME REGION THAT INTERACTS WITH DNA

ABSTRACT

Affinity modification of EcoRII DNA methyltransferase (M·EcoRII) by DNA duplexes containing oxidized 2′-O-β-D-ribofuranosylcytidine (Crib*) or 1-(β-D-galactopyranosyl)thymine (Tgal*) residues was performed. Cross-linking yields do not change irrespective of whether active Crib* replaces an outer or an inner (target) deoxycytidine within the EcoRII recognition site. Chemical hydrolysis of M·EcoRII in the covalent cross-linked complex with the Tgal*-substituted DNA indicates the region Gly²⁶⁸-Met³⁹¹ of the methylase that is likely to interact with the DNA sugar-phosphate backbone. Both specific and non-specific DNA interact with the same M·EcoRII region. Our results support the theoretically predicted DNA binding region of M·EcoRII.     

Induction of a deoxycytidineless state in cultured mammalian cells by bromodeoxyuridine

Bromodeoxyuridine triphosphate is an allosteric inhibitor of ribonucleotide reductase, and bromodeoxyuridine can therefore kill cells by starving them for deoxycytidine nucleotides. The toxicity of bromodeoxyuridine for some cell lines is reduced many fold when deoxycytidine is also present. For example, wild type 3T6 cells can be grown serially in 1.5 × 10^?4 Molar bromodeoxyuridine and 2 × 10^?4 Molar deoxycytidine, remain healthy, and incorporate bromodeoxyuridine extensively into cellular DNA. Some of the numerous effects of this drug on the behavior of cells and viruses may be due to a deoxycytidineless state, rather than to the incorporation of the bromodeoxyuridine into DNA.     

Radioimmunoassays of plasma thymidine,uridine, deoxyuridine,and cytidine/deoxycytidine

Radioimmunoassay techniques have been developed for the assay of thymidine, uridine, deoxyuridine, and deoxycytidine. Plasma levels of the first three nucleosides have been measured, and an upper limit has been determined for the plasma concentration of deoxycytidine. The assays involve displacement of a [³H]pyrimidine nucleoside from the appropriate labeled rabbit immunoglobulin. By assaying a mixture of uridine and deoxyuridine in the presence and absence of borax, the concentrations of both nucleosides have been measured. In seven healthy adults, plasma levels of uridine were 21.1 ± 8.4 μm (mean ± SD) and of deoxyuridine were 0.62 ± 0.39 μm. In cancer patients, thymidine levels were 7.5 ± 2.7 × 10^?7m. The upper limit for plasma deoxycytidine levels in six healthy adults was 0.71 ± 0.1 μm.     

Stereochemical Preference of 2'‐Deoxycytidine for Chiral Bis(diamido)‐bridged Basket Resorcin[4]arenes

Bis(diamido)‐bridged basket resorcin[4]arene (all-S)-1 and its (all-R)-1 enantiomer proved able to interact with 2'‐deoxycytidine ( 2 ) and pyrimidine nucleoside analogs in dimethyl sulfoxide (DMSO) solution. In such a solvent, the resorcinarene hosts adopt a preferential 1,3‐alternate‐like conformation, with a larger cavity delimited by two syn 3,5‐dimethoxyphenyl moieties, and two external pockets, each delimited by the other 3,5‐dimethoxyphenyl group and its diamido arm (the wing). Complexation phenomena were investigated by nuclear magnetic resonance (NMR) methods, including ¹H NMR DOSY and 1D ROESY experiments, and molecular modeling. Heteroassociation constants of [ (all-S)-1·2 ] and [ (all-R)-1·2 ] diastereoisomeric complexes were obtained from diffusion data by single point measurements, and from nonlinear fitting of ¹H NMR chemical shifts. Selective proton relaxation rate measurements allowed us to significantly discriminate the two complexes by identifying two different interaction sites of the guest in the resorcin[4]arene host, depending on its configuration. Chirality 25:840–851, 2013. © 2013 Wiley Periodicals, Inc.     

Identification, Development, and Regional Distribution of Ribonucleotide Reductase in Adult Rat Brain

The development and regional distribution of ribonucleotide reductase (EC 1.17.4.1) were determined in rat brain. Ribonucleotide reductase was partially purified by ammonium sulfate fractionation (20-40% saturation). Enzyme activity was measured by a specific radiochemical assay. This method involved the reduction of [¹⁴C]cytidine diphosphate (CDP) to [¹⁴C]deoxy-cytidine diphosphate with subsequent hydrolysis and separation of the product ([¹⁴C]deoxycytidine) from substrate ([¹⁴C]cytidine) by Dowex-1-borate ion-exchange chro-matography. The specific activity of ribonucleotide reductase in whole brain of newborn rats was 3.78 ± 0.55 units (pmol/h)/mg protein (SEM; n = 6) and declined to 0.17 ± 0.01 units/mg protein (n = 7) at 10-12 weeks of age, with a further decline to 0.11 ± 0.01 units/mg protein (n = 3) at 1 year. Ribonucleotide reductase activity in rat liver decreased from 4.58 ± 0.62 units/mg protein (n = 3) in newborn animals to 0.06 ± 0.01 units/mg protein (n = 7) at 10-12 weeks and was present at trace levels at 6 months of age. The decline in specific activity with age was not due to a change in the K_m for CDP. The K_m for CDP in brain of newborn and adult rats was 80-90 μM. In 10- to 12-week-old rats, the specific activity of ribonucleotide reductase was similar in the various regions of the brain tested except for the brainstem, which had 50% lower specific activity than the whole brain. These results indicate that ribonucleotide reductase activity is present and widely distributed in adult rat brain.     

Synthesis of 2′-Deoxyformycin B and 2′-Deoxyoxoformycin B

Abstract

3-β-D-Ribofuranosylpyazolo[4,3-d]pyrimidines (formycins)¹ modified in the heteroaromatic moiety are of biological interest as analogues of adenosine and guanosine, and have been the objects of intensive synthetic chemical effort by several groups.^2-9 2′-Deoxynucleosides^2c,2d,7b,13 and other analogties of the formycins modified in the sugar moiety^10-12 are also of potential interest, but have been less extensively studied. Examples of the 2′-deoxyribonucleoside type known to date include the 2′-deoxy-6-thioguanosine analogue 1, the 2′-deoxyadenosine (dAdo) analogue 2 (2′-deoxyformycin A),^10,13 and the 2-chloro-2′-deoxyadenosine analogue 3.^7b Compound 2 was found to be 10-15 times more potent than 2′-deoxyadenosine as an inhibitor of the growth of S49 cells, a murine lymphoma line of T-cell origin.¹³ Activity depended on 5′- phosphorylation, since mutants lacking the enzymes adenosine kinase (AK) and deoxycytidine kinase (dCK) were insensitive to the drug. Furthermore, activity was comparable in the presence and absence of an AK inhibitor, suggesting that 2, unlike dAdo, may be a poor substrate for adenosine deaminase. That 5′-phosphorylation of 2 was mediated by AK rather than dCK was indicated by the fact that miitants lacking only dCK retained sensitivity. This contrasted with the behavior of dAdo, which is known to be n substrate for both AK and dCK.¹⁴     

Incorporation of uracil into the growing strand of adenovirus 12 DNA.

The amount of rapidly labeled short DNA chains in adenovirus 12(Ad12)-infected cells was markedly increased in the presence of either uridine or deoxycytidine which could be converted to dUTP. When the infected cells were labeled with [³H]uridine or [³H] deoxycytidine and the labeled nucleotides in the short DNA chains from the Hirt supernatant were analysed by thin-layer chromatography, approximately 90 or 20% of the label was detected in dUTP. These results suggest that at least a portion of short DNA chains formed during Ad12 DNA replication is derived from an excision-repair mechanism of uracil containing nascent strands.