Open AccessFlow cytometric quantitation of nucleoside transporter sites on human leukemic cells
Author(s) -
Jamieson Gary P.,
Brocklebank Anne M.,
Snook Marie B.,
Sawyer William H.,
Buolamwini John K.,
Paterson Alan R. P.,
Wiley James S.
Publication year - 1993
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/cyto.990140107
Subject(s) - flow cytometry , nucleoside , nucleoside transporter , transporter , chemistry , microbiology and biotechnology , biology , biochemistry , gene
Abstract Quantitation of equilibrative, nitrobenzylthioinosine (NBMPR) sensitive ( es ) nucleoside transporters on blast cells isolated from patients with acute myeloblastic leukemia is useful in predicting intracellular accumulation of the antileukemic nucleoside drug, cytosine arabinoside. We previously reported the synthesis of a fluorescein‐labeled ligand for the es nucleoside transporter, 5‐(SAENTA‐x2)‐fluorescein. This paper reports the synthesis of 5‐(SAENTA‐x8)‐fluorescein in which the linkage between fluorescein and nucleoside ligand has been increased from 2 atoms to 8 atoms. This new ligand had a sixfold increase in affinity (K d 0.9 ± 0.1 nM) as well as an 86% increase in the cell associated fluorescence output compared to its prototype 5‐(SAENTA‐x2)‐fluorescein. The fluorescence signal arising from 5‐(SAENTA‐x8)‐fluorescein specifically bound to freshly isolated and cultured leukemic myeloblasts was converted to molecules of equivalent soluble fluorescein (MESF) using standardized fluorescein microbeads and compared with the number of es nucleoside transporter sites assayed concurrently by [ 3 H]NBMPR equilibrium binding analysis. A high correlation between the two assays was observed (r = 0.98), which enabled the cell‐bound fluorescence output of 5‐(SAENTA‐x8)‐fluorescein to be expressed in numbers of es nucleoside transporter sites per cell. The improved properties of 5‐(SAENTA‐x8)‐fluorescein over those of its prototype molecule make it a suitable reagent for flow cytometric quantitation of nucleoside transporter expression on leukemic cells isolated from patient samples. © 1993 Wiley‐Liss, Inc.