Cytosine arabinoside affects multiple cellular factors and induces drug resistance in human lymphoid cells

Sarkar, Malancha; Han, Tieran; Damaraju, Vijaya L.; Carpenter, Pat; Cass, Carol E.; Agarwal, Rekha

doi:10.1016/j.bcp.2005.05.014

Cited by 26 publications

(14 citation statements)

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“…Furthermore, expression and activity of hENT1 were identified as factors that conferred resistance to the cytosine analog cytarabine in AML patients and in cancer cell lines (Galmarini et al, 2002;Sarkar et al, 2005;Zimmerman et al, 2009). Improvement of drug transport was also a driving factor in the development of an elaidic acid derivative of 5-azacytidine (CP-4200; 5-azacytidine-59-elaidate) that contains a fatty acid moiety to facilitate the nucleoside transporter-independent uptake of the parent drug.…”

Section: Introductionmentioning

confidence: 99%

The Role of Human Equilibrative Nucleoside Transporter 1 on the Cellular Transport of the DNA Methyltransferase Inhibitors 5-Azacytidine and CP-4200 in Human Leukemia Cells

et al. 2013

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The nucleoside analog 5-azacytidine is an archetypical drug for epigenetic cancer therapy, and its clinical effectiveness has been demonstrated in the treatment of myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). However, therapy resistance in patients with MDS/AML remains a challenging issue. Membrane proteins that are involved in drug uptake are potential mediators of drug resistance. The responsible proteins for the transport of 5-azacytidine into MDS/AML cells are unknown. We have now systematically analyzed the expression and activity of various nucleoside transporters. We identified the human equilibrative nucleoside transporter 1 (hENT1) as the most abundant nucleoside transporter in leukemia cell lines and in AML patient samples. Transport assays using [14 C]5-azacytidine demonstrated Na 1 -independent uptake of the drug into the cells, which was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBTI), a hENT1 inhibitor. The cellular toxicity of 5-azacytidine and its DNA demethylating activity were strongly reduced after hENT1 inhibition. In contrast, the cellular activity of the 5-azacytidine derivative 5-azacytidine-59-elaidate (CP-4200), a nucleoside transporter-independent drug, persisted after hENT1 inhibition. A strong dependence of 5-azacytidine-induced DNA demethylation on hENT1 activity was also confirmed by array-based DNA methylation profiling, which uncovered hundreds of loci that became demethylated only when hENT1-mediated transport was active. Our data establish hENT1 as a key transporter for the cellular uptake of 5-azacytidine in leukemia cells and raise the possibility that hENT1 expression might be a useful biomarker to predict the efficiency of 5-azacytidine treatments. Furthermore, our data suggest that CP-4200 may represent a valuable compound for the modulation of transporter-related 5-azacytidine resistances.

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Section: Introductionmentioning

confidence: 99%

The Role of Human Equilibrative Nucleoside Transporter 1 on the Cellular Transport of the DNA Methyltransferase Inhibitors 5-Azacytidine and CP-4200 in Human Leukemia Cells

et al. 2013

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“…Using H9 lymphoid cell lines, Sarkar et al (2005) have shown that compared with the ara-C-sensitive cell line, the resistant cell line accumulated less ara-CTP and had significantly lower mRNA and/or protein expression of DCK and hENT1 (Sarkar et al, 2005). When treated with ara-C, patients whose leukemic cells had higher DCK expression demonstrated longer event-free survival than patients whose leukemic cells had lower DCK expression (Galmarini et al, 2002).…”

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confidence: 99%

Pharmacogenetics of Deoxycytidine Kinase: Identification and Characterization of Novel Genetic Variants

Lamba

Crews

Pounds

et al. 2007

J Pharmacol Exp Ther

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Deoxycytidine kinase (DCK) is a rate-limiting enzyme in the activation of nucleoside analogs such as cytarabine (ara-C), gemcitabine, clofarabine, and others. The present study was undertaken to identify and to determine the functional consequences of genetic variants in DCK. We sequenced 1.5 kilobases of the DCK proximal promoter and all seven coding exons in International HapMap Project panels (n ϭ 90 each) with European (Centre d' Etude du Polymorphisme Humain; CEPH) or African (Yoruba people in Ibadan, Nigeria; YRI) ancestry. Sixty-four genetic polymorphisms, including three nonsynonymous coding changes (I24V, A119G, and P122S) were identified. Compared with DCK-wild-type (WT) protein, the activity of the recombinant DCK24Val, DCK119Gly, and DCK122Ser proteins was 85 Ϯ 5, 66 Ϯ 3, and 43 Ϯ 4%, respectively. DCK119Gly and DCK122Ser mutants had lower K m (p Ͻ 0.01) and V max (p Ͻ 0.001) compared with DCK-WT protein. Lymphoblast cell lines from subjects heterozygous for the coding changes had significantly lower DCK activity compared with homozygous WT subjects. Ethnic differences were observed, with African ancestry subjects demonstrating significantly higher DCK mRNA expression compared with subjects with European ancestry. In both CEPH and YRI subjects, the C allele of a 3Ј-untranslated region singlenucleotide polymorphism (SNP) (35708 CϾT) was significantly associated with lower DCK mRNA expression. This SNP was strongly linked with other intronic SNPs, forming a major haplotype block in both ethnic groups. In an exploratory analysis, the 35708C allele was also associated with lower blast ara-C-5Ј-triphosphate (ara-CTP) levels in acute myeloid leukemia patients receiving ara-C as continuous infusion. These results suggest that genetic variation in DCK influences its activity and expression and may predict the variability observed in intracellular levels of the ara-C active metabolite ara-CTP.

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“…It shows wide substrate selectivity, accepting both purine and pyrimidine nucleosides, and has a 2:1 Na ϩ /nucleoside coupling ratio, allowing it to concentrate nucleosides intracellularly 10 times more efficiently than CNT1 or CNT2, which show a 1:1 Na ϩ /nucleoside stoichiometry (Ritzel et al, 2001). Its role in drug transport is not well known, being involved in the uptake of cladribine and fludarabine (Mangravite et al, 2003) and of cytosine arabinoside (Sarkar et al, 2005), as well as other non-nucleosidic drugs, such as the anthracycline pirarubicin (Nagai et al, 2005). CNT3 has also been shown to transport gemcitabine and the antiviral drugs azidothymidine and ribavirin (Hu et al, 2006;Yamamoto et al, 2007).…”

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confidence: 99%

Functional Characterization of a Nucleoside-Derived Drug Transporter Variant (hCNT3_C602R) Showing Altered Sodium-Binding Capacity

Errasti-Murugarren¹,

Cano-Soldado²,

Pastor‐Anglada³

et al. 2007

Mol Pharmacol

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A novel cloned polymorphism of the human concentrative nucleoside transporter hCNT3 was described and functionally characterized. This variant consists of a T/C transition leading to the substitution of cysteine 602 by an arginine residue in the core of transmembrane domain 13. The resulting hCNT3 C602R protein has the same selectivity and affinity for natural nucleosides and nucleoside-derived drugs as hCNT3 but much lower concentrative capacity. The insertion of the transporter into a polarized membrane seems unaffected in the variant. In a preliminary survey of a typical Spanish population, this variant showed an allelic frequency of 1%. The functional impairment of the hCNT3 C602R polymorphism is attributable to the presence of an arginine rather than the loss of a cysteine at position 602, because an engineered hCNT3 protein with a serine residue at this position (hCNT3 C602S ) and hCNT3 have similar kinetic parameters. The sodium activation kinetic analysis of both transporters revealed a variation in the affinity for sodium and a shift in the Hill coefficient that could be consistent with a stoichiometry of 2:1 and 1:1 sodium/nucleoside, for hCNT3 and hCNT3 C602R , respectively. In conclusion, the presence of an arginine residue in the core of transmembrane domain 13 is responsible for the different sodium affinity showed by the polymorphic transporter compared with the reference transporter. Individuals with the hCNT3 C602R variant might show a lower nucleoside and nucleoside analog concentrative capacity, which could be clinically relevant.

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Cytosine arabinoside affects multiple cellular factors and induces drug resistance in human lymphoid cells

Cited by 26 publications

References 40 publications

The Role of Human Equilibrative Nucleoside Transporter 1 on the Cellular Transport of the DNA Methyltransferase Inhibitors 5-Azacytidine and CP-4200 in Human Leukemia Cells

The Role of Human Equilibrative Nucleoside Transporter 1 on the Cellular Transport of the DNA Methyltransferase Inhibitors 5-Azacytidine and CP-4200 in Human Leukemia Cells

Pharmacogenetics of Deoxycytidine Kinase: Identification and Characterization of Novel Genetic Variants

Functional Characterization of a Nucleoside-Derived Drug Transporter Variant (hCNT3_C602R) Showing Altered Sodium-Binding Capacity

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Cytosine arabinoside affects multiple cellular factors and induces drug resistance in human lymphoid cells

Cited by 26 publications

References 40 publications

The Role of Human Equilibrative Nucleoside Transporter 1 on the Cellular Transport of the DNA Methyltransferase Inhibitors 5-Azacytidine and CP-4200 in Human Leukemia Cells

The Role of Human Equilibrative Nucleoside Transporter 1 on the Cellular Transport of the DNA Methyltransferase Inhibitors 5-Azacytidine and CP-4200 in Human Leukemia Cells

Pharmacogenetics of Deoxycytidine Kinase: Identification and Characterization of Novel Genetic Variants

Functional Characterization of a Nucleoside-Derived Drug Transporter Variant (hCNT3C602R) Showing Altered Sodium-Binding Capacity

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Product

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Functional Characterization of a Nucleoside-Derived Drug Transporter Variant (hCNT3_C602R) Showing Altered Sodium-Binding Capacity