The expression of P-glycoprotein encoded by the multidrug resistance (MDR1) gene is associated with the emergence of the MDR phenotype in cancer cells. Human MDR1 and its rodent homolog mdr1a and mdr1b are frequently overexpressed in liver cancers. However, the underlying mechanisms are largely unknown. The hepatocarcinogen 2-acetylamino¯uorene (2-AAF) e ciently activates rat mdr1b expression in cultured cells and in Fisher 344 rats. We recently reported that activation of rat mdr1b in cultured cells by 2-AAF involves a cis-activating element containing a NFkB binding site located 7167 to 7158 of the rat mdr1b promoter. 2-AAF activates IkB kinase (IKK), resulting in degradation of IkBb and activation of NF-kB. In this study, we report that 2-AAF could also activate the human MDR1 gene in human hepatoma and embryonic ®broblast 293 cells. Induction of MDR1 by AAF was mediated by DNA sequence located at 76092 which contains a NF-kB binding site. Treating hepatoma cells with 2-AAF activated phosphoinositide 3-kinase (PI3K) and its downstream e ectors Rac1, and NAD(P)H oxidase. Transient transfection assays demonstrated that constitutively activated PI3K and Rac1 enhanced the activation of the MDR1 promoter by 2-AAF. Treatment of hepatoma cells with 2-AAF also activated another PI3K downstream e ector Akt. Transfection of recombinant encoding a dominant activated Akt also enhanced the activation of MDR1 promoter activation by 2-AAF. These results demonstrated that 2-AAF up-regulates MDR1 expression is mediated by the multiple e ectors of the PI3K signaling pathway.

It has been implicated that reactive oxygen species (ROS) play important roles in modulating tumor progression. However, the mechanisms by which redox-regulated tumor progression are largely unknown. We previously demonstrated that reduced intracellular redox conditions could be achieved in stably transfected small cell lung cancer cells with gamma-glutamylcysteine synthetase (gamma-GCSh) cDNA which encodes a rate-limiting enzyme in the biosynthesis of glutathione (GSH), a major physiological redox regulator. In the present study, using DNA microarray analyses, we compared the expression profiles between the gamma-GCSh-transfected cells and their nontransfected counterpart. We observed downregulation of several matrix metalloproteinases (MMPs), i.e., MMPI and MMP3, and MMP10 in the transfected cells. Dot blot and Northern blot hybridizations confirmed that, among the 18 MMP gene family members and four tissue inhibitors of matrix metalloprotein family (TIMP) analyzed, the expression levels of these three MMPs were consistently reduced. Transiently increased gamma-GCSh expression using tetracycline-inducible gamma-GCSh adenoviral expression system also showed down-regulation of MMP3 and MMP10, but not MMP1. Our results demonstrated that redox regulation of MMP1, MMP3 and MMP10 expression depend upon different modes of redox manipulation. These results bear implication that antioxidant modulation of antitumor progression may be contributed at least in part by the downregulation of a subset of metrix metalloproteins.

Gamma-glutamylcysteine synthetase (␥-GCS) is a heterodimer consisting of heavy (␥-GCSh) and light (␥-GCSl) subunits. ␥-GCS catalyzes the rate-limiting de novo biosynthesis of glutathione (GSH), an abundant physiological antioxidant that plays important roles for regulating oxidative stress. Expression of ␥-GCSh and ␥-GCSl are sensitive to oxidative stress. To investigate whether expression of ␥-GCS is correlated with tumor progression, we used immunohistochemical approaches to examine 16 human colorectal adenomas and resected 57 carcinomas from untreated patients. In adjacent normal colorectal epithelium, levels of ␥-GCSh expression were low. Strong cytoplasmic staining for ␥-GCSh was detected in 3 (18.8%) adenoma and 48 (84.2%) carcinomas. The frequency of ␥-GCSh expression in carcinoma was significantly higher than in adenoma (p<0.0001). We used RNase protation assay and Western blot to determine levels of ␥-GCSh mRNA and protein from 10 pairs of matched carcinomas with adjacent normal controls. Elevated expression of both ␥-GCSh mRNA and protein were found in 6 cases, suggesting that transcriptional and/or posttranscriptional regulation play an important role in the upregulation of ␥-GCS during colorectal carcinogenesis. We also examined the expression of another redox-regulated gene, multidrug resistance protein 1 (MRP1). Strong staining for MRP1 was detected in 1 (6.3%) adenoma and 40 (70.2%) carcinomas. The frequency of MRP1 expression in carcinoma was significantly higher than in adenoma ( p<0.0001). Nuclear p53 expression was detected in 30 (52.6%) of carcinomas. There is a significant correlation between ␥-GCSh and MRP1 expression (p‫)310.0؍‬ but not between ␥-GCSh and p53. Since ␥-GCS is a sensor of oxidative stress, these results are consistent with the notion that oxidative stress is associated with colorectal tumor progression. © 2002 Wiley-Liss, Inc. Key words: ␥-GCSh; MRP1; colorectal neoplasmGlutathione (GSH) plays an important role in regulating intracellular redox conditions. Biosynthesis of GSH involves 2 enzymatic reactions: the first reaction involves the synthesis of glutamylcysteine through ␥-peptide bond formation between glutamate and cysteine and is catalyzed by the rate-limiting enzyme gamma-glutamylcysteine synthetase. 1 The second reaction is catalyzed by GSH synthetase, which conjugates glycine to glutamylcysteine. The mammalian ␥-GCS holoenzyme is a heterodimer that consists of a 73 kDa heavy subunit (␥-GCSh) and a 28 kDa light subunit (␥-GCSl). [2][3][4][5] The steady-state levels of ␥-GCSh and ␥-GCSl mRNA are increased in cultured cells exposed to prooxidants (␤-naphthoflavone, menadione, tert-butylhydorquinone and pyrrolidine dithiocarbamate, 6,7 ), antitumor agents (cisplatin, 8 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3 nitrosourea 9 ), cytokines (interleukin 1␤ 10 ), etc. These cytotoxic agents are known to generate reactive oxygen species (ROS) and exert various degrees of oxidative stress to the cells. In all cases, induction of ␥-GCS is accompanied by the...

These results indicate that not only esophageal squamous cell carcinomas but also normal esophageal epithelium show strong telomerase activity. Thus, telomerase activity may not be a good marker for the detection of carcinoma in the esophagus.