Cellular dNTP Research Articles

Activity of ribonucleotide reductase helps determine how cells repair DNA double strand breaks

Mammalian cells can choose either nonhomologous end joining (NHEJ) or homologous recombination (HR) for repair of chromosome breaks. Of these two pathways, HR alone requires extensive DNA synthesis and thus abundant synthesis precursors (dNTPs). We address here if this differing requirement for dNTPs helps determine how cells choose a repair pathway. Cellular dNTP pools are regulated primarily by changes in ribonucleotide reductase activity. We show that an inhibitor of ribonucleotide reductase (hydroxyurea) hypersensitizes NHEJ-deficient cells, but not wild type or HR-deficient cells, to chromosome breaks introduced by ionizing radiation. Hydroxyurea additionally reduces the frequency of irradiated cells with a marker for an early step in HR, Rad51 foci, consistent with reduced initiation of HR under these conditions. Conversely, promotion of ribonucleotide reductase activity protects NHEJ-deficient cells from ionizing radiation. Importantly, promotion of ribonucleotide reductase activity also increases usage of HR in cells proficient in both NHEJ and HR at a targeted chromosome break. Activity of ribonucleotide reductase is thus an important factor in determining how mammalian cells repair broken chromosomes. This may explain in part why G1/G0 cells, which have reduced ribonucleotide reductase activity, rely more on NHEJ for DSB repair.

Reduced dNTP Binding Affinity of 3TC-resistant M184I HIV-1 Reverse Transcriptase Variants Responsible for Viral Infection Failure in Macrophage

We characterized HIV-1 reverse transcriptase (RT) variants either with or without the (-)-2',3'-deoxy-3'-thiacytidine-resistant M184I mutation isolated from a single HIV-1 infected patient. First, unlike variants with wild-type M184, M184I RT variants displayed significantly reduced DNA polymerase activity at low dNTP concentrations, which is indicative of reduced dNTP binding affinity. Second, the M184I variant displayed a approximately 10- to 13-fold reduction in dNTP binding affinity, compared with the Met-184 variant. However, the k(pol) values of these two RTs were similar. Third, unlike HIV-1 vectors with wild-type RT, the HIV-1 vector harboring M184I RT failed to transduce cell types containing low dNTP concentrations, such as human macrophage, likely due to the reduced DNA polymerization activity of the M184I RT under low cellular dNTP concentration conditions. Finally, we compared the binary complex structures of wild-type and M184I RTs. The Ile mutation at position 184 with a longer and more rigid beta-branched side chain, which was previously known to alter the RT-template interaction, also appears to deform the shape of the dNTP binding pocket. This can restrict ground state dNTP binding and lead to inefficient DNA synthesis particularly at low dNTP concentrations, ultimately contributing to viral replication failure in macrophage and instability in vivo of the M184I mutation.

Structure of the orthorhombic form of human inosine triphosphate pyrophosphatase.

The structure of human inosine triphosphate pyrophosphohydrolase (ITPA) has been determined using diffraction data to 1.6 A resolution. ITPA contributes to the accurate replication of DNA by cleansing cellular dNTP pools of mutagenic nucleotide purine analogs such as dITP or dXTP. A similar high-resolution unpublished structure has been deposited in the Protein Data Bank from a monoclinic and pseudo-merohedrally twinned crystal. Here, cocrystallization of ITPA with a molar ratio of XTP appears to have improved the crystals by eliminating twinning and resulted in an orthorhombic space group. However, there was no evidence for bound XTP in the structure. Comparison with substrate-bound NTPase from a thermophilic organism predicts the movement of residues within helix alpha1, the loop before alpha6 and helix alpha7 to cap off the active site when substrate is bound.

Modification of Human Immunodeficiency Virus Type 1 Reverse Transcriptase to Target Cells with Elevated Cellular dNTP Concentrations

Retroviruses and DNA viruses utilize cellular dNTPs as substrates for their DNA polymerases during viral replication in infected cells. However, because of S phase-dependent dNTP biosynthesis, the availability of cellular dNTPs significantly varies among cell types (e.g. dividing versus nondividing cells and normal versus tumor cells). Here we tested whether alterations in the dNTP utilization efficiency and dNTP binding affinity of viral DNA polymerases can switch viral infection specificity to cell types with different dNTP concentrations. We employed an HIV-1 reverse transcriptase (RT) mutant (Q151N), which is catalytically active only at high dNTP concentrations because of its reduced dNTP binding affinity. Indeed, the modified HIV-1 vector harboring the Q151N mutant RT preferentially transduced tumor cells containing higher cellular dNTP concentrations than primary cells (e.g. human lung fibroblasts (HLFs) and human keratinocytes). Although the wild type HIV-1 vector transduced both HLFs and tumor cells, the Q151N vector failed to transduce HLFs and keratinocytes but efficiently transduced tumor cells. Pretreatment of HLFs with deoxynucleosides, which increase cellular dNTP pools, enabled the mutant vector to transduce HLFs, suggesting that the transduction failure of the RT mutant vector to primary cells is because of inefficient reverse transcription in low cellular dNTP environments. We also observed that the Q151N vector expressing herpes simplex virus-thymidine kinase renders tumor cells sensitive to gancyclovir. This study validates a novel strategy in which modifications of viral DNA polymerases in various vector systems allow the delivery of target genes exclusively to tumor cells exploiting elevated cellular dNTP concentration as a tumor cell-specific host factor.

Regulation of mtDNA Copy Number by the ATM/ATR Signaling Pathway

Mitochondrial DNA (mtDNA) encodes essential protein components of the mitochondrial oxidative phosphorylation complexes and is maintained in cells in multiple copies (usually 100–10,000 copies/cell). However, mtDNA copy number is strictly regulated in a tissue-specific manner by largely unknown mechanisms. We recently discovered that Mec1/Rad53 nuclear checkpoint pathway controls mtDNA copy number in yeast. The key target of this pathway is the enzyme ribonucleotide reductase (RNR), which catalyzes the rate-limiting step in de novo deoxynucleoside triphosphate (dNTP) synthesis. The homologous pathway in humans is the ATM/ATR DNA damage checkpoint pathway, which activates RNR via an inducible small subunit of the enzyme called p53R2. Our recent results demonstrate that this pathway is also involved in regulating mtDNA copy in mammalian cells, by acting as novel de novo pathway for the synthesis of dNTPs for replication and maintenance of mtDNA outside of S-phase and during DNA damage. Human mtDNA depletion syndromes are a class of diseases that result from an inability to maintain proper mtDNA levels in tissues, a subset of which result from disruptions in cellular dNTP metabolism. Altogther, our results suggest that disruptions of the ATM/ATR pathway may well be the cause of other mtDNA depletion syndromes and that aberrant mtDNA copy number regulation may underlie the complex pathology of Ataxia-Telangiectasia, a neurodegenerative disease caused by loss-of-function mutations in the ATM (Ataxia-Telangiectasia Mutated) protein kinase.

Macrophage Tropism of HIV-1 Depends on Efficient Cellular dNTP Utilization by Reverse Transcriptase

Retroviruses utilize cellular dNTPs to perform proviral DNA synthesis in infected host cells. Unlike oncoretroviruses, which replicate in dividing cells, lentiviruses, such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus, are capable of efficiently replicating in non-dividing cells (terminally differentiated macrophages) as well as dividing cells (i.e. activated CD4+ T cells). In general, non-dividing cells are likely to have low cellular dNTP content compared with dividing cells. Here, by employing a novel assay for cellular dNTP content, we determined the dNTP concentrations in two HIV-1 target cells, macrophages and activated CD4+ T cells. We found that human macrophages contained 130-250-fold lower dNTP concentrations than activated human CD4+ T cells. Biochemical analysis revealed that, unlike oncoretroviral reverse transcriptases (RTs), lentiviral RTs efficiently synthesize DNA even in the presence of the low dNTP concentrations equivalent to those found in macrophages. In keeping with this observation, HIV-1 vectors containing mutant HIV-1 RTs, which kinetically mimic oncoretroviral RTs, failed to transduce human macrophages despite retaining normal infectivity for activated CD4+ T cells and other dividing cells. These results suggest that the ability of HIV-1 to infect macrophages, which is essential to establishing the early pathogenesis of HIV-1 infection, depends, at least in part, on enzymatic adaptation of HIV-1 RT to efficiently catalyze DNA synthesis in limited cellular dNTP substrate environments.

Identification of Phosphorylation Sites on the Yeast Ribonucleotide Reductase Inhibitor Sml1

Sml1 is a small protein in Saccharomyces cerevisiae which inhibits the activity of ribonucleotide reductase (RNR). RNR catalyzes the rate-limiting step of de novo dNTP synthesis. Sml1 is a downstream effector of the Mec1/Rad53 cell cycle checkpoint pathway. The phosphorylation by Dun1 kinase during S phase or in response to DNA damage leads to diminished levels of Sml1. Removal of Sml1 increases the population of active RNR, which raises cellular dNTP levels. In this study using mass spectrometry and site-directed mutagenesis, we have identified the region of Sml1 phosphorylation to be between residues 52 and 64 containing the sequence GSSASASASSLEM. This is the first identification of a phosphorylation sequence of a Dun1 biological substrate. This sequence is quite different from the consensus Dun1 phosphorylation sequence reported previously from peptide library studies. The specific phosphoserines were identified to be Ser(56), Ser(58), and Ser(60) by chemical modification of these residues to S-ethylcysteines followed by collision activated dissociation. To investigate further Sml1 phosphorylation, we constructed the single mutants S56A, S58A, S60A, and the triple mutant S56A/S58A/S60A and compared their degrees of phosphorylation with that of wild type Sml1. We observed a 90% decrease in the relative phosphorylation of S60A compared with that of wild type, a 25% decrease in S58A, and little or no decrease in the S56A mutant. There was no observed phosphate incorporation in the triple mutant, suggesting that Ser(56), Ser(58), and Ser(60) in Sml1 are the sites of phosphorylation. Further mutagenesis studies reveal that Dun1 kinase requires an acidic residue at the +3 position, and there is cooperativity between the phosphorylation sites. These results show that Dun1 has a unique phosphorylation motif.

Deoxyribonucleoside 2'- or 3'-mixed disulfides: prodrugs to target ribonucleotide reductase and/or to inhibit HIV reverse transcription.

Herein, we report the design, synthesis, and biological effects of nucleosides bearing a disulfide function on the sugar ring as prodrugs of potentially active mercaptonucleotides that can target ribonucleotide reductase or reverse transcriptase. We show that cytidine derivatives efficiently reduce dNTP pools in human CEM/SS cells and that 3'-deoxythymidin-3'-yl methyl disulfide is able to interfere with both cellular dNTP synthesis and HIV reverse transcription.

Suppression of mismatched mutation by p53: a mechanism for guarding genomic integrity

The tumor suppressor p53 plays an important role in guarding the genomic integrity of the cells. The 3'-->5' exonuclease activity of p53 has recently been recognized as a novel biochemical function of this molecule, and has been shown to preferentially excise mismatched nucleotides from DNA and enhance the DNA replication fidelity of polymerase alpha in vitro. The present study further investigated the role of this biochemical function in whole cells by testing the possibility that p53 may reduce mismatched mutations in cells under a stress of DNA replication errors. Cells with different states of p53 expression, either endogenously or ectopically, were exposed to hydroxyurea to induce an imbalance of cellular dNTP pools and cause replication errors. The rates of mutation at the hypoxanthine guanine phosphoribosyltransferase ( HPRT) gene were determined by selecting colonies of HPRT- mutants. Incubation of cells with hydroxyurea induced a similar degree of dNTP pool imbalance in each cell line, but caused significantly more mutations in cells lacking p53 protein expression. The mutation frequency was significantly reduced by introduction of a wild-type p53 expression vector into the p53-null cells. Analysis of the mutants demonstrated that the clones were devoid of HPRT enzyme activity, but appeared to transcribe full-length HPRT mRNA. These data suggest that p53 is able to reduce mutations caused by misincorporation of deoxynucleotides. Thus, the preferential removal of mismatched nucleotides from DNA by p53 may be a mechanism to maintain genomic integrity. Defect in this biochemical function of p53 may contribute to genetic instability associated with cancer development and progression.

LCD1: an essential gene involved in checkpoint control and regulation of the MEC1 signalling pathway in Saccharomyces cerevisiae.

We identified YDR499W as a Saccharomyces cerevisiae open reading frame with homology to several checkpoint proteins, including S. cerevisiae Rfc5p and Schizosaccharomyces pombe Rad26. Disruption of YDR499W (termed LCD1) results in lethality that is rescued by increasing cellular deoxyribonucleotide levels. Cells lacking LCD1 are very sensitive to a range of DNA-damaging agents, including UV irradiation, and to the inhibition of DNA replication. LCD1 is necessary for the phosphorylation and activation of Rad53p in response to DNA damage or DNA replication blocks, and for Chk1p activation in response to DNA damage. LCD1 is also required for efficient DNA damage-induced phosphorylation of Rad9p and for the association of Rad9p with the FHA2 domain of Rad53p after DNA damage. In addition, cells lacking LCD1 are completely defective in the G(1)/S and G(2)/M DNA damage checkpoints. Finally, we reveal that endogenous Mec1p co-immunoprecipitates with Lcd1p both before and after treatment with DNA-damaging agents. These results indicate that Lcd1p is a pivotal checkpoint regulator, involved in both the essential and checkpoint functions of the Mec1p pathway.

Effects of Gemcitabine in Normaland Transformed Human Lung Cell Cultures: Cytotoxicity and Increase in Radiation Sensitivity

Several anticancer drugs increase cell sensitivity to irradiation. Gemcitabine (2', 2' difluorodeoxycytidine) decreases the cellular dNTP pools and thus significantly increases the sensitivity to the DNA damaging effects of low-dose radiation. In this study we have investigated whether gemcitabine may play a role as radiosensitizer also in lung adenocarcinoma treatment. We studied this nucleoside analogue in normal and transformed human cell lines (fetal lung and lung adenocarcinoma). After drug treatment, cell lines were irradiated with different doses. Cell damage following drug treatment and/or irradiation was assessed by measuring intracellular ATP level and by the colony forming assay. The two cell lines significantly differed in their sensitivity to the toxic effects of the drug; the normal cell line was much more resistant than its transformed counterpart. This difference was observed in both assays, although it was more evident in the colony forming assay. A low radiation dose (50-100 cGy) did not cause any significant damage to transformed cells; normal cells were more resistant and doses up to 500 cGy caused little damage. However, when transformed cells were pretreated for three hours with gemcitabine, even a nontoxic concentration of the drug (1-10 nM) caused a marked sensitization of the cells to irradiation (50-100 cGy). The radiosensitizing effect of gemcitabine could be observed also in normal cells, although these cells were more resistant to the damaging effects of both anticancer treatments. This study demonstrates that gemcitabine, a chemotherapeutic agent already used in the clinic, could be proposed as a radiosensitizer for radiation therapy of lung adenocarcinoma, having a clearly potentiating effect on lowdose radiation.

Differential Incorporation of Ara-C, Gemcitabine, and Fludarabine Into Replicating and Repairing DNA in Proliferating Human Leukemia Cells

The major actions of nucleoside analogs such as arabinosylcytosine (ara-C) and fludarabine occurs after their incorporation into DNA, during either replication or repair synthesis. The metabolic salvage and DNA incorporation of the normal nucleoside, deoxycytidine, is functionally compartmentalized toward repair synthesis in a process regulated by ribonucleotide reductase. The aim of this study was to investigate the metabolic pathways by which nucleoside analogs that do (fludarabine, gemcitabine) or do not (ara-C) affect ribonucleotide reductase are incorporated into DNA in proliferating human leukemia cells. Using alkaline density-gradient centrifugation to separate repaired DNA from replicating DNA and unreplicated parental DNA strands, approximately 60% of ara-C nucleotide in DNA was incorporated by repair synthesis in CCRF-CEM cells; the remainder was incorporated by replication. In contrast, fludarabine and gemcitabine, nucleosides that inhibit ribonucleotide reductase and decreased deoxynucleotide pools, were incorporated mainly within replicating DNA. Hydroxyurea also depleted deoxynucleotide pools and increased the incorporation of ara-C into DNA by replicative synthesis. Stimulation of DNA repair activity by UV irradiation selectively enhanced the incorporation of all nucleosides tested through repair synthesis. These findings suggest that the pathways by which therapeutically useful nucleoside analogs are incorporated into DNA are affected by cellular dNTP pools from de novo synthesis and by the relative activities of DNA repair and replication. The antitumor activity of these drugs may be enhanced by combination with either ribonucleotide reductase inhibitors to increase their incorporation into replicating DNA or with agents that induce DNA damage and evoke the DNA repair process.

Differential Incorporation of Ara-C, Gemcitabine, and Fludarabine Into Replicating and Repairing DNA in Proliferating Human Leukemia Cells

AbstractThe major actions of nucleoside analogs such as arabinosylcytosine (ara-C) and fludarabine occurs after their incorporation into DNA, during either replication or repair synthesis. The metabolic salvage and DNA incorporation of the normal nucleoside, deoxycytidine, is functionally compartmentalized toward repair synthesis in a process regulated by ribonucleotide reductase. The aim of this study was to investigate the metabolic pathways by which nucleoside analogs that do (fludarabine, gemcitabine) or do not (ara-C) affect ribonucleotide reductase are incorporated into DNA in proliferating human leukemia cells. Using alkaline density-gradient centrifugation to separate repaired DNA from replicating DNA and unreplicated parental DNA strands, approximately 60% of ara-C nucleotide in DNA was incorporated by repair synthesis in CCRF-CEM cells; the remainder was incorporated by replication. In contrast, fludarabine and gemcitabine, nucleosides that inhibit ribonucleotide reductase and decreased deoxynucleotide pools, were incorporated mainly within replicating DNA. Hydroxyurea also depleted deoxynucleotide pools and increased the incorporation of ara-C into DNA by replicative synthesis. Stimulation of DNA repair activity by UV irradiation selectively enhanced the incorporation of all nucleosides tested through repair synthesis. These findings suggest that the pathways by which therapeutically useful nucleoside analogs are incorporated into DNA are affected by cellular dNTP pools from de novo synthesis and by the relative activities of DNA repair and replication. The antitumor activity of these drugs may be enhanced by combination with either ribonucleotide reductase inhibitors to increase their incorporation into replicating DNA or with agents that induce DNA damage and evoke the DNA repair process.

Gemcitabine and radiosensitization in human tumor cells.

Gemcitabine is a nucleoside analogue with excellent clinical activity against solid tumors. Within the cell, gemcitabine is rapidly phosphorylated to its active di- and triphosphate metabolites. Cytotoxicity with gemcitabine appears to be related to multiple effects on DNA replication, where gemcitabine triphosphate can serve as both an inhibitor and substrate for DNA synthesis. Gemcitabine diphosphate inhibits ribonucleotide reductase, producing decreases in cellular dNTP pool levels in a cell-specific manner. These two major characteristics of gemcitabine, reduction in cellular dNTP pools and incorporation into DNA, are features of other antimetabolites antitumor agents which also exhibit radiosensitizing properties. Based on these favorable metabolic characteristics and the clinical activity of gemcitabine in tumor types which are commonly treated with radiation, the ability of gemcitabine to enhance X-radiation induced cytotoxicity was evaluated. Gemcitabine has been shown to be a potent radiosensitizer in a variety of tumor cell lines, including HT-29 colorectal carcinoma, pancreatic cancer, breast, non-small cell lung and head and neck cancer cell lines. Gemcitabine was most effective as a radiosensitizer when administered at least 2 hours prior to irradiation. For most cell lines, radiosensitization was evident at non-cytotoxic concentrations. The extent of radiosensitization increased with both increasing gemcitabine concentration and duration of exposure. Radiosensitization did not require redistribution of cells into a more radiosensitive phase of the cell cycle. The major metabolic effects observed under radiosensitizing conditions were the accumulation of high levels of gemcitabine triphosphate, and a selective decrease in the cellular dATP pool. The pattern of dATP decrease paralleled the increase in radiosensitization, whereas the level of gemcitabine triphosphate was not associated with the enhanced sensitivity to radiation. Compared to other radiosensitizers, the advantage of gemcitabine is that is can induce radiosensitization at concentrations that are 1000 times lower than typical plasma levels obtained with this drug. These studies will be used as guidelines for developing clinical trials of gemcitabine with radiation.

Regulation of the salvage pathway of deoxynucleotides synthesis in apoptosis induced by growth factor deprivation

Here we describe changes in dNTP metabolism that precede DNA fragmentation in a model of apoptosis driven by deprivation of the cytokine interleukin 3 (IL-3). In haemopoietic BAF3 cells, IL-3 withdrawal leads to a rapid decrease in the size of dATP, dTTP and dGTP pools without affecting dCTP levels. This imbalance in dNTP pools precedes DNA fragmentation and is accompanied by down-regulation of enzymes controlling the de novo and salvage pathways of dNTP synthesis, ribonucleotide reductase and thymidine kinase (TK) respectively. Readdition of IL-3 results in a rapid, protein synthesis-independent restoration of normal dNTP pools, enhanced TK activity and increased precursor incorporation through the salvage pathway. Up-regulation of TK activity after IL-3 readdition is prevented by the protein kinase C (PKC) inhibitor staurosporin, but not by tyrosine kinase inhibitors. Furthermore activation of PKC by phorbol esters mimics the stimulatory effect of IL-3 on TK activity, suggesting that PKC might be involved in regulating this effect. These results indicate that regulation by IL-3 of the salvage pathway of dNTP synthesis plays a role in the maintenance of cellular dNTP pool balance and suggests that alterations in dNTP metabolism after IL-3 deprivation could be a relevant event in the commitment of haemopoietic cells to apoptosis.

Effects of 3'-deoxynucleoside 5'-triphosphate concentrations on chain termination by nucleoside analogs during human immunodeficiency virus type 1 reverse transcription of minus-strand strong-stop DNA

We have compared the effects of nucleoside analogs in quiescent and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) exposed to human immunodeficiency virus type 1 (HIV-1) with those of their triphosphorylated derivatives in cell-free HIV-1 reverse transcription assays. We observed a substantial decrease in synthesis of early minus-strand proviral DNA products in HIV-1-infected, quiescent PBMC exposed to each of 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddI), and 2',3'-dideoxy-3'-thiacytidine (3TC), in comparison with nontreated, infected controls. In contrast, no such diminution was observed when PHA-stimulated, HIV-1-infected PBMC were treated with the same drugs. This result was attributed to previously reported findings that PHA-stimulated PBMC possessed larger deoxynucleoside triphosphate (dNTP) pools than quiescent cells did. To further investigate this subject, a cell-free HIV-1 reverse transcription reaction involving HIV-1 RNA genomic template, recombinant purified HIV-1 reverse transcriptase, all four dNTPs and either tRNA3Lys or a deoxyoligonucleotide as primer was used to monitor chain termi-nation mediated by 2',3'-dideoxynucleoside triphosphates (ddNTPs) during synthesis of minus-strand strong-stop DNA. Augmented chain termination was observed with decreasing concentrations of both ddNTP and dNTP when the ratio of dNTP to ddNTP was fixed. We also found that both the number and strength of reverse transcription pause sites were increased at low concentrations of dNTPs and when a deoxyoligonucleotide primer was used in place of the cognate primer, tRNA3Lys. Preferential incorporation of ddATP was observed dur-ing reverse transcription opposite a distinct pause site in a short synthetic RNA template. These results con-firm the notion that the antiviral activities of ddNTP are dependent on both cellular dNTP pools and the state of cellular activation. Pausing of HIV-1 reverse transcriptase during reverse transcription, altered by dNTP concentrations, may be a mechanism that controls the position and extent of incorporation of nucleoside analogs.

Intracellular Metabolism of 3′-Azido-3′-Deoxythymidine in the Presence of Ganciclovir or Foscarnet

Comparisons were made between the intracellular phosphorylation of 3′-Azido-3′-deoxythymidine (AZT) alone and in combination with ganciclovir (GCV) or foscarnet (PFA) in lymphocytes, uninfected fibroblasts and CMV-infected fibroblasts. The effects of AZT and the combinations of AZT with GCV or PFA on cellular dNTP pools were also examined. The phosphorylation of AZT was not altered by the presence of GCV or PFA in lymphocytes, and neither AZT nor the combinations of AZT with GCV or PFA changed the levels of cellular dNTP pools in these cells. AZT was phosphorylated to a greater extent in lymphocytes when compared to fibroblasts, but the proportion of AZT di- and triphosphates was greater in fibroblasts. The infection of fibroblasts with CMV enhanced AZT phosphorylation and increased the levels of cellular dNTP pools. GCV caused a specific reduction in AZT phosphorylation in CMV-infected fibroblasts, with a seven-fold drop in AZT triphosphate compared to AZT alone. GCV did not affect AZT phosphorylation in uninfected fibroblasts, nor did GCV reduce the dTTP pool compared to AZT alone. The effects of GCV upon AZT phosphorylation in CMV-infected cells may shed light on the antagonistic effects of GCV upon the anti-HIV activity of AZT, and are of importance for the development of effective combination therapies for the treatment of AIDS patients infected with CMV.

Determination of Pyrimidine Deoxynucleoside Triphosphates in Leukaemia Cell Extracts Containing 1-ß-D-Arabinofuranosylcytosine Triphosphate

Deoxynucleoside 5'-triphosphates (dNTPs) can be determined in cell extracts by high performance liquid chromatography after prior selective degradation of ribonucleoside 5'-triphosphates with sodium periodate and methylamine. When the method is used for the evaluation of deoxynucleoside triphosphates in 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP)-containing cell extracts, an additional peak coeluting with thymidine triphosphate (dTTP) is observed. This peak is due to the formation of a carboxylic acid derivative of ara-CTP by periodate oxidation, and it can lead to considerable overestimation of dTTP. Formation of this peak can be avoided by using alkaline reaction conditions (pH 7.5) and by changing the sequence of addition of the reagents used in the periodation procedure. By employing this modified protocol, cellular dNTP and ara-CTP levels can be monitored in extracts of leukaemic blasts during cytosine arabinoside treatment in two separate HPLC runs.

Interaction of arabinosyl nucleotides in K562 human leukemia cells

The objective of this investigation was to evaluate the ability of arabinosyl nucleotides to modulate the cellular metabolism of different arabinosyl nucleosides in K562 cells. The maximum rate of accumulation of the respective 5'-triphosphate (TP) was observed in cells incubated with 10 μM arabinosylcytosine (ara-C), 10 μM arabinosylguanine (ara-G), 300 μM arabinosyl-2-fluoroadenine (F-ara-A), and > 1000 μM arabinosyladenine (ara-A). Cell extract fractionation studies demonstrated that ara-C and F-ara-A were phosphorylated by dCyd kinase, whereas ara-A was phosphorylated by dCyd kinase and Ado kinase; ara-G phosphorylation was attributed to dGuo kinase. When nucleoside kinase was rate limiting to arabinosyl nucleotide accumulation, cells preloaded with F-ara-ATP showed increased rates of ara-CTP and ara-GTP accumulation, whereas cells preloaded with ara-CTP had decreased rates of F-ara-ATP and ara-GTP accumulation. Preloading cells with ara-GTP had little effect on arabinosyl nucleoside triphosphate accumulation. F-ara-ATP accumulation was inhibited in cells containing all other arabinosyl nucleotides, whereas ara-ATP metabolism was not affected by preincubation with any other arabinosyl nucleoside. Cells incubated with ara-C and ara-G had a general rise in dNTP, whereas F-ara-A incubation was associated with a decrease in cellular dNTP. The differential effects of arabinosyl nucleotides and cellular metabolism of other arabinosyl nucleosides are due to phosphorylation by distinct nucleoside kinases that likely have characteristic sensitivities to cellular dNTP levels.

The competitive miscoding of O6-methylguanine and O6-ethylguanine and the possible importance of cellular deoxynucleoside 5′-triphosphate pool sizes in mutagenesis and carcinogenesis

Using initiated poly(dG,O 6-RdG) and poly(dA,O 6-RdG) polynucleotides as templates for DNA polymerase I in vitro the promutagenic potential of O 6-MeG and O 6-EtG has been confirmed, together with the possibility of minor miscoding pathways for O 6-RG. These lead to the incorporation of dAMP and dGMP, which could give rise to some of the limited number of transversions that have been observed arising from the action of alkylating agents. The results are compatible with the current knowledge of oncogenes, explaining the changes in base sequence that have been observed. The competition for the miscoding of O 6-RG which leads to the incorporation of dCMP in addition to the expected dTMP is also shown. The relative amounts of these two nucleotides incorporated depend upon the concentrations of the dCTP and dTTP in the assay. The mutagenic efficiency of O 6-MeG is constant at approx. 0.4 over a wide range of dTTP and dCTP concentrations and only increases when the dCTP in the assay ceases to saturate the polymerizing enzyme, indicating that the DNA polymerase I plays a role in determining the mutagenic efficiency of a modified base. Although the mutagenic efficiency of both O 6-MeG and O 6-EtG depends upon the relative concentrations of dTTP and dCTP in the assay, a reduction in the concentration of dCTP can be more effective at increasing the mutagenic efficiency than a corresponding increase in the concentration of dTTP. These results indicate the importance of cellular dNTP pools in determining the cellular response to agents.