We recently reported that GS-X pump activity, as assessed by ATP-dependent transport of the glutathione-platinum complex and leukotriene C4, and intracellular glutathione (GSH) levels were remarkably enhanced in cis-diamminedichloroplatinum(II) (cisplatin)-resistant human leukemia HL-60 cells (Ishikawa, T., Wright, C. D., and Ishizuka, H. (1994) J. Biol. Chem. 269, 29085-29093). Now, using Northern hybridization and RNase protection assay, we provide evidence that the multidrug resistance-associated protein (MRP) gene, which encodes a human GS-X pump, is expressed at higher levels in cisplatin-resistant (HL-60/R-CP) cells than in sensitive cells, whereas amplification of the MRP gene is not detected by Southern hybridization. Culturing HL-60/R-CP cells in cisplatin-free medium resulted in reduced MRP mRNA levels, but these levels could be induced to rise within 30 h by cisplatin and heavy metals such as arsenite, cadmium, and zinc. The increased levels of MRP mRNA were closely related with enhanced activities of ATP-dependent transport of leukotriene C4 (LTC4) in plasma membrane vesicles. The glutathione-platinum (GS-Pt) complex, but not cisplatin, inhibited ATP-dependent LTC4 transport, suggesting that the MRP/GS-X pump transports both LTC4 and the GS-Pt complex. Expression of gamma-glutamylcysteine synthetase in the cisplatin-resistant cells was also co-induced within 24 h in response to cisplatin exposure, resulting in a significant increase in cellular GSH level. The resistant cells exposed to cisplatin were cross-resistant to melphalan, chlorambucil, arsenite, and cadmium. These observations suggest that elevated expression of the MRP/GS-X pump and increased GSH biosynthesis together may be important factors in the cellular metabolism and disposition of cisplatin, alkylating agents, and heavy metals.

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 ␥-...

Previous studies have demonstrated that treating cultured cells with cisplatin (CDDP) up-regulated the expression of glutathione (GSH) and its de novo rate-limiting enzyme glutamate-cysteine ligase (GCL), which consists of a catalytic (GCLC) and a modifier (GCLM) subunit. It has also been shown that many CDDPresistant cell lines exhibit high levels of GCLC/GCLM and GSH. Because the GSH system is the major intracellular regulator of redox conditions that serve as an important detoxification cytoprotector, these results have been taken into consideration that elevated levels of GCL/GSH are responsible for the CDDP resistance. In contrast to this context, we demonstrated here that overexpression of GSH by transfection with an expression plasmid containing the GCLC cDNA conferred sensitization to CDDP through up-regulation of human copper transporter (hCtr) 1, which is also a transporter for CDDP. Depleting GSH levels in these transfected cells reversed CDDP sensitivity with concomitant reduction of hCtr1 expression. Although rates of copper transport were also up-regulated in the transfected cells, these cells exhibited biochemical signature of copper deficiency, suggesting that GSH functions as an intracellular copper-chelator and that overexpression of GSH can alter copper metabolism. More importantly, our results reveal a new role of GSH in the regulation of CDDP sensitivity. Overproduction of GSH depletes the bioavailable copper pool, leading to upregulation of hCtr1 and sensitization of CDDP transport and cell killing. These findings also have important implications in that modulation of the intracellular copper pool may be a novel strategy for improving chemotherapeutic efficacy of platinumbased antitumor agents.

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There are several reports on Sonogashira couplings, but most of the reported reactions have employed aryl or alkenyl halides as coupling partners. Therefore, Sonogashira coupling is unsuitable for alkyl loadings, especially tertiary alkyl groups. In this research, we found that a copper catalyst is effective for a reaction between a terminal alkyne and an α-bromocarbonyl compound to form a quaternary carbon having alkynyl group at room temperature. Control experiments revealed that a copper acetylide is a key intermediate.

An intrapulmonary teratoma (IPT), multiloculated and bronchiectatic, with two polyps inside a 23-year-old man is reported. The IPT, a very rare benign cystic lesion, was communicating with segmental bronchus and was removed by a segmental resection from the upper lobe of the left lung. The teratoma contained various kinds of primordial derivatives, such as mesoderm, ectoderm, and endoderm. Though 65 cases of IPT have been reported in the literature (1839-1996), in the present case there were over 15 germ derivatives, the largest number reported to date. The tumor contained thymic tissue, apart from mediastinum, which may be significant in relation to the pathogenesis of IPT. Clinical manifestations, age, and gender distributions and the kind of germ cell derivatives are discussed.

No abstract

Iron is one of the most attractive catalysts, especially for aromatic C–H functionalizations. However, stoichiometric amounts of oxidants and strong carbanions are required, and C–H tertiary alkylation, especially with electron-deficient alkyl groups, is unexplored. In this paper, we describe the development of iron-catalyzed selective C–H tertiary alkylations with heteroaromatics, in which an iron salt acts as a single-electron source and enhances the reactivity of a tertiary alkyl radical generated from α-bromocarbonyl compounds. Our established methodology was demonstrated in the efficient synthesis of various quaternary carbon atoms under very simple conditions.