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We describe the fate of the polyene antibiotic amphotericin B (AmB) after its interaction with Chinese hamster ovary (CHO) cells. The global uptake of AmB by these cells was measured at 37 degrees C after a 1-h incubation in the presence of 5% fetal bovine serum. It increased with the total concentration of drug and reached a plateau of approximately 1 nmol/mg of cell protein for an external concentration of 25 microM. The same experiment performed at 5 degrees C revealed a drastic decrease in uptake. The distribution of the drug among plasma membranes, endosomes, and lysosomes was then investigated after the separation of the postnuclear fractions by a Percoll gradient. After a 10-min incubation, AmB was found only in the plasma membrane fraction, regardless of the drug concentrations used (5 to 100 microM). After 60 min, at low drug concentrations (5 and 10 microM) AmB was found to be incorporated mainly in plasma and lysosomal fractions. At high concentrations (50 microM) AmB accumulated in endosomal fractions and plasma membranes. At intermediate concentrations (25 microM) AmB was distributed among the three fractions. When the same experiment was carried out at 5 degrees C, AmB was associated only with the plasma membrane even after 60 min, which was consistent with the absence of endocytotic process at low temperature. The effect of AmB on the endocytic process resulted in the increased uptake of sulforhodamine B, a fluid-phase marker of endocytosis, as well as by the accumulation of sulforhodamine in spots scattered in the cytoplasms of AmB-treated cells, in contrast to the accumulation around the nuclei observed in the control cells. These results are interpreted as indicating that AmB is internalized by the cells through endocytosis and that high concentrations of the drug block the fusion between endosomes and/or the fusion between endosomes and lysosomes.

The endocytic process in CHO cells, a toxic pathway of the polyene antibiotic amphotericin B