Abstract
The ATP-dependent glutathione S-conjugate export pump, named GS-X pump, has been shown to eliminate a potentially cytotoxic glutathione-platinum (GS.Pt) complex from tumor cells, thereby modulating glutathione (GSH)-associated resistance to cis-diamminedichloroplatinum(II) (cisplatin) (Ishikawa, T., and Ali-Osman, F. (1993) J. Biol. Chem. 268, 20116-20125). The present study provides evidence that the GS-X pump is functionally overexpressed in cisplatin-resistant human promyelocytic leukemia HL-60 (HL-60/R-CP) cells, in which the cellular GSH level was substantially enhanced. Indeed, the rate of ATP-dependent transport of the GS.Pt complex, measured with plasma membrane vesicles, was about 4-fold greater in HL-60/R-CP cells than in HL-60 cells. Three membrane proteins with apparent molecular masses of 200, 110, and 70 kDa were overexpressed in HL-60/R-CP cells, whereas P-glycoprotein (MDR1) was not immunologically detected in the membrane preparations from resistant and sensitive cells. Unlike in HL-60 cells, increased numbers of intracellular vesicles were observed in the cytoplasm of HL-60/R-CP cells. Fluorescence microscopy with syn-(CICH2,CH3)-1,5-diazabicyclo-[3.3.0]-octa-3,6-dione-2,8-dione (monochlorobimane) revealed that the fluorescent glutathione S-conjugate accumulated in intracellular vesicles of the cisplatin-resistant cells in an energy-dependent manner. The GS-X pump is suggested to contribute to vesicle-mediated excretion of GSH-drug conjugates from cells. In addition, both HL-60 and HL-60/R-CP cells underwent cell differentiation in response to 12-O-tetradecanoylphorbol-13-acetate, retinoic acid, and dimethyl sulfoxide, resulting in proliferation arrest as well as a remarkable decrease in the c-myc mRNA levels. After cell differentiation, a significant decrease was observed in the activity of ATP-dependent transport of the GS.Pt complex in membrane vesicles prepared from both HL-60/R-CP and HL-60 cells. These results suggest that the expression of the GS-X pump in both cisplatin-resistant and -sensitive cells is related to cell proliferation.
Highlights
TheATP-dependent glutathione S-conjugate export one S-conjugates, cysteinyl leukotrienes, and certain organic pump, namedGS-Xpump, has been shownto eliminate a anions from normal and cancer cells [1]
HLSO/RCP cells, whereas P-glycoprotein (MDR1) was drug conjugates are potentially cytotoxic suggests that the eliminot immunologically detected in the membrane preparations from resistantand sensitive cells.Unlike in HL-60 cells, increased numbers of intracellular vesicles were observed in the cytoplasm ofHL-GO/R-CP cells
We have recently shown that cisplatin reacts with intracellular GSH and that the resulting glutathione-platinum (GSPt) complex is actively exported from leukemia cells via the GS-X pump [7].Since the GSPtcomplex is a potential inhibitor for protein synthesis, the function of the GS-X pump is considered to modulate the resistance of human cancers to cisplatin [7, 8]
Summary
Cisplatin-resistant HL-60 Cells-A cisplatin-resistant variant of human leukemia HL-60 cells was established by maintaining the cells with increasing concentrations (0.5-17 w)of cisplatin over 10 months. In order to determine the ATP-dependent transport of the GSPt complex in cisplatin-sensitive and-resistant cells, plasma membrane vesicles were prepared from HL-60 and HL-GO/R-CP cells by sucrose-density gradient centrifugation. The rateof the ATP-dependent transportof the GSPtcomplex was rbout4-fold greater in the membrane vesicles from HL-GO/R-CP cells than in those from HL-60 cells, whereas the apparenKt ,,, value for the GSPt complex was unchanged (130 w for themembrane vesicle preparations from resistant and sensitive cells). ATP-dependent transport of LTC,, an endogenous substrate of the GS-X pump (11, was 5-fold higher in the membrane vesicles from HL-GO/R-CP cells than in thosefrom HL-60 cells (data not shown). These results suggest that the GS-X pump was functionally overexpressed in the cisplatin-resistanct ells
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