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Vesicular anthracycline accumulation in doxorubicin-selected U-937 cells:
participation of lysosomes
SJ Hurwitz, M Terashima, N Mizunuma and CA Slapak
Division of Cancer Pharmacology, The Dana-Farber Cancer Institute and The
Harvard Medical School, Boston, MA, USA.
The U-A10 cell line, a doxorubicin-selected variant of human U-937 myeloid
leukemia cells, exhibits a redistribution of anthracyclines into a expanded
vesicular compartment. The acidic nature of this compartment was confirmed
by vital staining with a pH sensitive dye, LysoSensor yellow/blue DND-160.
Identification of the vesicular compartment was performed by
immunofluorescence analysis. Staining for the LAMP-1 and LAMP-2 antigens
showed that the vesicles are enlarged lysosomes that are eccentrically
placed near the nucleus of U-A10 cells. By contrast, the expression of the
multidrug resistance- associated protein and the P-glycoprotein was
observed predominately on the plasma membrane of the drug-resistant cells.
The accumulation of daunorubicin into cellular compartments was quantified
using radiolabeled drug. Exposing cells to 3[H]-daunorubicin and then
isolating intact nuclei showed that nuclei from U-A10 cells accumulated
twofold to threefold less anthracycline than nuclei from U-937 cells.
However, when nuclei were isolated first and then exposed to 3[H]-
daunorubicin, little difference in net nuclear drug accumulation was
detected. Cytoplasts prepared from U-A10 and U-937 cells were exposed to
3[H]-daunorubicin to measure cytoplasmic drug accumulation. At external
daunorubicin concentrations of 100 ng/mL or higher, cytoplasts from U-A10
cells accumulated significantly more daunorubicin than cytoplasts from
U-937 cells. Moreover, studies with the lysosomotropic agent chloroquine
showed that U-A10 cells accumulated twofold more chloroquine and showed
twofold enhanced sensitivity to this agent as compared with parental U-937
cells. Fluorescence microscopy showed that chloroquine affects vesicular
anthracycline sequestration in U-A10 cells with an associated increase in
daunorubicin nuclear fluorescence. Although chloroquine did not alter
anthracycline cytotoxicity in parental cells, it restored daunorubicin and
doxorubicin sensitivity to U-A10 cells. Taken together, these studies
demonstrate that U-A10 cells exhibit a redistribution of the lysosomal
compartment. The trapping of drug into an expanded acidic vesicular
compartment results in decreased nuclear drug accumulation and decreased
cytotoxicity. Lysosomotropic agents, such as chloroquine, warrant further
study as modulators of this acquired drug-resistance phenotype.
Volume 89,
Issue 10,
pp. 3745-3754,
05/15/1997
Copyright © 1997 by The American Society of Hematology
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