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.
Andersson
B S
BS
Beran
M
M
Barlogie
B
B
Van
N T
NT
McCredie
K B
KB
D000677:Amsacrine / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007364:Intercalating Agents / Q000378:metabolism; D007951:Leukemia, Myeloid / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000970:Antineoplastic Agents; D002250:Carbon Radioisotopes; D007364:Intercalating Agents; D000677:Amsacrine