Equilibrative nucleoside transporter-2 (hENT2) protein expression correlates with ex vivo sensitivity to fludarabine in chronic lymphocytic leukemia (CLL) cells (original) (raw)
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Blood, 2005
Quantitative analysis of nucleoside transporter and metabolism gene http://bloodjournal.hematologylibrary.org/content/105/2/767.full.html Updated information and services can be found at: (1930 articles) Signal Transduction (4217 articles) Neoplasia (1086 articles) Gene Expression Articles on similar topics can be found in the following Blood collections http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub\_requests Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml Information about subscriptions and ASH membership may be found online at: Resistance to fludarabine is observed in the clinic, and molecular predictive assays for benefit from chemotherapy are required. Our objective was to determine if expression of nucleoside transport and metabolism genes was associated with response to fludarabine therapy in patients with chronic lymphocytic leukemia (CLL). CLL cells from 56 patients were collected prior to treatment with fludarabine. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on sample RNA to determine the relative levels of mRNA of 3 nucleoside transporters that mediate fludarabine uptake (human equilibrative nucleoside transporter 1 [hENT1], human equilibrative nucleoside transporter 2 [hENT2], and human concentrative nucleoside transporter 3 [hCNT3]), deoxycytidine kinase (dCK), and 3 5-nucleotidases (ecto-5nucleotidase [CD73], deoxynucleotidase-1 [dNT-1], and cytoplasmic high-K m 5-nucleotidase [CN-II]). Two-dimensional hierarchical cluster analysis of gene expression identified 2 distinct populations of CLL. Cluster 2 patients experienced a 3.4-fold higher risk of disease progression than cluster 1 patients (P ؍ .0058, log-rank analysis). Furthermore, independent analysis of the individual genes of interest revealed statistically significant differences for risk of disease progression (adjusted hazard ratios [HRs]) with underex-pression of dNT-1 (HR ؍ 0.45; P ؍ .042), CD73 (HR ؍ 0.40; P ؍ .022), and dCK (HR ؍ 0.0.48; P ؍ .035), and overexpression of hCNT3 (HR ؍ 4.7; P ؍ .0007) genes. Subjects with elevated hCNT3 expression experienced a lower complete response rate to fludarabine therapy (11% vs 69%; P ؍ .002). No hCNT3-mediated plasma membrane nucleoside transport was detected in CLL samples expressing hCNT3 message, and hCNT3 protein was localized to the cytoplasm with immunohistochemical and confocal microscopy. (Blood. 2005;105:767-774)
Fludarabine uptake mechanisms in B-cell chronic lymphocytic leukemia
Blood, 2003
Nucleoside derivatives are currently used in the treatment of hematologic malignancies. Although intracellular events involved in the pharmacologic action of these compounds have been extensively studied, the role of plasma membrane transporters in nucleoside-derived drug bioavailability and action in leukemia cells has not been comprehensively addressed. We have monitored the amounts of mRNA for the 5 nucleoside transporter isoforms cloned so far (CNT1, CNT2, CNT3, ENT1, and ENT2) in several human cell types and in normal human leukocytes. We then examined the expression patterns of these plasma membrane proteins in patients with chronic lymphocytic leukemia (CLL) and correlated them with in vitro fludarabine cytotoxicity. Despite a huge individual variability in the mRNA amounts for every transporter gene expressed in CLL cells (CNT2, CNT3, ENT1, and ENT2), no relationship between mRNA levels and in vitro fludarabine cytotoxicity was observed. Fludarabine accumulation in CLL cells...
Cancer Chemotherapy and Pharmacology, 2009
Purpose Interpatient variability in renal elimination of fludarabine (9-β-D-arabinosyl-2-fluoroadenine) by renal human nucleoside transporters (hNTs) may contribute to unpredictable toxicities including rare nephrotoxicities. This study assessed relationships between hNT levels and fludarabine uptake and cytotoxicity in cultures of human renal proximal tubule cells (hRPTCs) that produce multiple transporter types. Methods hRPTC cultures were established from ten different individuals and their hNT characteristics were assessed by measuring RNA expression by TaqMan™ reverse transcriptase polymerase chain reaction, protein abundance by quantitative immunoblotting of cell surface protein preparations, and uptake by radiolabeled nucleoside uptake assays. Fludarabine cytotoxicity against hRPTC cultures was quantified using methoxyphenyl tetrazolium inner salt (MTS) assays. Results RNA, protein and activities for human equilibrative NT 1 (hENT1) and 2 (hENT2) and human concentrative NT 3 (hCNT3) were identified in cultures of hRPTCs from ten different individuals. Significant differences in hCNT3 activities were exhibited among hRPTC cultures and correlated positively with cell surface levels of hCNT3 protein, but did not correlate with hCNT3 mRNA levels. Conclusions Observed differences in hCNT3-mediated uptake activities, hNT-mediated fludarabine uptake activities, and fludarabine cytotoxicities correlated positively with each other, suggesting that hCNT3 is a primary determinant of fludarabine uptake and cytotoxicity in hRPTC cultures. Variations in hCNT3 abundance in renal proximal tubules, and hence nucleoside reabsorption, may explain interpatient variability in fludarabine’s pharmacokinetics and toxicities.
Journal of Biological Chemistry, 2005
The first mammalian examples of the equilibrative nucleoside transporter family to be characterized, hENT1 and hENT2, were passive transporters located predominantly in the plasma membranes of human cells. We now report the functional characterization of members of a third subgroup of the family, from human and mouse, which differ profoundly in their properties from previously characterized mammalian nucleoside transporters. The 475-residue human and mouse proteins, designated hENT3 and mENT3, respectively, are 73% identical in amino acid sequence and possess long N-terminal hydrophilic domains that bear typical (DE)XXXL(LI) endosomal/lysosomal targeting motifs. ENT3 transcripts and proteins are widely distributed in human and rodent tissues, with a particular abundance in placenta. However, in contrast to ENT1 and ENT2, the endogenous and green fluorescent proteintagged forms of the full-length hENT3 protein were found to be predominantly intracellular proteins that co-localized, in part, with lysosomal markers in cultured human cells. Truncation of the hydrophilic N-terminal region or mutation of its dileucine motif to alanine caused the protein to be relocated to the cell surface both in human cells and in Xenopus oocytes, allowing characterization of its transport activity in the latter. The protein proved to be a broad selectivity, low affinity nucleoside transporter that could also transport adenine. Transport activity was relatively insensitive to the classical nucleoside transport inhibitors nitrobenzylthioinosine, dipyridamole, and dilazep and was sodium ion-independent. However, it was strongly dependent upon pH, and the optimum pH value of 5.5 probably reflected the location of the transporter in acidic, intracellular compartments.
Differential Expression of Human Nucleoside Transporters in Normal and Tumor Tissue
Biochemical and Biophysical Research Communications, 2001
Responses to nucleoside analog drugs used in the treatment of cancers and viral infections can vary considerably between individuals. Genetic variability between individuals in their ability to transport drugs may be a contributory factor. Nucleoside transporters (NTs) move nucleosides and analog drugs across cell membranes. Four human NTs have been cloned: hENT1, hENT2, hCNT1, and hCNT2. Human NT expression profiles are not well defined; therefore, we undertook a comprehensive quantitative analysis of the differential expression of NTs within normal and tumor tissue. Results show tissue specific expression of the different NTs in normal tissue while matched normal/tumor tissue cDNA array data show considerable variability in all NT expression profiles from different individuals, in particular decreased expression in tumor tissue. Decreased NT expression in tumor tissue may contribute to reduced drug uptake and the development of resistance. These data suggest that nucleoside analog drug therapies may be optimized by determining individual NT expression profiles.
Nucleosides, Nucleotides & Nucleic Acids, 2020
Cellular uptake of clinically important deoxynucleoside analogs is mediated by nucleoside transporters including the human equilibrative nucleoside transporter 1 (hENT1) and the concentrative nucleoside transporter-1 (hCNT1). These transporters are responsible for influx of cytarabine and reduced hENT1 expression is a major resistance mechanism in acute myeloid leukemia. We determined hENT1 and hCNT1 protein expression by immunocytochemistry in 50 diagnostic pediatric acute myeloid leukemia patient samples. All samples expressed hENT1 [9/43 (21%) low; 26/43 (60%) medium and 8/43 (19%) high] and hCNT1 [2/42 (5%) low; 35/42 (83%) medium and 5/42 (12%) high] at the cell membrane and cytoplasm. Statistical analysis showed a non-significant relationship between survival and transporter expression and in vitro drug sensitivity. In conclusion, the nucleoside transporters hENT1 and hCNT1 are broadly expressed in pediatric acute myeloid leukemia at diagnosis.
British Journal of Cancer, 2005
Cytarabine (ara-C) is the most effective agent for the treatment of acute myeloid leukaemia (AML). Aberrant expression of enzymes involved in the transport/metabolism of ara-C could explain drug resistance. We determined mRNA expression of these factors using quantitative-real-time-PCR in leukemic blasts from children diagnosed with de novo AML. Expression of the inactivating enzyme pyrimidine nucleotidase-I (PN-I) was 1.8-fold lower in FAB-M5 as compared to FAB-M1/2 (P ¼ 0.007). In vitro sensitivity to deoxynucleoside analogues was determined using the MTT-assay. Human equilibrative nucleoside transporter-1 (hENT1) mRNA expression and ara-C sensitivity were significantly correlated (r p ¼ À0.46; P ¼ 0.001), with threefold lower hENT1 mRNA levels in resistant patients (P ¼ 0.003). hENT1 mRNA expression also seemed to correlate inversely with the LC 50 values of cladribine (r p ¼ À0.30; P ¼ 0.04), decitabine (r p ¼ À0.29; P ¼ 0.04) and gemcitabine (r p ¼ À0.33; P ¼ 0.02). Deoxycytidine kinase (dCK) and cytidine deaminase (CDA) mRNA expression seemed to correlate with in vitro sensitivity to gemcitabine (r p ¼ À0.31; P ¼ 0.03) and decitabine (r p ¼ 0.33; P ¼ 0.03), respectively. The dCK/PN-I ratio correlated inversely with LC 50 values for gemcitabine (r p ¼ À0.45, P ¼ 0.001) and the dCK/CDA ratio seemed to correlate with LC 50 values for decitabine (r p ¼ À0.29; 0.04). In conclusion, decreased expression of hENT1, which transports ara-C across the cell membrane, appears to be a major factor in ara-C resistance in childhood AML.