Fludarabine uptake mechanisms in B-cell chronic lymphocytic leukemia

Abstract

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 was mostly, if not exclusively, mediated by ENT-type transporters whose biologic activity was clearly correlated with fludarabine cytotoxicity, which reveals a role of ENT-mediated uptake in drug responsiveness in patients with CLL.

Nucleoside transporters in chronic lymphocytic leukaemia

“…hCNT1 was absent in all cell lines, although all of them expressed hCNT2 with great variability, and a few cell lines, in particular, NCEB, JVM-2 and NB4, also showed significant hCNT3 mRNA levels. 53 These observations are consistent with the classical kinetic data supporting the coexpression of CNTand ENT-type transporters in leukaemia cells. Indeed, hCNT1, although present in macrophages, 54,55 would be mostly expressed in epithelia.…”

“…The expression of these nucleoside transporters was also explored in cells from 22 CLL patients. 53 Interestingly, hCNT1 was not detected in any of the samples, in agreement with the data discussed above showing that this particular isoform was not found in a broad panel of human lymphoid cell lines. The other four genes did not cluster into patient subgroups.…”

Functional analysis of genetic variants in the human concentrative nucleoside transporter 3 (CNT3; SLC28A3)

“…2 These agents are currently considered the first drugs of choice in the treatment of a variety of lymphoproliferative disorders, such as chronic lymphocytic leukemia. 4,5 Recently, Molina-Arcas et al 6 showed that leukemic cancer cells had varying abilities to concentrate fludarabine intracellularly and this correlated with fludarabine cytotoxicity. These same cells had variable expression levels of nucleoside transporters, with CNT3 showing the highest interindividual variation.…”

“…These same cells had variable expression levels of nucleoside transporters, with CNT3 showing the highest interindividual variation. 6 Therefore, in this study we examined the potency of interaction of cladribine and fludarabine with CNT3 and the three common protein-altering variants, p. Ser5Asn, p. Tyr113Cys, and p. Ile328Val (Figure 4). No significant differences were observed in the potency of any of the compounds tested in inhibiting CNT3 reference and its three nonsynonymous polymorphisms (Table 3).…”

“…For example, recent studies have shown that variation in the intracellular transport of fludarabine correlated with its chemosensitivity. 6 In addition, analysis of expression levels of nucleoside transporters in leukemic cells suggests that CNT3 has the highest interindividual variability. 6 Gray et al 7 recently reported that several genetic variants of CNT1 exhibited altered interaction with gemcitabine, suggesting that common CNT1 variants may contribute to variation in systemic and intracellular levels of pyrimidine nucleoside analog drugs.…”