Expression of the MRP1 gene encoding the GS-X pump and of the ␥-GCSh gene encoding the heavy (catalytic) subunit of the ␥-glutamylcysteine synthetase is frequently elevated in many drug-resistant cell lines and can be co-induced by many cytotoxic agents. However, mechanisms that regulate the expression of these genes remain to be elucidated. We report here that like ␥-GCSh, the expression of MRP1 can be induced in cultured cells treated with pro-oxidants such as tert-butylhydroquinone, 2,3-dimethoxy-1,4-naphthoquinone, and menadione. Intracellular reactive oxygen intermediate (ROI) levels were increased in hepatoma cells treated with tert-butylhydroquinone for 2 h as measured by flow cytometry using an ROI-specific probe, dihydrorhodamine 123. Elevated GSH levels in stably ␥-GCSh-transfected cell lines down-regulated endogenous MRP1 and ␥-GCSh expression. ROI levels in these transfected cells were lower than those in the untransfected control. In the cell lines in which depleting cellular GSH pools did not affect the expression of the MRP1 and ␥-GCSh genes, only minor increased intracellular levels of ROIs were observed. These results suggest that intracellular ROI levels play an important role in the regulation of MRP1 and ␥-GCSh expression. Our data also suggest that elevated intracellular GSH levels not only facilitate substrate transport by the MRP1/GS-X pump as previously demonstrated, but also suppress MRP1 and ␥-GCSh expression.Human MRP1 (multidrug resistance protein) encoded by MRP1 was first isolated by molecular cloning from doxorubicinselected multidrug-resistant lung cancer cells (Ref. 1; reviewed in Ref. 2). Studies using plasma membrane vesicles prepared from MRP1-overproducing cell lines demonstrated increased ATP-dependent, high-affinity transport activities of cysteinyl leukotrienes (e.g. LTC 4 ) 1 (3, 4). Deletion of homologous MRP1 alleles in mice results in impaired response to inflammatory stimulus in these animals because LTC 4 is a potent mediator of the inflammation reaction (5). These findings suggest that MRP1 encodes the previously described GS-X (ATP-dependent glutathione S-conjugate export) pump (6). In addition to transporting LTC 4 and its related glutathione S-conjugates, naturally occurring organic conjugates, including 17␤-estradiol (17␤-D-glucuronide), and bile salt conjugates, including 6␣-glucuronosylhydrodeoxychlorate and 3␣-sulfatolithocholytaurine, are also good substrates for the MRP1/GS-X pump (7-9). There are also reports suggesting that GSH may serve as a cofactor in MRP1/GS-X pump-mediated drug transport (8, 11). In addition, the MRP1/GS-X pump is responsible for the release of GSSG from cells. This active export of GSSG is considered to be an important mechanism to maintain the reduced status of intracellular thiols under oxidative stress (12, 13). These observations underscore the importance of GSH for the function of MRP1.Biosynthesis of GSH is controlled by multiple enzyme systems (reviewed in Refs. 14 and 15). The first step of GSH biosynthesis, catalyzed by ␥-...

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Previous reports suggest that resistance to mitoxantrone in different tumor cell lines is unrelated to the overexpression of pglycoprotein. In order to determine the role of p-glycoprotein in the cellular pharmacology of mitoxantrone flow cytometry and confocal microscopy were used to study two human myeloid leukemia cell lines selected for resistance to mitoxantrone (HL-60MX2) and doxorubicin (HL-60DOX). To optimize the detection of intracellular mitoxantrone, we determined the maximum excitation (607 nm) and emission (684 nm) wavelength by fluorescence spectroscopy. The modified flow cytometric conditions using 568.2 nm laser emission for excitation and a 620 nm long pass filter for fluorescence collection resulted in a 1-log increase in sensitivity, compared with standard 488-nm laser excitation. Uptake and retention of mitoxantrone in the presence of verapamil, a calcium channel blocker known to inhibit p-glycoprotein, were analyzed. Our results showed no change in uptake and retention of the drug in pglycoprotein-negative mitoxantrone-resistant HL-60MX2 cells and in its sensitive parental line, HL-60s. In contrast, 3.1-and 2.4-fold increases were found in uptake and retention of mitoxantrone in p-glycoprotein-positive cells (HL-60DOX) incubated with verapamil. Confocal microscopy of intracellular drug distribution demonstrated reduced nuclear uptake, which could be reversed by verapamil, in HL-60DOX. A characteristic punctate pattern was observed for the intracytoplasmic drug distribution in HL-60DOX and HL-60MX2 cells and was partially modified by the presence of verapamil in HL-60DOX cells. Verapamil increased cytotoxicity of mitoxantrone two-fold in HL-60DOX cells, 1.4-fold in HL-60MX2, and had no effect in HL-60s. Our study demonstrates that the cellular pharmacology of mitoxantrone is affected by p-glycoprotein and can be reversed at least in part by verapamil. Other mechanisms of resistance however, seem to play a determinant role in the modulation of mitoxantrone cytotoxicity.

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The cytoplasmic progesterone receptor form human uterus has been purified to apparent homogeneity by a combination of ammonium sulfate fractionation and affinity chromatography. Affinity resins prepared by conventional means were compared to those prepared by a modified method. The latter give more reproducible results. A consistent finding was that low capacity resins gave the highest fold purification of the receptor. The pure receptor sedimented at 3.6 S on sucrose density gradient centrifugation, was eluted as a single band by 0.2 M KCl from DEAE-cellulose, and migrated as a single band of molecular weight 42 000 on NaDodSO4-polyacrylamide gel electrophoresis. Molecular weight determinations, obtained from Strokes' radii and sucrose gradient centrifugation, the receptors' behavior on ion exchange resins, and hormone binding specificity were all similar to those of the receptor found in crude cytosol. When the crude cytosol receptor was photoaffinity labeled by using 3H-labeled 17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione followed by NaDodSO4-polyacrylamide gel electrophoresis, only protein of Mr 42 000 was labeled. This is consistent with our previous findings that alkylation of the pure receptor using 11-deoxycorticosterone bromo[3H]acetate showed labeling of a single protein of Mr 42 000. These properties confirm that the identity and integrity of the receptor have been maintained throughout its purification.