Faculty Bibliography

Both water soluble and insoluble nickel compounds have been implicated in the etiology of human lung and nasal cancers. Water insoluble nickel compounds have been shown to enter cells by phagocytosis and are contained in cytoplasmic vacuoles, which are acidified thus accelerating the dissolution of soluble nickel from the particles. Using Newport Green, a dye that fluoresces when ionic nickel is bound, we have shown that following exposure (48-72 h) of human lung (A549) cells to NiS particles, most of the nickel is contained in the nucleus, while cells exposed to soluble NiCl2 exhibit most of the ions localized in the cytoplasm. This effect is consistent with previously published reports showing that short-term exposure of cells to crystalline nickel particles (1-3 days) is able to epigenetically silence target genes placed near heterochromatin, while similar short-term exposure to soluble nickel compounds are not able to induce silencing of genes placed near heterochromatin. However, a 3 week exposure of cells to soluble NiCl2 is also able to induce gene silencing. A similar effect was found in yeast cells where nickel was able to silence the URA-3 gene placed near (1.3 kb) a telomere silencing element, but not when the gene was placed farther away from the silencing element (2.0 kb). In addition to epigenetic effects, nickel compounds activate hypoxia signaling pathways. The mechanism of this effect involves the ability of either soluble or insoluble nickel compounds to block iron uptake leading to cellular iron depletion, directly affect iron containing enzymes, or both. This results in the inhibition of a variety of iron-dependent enzymes, such as aconitase and the HIF proline hydroxylases (PHD1-3). The inhibition of the HIF proline hydroxylases stabilizes the HIF protein and activates hypoxic signaling. Additional studies have shown that nickel and hypoxia decrease histone acetylation and increase the methylation of H3 lysine 9. These events are involved in gene silencing and hypoxia can also cause these effects in human cells. It is hypothesised that the state of hypoxia either by low oxygen tension or as a result of agents that signal hypoxia under normal oxygen tension (iron chelation, nickel and cobalt) results in low levels of acetyl CoA, which is a substrate for histone and other protein acetylation. This effect may in part be responsible for the gene silencing following nickel exposure and during hypoxia

Nickel is a widely distributed metal that is industrially applied in many forms. Accumulated epidemiological evidence confirms that exposures to nickel compounds are associated with increased nasal and lung cancer incidence, both in mostly occupational exposures. Although the molecular mechanisms by which nickel compounds cause cancer are still under intense investigation, the carcinogenic actions of nickel compounds are thought to involve oxidative stress, genomic DNA damage, epigenetic effects, and the regulation of gene expression by activation of certain transcription factors related to corresponding signal transduction pathways. The present review summarizes our current knowledge on the molecular mechanisms of nickel carcinogenesis, with special emphasis on the role of nickel induced reactive oxygen species (ROS) and signal transduction pathways

The previous studies have demonstrated that vanadium exposure can cause a variety of biological effects. However, the mechanisms involved in the biological effects caused by vanadium are not well understood. Our previous studies have shown that exposure of mouse epidermal Cl 41 cells to vanadate stimulated the phosphorylation of both ERKs and p38K, and calcium signaling leading NFAT activation. In view of the evidence that ERKs and p38 kinase contribute to VEGF induction, we investigated in the present study the potential roles of ERKs, p38K, and calcium signaling in VEGF induction caused by vanadium exposure. Exposure of Cl 41 cells to vanadium led to VEGF induction in both time- and dose-dependent manners. Pre-treatment of Cl 41 cells with PD98059, an inhibitor of MEK1/2-ERKs pathway, but not SB202190, an inhibitor for p38K pathway, resulted in a dramatic inhibition of VEGF induction by vanadium. More interesting, pre-treatment of Cl 41 cells with intracellular calcium chelator, but not calcium channel blocker, resulted in a dramatic decrease in VEGF induction by vanadium. However, both PI-3K inhibitors and overexpression of Deltap85, a dominant negative PI-3K mutant, resulted in only a marginal decrease in VEGF induction by vanadium. Moreover, mTOR, as a downstream molecule of PI-3K, did not attribute to VEGF induction by vanadium because rapamycin pre-treatment did not show any inhibitory effect on VEGF induction. These results indicate that ERKs and intracellular stored calcium release play a critical role in VEGF induction by vanadium. PI-3K is partially involved in VEGF induction by vanadium, while p38K and mTOR are not involved. Those results will help us to understand the molecular mechanisms involved in vanadium-induced biological effects

Soluble nickel compounds are likely human carcinogens. The mechanism by which soluble nickel may contribute to carcinogenesis is unclear, though several hypotheses have been proposed. Here we verify the ability of nickel to enter the cell via the divalent metal ion transporter 1 (DMT1) and disturb cellular iron homeostasis. Nickel may interfere with iron at both an extracellular level, by preventing iron from being transported into the cell, and at an intracellular level, by competing for iron sites on enzymes like the prolyl hydroxylases that modify hypoxia inducible factor-1alpha (HIF-1alpha). Nickel was able to decrease the binding of the Von Hippel-Lindau (VHL) protein to HIF-1alpha, indicating a decrease in prolyl hydroxylase activity. The ability of nickel to affect various iron dependent processes may be an important step in nickel dependent carcinogenesis. In addition, understanding the mechanisms by which nickel activates the HIF-1alpha pathway may lead to new molecular targets in fighting cancer

Exposure to a highly nickel-polluted environment has the potential to cause a variety of adverse health effects, such as the respiratory tract cancers. Since numerous studies have demonstrated that nickel generally has weak mutagenic activity, research focus had turned to cell signalling activation leading to gene modulation and epigenetic changes as a plausible mechanism of carcinogenesis. Previous studies have revealed that nickel compounds can induce the expression of vascular endothelial growth factor (VEGF), which is a key mediator of angiogenesis both in physiological and pathologic conditions. In the present study, we investigated the potential roles of PI-3K, ERKs, p38 kinase and calcium signalling in VEGF induction by nickel in Cl 41 cells. Exposure of Cl 41 cells to nickel compounds led to VEGF induction in both time- and dose-dependent manners. Pre-treatment of Cl 41 cells with PI-3K inhibitor, wortmannin or Ly294002, resulted in a striking inhibition of VEGF induction by nickel compounds, implicating the role of PI-3K in the induction. However, mTOR, one of downstream molecules of PI-3K, may not contribute to the induction because pre-treatment of Cl 41 cells with its inhibitor, rapamycin, did not show obvious decrease in nickel-induced VEGF expression. Furthermore, pre-treatment of Cl 41 cells with MEK1/2-ERKs pathway inhibitor, PD98059, significantly inhibited VEGF induction by both NiCl2 and Ni3S2, whereas p38 kinase inhibitor, SB202190, did not impair the induction. Pre-treatment of Cl 41 cells with intracellular calcium chelator, but not calcium channel blocker, inhibited VEGF induction by nickel. Collectively these data demonstrate that PI-3K, ERKs and cytosolic calcium, but not p38 kinase, play essential roles in VEGF induction by nickel compounds

Nickel (Ni) compounds are well-established carcinogens and are known to initiate a hypoxic response in cells via the stabilization and transactivation of hypoxia-inducible factor-1 alpha (HIF-1alpha). This change may be the consequence of nickel's interference with the function of several Fe(II)-dependent enzymes. In this study, the effects of soluble nickel exposure on cellular iron homeostasis were investigated. Nickel treatment decreased both mitochondrial and cytosolic aconitase (c-aconitase) activity in A549 cells. Cytosolic aconitase was converted to iron-regulatory protein 1, a form critical for the regulation of cellular iron homeostasis. The increased activity of iron-regulatory protein 1 after nickel exposure stabilized and increased transferrin receptor (Tfr) mRNA and antagonized the iron-induced ferritin light chain protein synthesis. The decrease of aconitase activity after nickel treatment reflected neither direct interference with aconitase function nor obstruction of [4Fe-4S] cluster reconstitution by nickel. Exposure of A549 cells to soluble nickel decreased total cellular iron by about 40%, a decrease that likely caused the observed decrease in aconitase activity and the increase of iron-regulatory protein 1 activity. Iron treatment reversed the effect of nickel on cytosolic aconitase and iron-regulatory protein 1. To assess the mechanism for the observed effects, human embryonic kidney (HEK) cells over expressing divalent metal transporter-1 (DMT1) were compared to A549 cells expressing only endogenous transporters for inhibition of iron uptake by nickel. The inhibition data suggest that nickel can enter via DMT1 and compete with iron for entry into the cell. This disturbance of cellular iron homeostasis by nickel may have a great impact on the ability of the cell to regulate a variety of cell functions, as well as create a state of hypoxia in cells under normal oxygen tension. These effects may be very important in how nickel exerts phenotypic selection pressure to convert a normal initiated cell into a cancer cell

Besides its well-established role in wound healing and fibrinolysis, tissue-type plasminogen activator (t-PA) has been shown to contribute to cognitive processes and memory formation within the central nervous system, and to promote glutamate receptor-mediated excitotoxicity. The t-PA gene is expressed and regulated in neuronal cells but the regulatory transcriptional processes directing this expression are still poorly characterized. We have used DNase I-hypersensitivity mapping and in vivo foot printing to identify putative regulatory elements and transcription factor binding sites in two human neuroblastomal (KELLY and SK-N-SH) and one human glioblastomal (SNB-19) cell lines. Hypersensitive sites were found in the proximal promoter region of all cell lines, and within the first exon for KELLY and SNB-19 cells. Mapping of methylation-protected residues in vivo detected a cluster of protected residues corresponding to a cAMP response element (CRE) and Sp1 sites in the proximal promoter previously shown to be essential for basal expression in other cell types. Protected residues were also found at other sites, notably a kappaB element at position bp -3081 to -3072 that was partly protected in KELLY and SNB-19 cells. Analysis of transfected reporter constructs in KELLY and SNB-19 cells confirmed that this particular element is functionally significant in the transactivation of the t-PA promoter in both cell types. This study defines, by in vivo and in vitro methods, a previously undescribed kappaB site in the t-PA gene promoter that influences t-PA expression in neuronal cells.

Although nickel and cobalt compounds have been known to cause induction of the transcription factor hypoxia-inducible factor 1 (HIF-1) and activation of a battery of hypoxia-inducible genes in the cell, the molecular mechanisms of this induction remain unclear. The post-translational modification of HIF-1a, the oxygen-sensitive subunit of HIF-1, regulates stabilization, nuclear translocation, DNA binding activity, and transcriptional activity of the protein. Among the enzymes regulating the post-translational modification of HIF-la, the factor inhibiting HIF-1 (FIH-1) hydroxylates the protein at asparagine 803, suppressing the interaction of HIF-1a with transcription coactivators p300/CBP and reducing the transcriptional activity of the protein. ARD-1, the acetyltransferase, acetylates HIF-1a at lysine 532, which enhances the interaction of HIF-1a with pVHL. Therefore, FIH-1 and ARD-1 negatively regulate the transcriptional activity and the stability of HIF-1a. We examined the mRNA levels of FIH-l and ARD-1 genes after exposure nickel (II) or cobalt (II) to the cell and found that both genes were down-regulated by the chemical treatment, which may lead to reduced levels of both proteins and result in increased level of HIF-1 a and its transcriptional activity

Polycyclic aromatic hydrocarbons (PAHs) and their derivatives, such as benzo[a]pyrene (B[a]P), (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), and 5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE), are complete carcinogens. However, the tumor promotion effects of PAHs remain unclear. We therefore investigated the possible activation of activator protein-1 (AP-1) and nuclear factor-kappaB (NFkappaB) in mouse epidermal Cl41 cells after different PAHs treatments, including B[a]P, B[a]PDE, chrysene-1,2-diol-3,4-epoxid (CDE), and 5-MCDE. The results showed that B[a]PDE and 5-MCDE were able to activate AP-1 and NF-kappaB, whereas B[a]P showed only marginal effect on AP-1 activation, and B[a]P and CDE had no effect on NF-kappaB activation. Treatment with either B[a]PDE or 5-MCDE also resulted in mitogen-activated protein kinases (MAPKs) activation as well as inhibitory subunit kappa-B (IkappaBalpha) phosphorylation and degradation, whereas B[a]P and CDE had no effect. Pretreatment with PD98059, a specific inhibitor for extracellular signal-regulated protein kinases (ERKs) upstream kinase MEK1/2, or SB202190, a p38 kinase inhibitor, resulted in a dramatic inhibition of B[a]PDE-induced AP-1 transactivation. In addition, B[a]PDE-induced AP-1 activation was also inhibited by overexpressing a dominant negative mutant of JNK1 in the cells. All these suggest ERKs, c-jun N-terminal kinases (JNKs), and p38 kinase signal transduction pathways are required for AP-1 induction by B[a]PDE. Taken together, B[a]PDE and 5-MCDE are the active compounds of PAHs to initiate signaling pathways. Considering the important roles of AP-1 and NF-kappaB in tumor promotion, we speculated the activation of AP-1 and NF-kappaB by B[a]PDE and 5-MCDE may involve in their or their parent compounds' tumor promotion effects. This study may help in better understanding the tumor promotion effects of PAHs

Cap43 protein has been tested for metal binding domains. The protein, specifically induced by nickel compounds in cultured human cells, had a new mono-histidinic motif consisting of 10 amino acids repeated three times in the C-terminus. The 20-Ac-TRSRSHTSEG-TRSRSHTSEG (Thr(341)-Arg-Ser-Arg-Ser-His(346)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His (356)-Thr-Ser-Glu-Gly(360) - peptide 1) and the 30-Ac-TRSRSHTSEG-TRSRSHTSEG-TRSRSHTSEG (Thr(341)-Arg-Ser-Arg-Ser-His(346)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His (356)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His(366)-Thr-Ser-Glu-Gly(370) - peptide 2) amino acids sequence has been analyzed as a site for Ni(II) binding. A combined pH-metric and spectroscopic (UV-visible, CD, NMR) studies of Ni(II) binding to both fragments were performed. The 20-amino acid peptide can bind one and two metal ions while the 30-amino acid fragment one, two and three metal ions. At physiological pH, depending on the metal to ligand molar ratio, peptide 1 forms the Ni(2)L species while peptide 2 the NiL, Ni(2)L and Ni(3)L complexes where each metal ion is coordinated to the imidazole nitrogen atom of the histidine residue of the 10-amino acid fragment. Octahedral complexes at pH 8-9 and planar 4N complexes with (N(Im), 3N(-)) bonding mode at pH above 9, are formed. This work supports the existence of an interesting binding site at the COOH-terminal domain of the Cap43 protein