Two Distinct Molecular Mechanisms Underlying Cytarabine Resistance in Human Leukemic Cells

Abstract

To understand the mechanism of cellular resistance to the nucleoside analogue cytarabine (1-B-D-arabinofuranosylcytosine, AraC), two resistant derivatives of the human leukemic line CCRF-CEM were obtained by stepwise selection in different concentrations of AraC. CEM/4ÂAraC cells showed low AraC resistance, whereas CEM/20ÂAraC cells showed high resistance. Both cell lines showed similar patterns of crossresistance to multiple cytotoxic nucleoside analogues, with the exception that CEM/20ÂAraC cells remained sensitive to 5-fluorouridine and 2-deoxy-5-fluorouridine. Both cell lines were sensitive to 5-fluorouracil and to a variety of natural product drugs. Although both CEM/4ÂAraC and CEM/ 20ÂAraC cells displayed reduced intracellular accumulation of [ 3 H]AraC, only CEM/4ÂAraC cells showed reduced uptake of [ 3 H]uridine, which was used to assess nucleoside transport activities. Genes encoding proteins known to be involved in nucleoside transport, efflux, and metabolism were analyzed for the presence of mutations in the two cell lines. In CEM/4ÂAraC cells, independent mutations were identified at each allele of human equilibrative nucleoside transporter 1 (hENT1; SLC29A1), one corresponding to a single-nucleotide change in exon 4, the other being a complex intronic mutation disrupting splicing of exon 13. In contrast to CEM/20ÂAraC cells, CEM/4ÂAraC cells did not bind the hENT1/SLC29A1 ligand nitrobenzylmercaptopurine ribonucleoside and lacked detectable hENT1/SLC29A1 protein. In CEM/20ÂAraC cells, independent intronic mutations impairing splicing of exons 2 and 3 were found at each allele of the deoxycytidine kinase gene. These studies point to at least two distinct mechanisms of AraC resistance in leukemic cells. [Cancer Res 2008;68(7):2349-57]

Host-Based Ribavirin Resistance Influences Hepatitis C Virus Replication and Treatment Response

“…Mutations in SLC29A1 could alter splicing, which could affect ENT1 protein stability, localization, and activity. Splice variants that inactivate ENT1 or alter RBV uptake have been identified (4,19). Although no differences were found in ENT1 RNA or protein levels in the RBV S and RBV R cells examined here, subtle changes such as mutations or altered splicing could have occurred in RBV R cells which may have been masked in our ENT1 protein and RNA analysis.…”

SLC29A1 single nucleotide polymorphisms as independent prognostic predictors for survival of patients with acute myeloid leukemia: an in vitro study

“…DCK can be inhibited by increased dCTP pool through a negative feedback, and increased CDA function leads to increase in the deamination of Ara-C to AraU [19][20][21]. Decreased Ara-C transport over the cell membrane into the cytoplasm [6,22] or inactivation of DCK [23,24] can both offset the cytotoxic function of Ara-C. Our previous study showed continuous exposure to Ara-C could induce drug resistance with decreased transcription level of DCK and SLC29A1 as well as elevated mRNA expression of CDA (data not shown). Genetic variations, particularly SNPs, have been identified in these genes involved in Ara-C transport and metabolism [25][26][27].…”

A Bioluminescent Microbial Biosensor for In Vitro Pretreatment Assessment of Cytarabine Efficacy in Leukemia

“…5, A and B). The diagnostic discriminator, assuming that generation of Ara-CTP in AML cells is the major cause of cell death (4,5 ), is the maximal difference in light output between the IPTG (LI) and IPTG ϩ AP (LIP) curves that are shown in Fig. 5E.…”