Sensitivity of Acute Leukemia Cells to Cytarabine Is a Correlate of Cellular es Nucleoside Transporter Site Content Measured by Flow Cytometry With SAENTA-Fluorescein

Abstract

Cytarabine (araC) is converted to araC 5′-triphosphate after entering leukemia cells as a substrate for nucleoside transport processes. This study tested the relationship between araC cytotoxicity, measured in an in vitro tetrazolium dye reduction assay of cell viability, and the cellular abundance of es nucleoside transport elements, assayed by a flow cytometric method that used the es-specific stain, 5-(SAENTA-x8)-fluorescein (5-(Sx8)-F), in cultured leukemia cells and in myeloblasts and lymphoblasts (blasts) from leukemia patients. Cellular es site abundance (Bmax value for 5-(Sx8)-F binding) varied sixfold among nine leukemic myeloblast samples from patients. In cultured OCI/AML-2 myeloblasts and CCRF-CEM T-lymphoblasts, and in fresh leukemic blasts, es sites were fractionally blocked by treatment with graded concentrations of nitrobenzylthioinosine (NBMPR), an inhibitory es site ligand, to simulate the variation in es expression found in leukemic blasts from patients with acute myeloid leukemia. When the cytotoxicity of a single concentration of araC was determined in NBMPR-treated leukemia cells, cell kill correlated closely with the intensity of 5-(Sx8)-F fluorescence (r = .92 to .99), a measure of the cell surface abundance of functional es nucleoside transporter sites. Concentrations of NBMPR that achieved half-maximal reduction (4.3 to 12 nmol/L) of cellular 5-(Sx8)-F fluorescence (measured by flow cytometry) approximated IC50 values (1 to 10 nmol/L) previously found for inhibition by NBMPR of es-mediated nucleoside fluxes in several cell types, supporting the view that 5-(Sx8)-F interacted with the es transporter. The correlation of araC cytotoxicity and the Bmax for 5-(Sx8)-F binding to es sites in cultured leukemia cells and in leukemic blasts from acute leukemia patients (r = .95) suggests that the flow cytometry assay of es capacity may be useful in predicting clinical response to araC.

Identification of a novel point mutation in ENT1 that confers resistance to Ara‐C in human T cell leukemia CCRF‐CEM cells

“…ENT‐mediated transport of nucleoside analogs is often the rate‐limiting step for drug‐induced cell cytotoxicity [6]. Thus, it is not surprising that studies have found a positive correlation between the expression of hENT1 and cancer cell sensitivity to the nucleoside analogs Ara‐C [19] and gemcitabine [20]. The reduction in hENT1 expression is a common mechanism for resistance to antimetabolite treatment of cancer [7,21].…”

Nucleoside Transporter Proteins

“…This was done mostly in lymphoproliferative malignancies, as these diseases allow the simultaneous measurement of the ex vivo sensitivity to drugs used in patient therapy. Several studies have reported a correlation between hENT1 expression and cytarabine sensitivity in acute myeloid and lymphoid leukaemia (AML and ALL) [81][82][83]. In chronic lymphocitic leukaemia (CLL) a significant correlation between fludarabine uptake via ENT carriers, hENT2 protein expression and ex vivo sensitivity was detected [78,84].…”

Towards targeted therapy for infant acute lymphoblastic leukaemia

“…Sensitivity to Ara‐C in infant ALL appeared not to be associated with rearrangements of the MLL gene, as both MLL rearranged and MLL germ line infant ALL cases appeared equally sensitive to this drug in vitro (Stam et al , 2005). The remarkable sensitivity to Ara‐C seems most likely to be due to the elevated expression of the human equilibrative nucleoside transporter 1 (hENT1) (Stam et al , 2003), on which Ara‐C is mainly dependent to permeate the cell membrane (Wiley et al , 1982; White et al , 1987; Gati et al , 1997). However, at high‐dose Ara‐C regimens generating high extracellular drug concentrations, Ara‐C also enters the cell by passive diffusion and membrane transport via ENT1 is circumvented.…”