Exposure of human and rodent cells to a wide variety of chemoprotective compounds confers resistance against a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of protective enzymes like glutathione S-transferases (GST). Antioxidant responsive elements (AREs) mediate the transcriptional induction of a battery of genes which comprise much of this chemoprotective response system. Past studies identified a necessary ARE ''core'' sequence of RTGACnnnGC, but this sequence alone is insufficient to mediate induction. In this study, the additional sequences necessary to define a sufficient, functional ARE are identified through systematic mutational analysis of the murine GST Ya ARE. Introduction of the newly identified necessary nucleotides into the regions flanking a nonresponsive, ARE-like, GST-Mu promoter sequence produced an inducible element. A screen of the GenBank database with the newly identified ARE consensus identified 16 genes which contained the functional ARE consensus sequence in their promoters. Included within this group was an ARE sequence from the murine ferritin-L promoter that mediated induction when tested. In an electrophoretic mobility-shift assay, the ferritin-L ARE was bound by ARE-binding protein 1, a protein previously identified as the likely mediator of the chemoprotective response. A three-level ARE classification system is presented to account for the distinct induction strengths observed in our mutagenesis studies. A model of the ARE as a composite regulatory site, where multiple transcription factors interact, is presented to account for the complex characteristics of AREmediated chemoprotective gene expression.

Methylglyoxal is an ␣-ketoaldehyde and dicarbonyl formed in cells as a side product of normal metabolism. Endogenously produced dicarbonyls, such as methylglyoxal, are involved in numerous pathogenic processes in vivo, including carcinogenesis and advanced glycation end-product formation; advanced glycation end-products are contributors to the pathophysiology of aging and chronic diabetes. Despite recent advances in understanding of the systemic effects of methylglyoxal, the full significance of this compound remains unknown. Herein we provide evidence that the majority of the methylglyoxal present in vivo is bound to biological ligands. The basis for our finding is an experimental approach that provides a measure of the bound methylglyoxal present in living systems, in this instance Chinese hamster ovary cells; with our approach, as much as 310 M methylglyoxal was detected, 100-to 1,000-fold more than observed previously in biological systems. Several artifacts were considered before concluding that the methylglyoxal was associated with cellular structures, including phosphate elimination from triose phosphates, carbohydrate degradation under the assay conditions, and interference from the derivatizing agent used as part of the assay procedure. A major source of the recovered methylglyoxal is most probably modified cellular proteins. With methylglyoxal at about 300 M, 0.02% of cellular amino acid residues could be modified. As few as one or two ''hits'' with methylglyoxal per protein molecule have previously been reported to be sufficient to cause protein endocytosis and subsequent degradation. Thus, 5-10% of cellular proteins may be modified to physiologically significant levels.

Nrf2 is a key transcription factor in the cellular response to oxidative stress. In this study we first identify two phosphorylated forms of endogenous human Nrf2 after chemically-induced oxidative stress and provide evidence that protein kinase CK2-mediated sequential phosphorylation plays potential role in Nrf2 activation and degradation. Human Nrf2 has a predicted molecular mass of 66 kDa. However, immunoblots showed that two bands at 98 and 118 kDa, which are identified as phosphorylated forms, are increased in response to Nrf2 inducers. In addition, human Nrf2 was found to be a substrate for CK2 which mediated two steps of phosphorylation, resulting in two forms of Nrf2 migrating with differing Mr at 98 kDa (Nrf2-98) and 118 kDa (Nrf2-118). Our results support a role in which calmodulin binding regulates CK2 activity, in that cold (25 °C) in Ca 2+ -free media (cold/Ca 2+ -free) decreased both cellular calcium levels and CK2-calmodulin binding and induced Nrf2-118 formation, the latter of which was prevented by CK2 specific inhibitors. Gel-shift assays showed that the Nrf2-118 generated under cold/Ca 2+ -free conditions does not bind to the antioxidant response element, indicating that Nrf2-98 has transcriptional activity. In contrast, Nrf2-118 is more susceptible to degradation. These results provide evidence for phosphorylation by CK2 as a critical controlling factor in Nrf2-mediated cellular antioxidant response.

Phase II drug-metabolizing enzymes, such as glutathione S-transferase and quinone reductase, play an important role in the detoxification of chemical carcinogens. The induction of these detoxifying enzymes by a variety of agents occurs at the transcriptional level and is regulated by a cis-acting element, called the antioxidant response element (ARE) or electrophile-response element. In this study, we identified a signaling kinase pathway that negatively regulates ARE-mediated gene expression. Treatment of human hepatoma HepG2 and murine hepatoma Hepa1c1c7 cells with tert-butylhydroquinone (tBHQ) stimulated the activity of p38, a member of mitogen-activated protein kinase family. Inhibition of p38 activation by its inhibitor, SB203580, enhanced the induction of quinone reductase activity and the activation of ARE reporter gene by tBHQ. In contrast, SB202474, a negative analog of SB203580, had little effect. Consistent with this result, interfering with the p38 kinase pathway by overexpression of a dominant-negative mutant of p38 or MKK3, an immediate upstream regulator of p38, potentiated the activation of the ARE reporter gene by tBHQ, whereas the wild types of p38 and MKK3 diminished such activation. In addition, inhibition of p38 activity augmented the induction of ARE reporter gene activity by tert-butylhydroxyanisole, sulforaphane, and ␤-naphthoflavone. Thus, p38 kinase pathway functions as a negative regulator in the AREmediated induction of phase II detoxifying enzymes.

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Expression of PDGF-B, the gene encoding the plateletderived growth factor B chain, has been implicated as a participant in an autocrine growth loop in the human osteosarcoma cell line U2-OS. In previous work, we identified a primary site in the PDGF-B promoter, the SIS proximal element (SPE), which is critical for transcription of the PDGF-B gene in U2-OS cells. We also identified Sp1 as one of the SPE-binding proteins in U2-OS nuclear extracts. In the present work, we have identified another SPE-binding protein to be Sp3. Gel mobility shift assays showed that both Sp1 and Sp3 require the CACCC motif within the SPE for binding. In vitro transcription assays showed that Sp1 or/and Sp3 is necessary for transcription of the PDGF-B gene. Cotransfection experiments functionally demonstrated that Sp1 and Sp3 can independently or additively activate the PDGF-B promoter through the SPE as well as a synthetic promoter. However, the CACCC motif within the SPE is not the only site within the minimal PDGF-B promoter through which Sp1/Sp3 acts; additional nested deletion analyses showed that multiple cis-acting elements within the minimal promoter are required for full level transcription of the PDGF-B gene in U2-OS cells. Platelet-derived growth factor (PDGF)1 is a potent mitogen and chemotactant for cells that express functional PDGF receptors, typically cells of mesenchymal origin (Antoniades, 1991;Deuel et al., 1982). It is a homo-or heterodimeric protein consisting of two related polypeptide chains, A and B (Johnsson et al., 1982). The three isoforms of PDGF (AA, AB, and BB) stimulate biological responses by binding to two cell surface PDGF receptors, ␣ and ␤ (Claesson-Welsh, 1994).A functional, contributory role for PDGF-B in the development and maintenance of cancer cells is supported by several observations: (i) the B chain gene of PDGF was identified as the cellular homolog of the simian sarcoma virus oncogene, v-sis (Doolittle et al., 1983;Waterfield et al., 1983), (ii) when under the control of a strong promoter, both v-sis and its cellular homolog PDGF-B caused the transformation of mouse 3T3 cells or human fibroblasts Clarke et al., 1984;Gazit et al., 1984;Stevens et al., 1988), (iii) the cellular PDGF-B gene has been shown to be constitutively expressed in a large percentage of human tumor cells, cells of both mesenchymal and epithelial origin, whereas it was not detectably expressed in the normal counterparts of these cells (Eva et al., 1982;Peres et al., 1987;Maxwell et al., 1990), and (iv) expression of a recombinant, dominant-negative mutant of PDGF-B in PDGF-B-expressing astrocytoma cells resulted in a substantial reduction in the tumorigenic growth of the cells (Shamah, 1993). The above evidence, especially the fact that high level expression of the cellular form of the sis oncogene is sufficient for oncogenesis, implies that loss of its transcriptional regulation provides a contributory step in neoplastic transformation.In U2-OS, a human osteosarcoma cell line, coexpression of the PDGF-B gene as well as ...

Increased levels of glutathione S-transferase (GST; RX:glutathione R-transferase; EC 2.5.1.18) mRNA, protein, and activity in tumor biopsy samples and in drugresistant cultured cells are associated with resistance to anticancer drugs. We report that each of three full-length cloned GST cDNAs, that for X7 (acidic), Ya (basic), and Yb1 (neutral), can confer drug resistance when expressed in cultured mammalian cells. In one approach, stably transfected mouse C3H/10T½ cells that express GST ar, Ya, or Ybj were cloned and analyzed for drug resistance in colony-forming assays. Transiently transfected COS cells that were sorted on a fluorescence-activated cell sorter were used in the second approach to avoid interclonal variation in factors other than the recombinant GST and to show that reversion of transient GST expression correlated with loss of drug resistance. A sorting technique, developed to separate the 20% of the electroporated COS cell population that transiently expressed GST ir, Ya, or Yb, from the nonexpressing population, was based on a GST-catalyzed intracellular cotijugation of glutathione to the fluorescent labeling reagent monochlorobimane. GST Ya conferred the greatest increase in resistance to chlorambucil and melphalan (1.3-to 2.9-fold), Yb, conferred the greatest increase in resistance to cisplatin (1.5-fold), and ir conferred the greatest increase in resistance to a racemic mixture of 7fi,8a-dihydroxy-9a, 10ar-epoxy-7,8,9,10-tetrahydrobenzo[alpyrene and 7a,8p-dihydroxy-9fi,108-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and doxorubicin (1.5-and 1.3-fold) relative to controls. These resistance values to alkylating agents are commensurate with values observed clinically. Cytotoxicity curves representing recombinant GST' populations were significantly different from their controls with P values ranging from 0.005 to 0.0001. No resistance to vinblastine was detected. Conferred drug resistance was proportional to the magnitude of GST Ya expression, and reversion of transient expression in GST Ya+ COS cell clones to a GST Ya-phenotype was associated with total loss of drug resistance.A major obstacle to the effective treatment of cancer is the inability to eliminate tumor cell subpopulations that have an intrinsic or acquired resistance to anticancer drugs. Various cellular defense mechanisms have developed that may be important in conferring protection against cytotoxicity induced by anticancer drugs. Decreased intracellular drug accumulation, elevated DNA repair rates, altered topoisomerase activity, and enhanced drug detoxification are associated with development of the drug-resistant phenotype (1-3).The glutathione S-transferases (GST; RX:glutathione Rtransferase; EC 2.5.1.18) are a family of dimeric isozymes that can confer resistance to (detoxify) xenobiotic molecules by several mechanisms (1-3). In one mechanism, GSTs catalyze the covalent addition of the tripeptide glutathione to electrophilic molecules, including products of the cytochrome P-450 mixed-function oxidases, yielding conjugat...