Analysis of nuclear m-AMSA content by DNA fluorochrome competition

Spectrofluorometry and flow cytometry were used to measure cellular uptake of the anti-leukemic drug 4'-(9-acridinylamino) methane sulfon-m-anisidide (m-AMSA). Because of its very low intrinsic fluorescence, we used m-AMSA to quench DNA-fluorescence induced by the vital DNA fluorochrome Hoechst 33342. Maximum fluorescence was obtained with cells incubated in the fluorochrome alone. Subsequent incubation of cells in increasing concentrations of m-AMSA resulted in a gradual decrease in fluorescence. Upon incubation in drug-free medium, the quenching phenomenon was reversible, consistent with rapid exit of m-AMSA from the cells. The novel competitive fluorescence assay for cellular uptake for m-AMSA showed a better correlation to nuclear accumulation of the drug, than to its overall cellular accumulation, which may be important in assessment of cellular resistance to m-AMSA, with possible low nuclear accumulation of the drug. When this competitive fluorescence technique for measurement of cellular m-AMSA concentration was applied in flow-cytometric setting, subpopulations of normal human white blood cells were detected with distinctly different fluorescence patterns, indicating differences in cellular m-AMSA uptake. The potential use of this technique is to detect differences between cell subpopulations with different drug uptake abilities.