CHARACTERIZATION OF H-ATPASE-DEPENDENT ACTIVITY OF MULTI DRUG RESISTANCE-ASSOCIATED PROTEIN IN HOMOHARRINGTONINE-RESISTANT HUMAN LEUKEMIC K562 CELLS()

Multidrug resistance (MDR), caused by overexpression of either P-glyco protein or the multidrug resistance-associated protein (MRP), is chara cterized by a decreased cellular drug accumulation due to an enhanced drug efflux. Many studies on cells overexpressing MRP and/or Pgp, have shown a concentration of the drug inside cytoplasmic acidic vesicles followed by an exocytotic process. In this study, we examined the effe cts of 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole or NBD (a H+-ATPase pu mp inhibitor), buthionine sulphoximine or BSO tan inhibitor of glutath ione (GSH) biosynthesis) and verapamil or VPL (a calcium channel block er) on the subcellular distribution of daunorubicin or DNR in K562 cel ls overexpressing MRP (K-H30) and Pgp (K-H300) and A549 cells overexpr essing spontaneously MRP. Nucleo-cytoplasmic distribution of DNR was c arried out using scanning confocal microspectrofluorometry. This techn ique allows determination of nuclear accumulation of anthracyclines. O ur results show that nuclear accumulation of DNR in K-H30 and A549 cel ls was increased by NBD, BSO and VPL while in K-H300 cells, only VPL w as able to increase nuclear accumulation of DNR. Similarly, NBD, BSO a nd VPL could reverse DNR resistance in K-H30 cells whereas, in K-H300 cells, only VPL increased the sensitivity of these cells. These data s uggest a requirement for GSH in MRP-mediated resistance and suggest th at even if vesicular sequestration can happen in cells overexpressing MRP and Pgp proteins, probably only the MRP protein is able to extrude the drug through intracellular vesicles and efflux. Finally, NBD and BSO might be a useful agents in facilitating discrimination between Pg p and MRP phenotypes and prognosis in patients.