Cadmium (Cd), a human carcinogen, can induce apoptosis in various cell types. Three major mitogen-activated protein kinases (MAPKs), c-JUN N-terminal kinase (JNK), p38 and extracellular signal-regulated kinase (ERK), have been shown to regulate apoptosis. In this study we explore the ability of Cd to activate JNK, p38 and ERK, including their effects on Cd-mediated growth inhibition and apoptosis in a human non-small cell lung carcinoma cell line, CL3. The kinase activity of JNK was induced dose-dependently by 30-160 microM CdCl(2). High cytotoxic doses of Cd (130-160 microM) markedly activated p38, but low Cd doses did not. Conversely, the activities of ERK1 and ERK2 were decreased by low cytotoxic doses of Cd (

The dual-specificity MAPK phosphatase MKP-1/CL100/DUSP1 is an inducible nuclear protein controlled by p44/42 MAPK (ERK1/2) in a negative feedback mechanism to inhibit kinase activity. Here, we report on the molecular basis for a novel positive feedback mechanism to sustain ERK activation by triggering MKP-1 proteolysis. Active ERK2 docking to the DEF motif (FXFP, residues 339 -342) of N-terminally truncated MKP-1 in vitro initiated phosphorylation at the

Sustained extracellular signal-regulated kinase 1/2 (ERK1/2) activation does not always correlate with its upstream Ras-Raf-mitogen-activated protein kinase kinase 1/2 (MKK1/2) signal cascade in cancer cells, and the mechanism remains elusive. Here we report a novel mechanism by which sustained ERK1/2 activation is established. We demonstrate that Pb(II), a carcinogenic metal, persistently induces ERK1/2 activity in CL3 human lung cancer cells and that Ras-Raf-MKK1/2 signaling cannot fully account for such activation. It is intriguing that Pb(II) treatment reduces mitogenactivated protein kinase phosphatase 1 (MKP-1) protein levels in time-and dose-dependent manners, which correlates with sustained ERK1/2 activation, and that Pb(II) also induces mRNA and de novo protein synthesis of MKP-1. In Pb ( Members of the family of mitogen-activated protein kinase (MAPK) 1 proteins are vital intracellular signaling components that become phosphorylated and activated in response to a wide diversity of extracellular stimuli, including growth factors, cytokines, and environmental stresses (reviewed in Refs. 1-5). MAPKs are activated through a three-kinase module composed of a MAPK, a MAPK kinase (MKK), and a MKK kinase (MKKK). These MAPK modules are connected to cell surface receptors and activated via interaction with a family of small GTPases and MKKK kinases. Activated MAPKs phosphorylate many substrates, including cytoskeletal proteins, other kinases, phosphatases, enzymes, and transcription factors, thereby orchestrating several cellular alterations including proliferation, differentiation, survival, and apoptosis. The duration and strength of MAPK activation also affects these biological outcomes. Three major MAPK subfamilies have been extensively studied, i.e. the extracellular signal-regulated kinases (ERK1/2), the c-Jun N-terminal kinases (JNKs), and the p38 kinases. Activation of a particular MAPK signal must be controlled with high specificity and efficiency to achieve precise physiological regulation. The recent discovery of specific docking sites among the members of the MAPK cascades provide a mechanism that explains how specific and efficient signaling is established. For instance, a cluster of positively charged amino acids followed by an LXL motif called the D domain (or the kinase interaction motif, KIM) has been identified in MKKs, MAPK phosphatases (MKPs), and several MAPK substrates (6 -8). The D domain binds specifically to an acidic domain (common docking domain) within a docking groove of MAPKs (6 -8). Another docking site found in many ERK substrates is called the DEF motif (docking site for ERK, FXFP) (8, 9). These docking interactions facilitate phosphorylation of substrates by MAPKs on specific Ser or Thr residues followed by a Pro residue ((S/T)P sites).The small GTPase, MKKK, and MKK in the ERK pathway are known to be Ras, Raf, and MKK1/2, respectively (1-5). Activation of ERK1/2 requires a dual-phosphorylation by MKK1/2 on the Thr and Tyr residues of TEY sites within the activation loop, wherea...

Metamaterials have unprecedented properties that facilitate the development of advanced devices and machines. However, their interconnected building structures limit their applications, especially in the fields that require large deformation, rich programmability and efficient shape‐reconfigurability. To break this limit and exploit more potentialities of metamaterials, an innovative material design strategy is proposed, named mechanical pixel (MP) array design. Similar to a screen that displays images by adjusting the colors of pixels, the metamaterials can form and reconfigure 3D morphologies by tuning the heights (lengths) of the MPs in the array. The strategy is demonstrated in a multistable metamaterial by experimental tests, theoretical analysis, and numerical simulations. Using this strategy, a large macroscopic shear deformation is obtained, and remarkable enhancements in the mechanical programmability, shape‐reconfigurability and adaptability, and reusable shock‐resistance are exhibited. Moreover, mechanical design and property prediction for the metamaterials are both greatly simplified due to the pixelated design. For a piece of the 3D pixel metamaterial with m n‐unit MPs, the number of programmable displacement–force curves increases from n+1 to 2m∙n+1, and the number of stable morphologies grows from n+1 to at least (n+1)m. This strategy can be used to enhance the merits and further excavate the potential of versatile metamaterials.

Kinds of mutations formed when a shuttle vector containing adducts of (±+)-7,8,8a-dihydroxy-9a,10a-epoxy-7,8,9,10- ABSTRACTWe have investigated the kinds of mutations induced when a shuttle vector containing covalently bound residues of (±)-7p,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) replicates in human cells. A human embryonic kidney cell line, 293, was used as the eukaryotic host. The target gene for mutation analysis, supF, codes for a tyrosine suppressor tRNA and is strategically located between the origin of replication of the plasmid in Escherichia coli and the gene for a selectable marker, so that the possibility of recovering supF mutants containing gross rearrangements is low. The frequency of supF mutants obtained when untreated plasmid replicated in 293 cells was 1.

No abstract

In this study we have explored the involvement of oxidative stress in Cr(VI)-induced JNK, p38 and ERK signaling pathways and their effects on Cr(VI) cytotoxicity in human non-small cell lung carcinoma CL3 cells. Exposure to K(2)Cr(2)O(7) markedly activated JNK and p38 and moderately activated ERK in a dose- (10-80 microM) and time-dependent (1-12 h) manner. The activated p38 decreased markedly and rapidly and the activated JNK decreased gradually when Cr(VI) was removed from the medium. Post-incubation of Cr(VI)-treated cells with H(2)O(2) increased the activities of JNK and p38, but not ERK. Co-administering Cr(VI) with 3-amino-1,2, 4-triazole (3AT), a catalase inhibitor, enhanced p38 activation, but did not influence JNK and ERK activation by Cr(VI). Conversely, co-administering Cr(VI) with mannitol, a hydroxyl radical scavenger and a Cr(V) chelator, reduced p38 activation and increased JNK and ERK activation by Cr(VI). These results indicate that p38 activation by Cr(VI) is positively correlated with oxidative stress, while JNK activity can be enhanced by either a quencher (mannitol) or activator (H(2)O(2)) of redox reactions in Cr(VI)-exposed CL3 cells. However, both 3AT and mannitol reduced the cytotoxicity of Cr(VI), but H(2)O(2) did not. The JNK activated by Cr(VI) was decreased (approximately 50%) by expression of a kinase-defective form of MKK7 (MKK7A) but not that of MKK4 (MKK4KR), suggesting that activation of JNK by Cr(VI) is mediated through MKK7. SB202190, a specific inhibitor of p38, markedly decreased JNK but did not change ERK activation by Cr(VI). PD98059, a specific inhibitor of ERK kinases MKK1/2, blocked ERK and p38 but did not alter JNK activation by Cr(VI). Neither the specific kinase inhibitors nor expression of MKK7A altered Cr(VI)-induced cytotoxicity. Together, these results suggest that activation of the JNK, p38 and ERK pathways by Cr(VI) is mediated through diverse redox mechanisms, yet their activation does not correlate with Cr(VI) cytotoxicity.

The capability of Cr(III) to induce DNA lesions generated by oxidative damage was investigated in this study by examining the formation of 8-hydroxydeoxyguanosine (8-OHdG) in calf thymus DNA by CrCl3 and/or H2O2 in 10 mM phosphate buffer. In the presence of 0.5 mM H2O2, the formation of 8-OHdG markedly increased with increasing CrCl3 concentration. In contrast, H2O2 or CrCl3 alone did not cause any increase in 8-OHdG level above background. The amount of 8-OHdG induced by CrCl3 plus H2O2 was time dependent; its generation increased linearly over an incubation period of 90 min. The formation of 8-OHdG was unfavorable in an acidic solution (pH < 6); the highest level of 8-OHdG was observed at pH 7-8. Scavengers of reactive oxygen species markedly inhibited the formation of 8-OHdG by CrCl3 plus H2O2; the inhibition effect was sodium azide > D-mannitol > Tris-HCl at an equal concentration. The induction of 8-OHdG by CrCl3 plus H2O2 remained unchanged in D2O. Moreover, an addition of catalase (2.2 U/ml) to the reaction mixture completely inhibited the formation of 8-OHdG by CrCl3/H2O2, whereas only 22% of that formation was inhibited by superoxide dismutase (11 U/ml). A large amount of bovine serum albumin (1.1 mg/ml) could reduce the formation of 8-OHdG by CrCl3 plus H2O2, thereby implying that Cr(III)-mediated DNA-protein crosslinks are unfavorable for 8-OHdG formation. Furthermore, ascorbate could prevent the formation of 8-OHdG by CrCl3 plus H2O2; the extent of prevention increased with increasing ascorbate concentration (10 microM-3 mM). Thus, ascorbate acts as a free radical scavenger in the CrCl3/H2O2 system. The above findings suggest that Cr(III)/H2O2 could generate oxidative damage to DNA, possibly through a Fenton-like reaction, i.e. Cr(III)+H2O2-->Cr(IV)+.OH+OH-. This study also indicates that Cr(III), previously considered as the ultimate kinetically stable species of Cr(VI) metabolites, is capable of inducing carcinogenic lesions through interaction with a cellular oxygen species.