DNA-protein cross-links induced by nickel compounds in intact cultured mammalian cells

The carcinogenic activity of crystalline NiS has been attributed to phagocytosis and intracellular dissolution of the particles to yield Ni2+ which is thought to enter the nucleus and damage DNA. In this study the extent and type of DNA damage in Chinese hamster ovary CHO cells treated with various nickel compounds was assessed by alkaline elution. Both insoluble (crystalline NiS) and soluble (NiCl2) nickel compounds induced single strand breaks and DNA protein cross-links. The single strand breaks were repaired relatively quickly but the DNA-protein cross-links were present and still accumulating 24 h after exposure to nickel. Single strand breakage occurred at both non-cytotoxic and cytotoxic concentrations of nickel, however, DNA-protein cross-linking was absent when cells were exposed to toxic nickel levels. The concentration of nickel that induced DNA-protein cross-linking correlated with those metal concentrations that reversibly inhibited cellular replication.     

Molecular biology of nickel carcinogenesis

A review of the molecular mechanisms of nickel carcinogenesis has been compiled. This work is based upon approximately 20 years of research conducted in my laboratory. Molecular mechanisms of nickel carcinogenesis are considered from the pointofview of the uptake of nickel, both soluble and insoluble particles in cells, its dissolution and its effects on heterochromatin. Molecular mechanisms by which nickel induces gene silencing in cells by DNA hypermethylation in mammalian cells and by inhibiting histone acetylation in yeast cells are also discussed.     

Soluble nickel interferes with cellular iron homeostasis

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-1α (HIF-1α). Nickel was able to decrease the binding of the Von Hippel–Lindau (VHL) protein to HIF-1α, 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-1α pathway may lead to new molecular targets in fighting cancer.     

Analysis of ARD1 function in hypoxia response using retroviral RNA interference

Cellular hypoxia response is regulated at the level of hypoxia-inducible factor (HIF) activity. A number of recently identified oxygen sensors are HIF-modifying enzymes that respond to low oxygen by altering HIF modification and thus lead to its activation. In addition to the HIF proline hydroxylases and asparagine hydroxylases, ARD1 is recently described as a HIF-1alpha acetylase that regulates its stability. We found that ARD1 is down-regulated in a number of cell lines in response to hypoxia and hypoxia mimic compounds. After surveying these lines for erythropoietin production and retroviral transfection efficiency, we chose to use HepG2 cells to study the function of ARD1. ARD1 short hairpin RNA delivered by a retroviral vector caused >80% reduction in ARD1 message. We observed decreases in erythropoietin and vascular endothelial growth factor protein production, whereas there was no change in the HIF-1alpha protein level. A gene chip analysis of HepG2 cells transduced with virus expressing ARD1 short hairpin RNA under normoxia and hypoxia conditions or with virus overexpressing recombinant ARD1 confirmed that inhibition of ARD1 does not cause activation of HIF and downstream target genes. However, this analysis revealed that ARD1 is involved in cell proliferation and in regulating a series of cellular metabolic pathways that are regulated during hypoxia response. The role of ARD1 in cell proliferation is confirmed using fluorescence labeling analysis of cell division. From these studies we conclude that ARD1 is not required to suppress HIF but is required to maintain cell proliferation in mammalian cells.     

The histone deacetylase inhibitor trichostatin A reduces nickel-induced gene silencing in yeast and mammalian cells

We have previously reported that nickel (Ni)-silenced expression of the URA3 gene in yeast (Saccharomyces cerevisiae) and gpt transgene in G12 Chinese hamster cells. In both cases, close proximity to a heterochromatic region was required for gene silencing. Yeast exposed to Ni exhibited reduced acetylation of the lysine residues in the N-terminal tail of histone H4. Ni-induced silencing of the gpt gene in mammalian cells involved hypermethylation of promoter region DNA. Yeast do not employ DNA methylation to silence gene expression. To determine if histone deacetylation participates in Ni-induced silencing of the URA3 and gpt genes, we exposed yeast and G12 hamster cells to the histone deacetylase inhibitor trichostatin A (TSA) prior to and concurrently with Ni. Treatment of yeast cells with 0.2-0.6mM NiCl(2) resulted in reduced expression of the URA3 gene as assessed by increased resistance to 1g/l 5-fluorotic acid (5-FOA). This effect was lessened when yeast were pre-treated with 50 microg TSA/ml. Similarly, treatment of G12 cells with 5 ng/ml TSA during and after exposure to 0.3 microg Ni(3)S(2)/cm(2) reduced silencing of the gpt gene as gauged by resistance to 10 microg/ml 6-thioguanine (6-TG). The ability of TSA alone and in combination with the DNA-demethylating agent (5-AzaC) to reactivate the gpt gene in Ni-silenced variants was also assessed. Although treatment with 100 ng/ml TSA for 48 h was partially effective in reactivating the gpt gene, treatment with 5 microM 5-AzaC was more efficacious. The greatest gpt gene reversion frequencies were observed following a sequential 5-AzaC/TSA treatment. Taken all together, our data from mammalian cells suggests that both DNA methylation and histone deacetylation participate in Ni-induced silencing of the gpt gene with DNA hypermethylation playing the more dominant role in maintaining the silenced state.     

Nickel ions increase histone H3 lysine 9 dimethylation and induce transgene silencing

We have previously reported that carcinogenic nickel compounds decreased global histone H4 acetylation and silenced the gpt transgene in G12 Chinese hamster cells. However, the nature of this silencing is still not clear. Here, we report that nickel ion exposure increases global H3K9 mono- and dimethylation, both of which are critical marks for DNA methylation and long-term gene silencing. In contrast to the up-regulation of global H3K9 dimethylation, nickel ions decreased the expression and activity of histone H3K9 specific methyltransferase G9a. Further investigation demonstrated that nickel ions interfered with the removal of histone methylation in vivo and directly decreased the activity of a Fe(II)-2-oxoglutarate-dependent histone H3K9 demethylase in nuclear extract in vitro. These results are the first to show a histone H3K9 demethylase activity dependent on both iron and 2-oxoglutarate. Exposure to nickel ions also increased H3K9 dimethylation at the gpt locus in G12 cells and repressed the expression of the gpt transgene. An extended nickel ion exposure led to increased frequency of the gpt transgene silencing, which was readily reversed by treatment with DNA-demethylating agent 5-aza-2'-deoxycytidine. Collectively, our data strongly indicate that nickel ions induce transgene silencing by increasing histone H3K9 dimethylation, and this effect is mediated by the inhibition of H3K9 demethylation.     

Effect of metal ions on HIF-1alpha and Fe homeostasis in human A549 cells

Several metals are carcinogenic but little is known about the mechanisms by which they cause cancer. A pathway that may contribute to metal ion induced carcinogenesis is by hypoxia signaling, which involves a disruption of cellular iron homeostasis by competition with iron transporters or iron-regulated enzymes. To examine the involvement of iron in the hypoxia signaling activity of these metal ions we investigated HIF-1alpha protein stabilization, IRP-1 activity, and ferritin protein levels in human lung carcinoma A459 cells exposed to various agents in serum- and iron-free salt-glucose medium (SGM) or in normal complete medium. We also studied the effects of excess exogenous iron on these responses induced by nickel ion exposure. Our results show the following: (1) SGM enhanced metals-induced HIF-1alpha stabilization and IRP-1 activation (e.g., nickel and cobalt ions). (2) If SGM was reconstituted with a slight excess level (25 microM of FeSO(4)) of iron, this enhancing ability was significantly decreased. (3) The effect of a high level of exogenous iron (500 microM of FeSO(4)) on metal-induced hypoxia and iron metabolism was highly dependent on the order of addition. If treatment with the Fe and metal ions was simultaneous (co-treatment), the effects of nickel ion exposure were overwhelmed, since the added Fe reversed HIF-1alpha stabilization, decreased IRP-1 activity, and increased ferritin level. Pre-treatment with iron was not able to reverse the responses caused by nickel ion exposure. These results imply that it is important to consider the available iron concentration and suitable exposure design when studying metal-induced hypoxia or metal-induced disruption of Fe homeostasis.