Development and validation of an LC-MS/MS quantitative method for endogenous deoxynucleoside triphosphates in cellular lysate.

The endogenous deoxynucleoside triphosphate (dNTP) pool includes deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and thymidine triphosphate (TTP). The endogenous dNTP pool is regulated by complex enzymatic pathways that can be targeted by drugs, such as antimetabolites. In addition, these components compete with antiviral nucleos(t)ide analog triphosphates, contributing to the mechanism of pharmacologic action. This communication describes the development and validation of a sensitive method to quantify the intracellular dNTP pool in cellular lysates. The extraction process was optimized for dNTPs using an indirect strategy - the separation of mono-, di- and triphosphate moieties by strong anion exchange, dephosphorylation of target fractions to molar equivalent nucleosides - followed by sensitive quantitation using liquid chromatography-tandem mass spectrometry. The validated analytical range was 50-2500 fmol/sample for each dNTP. The assay was used to quantify dNTPs in peripheral blood mononuclear cells from 40 clinical research participants (n = 279 samples). Median (interquartile range) concentrations were 143 (116, 169) for dATP, 737 (605, 887) for dCTP, 237 (200, 290) for dGTP and 315 (220, 456) for TTP, in femtomoles per million cells. This method allows for future studies of endogenous dNTP disposition in subjects receiving antimetabolites or nucleos(t)ide analogs, or for other clinical scenarios.

Fluorescent xDNA nucleotides as efficient substrates for a template-independent polymerase.

Template independent polymerases, and terminal deoxynucleotidyl transferase (TdT) in particular, have been widely used in enzymatic labeling of DNA 3'-ends, yielding fluorescently-labeled polymers. The majority of fluorescent nucleotides used as TdT substrates contain tethered fluorophores attached to a natural nucleotide, and can be hindered by undesired fluorescence characteristics such as self-quenching. We previously documented the inherent fluorescence of a set of four benzo-expanded deoxynucleoside analogs (xDNA) that maintain Watson-Crick base pairing and base stacking ability; however, their substrate abilities for standard template-dependent polymerases were hampered by their large size. However, it seemed possible that a template-independent enzyme, due to lowered geometric constraints, might be less restrictive of nucleobase size. Here, we report the synthesis and study of xDNA nucleoside triphosphates, and studies of their substrate abilities with TdT. We find that this polymerase can incorporate each of the four xDNA monomers with kinetic efficiencies that are nearly the same as those of natural nucleotides, as measured by steady-state methods. As many as 30 consecutive monomers could be incorporated. Fluorescence changes over time could be observed in solution during the enzymatic incorporation of expanded adenine (dxATP) and cytosine (dxCTP) analogs, and after incorporation, when attached to a glass solid support. For (dxA)(n) polymers, monomer emission quenching and long-wavelength excimer emission was observed. For (dxC)(n), fluorescence enhancement was observed in the polymer. TdT-mediated synthesis may be a useful approach for creating xDNA labels or tags on DNA, making use of the fluorescence and strong hybridization properties of the xDNA.

Simultaneous determination of deoxycytidine diphosphate and deoxycytidine triphosphate by capillary electrophoresis with transient isotachophoretic stacking: a sensitive monitoring method for ribonucleotide reductase activity.

A simple and rapid capillary electrophoretic method was developed for simultaneous determination of sub-micromolar 2'-deoxycytidine 5'-diphosphate (dCDP) and 2'-deoxycytidine 5'-triphosphate (dCTP) levels in enzyme assays without using radioactively labeled substrates. The separation was performed at 25°C using MES in the BGE as the terminating ion, the chloride ions in the sample buffer as the leading ion, and PEG 4000 in the BGE as the EOF suppressor for sample stacking by transient isotachophoresis (tITP). Several parameters affecting the separation were investigated, including the pH of the BGE, the concentration of sodium chloride in the sample buffer, and the concentrations of MES and PEG 4000 in the running buffer. Good separation with high separation efficiency was achieved within 6 min under optimal conditions. In comparison with the simple CZE method, the present tITP-CZE method enabled a 150-fold increase in the injection time without any decrease in resolution and the sensitivity was enhanced up to two orders of magnitude with the new method. The linear range of the method was 0.1-10 µM for dCDP and dCTP. The limits of detection of dCDP and dCTP were 85 and 73 nM, respectively. The proposed method was successfully applied for the activity assay of ribonucleotide reductase from Hep G2 and Sf9 cells.

Quantitation of cellular deoxynucleoside triphosphates.

Eukaryotic cells contain a delicate balance of minute amounts of the four deoxyribonucleoside triphosphates (dNTPs), sufficient only for a few minutes of DNA replication. Both a deficiency and a surplus of a single dNTP may result in increased mutation rates, faulty DNA repair or mitochondrial DNA depletion. dNTPs are usually quantified by an enzymatic assay in which incorporation of radioactive dATP (or radioactive dTTP in the assay for dATP) into specific synthetic oligonucleotides by a DNA polymerase is proportional to the concentration of the unknown dNTP. We find that the commonly used Klenow DNA polymerase may substitute the corresponding ribonucleotide for the unknown dNTP leading in some instances to a large overestimation of dNTPs. We now describe assay conditions for each dNTP that avoid ribonucleotide incorporation. For the dTTP and dATP assays it suffices to minimize the concentrations of the Klenow enzyme and of labeled dATP (or dTTP); for dCTP and dGTP we had to replace the Klenow enzyme with either the Taq DNA polymerase or Thermo Sequenase. We suggest that in some earlier reports ribonucleotide incorporation may have caused too high values for dGTP and dCTP.

Quantification of meCCNU-induced dG-dC crosslinks in oligonucleotide duplexes by liquid chromatography/electrospray ionization tandem mass spectrometry.

Chloroethynitrosoureas (CENUs) are important alkylating agents widely used in the treatment of cancers. Decomposition of CENUs generates active electrophilic ions that damage DNA, including the formation of dG-dC crosslinks which represents the most important cytotoxic mechanism of CENUs. In this work, a high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) method was employed to analyze the dG-dC crosslinks induced by 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (meCCNU, Semustine). The direct quantitation of dG-dC crosslinks in oligonucleotide duplexes was achieved by the selected reaction monitoring (SRM) mode using synthesized (15) N(3) -labeled dG-dC as an internal standard. Methods of enzymatic digestion and HPLC separation were developed for obtaining separation and reproducibility of the dG-dC peak in chromatograms. The limit-of-detection (LOD) was determined to be 0.08 nM and the limit-of-quantification (LOQ) was determined to be 0.16 nM. The linearity of the calibration curve was 0.9997 over the range of 0.08 to 32 nM. The precision and accuracy of the method ranged from 1.1 to 6.6% and 96 to 109%, respectively. The recovery of the dG-dC crosslink in the enzymatic hydrolysates from the oligonucleotide duplex was determined to be from 91 to 106%. The results of the validation study indicate that the method is suitable for quantifying dG-dC crosslinks in DNA. Consequently, this method was used to determine meCCNU-induced dG-dC crosslinks in four duplexes with different GC contents. The results showed that the crosslinking fraction (CF) increased as the GC content in the duplex increased, and a relatively low CF was observed in the early period of the reaction.

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.

Modified bases characterized in intact DNA by mass-analyzed ion kinetic energy spectrometry.

Pyrolysis of DNA into a chemical ionization source yields protonated bases and other base-containing ions. Kinetic energy spectra allow the characterization of the bases 5-methylcytosine and 1-methyladenine from underivatized salmon sperm DNA. Isomeric bases are distinguishable with this technique.

Modes of overinitiation, dnaA gene expression, and inhibition of cell division in a novel cold-sensitive hda mutant of Escherichia coli.

The chromosomal replication cycle is strictly coordinated with cell cycle progression in Escherichia coli. ATP-DnaA initiates replication, leading to loading of the DNA polymerase III holoenzyme. The DNA-loaded form of the beta clamp subunit of the polymerase binds the Hda protein, which promotes ATP-DnaA hydrolysis, yielding inactive ADP-DnaA. This regulation is required to repress overinitiation. In this study, we have isolated a novel cold-sensitive hda mutant, the hda-185 mutant. The hda-185 mutant caused overinitiation of chromosomal replication at 25 degrees C, which most likely led to blockage of replication fork progress. Consistently, the inhibition of colony formation at 25 degrees C was suppressed by disruption of the diaA gene, an initiation stimulator. Disruption of the seqA gene, an initiation inhibitor, showed synthetic lethality with hda-185 even at 42 degrees C. The cellular ATP-DnaA level was increased in an hda-185-dependent manner. The cellular concentrations of DnaA protein and dnaA mRNA were comparable at 25 degrees C to those in a wild-type hda strain. We also found that multiple copies of the ribonucleotide reductase genes (nrdAB or nrdEF) or dnaB gene repressed overinitiation. The cellular levels of dATP and dCTP were elevated in cells bearing multiple copies of nrdAB. The catalytic site within NrdA was required for multicopy suppression, suggesting the importance of an active form of NrdA or elevated levels of deoxyribonucleotides in inhibition of overinitiation in the hda-185 cells. Cell division in the hda-185 mutant was inhibited at 25 degrees C in a LexA regulon-independent manner, suggesting that overinitiation in the hda-185 mutant induced a unique division inhibition pathway.

Overexpression of ribonucleotide reductase as a mechanism of resistance to 2,2-difluorodeoxycytidine in the human KB cancer cell line.

In this study, human oropharyngeal epidermoid carcinoma KB cells that were resistant to 2,2-difluorodeoxycytidine (dFdCyd) were selected and designated the KB-Gem clone. The KB parental cell line IC50 was 0.3 microM dFdCyd, as compared with the KB-Gem clone IC50 of 32 microM dFdCyd. The KB-Gem clone demonstrated overexpression of ribonucleotide reductase (RR) M2 subunit mRNA (9-fold) and overexpression of M2 protein (2-fold); RR activity was 2.3-fold higher than the KB parental cell line. Both the dATP and dCTP pools of the KB-Gem clone increased 2-fold over the parental cell line, with no change in the dGTP and dTTP pools. Reverse transcriptase-PCR was used to clone the cDNA of deoxycytidine kinase (DCK). Resulting sequences revealed two silent mutations in the KB-Gem clone. The amino acid sequence of the DCK protein and mRNA expression remained unchanged. The KB-Gem clone's DCK enzyme activity was 56% of that of the parental cell line. After the endogenous dNTPs were removed with a G-25 column, no difference was evident between the enzyme activities of the KB-Gem clone and parental cells. Thus, contrary to previous hypotheses, DCK deficiency does not play the primary role in the resistance mechanism of dFdCyd, accepting a secondary role to the overexpression of the target gene, RR, and pool expansion.

Potential for selective enhancement of the in vivo metabolism of 1-beta-D-arabinofuranosylcytosine in rats by thymidine pretreatment.

In this study, the ability of deoxythymidine (dThd) to enhance selectively the metabolism of 1-beta-D-arabinofuranosylcytosine (ara-C) in rats bearing transplantable colon carcinoma was investigated. A steady-state plasma level of 375 microM dThd was achieved within 3 h after initiation of a 24-h infusion of dThd (7 g/kg/day) with a concomitant 80% reduction in circulating 2'-deoxycytidine levels. Complete recovery to control values occurred within 6 to 8 h after termination of the infusion. Under the conditions of dThd infusion, the intracellular levels of 2'-deoxycytidine 5'-triphosphate rose from 0.15 to 60 pmol/mg tumor tissue, from 2.5 to 15 pmol/mg intestinal tissue, and from 0.07 to 0.25 pmol/10(6) bone marrow cells. During the steady-state plasma concentration of dThd, the intracellular concentration of 2'-deoxycytidine 5'-triphosphate in tumor tissue was reduced by 50% at 6 h after the initiation of dThd treatment with a complete recovery 9 h thereafter. Differences in the capacity of tumor and host normal tissues to recover from the effects of dThd pretreatment were evaluated by measuring decreasing 1-beta-D-arabinofuranosylcytosine 5'-triphosphate formation with time following dThd infusion. The ability to accumulate 1-beta-D-arabinofuranosylcytosine 5'-triphosphate was reduced by 60 to 80% in normal tissues by 3 h after cessation of the dThd infusion but was decreased by only 15% in the tumor. These results suggested that delaying ara-C administration following dThd might result in less host toxicity while maintaining the antitumor effect. Sequential infusion of dThd (7 g/kg/day) for 24 h followed 3 h later by a 48-h infusion of ara-C (175 mg/kg/day), was as effective in reducing tumor mass as was dThd infusion immediately prior to ara-C and resulted in reduced host toxicity (less weight loss). The best schedule for the dThd-ara-C combination was two courses of alternating 24-h sequential infusions of dThd and ara-C with a 3-h delay in ara-C administration following dThd. These data show that under the conditions used, reductions in intracellular 2'-deoxythymidine 5'-triphosphate pools by dThd in vivo do not appear to correlate with the antitumor activity of the dThd-ara-C combination. Intracellular 1-beta-D-arabinofuranosylcytosine 5'-triphosphate accumulation, however, was prolonged in rat colon tumor compared to normal tissues, and selectivity of the dThd-ara-C combination in favor of the tumor could be achieved by schedule modification.

Modulation of 1-beta-D-arabinofuranosylcytosine metabolism by thymidine in human acute leukemia.

Twenty-seven patients with acute leukemia have been treated by sequential 6-day courses of thymidine (30 g/m2 by i.v. continuous infusion, days 1 and 4) and 1-beta-D-arabinofuranosylcytosine (ara-C) (200 mg/m2 by i.v. continuous infusion, days 2,3,5, and 6). Of 25 evaluable patients 4 achieved a complete remission: one of 9 for acute myelogenous leukemia; and 3 of 14 in the blastic crisis of chronic myelocytic leukemia. Six minor responses were also observed. Toxicity was mainly hematological and did not appear to be higher than that expected from ara-C alone. However, thymidine infusions gave rise to headache and somnolence. The clinical benefit of such treatment seems to be limited to the blastic crisis of chronic myelocytic leukemia. Parallel cytokinetic and biochemical studies were performed in order to assess the cytokinetic and metabolic changes induced by both drugs and to correlate them with the clinical response. Recruitment of cells into the S-phase fraction was observed following the first thymidine infusion in the two complete responders and in three of the five nonresponders studied. In contrast to this high pretherapeutic levels of S-phase fraction were observed in most minor responders and in some nonresponders with further decrease following the thymidine infusion. Recruitment of cells into S phase therefore appeared to be an important but not sufficient factor for prediction of complete response to ara-C. Responders in contrast to most nonresponders were characterized by a higher intracellular level of ara-C and its metabolites following the first 24-h infusion of the drug. Deoxythymidine triphosphate and deoxycytidine triphosphate pools were also measured before and during treatment in order to assess if nucleotide pool variations induced by the administration of thymidine can in fact correlate with the intracellular alteration in ara-C metabolism and with clinical response. The level of deoxycytidine triphosphate pools before treatment showed marked interpatient variations but did not correlate with response. As expected, thymidine infusion induced a rise in the deoxythymidine triphosphate pool and a decrease in deoxycytidine triphosphate. The pools, however, generally returned promptly to the pretherapeutic level 24 h after the end of the infusion of thymidine. There were no significant differences between responders and nonresponders in the modulation of these pools.

PCR amplification of GC-rich DNA regions using the nucleotide analog N4-methyl-2'-deoxycytidine 5'-triphosphate.

GC-rich DNA regions were PCR-amplified with Taq DNA polymerase using either the canonical set of deoxynucleoside triphosphates or mixtures in which the dCTP had been partially or completely replaced by its N4-methylated analog, N4-methyl-2'-deoxycytidine 5'-triphosphate (N4me-dCTP). In the case of a particularly GC-rich region (78.9% GC), the PCR mixtures containing N4me-dCTP produced the expected amplicon in high yield, while mixtures containing the canonical set of nucleotides produced numerous alternative amplicons. For another GC-rich DNA region (80.6% GC), the target amplicon was only generated by re-amplifying a gel-purified sample of the original amplicon with N4me-dCTP-containing PCR mixtures. In a direct PCR comparison on a highly GC-rich template, mixtures containing N4me-dCTP clearly performed better than did solutions containing the canonical set of nucleotides mixed with various organic additives (DMSO, betaine, or ethylene glycol) that have been reported to resolve or alleviate problems caused by secondary structures in the DNA. This nucleotide analog was also tested in PCR amplification of DNA regions with intermediate GC content, producing the expected amplicon in each case with a melting temperature (Tm) clearly below the Tm of the same amplicon synthesized exclusively with the canonical bases.

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.

Resistance to 9-beta-D-arabinofuranosyl-2-fluoroadenine due to reduced incorporation into DNA from competition by excess deoxyadenosine triphosphate: implications for different sensitivities to nucleoside analogues.

The cytotoxic action of the deoxyadenosine analogue 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) depends on the incorporation into DNA after being phosphorylated to F-ara-A triphosphate (F-ara-ATP) by deoxycytidine kinase (dCK). The mechanisms of resistance to F-ara-A were investigated in a newly established variant of L1210 mouse leukemia cells (L1210/F). L1210/F was more than 41-fold more resistant to F-ara-A than the parental cell line and had a 55% lower dCK activity. Interestingly, L1210/F showed a modest level of cross-resistance to deoxycytidine analogues phosphorylated by dCK, for instance, 1-beta-D-arabinofuranosylcytosine (ara-C). The comparative study of F-ara-A and ara-C demonstrated that the difference in the accumulation of their respective triphosphates was minor. In contrast, the incorporation of F-ara-A into DNA was strikingly suppressed compared with that of ara-C. In general, the high natural triphosphate levels interfere with corresponding analogue incorporation into DNA. The deoxyadenosine triphosphate (dATP) and deoxycytidine triphosphate pool sizes in L1210/F cells were increased by 4.9-fold and 1.9-fold, respectively, compared with the parental cells. Treatment with hydroxyurea increased the ratio of F-ara-ATP to dATP 2.1-fold and enhanced the action of F-ara-A in L1210/F. This is the first cell line to show that the profoundly defective incorporation of F-ara-A into DNA during competition with excess dATP confers a high degree of resistance to F-ara-A.

Deoxyribonucleoside triphosphate pools in human diploid fibroblasts and their modulation by hydroxyurea and deoxynucleosides.

Deoxyribonucleoside triphosphate (dNTP) pool levels were examined in synchronized and unsynchronized log phase cultures and in quiescent cultures of human diploid foreskin fibroblasts. dNTP levels were in good agreement with those previously published for human HeLa and lymphoblastic leukemia cells. dCTP and dGTP levels showed only a modest lowering in quiescent as compared to log-phase cells, but dATP and dTTP levels were reduced dramatically in quiescent cultures. Cells synchronized by serum starvation and assayed at the peak DNA synthetic phase (18-21 hr post release) showed substantially higher pools of all four dNTPs. Hydroxyurea treatment reduced only purine dNTPs in both log phase and confluent cells while increasing dTTP and dCTP pools. The effects of deoxynucleosides on dNTP pools were also examined and are discussed in light of current models regarding regulation of purified ribonucleotide reductase formulated from in vitro studies.

Combination chemotherapy studies with gemcitabine and etoposide in non-small cell lung and ovarian cancer cell lines.

Gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and etoposide (4'-demethylepipodo-phyllo-toxin-9-4,6-O-ethylidene-beta-D-g lucopyranoside, VP-16) are antineoplastic agents with clinical activity against various types of solid tumors. Because of the low toxicity profile of dFdC and the differences in mechanisms of cytotoxicity, combinations of both drugs were studied in vitro. For this purpose, we used the human ovarian cancer cell line A2780, its cis-diammine-dichloroplatinum-resistant and VP-16 cross-resistant variant ADDP, and two non-small cell lung cancer cell lines, Lewis Lung (LL, murine) and H322 (human). The interaction between the drugs was determined with the multiple drug effect analysis (fixed molar ratio) and with a variable drug ratio. In the LL cell line, the combination of dFdC and VP-16 at a constant molar ratio (dFdC:VP-16 = 1:4 or 1:0.125 after 4- or 24-hr exposure, respectively) was synergistic (combination index [CI], calculated at 50% growth inhibition = 0.7 and 0.8, respectively; CI <1 indicating synergism). After 24- and 72-hr exposure to both drugs at a constant ratio, additivity was found in the A2780, ADDP, and H322 cell lines (dFdC:VP-16 = 1:500 for both exposure times in these cell lines). When cells were exposed to a combination of dFdC and VP-16 for 24 or 72 hr, with VP-16 at its IC25 and dFdC in a concentration range, additivity was found in both the LL and H322 cells; synergism was observed in the A2780 and ADDP cells, which are the least sensitive to VP-16. Schedule dependency was found in the LL cell line; when cells were exposed to dFdC 4 hr prior to VP-16 (constant molar ratio, total exposure 24 hr), synergism was found (CI = 0.5), whereas additivity was found when cells were exposed to VP-16 prior to dFdC (CI = 1.6). The mechanism of interaction between the drugs was studied in more detail in the LL cell line; dFdCTP accumulation was 1.2-fold enhanced by co-incubation with VP-16, and was even more pronounced (1.4-fold) when cells were exposed to VP-16 prior to dFdC. dCTP levels were decreased by VP-16 alone as well as by the combination of both compounds, which may favor phosphorylation of dFdC, thereby increasing dFdCTP accumulation. DNA strand break (DSB) formation was increased for exposure to both compounds together compared to exposure to each compound separately, this effect being most pronounced when cells were exposed to VP-16 prior to dFdC (38% and 0% DSB for dFdC and VP-16 alone, respectively and 97% DSB for the combination). The potentiation in DSB formation might be a result of the inhibition of DNA repair by dFdC. Provided the right schedule is used, VP-16 is certainly a compound eligible for combination with dFdC.

Molecular effects of 2',2'-difluorodeoxycytidine (Gemcitabine) on DNA replication in intact HL-60 cells.

The ability of pH-step alkaline elution to isolate different size species of nascent DNA (nDNA) from intact cells was utilized to study the effects of 2',2'-difluorodeoxycytidine (dFdC) on DNA replication in HL-60 cells. Preincubation with dFdC caused a concentration-dependent decrease in overall [3H]thymidine incorporation into DNA, accompanied by an increase in the proportion of radiolabel accumulated in small nDNA fragments. Twenty-four hours following removal of dFdC, radiolabel progressed from smaller to larger fragments and into genomic-length DNA. At initial concentrations of exposures to dFdC or cytosine arabinoside (ara-C) that caused 50% lethality (LC50) to HL-60 cells (40 and 50 nM, respectively), slower and less complete transit of nDNA from small subreplicon-length fragments through larger intermediates to genomic-length DNA was observed for nDNA fragments containing incorporated [3H]dFdC than for fragments containing [3H]ara-C. This was accomplished with less [3H]dFdC incorporated into DNA than [3H]ara-C at these extracellular concentrations of drug. Pulse-chase studies, using higher concentrations of radiolabeled drug, similarly revealed that nDNA fragments containing incorporated dFdC, like those containing ara-C, progressed with respect to time into larger nDNA intermediates and ultimately into genomic-length DNA; however, such progression for nDNA fragments containing dFdC was less complete than for fragments containing ara-C. The radioactivity incorporated into DNA represented authentic dFdC, as determined by DNA degradation studies, and was stable in DNA for at least 48 hr after removal of extracellular [3H]dFdC. Some of the effects of dFdC on ribonucleotide reduction in HL-60 cells were assessed by measurement of the intracellular pools of dCTP and dGTP. The drug had a greater effect on pools of dGTP than of dCTP, with transient reductions in dGTP observed at concentrations that encompass the LC50 for dFdC. These studies suggest that the interaction with DNA synthesis is an important component of the cytotoxicity of dFdC in HL-60 cells. Because it is incorporated progressively through nDNA compartments and ultimately into genomic-length DNA, dFdC should be categorized as an agent that slows DNA elongation in the intact cell, and not as a chain terminator in the absolute sense.

Enhanced production of intracellular ara-CTP after sequential use of methotrexate and 1-beta-D-arabinofuranosylcytosine.

Synergistic antiproliferative effect has been proven in vitro when mouse leukemic cells were sequentially treated with MTX and ara-C. The mechanism of this combination effect not well elucidated but the intracellular uptake of ara-C was higher when cells were pre-exposed to MTX. In this experiment, the intracellular ara-CTP was measured by HPLC after MTX and ara-C were sequentially administered to BDF1 mice bearing L1210 leukemic cell, either being sensitive or resistant to MTX. When MTX at the dose of 12 mg/kg was preceded 6 h and 3 h to ara-C at the dose of 25 mg/kg, the intracellular levels of ara-CTP were found to be significantly higher as compared with those of ara-C alone as control group. At 1 h after ara-C, ara-CTP was measured about 165 and 130% of the control levels, respectively, and at 12 h, ara-CTP was over 4 times higher of control level with group of mice to which MTX was preceded 6 h prior to MTX. On the contrary, the enhancement of ara-CTP production was definitely diminished with MTX-resistant cells in the same administrative model. From our present experiment, the time sequential modulation of intracellular ara-CTP production by MTX was reconfirmed in vivo, and this modulation might depend upon the sensitivity of MTX of leukemic cells.

Analysis of the drug synergism between thymidine and arabinosyl cytosine using mouse S49 T lymphoma mutants.

The synergism between arabinosyl cytosine (araC) and thymidine is characterized using two mutant S49 T lymphoma cell populations with altered deoxyribonucleotide metabolism. AraC-1-6 cells are deficient in dCMP deaminase activity resulting in a secondary elevation of intracellular dCTP pools, whereas dGuo-200-1 cells have a mutation in the Ml subunit of ribonucleotide diphosphate reductase, which also results in elevation of dCTP levels. These two mutant cell populations are partially resistant to araC cytotoxicity as compared to the wild type cells. The resistance to araC is contributed to the elevation of dCTP levels in these mutants which prevent araC incorporation into the DNA due to feedback inhibition of deoxycytidine kinase. Addition of extracellular thymidine to dCMP deaminase deficient cells causes a decrease in dCTP levels and in parallel increase their sensitivity to araC. In contrast, extracellular thymidine does not reduce dCTP levels in the mutant cells with altered ribonucleotide reductase and no synergism between araC and thymidine is observed in these cells. The expansion of dTTP pools in the presence of thymidine is similar in the two mutants. These results suggest that the depletion of dCTP pools by thymidine is responsible for the synergistic action of thymidine on araC cytotoxicity and that dTTP does not directly enhance the incorporation of araC into the DNA of T lymphoma cells.