Glutathione (GSH) is an abundant cellular non-protein sulfhydryl that functions as an important protectant against reactive oxygen species and electrophiles, is involved in the detoxification of xenobiotics, and contributes to the maintenance of cellular redox balance. The rate-limiting enzyme in the de novo synthesis of glutathione is ␥-glutamylcysteine synthetase (GCS), a heterodimer consisting of heavy and light subunits expressing catalytic and regulatory functions, respectively. Exposure of HepG2 cells to ␤-naphthoflavone (␤-NF) resulted in a time-and dose-dependent increase in the steady-state mRNA levels for both subunits. In order to identify sequences mediating the constitutive and induced expression of the heavy subunit gene, a series of deletion mutants created from the 5 -flanking region (؊3802 to ؉465) were cloned into a luciferase reporter vector (pGL3-Basic) and transfected into HepG2 cells. Constitutive expression was maximally directed by sequences between ؊202 and ؉22 as well as by elements between ؊3802 to ؊2752. Glutathione (L-␥-glutamyl-L-cysteinyl-glycine, GSH), 1 a nonprotein sulfhydryl compound present in millimolar concentrations in virtually all cells, serves a myriad of cellular functions and plays a prominent role as an intracellular protectant (1, 2). GSH is an effective oxygen radical scavenger and serves as a critical co-factor in peroxide detoxification via a reaction catalyzed by glutathione peroxidase. Furthermore, conjugation with GSH is an integral step in the detoxification and elimination of diverse classes of toxic chemical compounds. The formation of hydrophilic glutathionyl conjugates is catalyzed by glutathione S-transferases, a family of isozymes that mediate the conjugation reaction in a substrate-dependent fashion (3). Long the object of interest from a toxicology perspective, the protective properties of GSH have assumed even further significance since GSH not only plays a critical role in protection of normal cells, but it has recently been implicated in protection of neoplastic cells from a number of chemotherapeutic agents that exert their cytotoxic effects via generation of reactive oxygen species or production of electrophilic intermediates (4, 5). The augmentation of GSH and GSH-related detoxification systems has also engendered considerable interest as a possible approach for the chemoprevention of cancer. Many chemical chemopreventive agents have been shown to exert an effect on GSH homeostasis or on other elements of GSH detoxification pathways (6 -8).Exposure of cells to a number of xenobiotic agents has been demonstrated to result in an increase in the total intracellular GSH content. In several cases (9 -16) where it has been examined, the increase in GSH has been attributed to an

During fetal prostate development, Sonic hedgehog (Shh) expression by the urogenital sinus epithelium activates Gli-1 expression in the adjacent mesenchyme and promotes outgrowth of the nascent ducts. Shh signaling is down-regulated at the conclusion of prostate ductal development. However, a survey of adult human prostate tissues reveals substantial levels of Shh signaling in normal, hyperplasic, and malignant prostate tissue. In cancer specimens, the Shh expression is localized to the tumor epithelium, whereas Gli-1 expression is localized to the tumor stroma. Tight correlation between the levels of Shh and Gli-1 expression suggests active signaling between the tissue layers. To determine whether Shh-Gli-1 signaling could be functionally important for tumor growth and progression, we performed experiments with the LNCaP xenograft tumor model and demonstrated that: 1). Shh expressed by LNCaP tumor cells activates Gli-1 expression in the tumor stroma, 2). genetically engineered Shh overexpression in LNCaP cells leads to increased tumor stromal Gli-1 expression, and 3). Shh overexpression dramatically accelerates tumor growth. These data suggest that hedgehog signaling from prostate cancer cells to the stroma can elicit the expression of paracrine signals, which promote tumor growth.

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gamma-Glutamylcysteine synthetase (GCS), the rate-limiting enzyme in the de novo synthesis of GSH, is a heterodimer, consisting of a catalytic (GCSh) and a regulatory subunit (GCSl). We previously demonstrated that the constitutive and beta-naphthoflavone (beta-NF)-induced expression of the GCSh gene is mediated by a distal antioxidant response element (ARE), ARE4, located 3.1 kb upstream of the transcriptional start site [Mulcahy, Wartman, Bailey and Gipp (1997) J. Biol. Chem. 272, 7445-7454]. ARE4 consists of a consensus ARE sequence (5'-GTGACTCAGCG-3') containing an embedded PMA-responsive element (TRE, underlined). The relative significance of the two overlapping response elements to constitutive and beta-NF-induced expression of the GCSh gene was determined by mutational analyses. The internal activator protein-1 (AP-1)-binding sequence mediated constitutive expression of promoter/reporter transgenes, but was not required for beta-NF responsiveness. In gel-shift experiments, the TRE was necessary for binding of proteins from nuclear extracts prepared from untreated HepG2 cells. In contrast, induction by beta-NF was dependent on an intact ARE sequence, particularly the terminal GC box of ARE4. The GC box of ARE4 was shown to be essential for both basal and beta-NF-induced expression of reporter constructs. This element also influenced binding of nuclear proteins to ARE4, specifically in extracts isolated from beta-NF-treated HepG2 cells. Because previous studies indicated that ARE4 may co-operate with a separate putative ARE, the role of the neighbouring sequence (ARE3), located 34 bases downstream of ARE4, was also evaluated. Mutation of this element within a GCSh promoter/reporter did not modify the basal or beta-NF-induced expression of the transgene, demonstrating that ARE3 does not influence the constitutive or beta-NF-induced expression of the GCSh gene.

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