INTRACELLULAR-LOCALIZATION, VESICULAR ACCUMULATION AND KINETICS OF DAUNORUBICIN IN SENSITIVE AND MULTIDRUG-RESISTANT GASTRIC-CARCINOMA EPG85-257 CELLS

In the human gastric carcinoma cell line EPG85-257P (parent) induction of resistance to daunorubicin (DAU) was achieved by selection with st epwise increased concentrations of the drug. The new vairant was named EPG85-257DAU and was shown to overexpress the mdr1 gene product 170 k Da P-glycoprotein (P-Gp) as demonstrated by immunocytochemistry and md r1-specific RT-PCR. To investigate the intracellular pathway of DAU th e subcellular distribution of this autofluorescent drug was studied in the resistant cells and compared to its chemosensitive counterpart EP G85-257P. When sensitive cells were exposed to DAU the drug rapidly ac cumulated in the nucleus until cell death. No redistribution of DAU to the cytoplasm was observed. In resistant cells exposed to the drug DA U also accumulated in the nucleus but to a lesser extent than in paren t cells. Following exposure, nuclear fluorescence was observed to decr ease over a time period of up to 48 h. Six hours after DAU exposure fo rmation of fluorescent vesicle formation started in the perinuclear re gion and increased continously. After 48 h nuclear fluorescence was no longer detectable and DAU was located exclusively in vesicles. During this period the vesicles moved from the region of origin to the cell periphery. A pulse chase experiment showed, that vesicles may contain DAU derived from the nucleus. Treatment of EPG85-257DAU cells with DAU in conjunction with the chemosensitizer cyclosporin A (CsA) increased nuclear fluorescence without impairing vesicle formation. Disruption of microtubules by nocodazole led to an accumulation of vesicles in th e perinuclear region indicating that microtubules are involved in vesi cular transport. Treatment of EPG85-257DAU cells with the actin disrup ter cytochalasin B led to accumulation of vesicles in the cell periphe ry indicating that actin may be involved in exocytosis. Uptake and eff lux of DAU and rhodamin (RH) were determined in sensitive and resistan t cells using a fluorescence activated cell sorter. Uptake of both com pounds was distinctly lower in resistant than in sensitive cells. When resistant cells preloaded for 2 h with RH subsequently were incubated in drug free medium the substance was rapidly released indicating tra nsmembrane transport by P-Gp. In contrast, despite expression of P-Gp in resistant cells no considerable release of DAU was observed for up to 2 h under the same experimental protocol. This indicates that in re sistant cells intracellular DAU at least in part may be inaccessible f or P-Gp and that vesicular drug transport appears to contribute to DAU resistance by removing intracellular DAU via exocytosis.