Enzymatic Biotransformation of Ginsenoside Rb2 into Rd by Recombinant α-L-Arabinopyranosidase from Blastococcus saxobsidens.

In this study, we used a novel α-L-arabinopyranosidase (AbpBs) obtained from ginsenoside-converting Blastococcus saxobsidens that was cloned and expressed in Escherichia coli BL21 (DE3), and then applied it in the biotransformation of ginsenoside Rb2 into Rd. The gene, termed AbpBs, consisting of 2,406 nucleotides (801 amino acid residues), and with a predicted translated protein molecular mass of 86.4 kDa, was cloned into a pGEX4T-1 vector. A BLAST search using the AbpBs amino acid sequence revealed significant homology with a family 2 glycoside hydrolase (GH2). The over-expressed recombinant AbpBs in Escherichia coli BL21 (DE3) catalyzed the hydrolysis of the arabinopyranose moiety attached to the C-20 position of ginsenoside Rb2 under optimal conditions (pH 7.0 and 40°;C). Kinetic parameters for α-Larabinopyranosidase showed apparent Km and Vmax values of 0.078 ± 0.0002 micrometer and 1.4 ± 0.1 µmol/min/mg of protein against p-nitrophenyl-α-L-arabinopyranoside. Using a purified AbpBs (1 µg/ml), 0.1% of ginsenoside Rb2 was completely converted to ginsenoside Rd within 1 h. The recombinant AbpBs could be useful for high-yield, rapid, and low-cost preparation of ginsenoside Rd from Rb2.

Determination and quantification of intracellular fludarabine triphosphate, cladribine triphosphate and clofarabine triphosphate by LC-MS/MS in human cancer cells.

Purine nucleoside analogues are widely used in the treatment of haematological malignancies, and their biological activity is dependent on the intracellular accumulation of their triphosphorylated metabolites. In this context, we developed and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to study the formation of 5'-triphosphorylated derivatives of cladribine, fludarabine, clofarabine and 2'-deoxyadenosine in human cancer cells. Br-ATP was used as internal standard. Separation was achieved on a hypercarb column. Analytes were eluted with a mixture of hexylamine (5 mM), DEA (0.4%, v/v, pH 10.5) and acetonitrile, in a gradient mode at a flow rate of 0.3mLmin-1. Multiple reactions monitoring (MRM) and electrospray ionization in negative mode (ESI-) were used for detection. The application of this method to the quantification of these phosphorylated cytotoxic compounds in a human follicular lymphoma cell line, showed that it was suitable for the study of relevant biological samples.

Interaction of anticancer drug clofarabine with human serum albumin and human α-1 acid glycoprotein. Spectroscopic and molecular docking approach.

The binding interaction between clofarabine, an important anticancer drug and two important carrier proteins found abundantly in human plasma, Human Serum Albumin (HSA) and α-1 acid glycoprotein (AAG) was investigated by spectroscopic and molecular modeling methods. The results obtained from fluorescence quenching experiments demonstrated that the fluorescence intensity of HSA and AAG is quenched by clofarabine and the static mode of fluorescence quenching is operative. UV-vis spectroscopy deciphered the formation of ground state complex between anticancer drug and the two studied proteins. Clofarabine was found to bind at 298K with both AAG and HSA with the binding constant of 8.128×103 and 4.120×103 for AAG and HSA, respectively. There is stronger interaction of clofarabine with AAG as compared to HSA. The Gibbs free energy change was found to be negative for the interaction of clofarabine with AAG and HSA indicating that the binding process is spontaneous. Binding of clofarabine with HSA and AAG induced ordered structures in both proteins and lead to molecular compaction. Clofarabine binds to HSA near to drug site II. Hydrogen bonding and hydrophobic interactions were the main bonding forces between HSA-clofarabine and AAG-clofarabine as revealed by docking results. This study suggests the importance of binding of anticancer drug to AAG spatially in the diseases like cancers where the plasma concentration of AAG increases many folds. Design of drug dosage can be adjusted accordingly to achieve optimal treatment outcome.

Efficient Fludarabine-Activating PNP From Archaea as a Guidance for Redesign the Active Site of E. Coli PNP.

The combination of the gene of purine nucleoside phosphorylase (PNP) from Escherichia coli and fludarabine represents one of the most promising systems in the gene therapy of solid tumors. The use of fludarabine in gene therapy is limited by the lack of an enzyme that is able to efficiently activate this prodrug which, consequently, has to be administered in high doses that cause serious side effects. In an attempt to identify enzymes with a better catalytic efficiency than E. coli PNP towards fludarabine to be used as a guidance on how to improve the activity of the bacterial enzyme, we have selected 5'-deoxy-5'-methylthioadenosine phosphorylase (SsMTAP) and 5'-deoxy-5'-methylthioadenosine phosphorylase II (SsMTAPII), two PNPs isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. Substrate specificity and catalytic efficiency of SsMTAP and SsMTAPII for fludarabine were analyzed by kinetic studies and compared with E. coli PNP. SsMTAP and SsMTAPII share with E. coli PNP a comparable low affinity for the arabinonucleoside but are better catalysts of fludarabine cleavage with k(cat)/K(m) values that are 12.8-fold and 6-fold higher, respectively, than those reported for the bacterial enzyme. A computational analysis of the interactions of fludarabine in the active sites of E. coli PNP, SsMTAP, and SsMTAPII allowed to identify the crucial residues involved in the binding with this substrate, and provided structural information to improve the catalytic efficiency of E. coli PNP by enzyme redesign.

The readout of base-pair information in adenine-thymine α-D-Arabinonucleosides.

Structurally modified nucleosides are central players in the field of nucleic acid chemistry. Adenine-thymine (AT) pyrimido[4,5-d]pyrimidine furanosyl and pyranosyl arabinonucleosides have been synthesized for the first time. Single-crystal X-ray diffraction analysis reveals novel base pairs that, in synergy with the sugar residues, direct the emergence of distinct networks containing channels and cavities. The microscopic noncovalent connections can be translated into macroscopic levels in which robust organogels are formed by the furanoside but not the pyranoside. The influences of the sugars are also displayed by the different shaped superstructures of the free nucleosides in solution. The readout of the information in the base moiety is therefore tailored by the sugar configuration, and the interplays exert subtle effects on the structures, from solid to gel and to the solution state. The potential for forming these appealing base pairs and higher structures enables these intriguing nucleosides to serve as unique building blocks in various areas or to construct innovative nucleic acid structures.

Reduced drug incorporation into DNA and antiapoptosis as the crucial mechanisms of resistance in a novel nelarabine-resistant cell line.

BACKGROUND: Nine-beta-D-arabinofuranosylguanine (ara-G), an active metabolite of nelarabine, enters leukemic cells through human Equilibrative Nucleoside Transporter 1, and is then phosphorylated to an intracellular active metabolite ara-G triphosphate (ara-GTP) by both cytosolic deoxycytidine kinase and mitochondrial deoxyguanosine kinase. Ara-GTP is subsequently incorporated into DNA, thereby inhibiting DNA synthesis. METHODS: In the present study, we developed a novel ara-G-resistant variant (CEM/ara-G) of human T-lymphoblastic leukemia cell line CCRF-CEM, and elucidated its mechanism of ara-G resistance. The cytotoxicity was measured by using the growth inhibition assay and the induction of apoptosis. Intracellular triphosphate concentrations were quantitated by using HPLC. DNA synthesis was evaluated by the incorporation of tritiated thymidine into DNA. Protein expression levels were determined by using Western blotting. RESULTS: CEM/ara-G cells were 70-fold more ara-G-resistant than were CEM cells. CEM/ara-G cells were also refractory to ara-G-mediated apoptosis. The transcript level of human Equilibrative Nucleoside Transporter 1 was lowered, and the protein levels of deoxycytidine kinase and deoxyguanosine kinase were decreased in CEM/ara-G cells. The subsequent production of intracellular ara-GTP (21.3 pmol/107 cells) was one-fourth that of CEM cells (83.9 pmol/107 cells) after incubation for 6 h with 10 µM ara-G. Upon ara-G treatment, ara-G incorporation into nuclear and mitochondrial DNA resulted in the inhibition of DNA synthesis of both fractions in CEM cells. However, DNA synthesis was not inhibited in CEM/ara-G cells due to reduced ara-G incorporation into DNA. Mitochondrial DNA-depleted CEM cells, which were generated by treating CEM cells with ethidium bromide, were as sensitive to ara-G as CEM cells. Anti-apoptotic Bcl-xL was increased and pro-apoptotic Bax and Bad were reduced in CEM/ara-G cells. CONCLUSIONS: An ara-G-resistant CEM variant was successfully established. The mechanisms of resistance included reduced drug incorporation into nuclear DNA and antiapoptosis.

Cytarabine-resistant leukemia cells are moderately sensitive to clofarabine in vitro.

BACKGROUND/AIM: Clofarabine is transported into leukemic cells via the equilibrative nucleoside transporters (hENT) 1 and 2 and the concentrative nucleoside transporter (hCNT) 3, then phosphorylated by deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) to an active triphosphate metabolite. Cytarabine uses hENT1 and dCK for its activation. We hypothesized that cytarabine-resistant leukemia cells retain sensitivity to clofarabine. MATERIALS AND METHODS: Human myeloid leukemia HL-60 cells and cytarabine-resistant variant HL/ara-C20 cells were used in the present study. RESULTS: Despite 20-fold cytarabine resistance, the HL/ara-C20 cells exhibited only a 6-fold resistance to clofarabine compared to HL-60 cells. The intracellular concentration of the triphosphate metabolite of cytarabine was reduced to 1/10, and that of clofarabine was halved in the HL/ara-C20 cells. hENT1 and dCK were reduced, but hCNT3 and dGK were not altered in the HL/ara-C20 cells, which might contribute to their retained capability to produce intracellular triphosphate metabolite of clofarabine. CONCLUSION: Clofarabine was cytotoxic to leukemia cells that were resistant to cytarabine.

A sensitive LC-MS/MS method for quantifying clofarabine triphosphate concentrations in human peripheral blood mononuclear cells.

Clofarabine triphosphate is an intracellular active metabolite of clofarabine. In the present study, we developed and validated a rapid, sensitive, and selective liquid chromatography-tandem mass spectrometry method (LC-MS/MS) for quantifying clofarabine triphosphate concentrations in human peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from blood using the Ficoll gradient centrifugation method. Chromatographic separation was performed on a CN column using an isocratic mobile phase comprising acetonitrile/5mM ammonium acetate with 0.001% ammonium hydroxide (20/80, v/v) at a flow rate of 0.60 mL/min. Detection was carried out by MS/MS in the multiple reaction monitoring mode using a negative electrospray ionization interface. The method was validated in concentration ranges of 1.25-100 ng/10(7) cells with acceptable accuracy and precision using 50 µL of cell extract. Clofarabine triphosphate was stable in a series of stability studies with bench-top, auto-sampler, and repeated freeze-thaw cycles. The validated method was successfully used to measure the concentrations of clofarabine triphosphate in PBMCs from cancer patients treated with clofarabine.

Novel leukemic cell lines resistant to clofarabine by mechanisms of decreased active metabolite and increased antiapoptosis.

Clofarabine (CAFdA) is incorporated into leukemic cells by human equilibrative nucleoside transporters (hENT) 1 and 2 and human concentrative nucleoside transporter (hCNT) 3. CAFdA is then phosphorylated to the active metabolite CAFdA triphosphate (CAFdATP) by deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Two novel CAFdA-resistant variants were established and their mechanism of resistance was elucidated. The two variants (HL/CAFdA20, HL/CAFdA80) were 20-fold and 80-fold more CAFdA-resistant than HL-60, respectively. mRNA levels of hENT1, hENT2 and hCNT3 were 53.9, 41.8 and 17.7% in HL/CAFdA20, and 30.8, 13.9 and 7.9% in HL/CAFdA80, respectively, compared with HL-60. Thus, the total nucleoside transport capacity of CAFdA was reduced in both variants. dCK protein levels were 1/2 in HL/CAFdA20 and 1/8 in HL/CAFdA80 of that of HL-60. dGK protein levels were 1/2 and 1/3, respectively. CAFdATP production after 4-h incubation with 10 µM CAFdA was 20 pmol/10(7) cells in HL/CAFdA20 and 3 pmol/10(7) cells in HL/CAFdA80 compared with 63 pmol/10(7) cells in HL-60. The decreased CAFdATP production attenuated drug incorporation into both mitochondrial and nuclear DNA. In addition, the two variants were resistant to CAFdA-induced apoptosis due to Bcl2 overexpression and decreased Bim. A Bcl2 inhibitor, ABT737, acted synergistically with CAFdA to inhibit the growth with combination index values of 0.27 in HL/CAFdA20 and 0.23 in HL/CAFdA80, compared with 0.65 in HL-60. Thus, the mechanism of resistance primarily included not only reduced CAFdATP production, but also increased antiapoptosis. The combination of CAFdA and ABT737 may be effective against CAFdA resistance.

Cytotoxic purine nucleoside analogues bind to A1, A2A, and A3 adenosine receptors.

Fludarabine, clofarabine, and cladribine are anticancer agents which are analogues of the purine nucleoside adenosine. These agents have been associated with cardiac and neurological toxicities. Because these agents are analogues of adenosine, they may act through adenosine receptors to elicit their toxic effects. The objective of this study was to evaluate the ability of cytotoxic nucleoside analogues to bind and activate adenosine receptor subtypes (A(1), A(2A), A(2B), and A(3)). Radioligand binding studies utilizing Chinese hamster ovary cells, stably transfected with adenosine A(1), A(2A), or A(3) receptor subtype, were used to assess the binding affinities of these compounds, whereas adenylyl cyclase activity was used to assess the binding to A(2B) receptors. Clofarabine and cladribine both bound to the A(2A) receptor with a K (i) of 17 and 15 µM, respectively. Clofarabine was the only adenosine analogue to bind to the A(3) receptor with a K (i) of 10 µM, and none of these compounds bound to the A(2B) receptor. Results show that clofarabine, cladribine, and fludarabine bind to the A(1) receptor. In addition, clofarabine, cladribine, and fludarabine were A(1) agonists (IC(50) 3.1, 30, and 30 µM, respectively). Neither pyrimidine nucleoside analogues gemcitabine nor cytarabine associated with any of the adenosine receptor subtypes (K (i) > 100µM). This is the first report of an interaction between all adenosine receptor subtypes and chemotherapeutic nucleoside analogues commonly used in the treatment of cancer. Therefore, activation of these receptors may be at least one mechanism through which fludarabine-associated toxicity occurs.

Clofarabine 5'-di and -triphosphates inhibit human ribonucleotide reductase by altering the quaternary structure of its large subunit.

Human ribonucleotide reductases (hRNRs) catalyze the conversion of nucleotides to deoxynucleotides and are composed of α- and ß-subunits that form active α(n)ß(m) (n, m = 2 or 6) complexes. α binds NDP substrates (CDP, UDP, ADP, and GDP, C site) as well as ATP and dNTPs (dATP, dGTP, TTP) allosteric effectors that control enzyme activity (A site) and substrate specificity (S site). Clofarabine (ClF), an adenosine analog, is used in the treatment of refractory leukemias. Its mode of cytotoxicity is thought to be associated in part with the triphosphate functioning as an allosteric inhibitor of hRNR. Studies on the mechanism of inhibition of hRNR by ClF di- and triphosphates (ClFDP and ClFTP) are presented. ClFTP is a reversible inhibitor (K(i) = 40 nM) that rapidly inactivates hRNR. However, with time, 50% of the activity is recovered. D57N-α, a mutant with an altered A site, prevents inhibition by ClFTP, suggesting its A site binding. ClFDP is a slow-binding, reversible inhibitor ( K(i)*; t(1/2) = 23 min). CDP protects α from its inhibition. The altered off-rate of ClFDP from E•ClFDP* by ClFTP (A site) or dGTP (S site) and its inhibition of D57N-α together implicate its C site binding. Size exclusion chromatography of hRNR or α alone with ClFDP or ClFTP, ± ATP or dGTP, reveals in each case that α forms a kinetically stable hexameric state. This is the first example of hexamerization of α induced by an NDP analog that reversibly binds at the active site.

Clofarabine for myelodysplastic syndromes.

INTRODUCTION: Treatment options in myelodysplastic syndromes (MDS) remain limited. The introduction of novel therapies that can improve response rates and survival outcomes in MDS remains a challenge. Clofarabine is a purine nucleoside analog that works primarily via inhibition of DNA biosynthesis and the ribonucleotide reductase enzyme with recent evidence suggesting that at low doses it may affect DNA methylation. It has been successfully used in the treatment of acute myeloid leukemia (AML) and is under investigation in MDS. AREAS COVERED: A PubMed search for articles pertaining to clofarabine was conducted and streamlined to only include data on MDS or AML that evolved from MDS. Also included were clofarabine-related response and safety data from presentations at the 52(nd) Annual American Society of Hematology Meeting in Orlando, Florida, USA. EXPERT OPINION: Clinical trials using clofarabine in MDS and MDS/myeloproliferative neoplasms have produced overall response rates of 31 - 43% including complete responders. Although myelosuppression is an important side effect, clofarabine is generally well tolerated in MDS. Clofarabine is currently available in an intravenous form with an oral formulation presently under investigation, either as a single agent or in combination therapy in MDS. Larger studies may help clarify the viability of clofarabine in the treatment of MDS patients.

Synergistic therapeutic effect in gastric cancer cells produced by oncolytic adenovirus encoding Drosophila melanogaster deoxyribonucleoside kinase.

Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK), a novel suicide kinase, was applied as a cancer gene therapeutic approach. To improve the antitumor effect of Dm-dNK and its substrate with the selectivity and safety control in consideration, the conditionally replicative gene-viral system ZD55-dNK, which includes the replicationselective adenovirus ZD55 armed with the Dm-dNK, was used to further explore the potential of this approach. When ZD55-dNK was combined with BVDU it produced a synergistic inhibitive effect of adenovirus replication in vitro while maintaining specific cancer cell killing activity. From a clinical standpoint, this approach is promising for its considerably low toxic side effects.

Deoxycytidine kinase modulates the impact of the ABC transporter ABCG2 on clofarabine cytotoxicity.

Purine nucleoside antimetabolites, such as clofarabine, are effective antileukemic agents. However, their effectiveness depends on an initial activation step in which they are monophosphorylated by deoxycytidine kinase (dCK). Some purine nucleoside antimetabolites and their monophosphate derivatives are exported by the ABC transporter ABCG2. Because clofarabine is a dCK substrate, and we show substantial variation in dCK and ABCG2 in myeloid leukemia, we hypothesized that the activity of dCK may modulate ABCG2-mediated resistance to clofarabine by regulating the formation of clofarabine monophosphate. We show that ABCG2 influence on clofarabine cytotoxicity was markedly influenced by dCK activity. When dCK expression was reduced by siRNA, clofarabine cytotoxicity was strongly reduced by enhanced ABCG2-mediated efflux. Conversely, dCK overexpression blunted ABCG2-mediated efflux of clofarabine by increasing the formation of clofarabine nucleotides. The use of an ABCG2 inhibitor confirmed that ABCG2 export of clofarabine is maximal when dCK levels are minimal. Analysis of intracellular clofarabine metabolites suggested that ABCG2 exported clofarabine more readily than clofarabine monophosphate. That ABCG2 primarily effluxes clofarabine, but not chlorfarabine-monophosphate, was confirmed by HPLC analysis of drug exported from ABCG2-overexpressing cells. Because the level and function of dCK and ABCG2 vary substantially among other types of cancer, these findings have important implications not only for clofarabine therapy but for purine nucleoside therapy in general. Therefore, we propose that addition of ABCG2 inhibitors would effectively increase the antitumor efficacy of purine nucleosides by blocking drug efflux that may be a significant mode of resistance when dCK levels are low.

Activation of guanine-ß-D-arabinofuranoside and deoxyguanosine to triphosphates by a common pathway blocks T lymphoblasts at different checkpoints.

The deoxyguanosine (GdR) analog guanine-ß-d-arabinofuranoside (araG) has a specific toxicity for T lymphocytes. Also GdR is toxic for T lymphocytes, provided its degradation by purine nucleoside phosphorylase (PNP) is prevented, by genetic loss of PNP or by enzyme inhibitors. The toxicity of both nucleosides requires their phosphorylation to triphosphates, indicating involvement of DNA replication. In cultured cells we found by isotope-flow experiments with labeled araG a rapid accumulation and turnover of araG phosphates regulated by cytosolic and mitochondrial kinases and deoxynucleotidases. At equilibrium their partition between cytosol and mitochondria depended on the substrate saturation kinetics and cellular abundance of the kinases leading to higher araGTP concentrations in mitochondria. dGTP interfered with the allosteric regulation of ribonucleotide reduction, led to highly imbalanced dNTP pools with gradual inhibition of DNA synthesis and cell-cycle arrest at the G1-S boundary. AraGTP had no effect on ribonucleotide reduction. AraG was in minute amounts incorporated into nuclear DNA and stopped DNA synthesis arresting cells in S-phase. Both nucleosides eventually induced caspases and led to apoptosis. We used high, clinically relevant concentrations of araG, toxic for nuclear DNA synthesis. Our experiments do not exclude an effect on mitochondrial DNA at low araG concentrations when phosphorylation occurs mainly in mitochondria.

A new high-performance liquid chromatography method determines low production of 9-beta-D-arabinofuranosylguanine triphosphate, an active metabolite of nelarabine, in adult T-cell leukemia cells.

The 9-beta-D-arabinofuranosylguanine (ara-G), an active compound of nelarabine, demonstrates potent cytotoxicity specifically on T-cell malignancies. In cells, ara-G is phosphorylated to ara-G triphosphate (ara-GTP), which is subsequently incorporated into DNA, thereby inhibiting DNA synthesis. Because ara-GTP is crucial to ara-G's cytotoxicity, the determination of ara-GTP production in cancer cells is informative for optimizing nelarabine administration. Here, we developed a new, sensitive isocratic-elution HPLC method for quantifying ara-GTP. Samples were eluted isocratically by using phosphate buffer at a constant flow rate. Ara-GTP was clearly separated from other nucleotides by using an anion-exchange column and it was quantitated by its peak area at 254 nm. The standard curve was linear with low variability and a sensitive detection limit (10 pmol). Furthermore, due to ara-G's specificity to T-cells we hypothesized that nelarabine might be effective against adult T-cell leukemia (ATL). The ara-GTP production was compared between T-lymphoblastic leukemia CCRF-CEM and ATL cell lines in vitro. When CEM cells were incubated with ara-G, the ara-GTP production increased in a concentration- and time-dependent manner. In contrast, 5 ATL cell lines accumulated lower ara-GTP in the same condition. While ara-G inhibited the growth of CEM cells with a 50% growth inhibition concentration of 2 microM, the inhibitory-concentration values were >1 mM in 8 of the 12 ATL cell lines. This ineffectiveness appeared to correspond with the low ara-GTP production. The present study is the first to evaluate the potential of ara-G against ATL cells; our results suggest that nelarabine would not be effective against ATL.

Synthesis and hybridization studies of alpha-configured arabino nucleic acids.

Synthesis of alpha-L-arabino- and alpha-D-arabino-configured pentofuranosyl nucleosides of four of the natural bases [thymine (ara-T), adenine (ara-A), cytosine (ara-C) and guanine (ara-G)] is reported together with hybridization properties of oligonucleotides containing alpha-L-ara-T and -A, alpha-D-ara-T and -A, and 2'-amino-alpha-L-ara-T monomers. 2'-O-Acetylated alpha-L-ara-T, -A, -C and -G, alpha-D-ara-T, -A, -C and -G, and N2'-acylated-alpha-L-ara-T phosphoramidite building blocks were synthesized and used together with standard DNA phosphoramidites for solid-phase synthesis of 18-mer oligonucleotides. Thermal denaturation experiments showed that incorporation of three or six of the arabino-configured monomers into DNA-oligonucleotides reduced the binding affinity towards antiparallel DNA/RNA complements. Fully modified alpha-L-ara-oligonucleotides did not hybridize with DNA/RNA complements, whereas hybridization of fully modified alpha-D-ara-oligonucleotides with complementary DNA/RNA in parallel strand orientation was confirmed.

Contribution of the drug transporter ABCG2 (breast cancer resistance protein) to resistance against anticancer nucleosides.

We have studied the potential contribution of ABCG2 (breast cancer resistance protein) to resistance to nucleoside analogues. In cells transfected with DNA constructs resulting in overexpression of human or mouse ABCG2, we found resistance against cladribine, clofarabine, fludarabine, 6-mercaptopurine, and 6-mercaptopurine riboside in both MDCKII and HEK293 cells and against gemcitabine only in HEK293 cells. With Transwell studies in MDCK cells and transport experiments with vesicles from Sf9 and HEK293 cells, we show that ABCG2 is able to transport not only the nucleotide CdAMP, like several other ATP-binding cassette transporters of the ABCC (multidrug resistance protein) family, but also the nucleoside cladribine itself. Expression of ABCG2 in cells results in a substantial decrease of intracellular CdATP, explaining the resistance against cladribine. The high transport rate of cladribine and clofarabine by ABCG2 deduced from Transwell experiments raises the possibility that this transporter could affect the disposition of nucleoside analogues in patients or cause resistance in tumors.

The role of human nucleoside transporters in cellular uptake of 4'-thio-beta-D-arabinofuranosylcytosine and beta-D-arabinosylcytosine.

4'-Thio-beta-D-arabinofuranosyl cytosine (TaraC) is in phase I development for treatment of cancer. In human equilibrative nucleoside transporter (hENT) 1-containing CEM cells, initial rates of uptake (10 microM; picomoles per microliter of cell water per second) of [3H]TaraC and [3H]1-beta-D-arabinofuranosyl cytosine (araC) were low (0.007 +/- 003 and 0.034 +/- 0.003, respectively) compared with that of [3H]uridine (0.317 +/- 0.048), a highactivity hENT1 permeant. In hENT1- and hENT2-containing HeLa cells, initial rates of uptake (10 microM; picomoles per cell per second) of [3H]TaraC, [3H]araC, and [3H]deoxycytidine were low (0.30 +/- 0.003, 0.42 +/- 0.03, and 0.51 +/- 0.11, respectively) and mediated primarily by hENT1 (approximately 74, approximately 65, and approximately 61%, respectively). In HeLa cells with recombinant human concentrative nucleoside transporter (hCNT) 1 or hCNT3 and pharmacologically blocked hENT1 and hENT2, transport of 10 microM[3H]TaraC and [3H]araC was not detected. The apparent affinities of recombinant transporters (produced in yeast) for a panel of cytosine-containing nucleosides yielded results that were consistent with the observed low-permeant activities of TaraC and araC for hENT1/2 and negligible permeant activities for hCNT1/2/3. During prolonged drug exposures of CEM cells with hENT1 activity, araC was more cytotoxic than TaraC, whereas coexposures with nitrobenzylthioinosine (to pharmacologically block hENT1) yielded identical cytotoxicities for araC and TaraC. The introduction by gene transfer of hENT2 and hCNT1 activities, respectively, into nucleoside transport-defective CEM cells increased sensitivity to both drugs moderately and slightly. These results demonstrated that nucleoside transport capacity (primarily via hENT1, to a lesser extent by hENT2 and possibly by hCNT1) is a determinant of pharmacological activity of both drugs.

Bromovinyl-deoxyuridine: A selective substrate for mitochondrial thymidine kinase in cell extracts.

Cellular models of mitochondrial thymidine kinase (TK2) deficiency require a reliable method to measure TK2 activity in whole cell extracts containing two interfering deoxyribonucleoside kinases, thymidine kinase 1 (TK1) and deoxycytidine kinase. We tested the value of the thymidine analog (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) as a TK2-specific substrate. With extracts of OSTTK1- cells containing TK2 as the only thymidine kinase and a highly specific TK2 inhibitor we established conditions to detect the low TK2 activity commonly present in cells. With extracts of TK1-proficient osteosarcoma cells and normal human fibroblasts we showed that BVDU, but not 1-(beta-d-arabinofuranosyl)thymine (Ara-T), discriminates TK2 activity even in the presence of 100-fold excess TK1. A comparison with current procedures based on TK2 inhibition demonstrated the better performance of the new TK2 assay. When cultured human fibroblasts passed from proliferation to quiescence TK2 activity increased by 3-fold, stressing the importance of TK2 function in the absence of TK1.