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.

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.

High spatial resolution nanoslit SERS for single-molecule nucleobase sensing.

Solid-state nanopores promise a scalable platform for single-molecule DNA analysis. Direct, real-time identification of nucleobases in DNA strands is still limited by the sensitivity and the spatial resolution of established ionic sensing strategies. Here, we study a different but promising strategy based on optical spectroscopy. We use an optically engineered elongated nanopore structure, a plasmonic nanoslit, to locally enable single-molecule surface enhanced Raman spectroscopy (SERS). Combining SERS with nanopore fluidics facilitates both the electrokinetic capture of DNA analytes and their local identification through direct Raman spectroscopic fingerprinting of four nucleobases. By studying the stochastic fluctuation process of DNA analytes that are temporarily adsorbed inside the pores, we have observed asynchronous spectroscopic behavior of different nucleobases, both individual and incorporated in DNA strands. These results provide evidences for the single-molecule sensitivity and the sub-nanometer spatial resolution of plasmonic nanoslit SERS.

Effect of hydroxyurea and dideoxyinosine on intracellular 3'-deoxyadenosine-5'-triphosphate concentrations in HIV-infected patients.

Hydroxyurea (HU) significantly enhances the antiretroviral effects of the adenosine analog reverse transcriptase inhibitor dideoxyinosine (ddI). This is believed to be due to a reduction in intracellular de-oxyadenosine triphosphate (dATP) concentrations resulting from HU-mediated inhibition of ribonucleotide reductase (RnR). The effect of combined HU-ddI treatment on intracellular dATP pools in vivo has not been examined. We measured intracellular dATP concentrations in peripheral blood mononuclear cells (PBMCs) from 69 HIV-infected patients receiving 1000 or 1500 mg HU daily for 14 days, 200 mg ddI twice daily for 14 days, or a combination of the two drugs. Median intracellular dATP concentrations decreased from base-line to day 14 by 46% in the ddI + 1000 mg HU arm and by 62% in the ddI + 1500 mg HU arm. When compared to the HU monotherapy arms, these changes proved statistically significant (p = 0.018; stratified Wilcoxon rank-sum test). These findings support reduced intracellular dATP as the mechanism of ddI-HU synergistic activity, and indicate that changes in intracellular nucleotides contribute to HU activity and toxicity in patients. Since a significant reduction in dATP was measurable only when ddI was combined with HU, the antiretroviral activity of ddI may be more complex than previously assumed.

Visualization of RecA filaments and DNA by fluorescence microscopy.

We have developed two experimental methods for observing Escherichia coli RecA-DNA filament under a fluorescence microscope. First, RecA-DNA filaments were visualized by immunofluorescence staining with anti-RecA monoclonal antibody. Although the detailed filament structures below submicron scale were unable to be measured accurately due to optical resolution limit, this method has an advantage to analyse a large number of RecA-DNA filaments in a single experiment. Thus, it provides a reliable statistical distribution of the filament morphology. Moreover, not only RecA filament, but also naked DNA region was visualized separately in combination with immunofluorescence staining using anti-DNA monoclonal antibody. Second, by using cysteine derivative RecA protein, RecA-DNA filament was directly labelled by fluorescent reagent, and was able to observe directly under a fluorescence microscope with its enzymatic activity maintained. We showed that the RecA-DNA filament disassembled in the direction from 5' to 3' of ssDNA as dATP hydrolysis proceeded.

Simultaneous quantitation of the nucleotide analog adefovir, its phosphorylated anabolites and 2'-deoxyadenosine triphosphate by ion-pairing LC/MS/MS.

The nucleotide analog adefovir is an important therapy for hepatitis B viral infection. The study of nucleoside/tide pharmacology has been hampered by difficulties encountered when trying to develop LC/MS/MS methods for these polar analytes. In an attempt to identify a more convenient, selective and sensitive alternative to the analysis of the metabolism of radiolabeled parent nucleotide traditionally used for in vitro cell culture studies, an LC/MS/MS method was developed for the quantitative detection of adefovir and its phosphorylated metabolites in cellular samples. Ion-pairing reversed phase LC using tetrabutylammonium (TBA) and ammonium phosphate had the best compromise between chromatographic separation and positive mode MS/MS detection. Using microbore reverse phase columns and a low flow acetonitrile gradient it was possible to quantitate adefovir, its metabolites and 2'-deoxyadenosine triphosphate. A cross-validation showed comparable levels of adefovir and its metabolites were determined using either LC/MS/MS or radioactivity detection. However, initial methods were conducted at high pH and utilized an acetonitrile step gradient causing unacceptable column life and unpredictable equilibration. Further method optimization lowered the concentration of TBA and phosphate, decreased pH and applied a linear gradient of acetonitrile. This work resulted in a method that was found to have sensitivity, accuracy and precision sufficient to be a useful tool in the study of the intracellular pharmacology of adefovir in vitro and may be more broadly applicable.

Deoxy-adenosine-monophosphate (dAMP) di-n-butylester induces apoptosis by increasing the dATP level in HL-60 cells.

Deoxy-ATP is a potent inducer of apoptosis. We intended to synthesize a lipophilic dAMP derivative which, according to our working hypothesis penetrates into the cell, is converted to dAMP by intracellular esterases and to dATP by nucleotide kinases. We synthesized dAMP-di-n-butylester (DAB) and tested it. We found that it fulfills the above-described expectations. DAB treatment decreases the viability of HL-60 cells, increases the dATP concentration and induces apoptogenic cytochrome c release from mitochondria with concomitant elevation of caspase-9 activity. Our results indicate that use of dAMP derivatives with masked phosphate may be a feasible approach for pharmacological elevation of intracellular dATP and induction of apoptosis.

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.

The ribonucleoside diphosphate reductase inhibitor (E)-2'-deoxy-(fluoromethylene)cytidine as a cytotoxic radiosensitizer in vitro.

(E)-2'-Deoxy-(fluoromethylene)cytidine (FMdC) is known as an inhibitor of ribonucleoside diphosphate reductase, a key enzyme in the de novo pathway of DNA synthesis. FMdC was tested as a modifier of radiation response in vitro on a human colon carcinoma cell line (WiDr), and the observed radiosensitization was confirmed on two human cervix cancer cell lines (C33-A and SiHa). Using the clonogenic assay, the effect ratio (ER) at a clinically relevant dose level of 2 Gy was 2.10 (50 nM FMdC), 1.70 (30 nM FMdC), and 1.71 (40 nM FMdC) for the three cell lines WiDr, C33-A, and SiHa, respectively. A more detailed analysis of the importance of timing and concentration of FMdC was done on the WiDr cell line alone, yielding an increased ER(2Gy) with increasing concentration and duration of exposure to the drug, ranging from 1.0 (6 h) to 1.8 (72 h) at 30 nM FMdC and from 1.2 (6 h) to 3.5 (24 h) at 300 nM. We investigated the effect of FMdC on the cellular deoxynucleotide triphosphate pool in WiDr cells and demonstrated a marked depletion of dATP and a significant rise of TTP levels. Cell cycle analysis showed early S-phase accumulation induced by FMdC alone, G2-M block induced by irradiation alone, and an increased accumulation of cells in G2-M if both modalities are used. Our data suggest that FMdC is a radiation response modifier in vitro on different cancer cell lines. The observed radiosensitization may in part be explained by alteration of the deoxynucleotide triphosphate pool, which is consistent with the effect of FMdC on ribonucleoside diphosphate reductase.

Cycle DNA sequencing with [alpha-35S]dATP demonstrates polymorphism of a surface antigen in malaria parasites from Sri Lankan patients.

Structural diversity in a 45 kDa surface antigen on Plasmodium falciparum merozoites (termed GYMSSA, MSP-2 or MSA-2) and other candidate molecules for developing a malaria vaccine need to be investigated in parasites obtained directly from patients in different malaria endemic countries. A double-stranded DNA sequencing method suitable for this purpose, and also for studying diversity in genes of other haploid cells, is described. A first round polymerase chain reaction (PCR) on DNA isolated from blood was carried out with a primer containing the GCN4 binding site to amplify and subsequently purify the coding region of the MSA-2 gene on GCN4 coated tubes. A second round PCR with more internal primers incorporating M13 forward and reverse primer sequences was then performed. Cycle sequencing was done with unlabelled M13 primers and [alpha-35S]dATP by the dideoxynucleotide procedure. Two different allelic forms of MSA-2 were identified in samples of Plasmodium falciparum from patients in Sri Lanka.

Deoxyadenosine toxicity in an adenosine deaminase-inhibited human CCRF-CEM T-lymphoblastoid cell line causes cell swelling.

The human T-lymphoblastoid cell line CCRF-CEM, pre-treated with 2'-deoxycoformycin, was used as a model for adenosine deaminase deficiency to investigate how 2'-deoxyadenosine exerts its cytotoxic effects. Incubation of these cells with 1 microM or 5 microM deoxyadenosine for 24 and 48 h caused an increase of up to 50% in their modal cell volume as measured by a Coulter Size Distribution Analyzer and this increase in cell volume was accompanied by an increase in their fragility and deformability. The swelling of cells was concomitant with the phosphorylation of deoxyadenosine and its intracellular accumulation as dATP. There was no evidence of osmotic imbalance or of inhibition of the Na+/K(+)-dependent ATPase activity as the intracellular concentrations (and the intracellular:extracellular ratios) of Na+, K+ and Ca2+ were essentially unchanged. Cytochalasin B (20 microM) also caused lymphoblasts to swell over a 6-h period and its effect on cell size was similar to that of either 1 microM or 5 microM deoxyadenosine over 24 or 48 h. Longer time-courses of incubation with cytochalasin B caused severe toxicity leading to the death and lysis of a significant proportion of the cells. Other drugs, such as colchicine, vincristine and vinblastine that are known to affect various components of the cytoskeleton also caused swelling of cells in a concentration- and time-dependent manner but there was no evidence that these effects were additive or synergistic with those of deoxyadenosine. Inhibition of DNA synthesis, either directly by aphidicolin or indirectly by hydroxyurea, was less cytotoxic than the effect caused by deoxyadenosine. We conclude that one of the toxic effects resulting from the excessive phosphorylation of deoxyadenosine and its accumulation as dATP in human T-lymphoblasts is not dependent on inhibition of DNA synthesis but may be caused by the disruption of the cytoskeleton in these cells.

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.

Ribonucleotide reductase-mediated increase in dATP improves cardiac performance via myosin activation in a large animal model of heart failure.

AIMS: Heart failure remains a leading cause of morbidity, hospitalizations, and deaths. We previously showed that overexpression of the enzyme ribonucleotide reductase (RNR) in cardiomyocytes increased levels of the myosin activator, 2-deoxy-ATP, catalysed enhanced contraction, and improved cardiac performance in rodent hearts. Here we used a swine model of myocardial infarction (MI) to test preliminarily a novel gene therapy for heart failure based on delivery of the human RNR enzyme complex under the control of a cardiac-specific promoter via an adeno-associated virus serotype 6 vector--designated as BB-R12. METHODS AND RESULTS: We induced heart failure following MI in Yucatan minipigs by balloon occlusion of the left anterior descending artery. Two weeks, later, pigs received BB-R12 at one of three doses via antegrade coronary infusion. At 2 months post-treatment, LVEF and systolic LV dimension (measured by echocardiography) improved significantly in the high-dose group, despite further deterioration in the saline controls. Haemodynamic parameters including LV end-diastolic pressure, +dP/dt, and -dP/dt all trended towards improvement in the high-dose group. We observed no difference in the histopathological appearance of hearts or other organs from treated animals vs. controls, nor did we encounter any safety or tolerability concerns following BB-R12 delivery. CONCLUSION: These pilot results suggest cardiac-specific gene therapy using BB-R12 may reverse cardiac dysfunction by myosin activation in a large-animal heart failure model with no observed safety concerns. Thus further research into the therapeutic potential of BB-R12 for patients with chronic heart failure appears warranted.

Heterogeneous repair of platinum-DNA adducts by protein extracts from mammalian tissues.

Cis-diamminedichloroplatinum (II) (cisplatin) is a mainstay of human cancer chemotherapy. In addition to its antitumour effects however, cisplatin is toxic to normal tissues, causing dose-limiting nephrotoxicity and neurotoxicity. On the other hand, myelosuppression is uncommon. In the light of data suggesting a role for DNA repair mechanisms as determinants of cellular cisplatin sensitivity, we postulated that varying DNA repair capacities between tissues could explain the patterns of organ toxicity seen in clinical practice. Using a novel cell-free assay of repair of cisplatin-DNA adducts, we find that the DNA repair capacity of protein extracts from different tissues varies significantly in our assay, but does not directly correlate with the organ toxicity profile of cisplatin.

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.

Biochemical mechanisms of deoxycoformycin toxicity in chronic leukemias.

The in-vitro effects of deoxycoformycin (dCF) on dATP, NAD, ATP and DNA strand breaks have been evaluated in the cells from 42 patients with various types of chronic lymphoid leukemia. These included 18 with B-cell chronic lymphoid leukemias of different types (BCL); 10 with hairy cell leukemia (HCL) and 14 with T-cell chronic lymphoid leukemias of different types (TCL). The dATP concentrations in HCL, BCL and TCL increased from means of 2.9, 1.8 and 3.0 to 100.3, 68.2 and 51.3 pmol/10(6) cells respectively after 2 h with 10(-5) M dCF and 10(-4)M deoxyadenosine. After 18-24 h, the NAD levels and total double-stranded DNA decreased to 37 and 12.5% (HCL) to 36 and 21.6% (BCL) and 40 and 20.5% (TCL) of control values respectively. Similar decreases were observed in ATP levels. The results do not suggest that these measurements in vitro would predict which patients with these disorders will respond to dCF therapy. Although HCL responds particularly well to dCF in vivo, no difference in the in-vitro effects of dCF studied here could be detected between cases of HCL and the other types of chronic leukemia.