(1/752) Comparison of the mechanism of cytotoxicity of 2-chloro-9-(2-deoxy-2- fluoro-beta-D-arabinofuranosyl)adenine, 2-chloro-9-(2-deoxy-2-fluoro- beta-D-ribofuranosyl)adenine, and 2-chloro-9-(2-deoxy-2,2-difluoro- beta-D-ribofuranosyl)adenine in CEM cells.
In an effort to understand biochemical features that are important to the selective antitumor activity of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine [Cl-F( upward arrow)-dAdo], we evaluated the biochemical pharmacology of three structurally similar compounds that have quite different antitumor activities. Cl-F( upward arrow)-dAdo was 50-fold more potent as an inhibitor of CEM cell growth than were either 2-chloro-9-(2-deoxy-2-fluoro-beta-D-ribofuranosyl)adenine [Cl-F( downward arrow)-dAdo] or 2-chloro-9-(2-deoxy-2, 2-difluoro-beta-D-ribofuranosyl)adenine [Cl-diF( upward arrow downward arrow)-dAdo]. The compounds were similar as substrates of deoxycytidine kinase. Similar amounts of their respective triphosphates accumulated in CEM cells, and the rate of disappearance of these metabolites was also similar. Cl-F( upward arrow)-dAdo was 10- to 30-fold more potent in its ability to inhibit the incorporation of cytidine into deoxycytidine nucleotides than either Cl-F( downward arrow)-dAdo or Cl-diF( upward arrow downward arrow)-dAdo, respectively, which indicated that ribonucleotide reductase was differentially inhibited by these three compounds. Thus, the differences in the cytotoxicity of these agents toward CEM cells were not related to quantitative differences in the phosphorylation of these agents to active forms but can mostly be accounted for by differences in the inhibition of ribonucleotide reductase activity. Furthermore, the inhibition of RNA and protein synthesis by Cl-F( downward arrow)-dAdo and Cl-diF( upward arrow downward arrow)-dAdo at concentrations similar to those required for the inhibition of DNA synthesis can help explain the poor antitumor selectivity of these two agents because all cells require RNA and protein synthesis. (+info)
(2/752) In vitro reactions of butadiene monoxide with single- and double-stranded DNA: characterization and quantitation of several purine and pyrimidine adducts.
We have previously shown that butadiene monoxide (BM), the primary metabolite of 1,3-butadiene, reacted with nucleosides to form alkylation products that exhibited different rates of formation and different stabilities under in vitro physiological conditions. In the present study, BM was reacted with single-stranded (ss) and double-stranded (ds) calf thymus DNA and the alkylation products were characterized after enzymatic hydrolysis of the DNA. The primary products were regioisomeric N-7-guanine adducts. N-3-(2-hydroxy-3-buten-1-yl)adenine and N-3-(1-hydroxy-3-buten-2-yl)adenine, which were depurinated from the DNA more rapidly than the N-7-guanine adducts, were also formed. In addition, N6-(2-hydroxy-3-buten-1-yl)deoxyadenosine and N6-(1-hydroxy-3-buten-2-yl)deoxyadenosine were detected and evidence was obtained that these adducts were formed by Dimroth rearrangement of the corresponding N-1-deoxyadenosine adducts, not while in the DNA, but following the release of the N-1-alkylated nucleosides by enzymatic hydrolysis. N-3-(2-hydroxy-3-buten-1-yl)deoxyuridine adducts, which were apparently formed subsequent to deamination reactions of the corresponding deoxycytidine adducts, were also detected and were stable in the DNA. Adduct formation was linearly dependent upon BM concentration (10-1000 mM), with adduct ratios being similar at the various BM concentrations. At a high BM concentration (750 mM), the adducts were formed in a linear fashion for up to 8 h in both ssDNA and dsDNA. However, the rates of formation of the N-3-deoxyuridine and N6-deoxyadenosine adducts increased 10- to 20-fold in ssDNA versus dsDNA, whereas the N-7-guanine adducts increased only slightly, presumably due to differences in hydrogen bonding in ssDNA versus dsDNA. These results may contribute to a better understanding of the molecular mechanisms of mutagenesis and carcinogenesis of both BM and its parent compound, 1,3-butadiene. (+info)
(3/752) Stochastic and nonstochastic post-transcriptional silencing of chitinase and beta-1,3-glucanase genes involves increased RNA turnover-possible role for ribosome-independent RNA degradation.
Stochastic and nonstochastic post-transcriptional gene silencing (PTGS) in Nicotiana sylvestris plants carrying tobacco class I chitinase (CHN) and beta-1,3-glucanase transgenes differs in incidence, stability, and pattern of expression. Measurements with inhibitors of RNA synthesis (cordycepin, actinomycin D, and alpha-amanitin) showed that both forms of PTGS are associated with increased sequence-specific degradation of transcripts, suggesting that increased RNA turnover may be a general feature of PTGS. The protein synthesis inhibitors cycloheximide and verrucarin A did not inhibit degradation of CHN RNA targeted for PTGS, confirming that PTGS-related RNA degradation does not depend on ongoing protein synthesis. Because verrucarin A, unlike cycloheximide, dissociates mRNA from ribosomes, our results also suggest that ribosome-associated RNA degradation pathways may not be involved in CHN PTGS. (+info)
(4/752) Nucleotide pool imbalance and adenosine deaminase deficiency induce alterations of N-region insertions during V(D)J recombination.
Template-independent nucleotide additions (N regions) generated at sites of V(D)J recombination by terminal deoxynucleotidyl transferase (TdT) increase the diversity of antigen receptors. Two inborn errors of purine metabolism, deficiencies of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP), result in defective lymphoid development and aberrant pools of 2'-deoxynucleotides that are substrates for TdT in lymphoid precursors. We have asked whether selective increases in dATP or dGTP pools result in altered N regions in an extrachromosomal substrate transfected into T-cell or pre-B-cell lines. Exposure of the transfected cells to 2'-deoxyadenosine and an ADA inhibitor increased the dATP pool and resulted in a marked increase in A-T insertions at recombination junctions, with an overall decreased frequency of V(D)J recombination. Sequence analysis of VH-DH-JH junctions from the IgM locus in B-cell lines from ADA-deficient patients demonstrated an increase in A-T insertions equivalent to that found in the transfected cells. In contrast, elevation of dGTP pools, as would occur in PNP deficiency, did not alter the already rich G-C content of N regions. We conclude that the frequency of V(D)J recombination and the composition of N-insertions are influenced by increases in dATP levels, potentially leading to alterations in antigen receptors and aberrant lymphoid development. Alterations in N-region insertions may contribute to the B-cell dysfunction associated with ADA deficiency. (+info)
(5/752) Theoretical calculations, synthesis and base pairing properties of oligonucleotides containing 8-amino-2'-deoxyadenosine.
Theoretical calculations on double and triple helices containing 8-amino-2'-deoxyadenosine were made to analyze the possible differences in base pairing properties between 8-aminoadenine and adenine. These calculations indicate a strong preferential stabilization of the triplex over the duplex when adenine is replaced by 8-aminoadenine. In addition, a protected phosphoramidite derivative of 8-amino-2'-deoxyadenosine was prepared for the introduction of 8-aminoadenine into synthetic oligonucleotides using the phosphite-triester approach. DNA triple helical structures are normally observed at acidic pH. However, when oligonucleotides carrying 8-aminoadenine are used, very stable triple helical structures can be observed even at neutral pH. Biological applications of triple helices could benefit from the use of 8-aminoadenine derivatives. (+info)
(6/752) A transfected cell model for the renal toxin transporter, rOCT2.
A cDNA for the organic cation transporter (rOCT2) of the rat kidney was inserted into the retroviral plasmid pLXSN. This plasmid was used to stably transfect NIH3T3 cells. The transfected cell line exhibited an enhanced rate of tetraethylammonium (TEA) uptake and efflux compared to wild-type NIH3T3 cells. Uptake of TEA by the transfected cells was markedly reduced upon incubation at 4 degrees C. When the extracellular pH was lowered from 8.1 to 5.9, uptake was also reduced, suggesting inhibition of rOCT2 by extracellular protons. The apparent K(m) for TEA in the transfected cells was 141 microM. The classical organic cation transport inhibitors, cyanine 863 and cimetidine, produced noncompetitive inhibition with apparent Ki values of 0.81 and 198 microM, respectively. Daunomycin, vinblastine, and the deoxyadenosine analogs, 2'-deoxytubercidin and 2-chlorodeoxyadenosine, did not appear to be substrates for rOCT2. However, the anticancer drug, cisplatin, competitively inhibited TEA uptake by rOCT2 with an apparent Ki value of 925 microM, suggesting that rOCT2 may play a role in its renal secretion. In summary, transfected NIH3T3 cells provide a facile system by which this and other organic ion transporters can be studied. (+info)
(7/752) Adenosine kinase inhibitors as a novel approach to anticonvulsant therapy.
Adenosine levels increase at seizure foci as part of a postulated endogenous negative feedback mechanism that controls seizure activity through activation of A1 adenosine receptors. Agents that amplify this site- and event-specific surge of adenosine could provide antiseizure activity similar to that of adenosine receptor agonists but with fewer dose-limiting side effects. Inhibitors of adenosine kinase (AK) were examined because AK is normally the primary route of adenosine metabolism. The AK inhibitors 5'-amino-5'-deoxyadenosine, 5-iodotubercidin, and 5'-deoxy-5-iodotubercidin inhibited maximal electroshock (MES) seizures in rats. Several structural classes of novel AK inhibitors were identified and shown to exhibit similar activity, including a prototype inhibitor, 4-(N-phenylamino)-5-phenyl-7-(5'-deoxyribofuranosyl)pyrrolo[2, 3-d]pyrimidine (GP683; MES ED50 = 1.1 mg/kg). AK inhibitors also reduced epileptiform discharges induced by removal of Mg2+ in a rat neocortical preparation. Overall, inhibitors of adenosine deaminase or of adenosine transport were less effective. The antiseizure activities of GP683 in the in vivo and in vitro preparations were reversed by the adenosine receptor antagonists theophylline and 8-(p-sulfophenyl)theophylline. GP683 showed little or no hypotension or bradycardia and minimal hypothermic effect at anticonvulsant doses. This improved side effect profile contrasts markedly with the profound hypotension, bradycardia, and hypothermia and greater inhibition of motor function observed with the adenosine receptor agonist N6-cyclopentyladenosine and opens the way to clinical evaluation of AK inhibitors as a novel, adenosine-based approach to anticonvulsant therapy. (+info)
(8/752) Determinants of nucleotide sugar recognition in an archaeon DNA polymerase.
Vent DNA polymerase normally discriminates strongly against incorporation of ribonucleotides, 3'-deoxyribonucleotides (such as cordycepin) and 2',3'-dideoxyribonucleotides. To explore the basis for this discrimination we have generated a family of variants with point mutations of residues in conserved Regions II and III and assayed incorporation of nucleo-tides with modified sugars by these variants, all of which were created in an exonuclease-deficient form of the enzyme. A Y412V variant incorporates ribonucleotides at least 200-fold more efficiently than the wild-type enzyme, consistent with Y412 acting as a 'steric gate' to specifically exclude ribonucleotides. The most striking variants tested involved changes to A488, a residue predicted to be facing away from the nucleotide binding site. The pattern of relaxed specificity at this position roughly correlates with the size of the substituted amino acid sidechain and affects a variety of modified nucleotide sugars. (+info)