Carcinogenesis is a multistep process involving mutation and the subsequent selective clonal expansion of the mutated cell. Chemical and physical agents including those that induce reative oxygen species can induce and/or modulate this multistep process. Several modes of action by which carcinogens induce cancer have been identified, including through production of reactive oxygen species (ROS). Oxidative damage to cellular macromolecules can arise through overproduction of ROS and faulty antioxidant and/or DNA repair mechanisms. In addition, ROS can stimulate signal transduction pathways and lead to activation of key transcription factors such as Nrf2 and NF-kB. The resultant altered gene expression patterns evoked by ROS contribute to the carcinogenesis process. Recent evidence demonstrates an association between a number of single nucleotide polymorphisms (SNPs) in oxidative DNA repair genes and antioxidant genes with human cancer susceptibility. These aspects of ROS biology will be discussed in the context of their relationship to carcinogenesis.

Transforming growth factor-β (TGF-β) exerts its effects on cell proliferation, differentiation and migration in part through its modulation of extracellular matrix components, such as fibronectin and plasminogen activator inhibitor-1 (PAI-1). Although the SMAD family of proteins recently has been shown to be a key participant in TGF-β signaling, other signaling pathways have also been shown to be activated by TGF-β. We report here that c-Jun N-terminal kinase (JNK), a member of the MAP kinase family, is activated in response to TGF-β in the human fibrosarcoma HT1080-derived cell line BAHgpt. Stable expression of dominant-negative forms of JNK1 and MKK4, an upstream activator of JNK, results in loss of TGF-β-stimulated fibronectin mRNA and protein induction, while having little effect on TGF-β-induced levels of PAI-1. The human fibronectin promoter contains three CRE elements, one of which has been shown to bind a c-Jun-ATF-2 heterodimer. Utilizing a GAL4 fusion trans-reporting system, we demonstrate a decrease in transactivating potential of GAL4-c-Jun and GAL4-ATF-2 in dominant-negative JNK1-and MKK4-expressing cells. Finally, we show that TGF-β-induced fibronectin synthesis is independent of Smad4. These results demonstrate that TGF-β-mediated fibronectin induction requires activation of JNK which in turn modulates the activity of c-Jun and ATF-2 in a Smad4-independent manner.

Using a genetic complementation approach we have identi®ed disabled-2 (Dab2), a structural homolog of the Dab1 adaptor molecule, as a critical link between the transforming growth factor b (TGFb) receptors and the Smad family of proteins. Expression of wildtype Dab2 in a TGFb-signaling mutant restores TGFb-mediated Smad2 phosphorylation, Smad translocation to the nucleus and Smad-dependent transcriptional responses. TGFb stimulation triggers a transient increase in association of Dab2 with Smad2 and Smad3, which is mediated by a direct interaction between the N-terminal phosphotyrosine binding domain of Dab2 and the MH2 domain of Smad2. Dab2 associates with both the type I and type II TGFb receptors in vivo, suggesting that Dab2 is part of a multiprotein signaling complex. Together, these data indicate that Dab2 is an essential component of the TGFb signaling pathway, aiding in transmission of TGFb signaling from the TGFb receptors to the Smad family of transcriptional activators.

The adaptor molecule Disabled-2 (Dab2) has been shown to link cell surface receptors to downstream signaling pathways. Using a small-pool cDNA screening strategy, we identify that the N-terminal domain of Dab2 interacts with Dishevelled-3 (Dvl-3), a signaling mediator of the Wnt pathway. Ectopic expression of Dab2 in NIH-3T3 mouse ®broblasts attenuates canonical Wnt/b-catenin-mediated signaling, including accumulation of b-catenin, activation of b-catenin/ T-cell-speci®c factor/lymphoid enhancer-binding factor 1-dependent reporter constructs, and endogenous cyclin D1 induction. Wnt stimulation leads to a timedependent dissociation of endogenous Dab2±Dvl-3 and Dvl-3±axin interactions in NIH-3T3 cells, while Dab2 overexpression leads to maintenance of Dab2±Dvl-3 association and subsequent loss of Dvl-3±axin interactions. In addition, we ®nd that Dab2 can associate with axin in vitro and stabilize axin expression in vivo. Mouse embryo ®broblasts which lack Dab2 exhibit constitutive Wnt signaling as evidenced by increased levels of nuclear b-catenin and cyclin D1 protein levels. Based on these results, we propose that Dab2 functions as a negative regulator of canonical Wnt signaling by stabilizing the b-catenin degradation complex, which may contribute to its proposed role as a tumor suppressor. IntroductionThe Wnt family of secreted glycoproteins play key roles in embryonic development, regulating cell proliferation, motility and cell fate (Cadigan and Nusse, 1997;Dale, 1998) and in adult tissues where aberrant Wnt signaling has been shown to contribute to a variety of human cancers (Polakis, 2000;Bienz and Clevers, 2000). Wnt ligand binding to its cognate receptors can stimulate several distinct signaling pathways including the canonical Wnt±b-catenin and non-canonical planar cell polarity± convergent extension (PCP±CE) pathways. Required for activation of both of these pathways is the common mediator dishevelled (Dvl) which interprets signals from various receptors and transmits them to different effector molecules. In the canonical pathway, activation of the frizzled and LRP5/6 co-receptors results in recruitment of Dvl which relays the signal to a complex composed of adenomatous polyposis coli (APC), axin, glycogen synthase kinase 3b (GSK3b) and b-catenin (Polakis, 2000;Wharton, 2003). In the absence of Wnt signaling, b-catenin is phosphorylated on N-terminal serine and threonine residues, targeting it for degradation by the ubiquitin±proteasome pathway (Kitagawa et al., 1999). In the presence of Wnt, association of Dvl with axin prevents GSK3b from phosphorylating b-catenin, leading to stabilization of b-catenin and its translocation to the nucleus. In the nucleus, b-catenin complexes with members of the T-cell-speci®c factor/lymphoid enhancerbinding factor 1 (TCF/LEF-1) transcription factor family to regulate target genes which include c-myc and cyclin D1 (He et al., 1998;Shtutman et al., 1999). PCP signaling, responsible for proper orientation of photoreceptor cells in Drosophila, and vertebrate CE, responsi...

Thioltransferase from human red blood cells (HRBC TTase), coupled to GSSG reductase, catalyzed glutathione (GSH)-dependent reduction of prototype substrates hydroxyethyl disulfide (HEDS) and sodium S-sulfocysteine as well as of other homo- and heterodisulfides, including the protein mixed disulfide albumin-S-S-cysteine. Whereas apparent KM values for the substrates varied over more than a 20-fold range, the Vmax values agreed quite closely, usually within less than a factor of 2, suggesting that initial interaction of oxidized substrate with enzyme is not rate determining. HRBC TTase was inactivated by iodoacetamide (IAA), and this was prevented by pretreatment with disulfides. The pH dependence of IAA inactivation gave a remarkably low apparent pKa of 3.5, which was independent of ionic strength (0.05-2 M). At pH 6, one radiolabeled carboxyamidomethyl moiety was bound to the enzyme after treatment with [14C]IAA. This unusual thiol reactivity suggests that the active-site cysteine moiety of the TTase may be involved in a hydrogen bond with a carboxylate moiety. In contrast, the pH dependence for GSH-dependent TTase catalysis of disulfide reduction displayed an inflection point near pH 8.0, also suggesting that the initial reaction of oxidized substrate with the active-site thiol is not involved in rate determination. Two substrate kinetic studies of HRBC TTase and rat liver TTase (e.g., [GSH] and [HEDS] varied independently) gave patterns of intersecting lines on double-reciprocal plots (1/v vs 1/S), indicating a sequential mechanism for the TTase reactions, rather than a ping-pong mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)