Hurwitz S J, Terashima M, Mizunuma N, Slapak C A
Division of Cancer Pharmacology, The Dana-Farber Cancer Institute and The Harvard Medical School, Boston, MA, USA.
Blood. 1997 May 15;89(10):3745-54.
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.
U-A10细胞系是一种经阿霉素筛选的人U-937髓系白血病细胞变体，表现出蒽环类药物重新分布到一个扩大的囊泡区室中。用pH敏感染料LysoSensor yellow/blue DND-160进行活细胞染色，证实了该区室的酸性性质。通过免疫荧光分析对囊泡区室进行鉴定。对LAMP-1和LAMP-2抗原进行染色显示，这些囊泡是增大的溶酶体，偏心地位于U-A10细胞的细胞核附近。相比之下，多药耐药相关蛋白和P-糖蛋白的表达主要在耐药细胞的质膜上观察到。使用放射性标记药物对柔红霉素在细胞区室中的积累进行定量。将细胞暴露于3[H]-柔红霉素，然后分离完整细胞核，结果显示U-A10细胞的细胞核积累的蒽环类药物比U-937细胞的细胞核少两倍至三倍。然而，当首先分离细胞核，然后暴露于3[H]-柔红霉素时，未检测到净核药物积累有显著差异。将从U-A10和U-937细胞制备的胞质体暴露于3[H]-柔红霉素以测量细胞质药物积累。在外部柔红霉素浓度为100 ng/mL或更高时，U-A10细胞的胞质体比U-937细胞的胞质体积累的柔红霉素明显更多。此外，用溶酶体促渗剂氯喹进行的研究表明，与亲代U-937细胞相比，U-A10细胞积累的氯喹多两倍，且对该药物的敏感性增强两倍。荧光显微镜检查显示，氯喹影响U-A10细胞中囊泡蒽环类药物的隔离，同时柔红霉素核荧光相应增加。虽然氯喹没有改变亲代细胞中蒽环类药物的细胞毒性，但它恢复了U-A10细胞对柔红霉素和阿霉素的敏感性。综上所述，这些研究表明U-A10细胞表现出溶酶体区室的重新分布。药物被困在扩大的酸性囊泡区室中导致核药物积累减少和细胞毒性降低。溶酶体促渗剂，如氯喹，作为这种获得性耐药表型的调节剂值得进一步研究。
