Isocratic HPLC analysis for the simultaneous determination of dNTPs, rNTPs and ADP in biological samples.

Information about the cellular concentrations of deoxyribonucleoside triphosphates (dNTPs) is instrumental for mechanistic studies of DNA replication and for understanding diseases caused by defects in dNTP metabolism. The dNTPs are measured by methods based on either HPLC or DNA polymerization. An advantage with the HPLC-based techniques is that the parallel analysis of ribonucleoside triphosphates (rNTPs) can serve as an internal quality control of nucleotide integrity and extraction efficiency. We have developed a Freon-free trichloroacetic acid-based method to extract cellular nucleotides and an isocratic reverse phase HPLC-based technique that is able to separate dNTPs, rNTPs and ADP in a single run. The ability to measure the ADP levels improves the control of nucleotide integrity, and the use of an isocratic elution overcomes the shifting baseline problems in previously developed gradient-based reversed phase protocols for simultaneously measuring dNTPs and rNTPs. An optional DNA-polymerase-dependent step is used for confirmation that the dNTP peaks do not overlap with other components of the extracts, further increasing the reliability of the analysis. The method is compatible with a wide range of biological samples and has a sensitivity better than other UV-based HPLC protocols, closely matching that of mass spectrometry-based detection.

A user-friendly, high-throughput tool for the precise fluorescent quantification of deoxyribonucleoside triphosphates from biological samples.

Cells maintain a fine-tuned, dynamic concentration balance in the pool of deoxyribonucleoside 5'-triphosphates (dNTPs). This balance is essential for physiological processes including cell cycle control or antiviral defense. Its perturbation results in increased mutation frequencies, replication arrest and may promote cancer development. An easily accessible and relatively high-throughput method would greatly accelerate the exploration of the diversified consequences of dNTP imbalances. The dNTP incorporation based, fluorescent TaqMan-like assay published by Wilson et al. has the aforementioned advantages over mass spectrometry, radioactive or chromatography based dNTP quantification methods. Nevertheless, the assay failed to produce reliable data in several biological samples. Therefore, we applied enzyme kinetics analysis on the fluorescent dNTP incorporation curves and found that the Taq polymerase exhibits a dNTP independent exonuclease activity that decouples signal generation from dNTP incorporation. Furthermore, we found that both polymerization and exonuclease activities are unpredictably inhibited by the sample matrix. To resolve these issues, we established a kinetics based data analysis method which identifies the signal generated by dNTP incorporation. We automated the analysis process in the nucleoTIDY software which enables even the inexperienced user to calculate the final and accurate dNTP amounts in a 96-well-plate setup within minutes.

Quantitation of deoxynucleoside triphosphates by click reactions.

The levels of the four deoxynucleoside triphosphates (dNTPs) are under strict control in the cell, as improper or imbalanced dNTP pools may lead to growth defects and oncogenesis. Upon treatment of cancer cells with therapeutic agents, changes in the canonical dNTPs levels may provide critical information for evaluating drug response and mode of action. The radioisotope-labeling enzymatic assay has been commonly used for quantitation of cellular dNTP levels. However, the disadvantage of this method is the handling of biohazard materials. Here, we described the use of click chemistry to replace radioisotope-labeling in template-dependent DNA polymerization for quantitation of the four canonical dNTPs. Specific oligomers were designed for dCTP, dTTP, dATP and dGTP measurement, and the incorporation of 5-ethynyl-dUTP or C8-alkyne-dCTP during the polymerization reaction allowed for fluorophore conjugation on immobilized oligonucleotides. The four reactions gave a linear correlation coefficient >0.99 in the range of the concentration of dNTPs present in 106 cells, with little interference of cellular rNTPs. We present evidence indicating that data generated by this methodology is comparable to radioisotope-labeling data. Furthermore, the design and utilization of a robust microplate assay based on this technology evidenced the modulation of dNTPs in response to different chemotherapeutic agents in cancer cells.

Quantification of Cellular Deoxyribonucleoside Triphosphates by Rolling Circle Amplification and Förster Resonance Energy Transfer.

The quantification of cellular deoxyribonucleoside triphosphate (dNTP) levels is important for studying pathologies, genome integrity, DNA repair, and the efficacy of pharmacological drug treatments. Current standard methods, such as enzymatic assays or high-performance liquid chromatography, are complicated, costly, and labor-intensive, and alternative techniques that simplify dNTP quantification would present very useful complementary approaches. Here, we present a dNTP assay based on isothermal rolling circle amplification (RCA) and rapid time-gated Förster resonance energy transfer (TG-FRET), which used a commercial clinical plate reader system. Despite the relatively simple assay format, limits of detection down to a few picomoles of and excellent specificity for each dNTP against the other dNTPs, rNTPs, and dUTP evidenced the strong performance of the assay. Direct applicability of RCA-FRET to applied nucleic acid research was demonstrated by quantifying all dNTPs in CEM-SS leukemia cells with and without hydroxyurea or auranofin treatment. Both pharmacological agents could reduce the dNTP production in a time- and dose-dependent manner. RCA-FRET provides simple, rapid, sensitive, and specific quantification of intracellular dNTPs and has the potential to become an advanced tool for both fundamental and applied dNTP research.

Absolute Quantification of RNA or DNA Using Acid Hydrolysis and Mass Spectrometry.

Accurate, traceable quantification of ribonucleotide or deoxyribonucleotide oligomers is achievable using acid hydrolysis and isotope dilution mass spectrometry (ID-MS). In this work, formic acid hydrolysis is demonstrated to generate stoichiometric release of nucleobases from intact oligonucleotides, which then can be measured by ID-MS, facilitating true and precise absolute quantification of RNA, short linearized DNA, or genomic DNA. Surrogate nucleobases are quantified with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow, using multiple reaction monitoring (MRM). Nucleobases were chromatographically resolved using a novel cation-exchange separation, incorporating a pH gradient. Trueness of this quantitative assay is estimated from agreement among the surrogate nucleobases and by comparison to concentrations provided for commercial materials or Standard Reference Materials (SRMs) from the National Institute of Standards and Technology (NIST). Comparable concentration estimates using NanoDrop spectrophotometry or established from droplet-digital polymerase chain reaction (ddPCR) techniques agree well with the results. Acid hydrolysis-ID-LC-MS/MS provides excellent quantitative selectivity and accuracy while enabling traceability to mass unit. Additionally, this approach can be uniquely useful for quantifying modified nucleobases or mixtures.

Simultaneous isotope dilution quantification and metabolic tracing of deoxyribonucleotides by liquid chromatography high resolution mass spectrometry.

Quantification of cellular deoxyribonucleoside mono- (dNMP), di- (dNDP), triphosphates (dNTPs) and related nucleoside metabolites are difficult due to their physiochemical properties and widely varying abundance. Involvement of dNTP metabolism in cellular processes including senescence and pathophysiological processes including cancer and viral infection make dNTP metabolism an important bioanalytical target. We modified a previously developed ion pairing reversed phase chromatography-mass spectrometry method for the simultaneous quantification and 13C isotope tracing of dNTP metabolites. dNMPs, dNDPs, and dNTPs were chromatographically resolved to avoid mis-annotation of in-source fragmentation. We used commercially available 13C15N-stable isotope labeled analogs as internal standards and show that this isotope dilution approach improves analytical figures of merit. At sufficiently high mass resolution achievable on an Orbitrap mass analyzer, stable isotope resolved metabolomics allows simultaneous isotope dilution quantification and 13C isotope tracing from major substrates including 13C-glucose. As a proof of principle, we quantified dNMP, dNDP and dNTP pools from multiple cell lines. We also identified isotopologue enrichment from glucose corresponding to ribose from the pentose-phosphate pathway in dNTP metabolites.

Method for Quantification of Ribonucleotides and Deoxyribonucleotides in Human Cells Using (Trimethylsilyl)diazomethane Derivatization Followed by Liquid Chromatography-Tandem Mass Spectrometry.

Investigation into intracellular ribonucleotides (RNs) and deoxyribonucleotides (dRNs) is important for studies of the mechanism of many biological processes, such as RNA and DNA synthesis and DNA repair, as well as metabolic and therapeutic efficacy of nucleoside analogues. However, current methods are still unsatisfactory for determination of nucleotides in complex matrixes. Here we describe a novel method for the determination of RN and dRN pools in cells based on fast derivatization with (trimethylsilyl)diazomethane (TMSD) followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Derivatization was accomplished in 3 min, and each derivatized nucleotide not only had a sufficient retention on reversed-phase column by introduction of methyl groups but also exhibited a unique ion transition which consequently eliminated mutual interference in LC-MS/MS. Chromatographic separation was performed on a C18 column with a simple acetonitrile-water gradient elution system, which avoided contamination and ion suppression caused by ion-pairing reagents. The developed method was fully validated and applied to the analysis of RNs and dRNs in cell samples. Moreover, results demonstrated that the applicability of this method could be extended to nucleoside analogues and their metabolites and could facilitate many applications in future studies.

Host SAMHD1 protein restricts endogenous reverse transcription of HIV-1 in nondividing macrophages.

BACKGROUND: SAM domain and HD domain containing protein 1 (SAMHD1) is a host anti-HIV-1 restriction factor known to suppress viral reverse transcription in nondividing myeloid cells by its dNTP triphosphorylase activity that depletes cellular dNTPs. However, HIV-2 and some SIV strains rapidly replicate in macrophages due to their accessory protein, viral protein X (Vpx), which proteosomally degrades SAMHD1 and elevates dNTP levels. Endogenous reverse transcription (ERT) of retroviruses is the extra-cellular reverse transcription step that partially synthesizes proviral DNAs within cell-free viral particles before the viruses infect new cells. ERT activity utilizes dNTPs co-packaged during budding from the virus-producing cells, and high ERT activity is known to enhance HIV-1 infectivity in nondividing cells. Here, since Vpx elevates cellular dNTP levels in macrophages, we hypothesize that HIV-2 should contain higher ERT activity than HIV-1 in macrophages, and that the Vpx-mediated dNTP elevation should enhance both ERT activity and infectivity of HIV-1 particles produced in macrophages. RESULTS: Here, we demonstrate that HIV-2 produced from human primary monocyte derived macrophages displays higher ERT activity than HIV-1 produced from macrophages. Also, HIV-1 particles produced from macrophages treated with virus like particles (VLPs) containing Vpx, Vpx (+), displayed large increases of ERT activity with the enhanced copy numbers of early, middle and late reverse transcription products within the viral particles, compared to the viruses produced from macrophages treated with Vpx (-) VLPs. Furthermore, upon the infection with an equal p24 amount to fresh macrophages, the viruses produced from the Vpx (+) VLP treated macrophages demonstrated higher infectivity than the viruses from the Vpx (-) VLP treated macrophages. CONCLUSIONS: This finding identifies the viral ERT step as an additional step of HIV-1 replication cycle that SAMHD1 restricts in nondividing myeloid target cells.

Exploring the Antitumor Mechanism of High-Dose Cytarabine through the Metabolic Perturbations of Ribonucleotide and Deoxyribonucleotide in Human Promyelocytic Leukemia HL-60 Cells.

Despite the apparent clinical benefits of high-dose cytarabine (Ara-C) over lower dose Ara-C in acute myeloid leukemia (AML) therapy, the mechanism behind high-dose Ara-C therapy remains uncertain. In this study, a LC-MS-based method was carried out to investigate the metabolic alteration of ribonucleotide and deoxyribonucleotide in human promyelocytic leukemia cells (HL-60) after treatment with Ara-C to reveal its antitumor mechanism. The metabolic results revealed that four nucleotides (ATP, ADP, CDP, and dCTP) could be used as potential biomarkers indicating the benefit of high-dose Ara-C over lower dose Ara-C treatment. Combining metabolic perturbation and cell cycle analysis, we conjectured that, apart from the acknowledged mechanism of Ara-C on tumor inhibition, high-dose Ara-C could present a specific action pathway. It was suggested that the pronounced rise in AMP/ATP ratio induced by high-dose Ara-C can trigger AMP-activated protein kinase (AMPK) and subsequently Forkhead Box, class O (FoxO), to promote cell cycle arrest. Moreover, the significant decrease in CDP pool induced by high-dose Ara-C might further accelerate the reduction of dCTP, which then aggravates DNA synthesis disturbance. As a result, all of these alterations led to heightened tumor inhibition. This study provides new insight in the investigation of potential mechanisms in the clinical benefits of high-dose Ara-C in therapy for AML.

Quantitative analysis of intracellular nucleoside triphosphates and other polar metabolites using ion pair reversed-phase liquid chromatography coupled with tandem mass spectrometry.

Simultaneous, quantitative determination of intracellular nucleoside triphosphates and other polar metabolites using liquid chromatography with electrospray ionization tandem mass spectrometry (LC-MS/MS) represents a bioanalytic challenge because of charged, highly hydrophilic analytes presented at a large concentration range in a complex matrix. In this study, an ion pair LC-MS/MS method using triethylamine (TEA)-hexafluoroisopropanol (HFIP) ion-pair mobile phase was optimized and validated for simultaneous and unambiguous determination of 8 nucleoside triphosphates (including ATP, CTP, GTP, UTP, dATP, dCTP, dGTP, and dTTP) in cellular samples. Compared to the the less volatile ion-pair reagent, triethylammonium acetate (100mM, pH 7.0), the combination of HFIP (100mM) and TEA (8.6mM) increased the MS signal intensity by about 50-fold, while retaining comparable chromatographic resolution. The isotope-labeled internal standard method was used for the quantitation. Lower limits of quantitation were determined at 0.5nM for CTP, UTP, dATP, dCTP, and dTTP, at 1nM for ATP, and at 5nM for GTP and dGTP. The intra- and inter-day precision and accuracy were within the generally accepted criteria for bioanalytical method validation (<15%). While the present method was validated for the quantitation of intracellular nucleoside triphosphates, it had a broad application potential for quantitative profiling of nucleoside mono- and bi-phosphates as well as other polar, ionic metabolic intermediates (including carbohydrate derivatives, carboxylic acid derivatives, co-acyl A derivatives, fatty acyls, and others) in biological samples.

Impact of histidine residue on chelating ability of 2'-deoxyriboadenosine.

Copper(II) complexes with a new chelator-type nucleoside-histidine modified 2'-deoxyriboadenosine (N-[(9-ß-D-2'-deoxyribofuranosylpurin-6-yl)-carbamoyl]histidine) were studied by potentiometric and spectroscopic (UV-visible, CD, EPR) techniques, in conjunction with computer modeling optimization. The ligand can act as bidentate or tridentate depending on pH range. In acidic pH a very stable dimeric complex Cu(2)L(2) predominates with coordination spheres of both metal ions composed of oxygen atoms from carboxylic groups, one oxygen atom from ureido group and two nitrogen atoms derived from purine base and histidine ring. Above pH 5, deprotonation of carbamoyl nitrogens leads to the formation of CuL(2), Cu(2)L(2)H(-1) and Cu(2)L(2)H(-2) species. The CuL(2)H(-1) and CuL(2)H(-2) complexes with three or four nitrogens in Cu(II) coordination sphere have been detected in alkaline medium. Our findings suggest that N-[(9-beta-D-2'-deoxyribofuranosylpurin-6-yl)-carbamoyl]histidine chelates copper(II) ions very efficiently. The resulting complex might be used as an alternative base-pairing mode in which hydrogen-bonded base pairs present in natural DNA are replaced by metal-mediated ones.

A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates.

Current methods for measuring deoxyribonucleoside triphosphates (dNTPs) employ reagent and labor-intensive assays utilizing radioisotopes in DNA polymerase-based assays and/or chromatography-based approaches. We have developed a rapid and sensitive 96-well fluorescence-based assay to quantify cellular dNTPs utilizing a standard real-time PCR thermocycler. This assay relies on the principle that incorporation of a limiting dNTP is required for primer-extension and Taq polymerase-mediated 5-3' exonuclease hydrolysis of a dual-quenched fluorophore-labeled probe resulting in fluorescence. The concentration of limiting dNTP is directly proportional to the fluorescence generated. The assay demonstrated excellent linearity (R(2) > 0.99) and can be modified to detect between ∼0.5 and 100 pmol of dNTP. The limits of detection (LOD) and quantification (LOQ) for all dNTPs were defined as <0.77 and <1.3 pmol, respectively. The intra-assay and inter-assay variation coefficients were determined to be <4.6% and <10%, respectively with an accuracy of 100 ± 15% for all dNTPs. The assay quantified intracellular dNTPs with similar results obtained from a validated LC-MS/MS approach and successfully measured quantitative differences in dNTP pools in human cancer cells treated with inhibitors of thymidylate metabolism. This assay has important application in research that investigates the influence of pathological conditions or pharmacological agents on dNTP biosynthesis and regulation.

Histone deacetylase inhibition modulates deoxyribonucleotide pools and enhances the antitumor effects of the ribonucleotide reductase inhibitor 3'-C-methyladenosine in leukaemia cells.

Histone deacetylase (HDAC) inhibitors are a new class of epigenetic agents that were reported to enhance the cytotoxic effects of classical anticancer drugs through multiple mechanisms. However, which of the possible drug combinations would be the most effective and clinically useful are to be determined. We treated the HL60 and NB4 promyelocytic leukaemia cells with a combination of the ribonucleotide reductase (RR) inhibitor 3'-C-methyladenosine (3'-Me-Ado) and several hydroxamic acid-derived HDAC inhibitors, including two recently synthesized molecules, MC1864 and MC1879, and the reference compound trichostatin A (TSA). The results showed significant growth inhibitory and apoptotic synergistic effects with the combinations. Hence, we evaluated the effects of the combinations on cell cycle distribution and on the level of several proteins involved in the apoptotic process (p21, caspase-3, Bcl-2, Bax, AIF). Since HDAC inhibitors increased the G1-S transition block induced by 3'-Me-Ado, an effect on RR activity was hypothesized. Indeed, the HPLC evaluation of intracellular deoxyribonucleotide (dNTP) pools showed that both TSA and MC1864 induced a decrease in dNTPs, even if with a somewhat different pattern, suggesting that RR inhibition contributes to the observed synergism. Furthermore, while TSA was shown to activate the intrinsic apoptotic pathway, MC1864 induced a dose-dependent increase in ROS and AIF levels. Moreover, the treatment with the radical scavenger N-acetylcysteine determined a significant inhibition of MC1864- but not TSA-mediated synergistic effects. Hence, our findings are consistent with a possible role of HDAC inhibitor mediated-ROS induction in RR inhibition and in the potentiation of RR inhibitor-mediated apoptosis.

Detection and separation of nucleoside-5'-monophosphates of DNA by conjugation with the fluorescent dye BODIPY and capillary electrophoresis with laser-induced fluorescence detection.

We investigated the separation and detection of the 5'-monophosphates of 2'-deoxynucleosides selectively conjugated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5'-phosphate group using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). BODIPY conjugates of the four common deoxynucleoside-5'-monophosphates (2'-deoxyguanosine-5'-monophosphate, 2'-deoxyadenosine-5'-monophosphate, 2'-deoxycytidine-5'-monophosphate, and thymidine-5'-monophosphate) were prepared and subjected to CE-LIF to serve as standard compounds for peak assignment and to develop separation conditions for the analysis of DNA. BODIPY conjugates were detected and resolved by CE-LIF after digestion of DNA or an oligonucleotide to 5'-monophosphates by nuclease P1 (NP 1) and fluorescence labeling without further purification step. Comparative analyses of calf-thymus DNA digested either with micrococcal nuclease/spleen phosphodiesterase to 3'-monophosphates or with NP 1 to 5'-monophosphates showed that both versions of the fluorescence postlabeling assay were equally efficient and sensitive. Moreover, using the same assay, 2'-deoxyuridine and 2'-deoxy-5methylcytidine were identified in bisulfite treated DNA after NP 1 digestion indicating that fluorescence postlabeling of 2'-deoxyribonucleoside-5'-monophosphates with BODIPY FL EDA and detection by CE-LIF has the potential to determine DNA damage and genomic DNA methylation.

Effect of gemcitabine and cis-platinum combinations on ribonucleotide and deoxyribonucleotide pools in ovarian cancer cell lines.

Gemcitabine (dFdC) and cisplatin (CDDP) act synergistically by an increase in platinum-DNA adduct formation. Since ribonucleotide (NTP) and deoxyribonucleotide (dNTP) levels are essential for DNA-synthesis and repair of DNA damage, we investigated whether disturbances might account for differences in effects between sensitive and resistant cell lines. The human ovarian cancer cell line A2780, its CDDP-resistant variant ADDP and its dFdC-resistant variant AG6000 were exposed for 24 h to dFdC or CDDP alone, or a combination causing moderate to strong growth inhibition. In AG6000 cells UTP levels were 2-fold lower and in ADDP cells almost 2-fold higher than in A2780 cells. Levels of dTTP, dATP and dCTP were 2-5-fold lower in the resistant cell lines. Drug treatment affected NTP and dNTP levels most pronounced in A2780 cells. dFdC alone, at 1.5 nM to 1 micro M increased ATP, GTP and CTP pools 1.2 to 2.0-fold, while 10 micro M dFdC increased UTP 2.5-fold. Combination of dFdC and CDDP increased all NTP levels at low dFdC and CDDP concentrations more than 1.2-fold, but at 20 micro M CDDP only CTP increased 2.4-fold. Only 1.5 nM dFdC increased all dNTP pools more than 1.6-fold, but at 0.1 and 1 micro M dFdC, dATP and dGTP decreased down to 10-fold, while dTTP increased 3-5-fold. CDDP and the combination increased all dNTP pools over 1.4 and 1.9-fold, respectively. In AG6000 cells dFdC and CDDP hardly affected the NTP and dNTP status, except at the high concentrations, which decreased ATP, GTP and UTP levels 1.2-1.8-fold. Both CDDP alone and the combination increased dTTP, dCTP and dATP pools up to 1.6-fold. In ADDP cells NTP and most dNTP levels were hardly affected, except dGTP levels which decreased to non-detectable levels. In conclusion, both dFdC and CDDP induce concentration and combination dependent changes in NTP and dNTP pools.

Identification of N-(deoxyguanosin-8-yl)-4-azobiphenyl by (32)P-postlabeling analyses of DNA in human uroepithelial cells exposed to proximate metabolites of the environmental carcinogen 4-aminobiphenyl.

DNA adducts formed in human uroepithelial cells (HUC) following exposure to N-hydroxy-4-aminobiphenyl (N-OH-ABP), the proximate metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP), were analyzed by the (32)P-postlabeling method. Two adducts detected by (32)P-postlabeling were previously identified as the 3',5'-bisphospho derivatives of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) and N-(deoxyadenosin-8-yl)-4-aminobiphenyl (dA-C8-ABP) (Frederickson S et al. [1992] Carcinogenesis 13: 955-961; Hatcher and Swaminathan [1995b] Carcinogenesis 16: 295-301). In contrast to the dG-C8-ABP adduct, which was 3'-dephosphorylated by nuclease P1, dA-C8-ABP was resistant to nuclease P1, thus providing an enrichment step before postlabeling. Autoradiography of the two-dimensional thin-layer chromatogram of the postlabeled products obtained following nuclease P1 digestion revealed several minor adducts, one of which has been identified in the present study. Postlabeling analyses following nuclease P1 digestion of the products obtained from the reaction of N-acetoxy-4-aminobiphenyl with deoxyguanosine-3'-monophosphate (dGp) demonstrated the presence of this minor adduct. The 3'-monophosphate derivative of the adduct was subsequently chromatographically purified and subjected to spectroscopic analyses. Based on proton NMR and mass spectroscopic analyses of the synthetic product, the chemical structure of the adduct has been identified as N-(deoxyguanosin-N(2)-yl)-4-azobiphenyl (dG-N==N-ABP). (32)P-Postlabeling analysis of the nuclease P1-enriched DNA hydrolysate of HUCs treated with N-OH-ABP or N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) showed the presence of the dG-N==N-ABP adduct. It was also detected in calf thymus DNA incubated with HUC cytosol and N-OH-ABP in the presence of acetyl-CoA, or incubated with HUC microsomes and N-OH-AABP. These results demonstrate that in the target cells for ABP carcinogenesis in vivo, N-OH-ABP and N-OH-AABP are bioactivated by acyltransferases to reactive arylnitrenium ions that covalently interact at the N2 position of deoxyguanosine in DNA.

Maintenance of ATP favours apoptosis over necrosis triggered by benzamide riboside.

A new synthetic drug, benzamide riboside (BR) exhibited strong oncolytic activity against leukemic cells in the 5-10 microM range. Higher BR-concentrations (20 microM) predominantly induced necrosis which correlated with DNA strand breaks and subsequent depletion of ATP- and dATP levels. Replenishment of the ATP pool by addition of adenosine prevented necrosis and favoured apoptosis. This effect was not a pecularity of BR-treatment, but was reproduced with high concentrations of all trans-retinoic acid (120 microM) and cyanide (20 mM). Glucose was also capable to suppress necrosis and to favour apoptosis of HL-60 cells, which had been treated with necrotic doses of BR and cyanide. Apoptosis eliminates unwanted cells without affecting the microenvironment, whereas necrosis causes severe inflammation of surrounding tissues due to spillage of cell fluids into the peri-cellular space. Thus, the monitoring and maintenance of cellular energy pools during therapeutic drug treatment may help to minimize nonspecific side effects and to improve attempted drug effects.

Analysis of melphalan adducts of 2'-deoxynucleotides in calf thymus DNA hydrolysates by capillary high-performance liquid chromatography-electrospray tandem mass spectrometry.

Melphalan is a bifunctional alkylating agent that covalently binds with intracellular nucleophilic sites. A methodology using electrospray mass spectrometry was developed to detect and identify DNA adducts. Alkylation sites within a particular nucleotide were examined using electrospray tandem mass spectrometry hyphenated to capillary liquid chromatography in combination with a column switching system. In the reaction mixtures resulting from the interaction of 2'-deoxynucleotides and melphalan several base-aklylated adducts were found. In the case of 2'-deoxyadenosine monophosphate, thymidine monophosphate and 2'-deoxyguanosine phosphate alkylation was observed in the mononucleotide reaction mixtures but not in the DNA-hydrolysates. Calf thymus DNA was reacted in vitro with melphalan. The DNA pellet was isolated and enzymatically hydrolyzed with the aid of Nuclease P1. In this hydrolysate both mono-alkylated 2'-deoxynucleotides and dinucleotides were found. The most important adduct found was identified as the N-7 alklylated dGMP adduct. The alkylated dinucleotides were identified as a pdApdT/melphalan and pdGpdC/melphalan the latter being the most important.

Interstrain variation in the deoxynucleotide composition of Cryptococcus neoformans: nucleotide composition of Cryptococcus neoformans.

The deoxynucleotide (dNMP) composition of ten strains of C. neoformans was analysed by 32P-labelling and two-dimensional thin-layer chromatography. This technique is very sensitive for detecting rare deoxynucleotide adducts and analogues (minor bases) in DNA. The results indicate considerable variation among strains in DNA nucleotide composition.

Indirect inhibition by antibiotics of nucleotide and deoxynucleotide biosynthesis in Plasmodium falciparum.

The effects of the antibiotics, doxycycline, azithromycin, ciprofloxacin and chloramphenicol, upon levels of nucleoside-5'-triphosphates (NTPs) and 2'-deoxynucleoside-5'-triphosphates (dNTPs) have been compared in the malarial parasite, Plasmodium falciparum, and in human CCRF-CEM leukemia cells. All 4 antibiotics had more severe effects upon levels of NTPs and dNTPs in P. falciparum compared with leukemia cells providing an explanation for their selective toxicity against malaria and their utility as antimalarial drugs. In bacteria, the first 3 drugs inhibit protein synthesis while ciprofloxacin inhibits topoisomerase II. The observed depletions of NTPs and dNTPs would be a secondary effect of the drug but may result in death of the parasite.