Flow cytometric quantitation of nucleoside transporter sites on human leukemic cells

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 (Kd 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 [3H]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.

Modulation of the equilibrative nucleoside transporter by inhibitors of DNA synthesis

“…Cells were resuspended at a concentration of 1 x 106ml-' in phenol red-free medium, and labelled with 5 nM 5-(SA-ENTA-x8)-fluorescein at room temperature (RT) for 10min in the dark, in the presence or absence of 2.5 iLM NBMPR as previously described (Jamieson et al, 1993 Dual staining with 5-(SAENTA-x8)-fluorescein and the vital DNA dye Hoechst-33342 enabled the investigation of changes in es expression throughout the cell cycle. Because competition was observed between Hoechst-33342 uptake and 5-(SAENTA-x8)-fluorescein binding, the concentration of Hoechst-33342 used was reduced from 10 to 1.0 iLM, and the concentration of 5-(SAENTA-x8)-fluorescein increased from 5 to 80 nM.…”

Synthesis and Biologic Study of IV-14, a New Ribonucleoside Radiotracer for Tumor Visualization

“…59-[(Fluorescein-5-ylcarbonyl)-6-aminohexanoyl-(2-aminoethyl)]-N 6 -(4-nitrobenzyl)-59-thioadenosine (SAENTA-x8-fluorescein; solution in 4% dimethyl sulfoxide) (17) was then added to a 50 nM final concentration, and incubation was continued for a further 20 min at room temperature. The cells were washed once with PBS, and the cellassociated fluorescence was measured using a FACSCalibur Flow Cytometer (BD Biosciences).…”

FITC-poly-D-lysine conjugates as fluorescent probes to quantify hapten-specific macrophage receptor binding and uptake kinetics

“…FITC-labeled microspheres (FCSC, San Juan, P.R.) with MESF in the range of ϳ50,000-ϳ200,000 were used to calibrate the flow cytometer to determine the number of binding sites per cell for each FITC-PDL 2 probe from a calibration plot of MESF versus mean fluorescence intensity of the beads (14,25).…”

“…FITC-labeled microspheres corresponding to an MESF ranging from ϳ50,000 to ϳ200,000 were used to calibrate the flow cytometer and construct a calibration plot of assigned MESF vs. relative fluorescence intensity of the microspheres (14,25). From the calibration plot the number of ligand molecules bound/cell was determined as ϳ195,000 Ϯ 5000 for FITC 12 PDL 2 , ϳ112,000 Ϯ 2000 for FITC 5 PDL 2 and ϳ1350 Ϯ 250 for FITC 34 PDL 2 using the method described by Schwartz et al (25).…”