Both soluble and insoluble nickel compounds have been implicated as human carcinogens. Among several mechanisms proposed to explain nickel carcinogenesis, the hypoxia-mimicking stress and aberrant epigenetic changes distinguish carcinogenic Ni compounds from a number of other known carcinogens. The work conducted in this laboratory indicates that these two distinct mechanisms may be linked by a common thread in involving the interference of Ni ions with iron function. Exposure of cells to soluble nickel compounds results in a decrease of cellular iron levels, which subsequently activate iron regulatory protein (IRP) binding to an iron response element and also lead to an inhibition of aconitase activity, since aconitase moonlights as IRP. This nickel-induced IRP activation subsequently stabilizes and increases transferrin receptor mRNA as well as decreases ferritin protein synthesis, which are both compensatory measures responding to decreased cellular iron levels. Hypoxia inducible factor 1 alpha (HIF-1 alpha) transcription factor is stabilized as a result of the depletion of cellular iron. The primary mechanism by which nickel ions stabilize HIF-1 alpha is by their inhibition of iron- and oxoglutarate-dependent dioxygenases that regulate the stability of HIF-1 alpha. Nickel ions are likely to displace and deplete iron from these enzymes. In addition, nickel ions are found to inhibit a putative iron- and oxoglutarate-dependent histone H3 lysine 9 demethylase in vitro, whose inactivation likely results in an increase of global histone H3 lysine 9 dimethylation which eventually triggers the epigenetic silencing of genes. Since this demethylase should utilize the same mechanism to bind iron as the dioxygenases that regulate the stability of HIFI alpha, nickel ions target these enzymes by interfering with the function of the iron moiety. Collectively, these findings unveil a common mechanism of nickel carcinogenesis that is related to the ability of nickel ions to interfere with iron binding and catalysis of the iron-dependent dioxygenases. These dioxygenases have important roles as diverse as regulating the stability of HIF-1 alpha transcription factor as regulating histone methylation which is the mark for gene silencing.

Epidemiological studies have demonstrated that nickel, chromium, arsenic, cadmium, and beryllium compounds are human carcinogens. Most of these epidemiological studies were done retrospectively in humans occupationally exposed to compounds of these metals. There are only a few environmental studies in humans exposed to any of these metal compounds in which possible speciation of the metal compound is considered. Of the compounds of the five metals, those of nickel, chromium, cadmium, and beryllium have been confirmed as carcinogens in vivo, in experimental animals. The genotoxicity and mutagenic activity of metals is discussed briefly; however, the reader is referred to more detail in chapters on individual metals and to Chapter 9, "Selected Molecular Mechanisms of Metal Toxicity and Carcinogenicity." Cell transformation in tissue culture and other mechanistic information is summarized for each metal ion.

p27 is a cyclin-dependent kinase (CDK) inhibitor that suppresses a cell's transition from G0 to S phase, therefore acting as a tumor suppressor. Our most recent studies demonstrate that upon arsenite exposure, p27 suppresses Hsp27 and Hsp70 expressions through the JNK2/c-Jun- and HSF-1-dependent pathways, suggesting a novel molecular mechanism underlying the tumor suppressive function of p27 in a CDK-independent manner. We found that p27-deficiency (p27−/−) resulted in the elevation of cyclooxygenase-2 (COX-2) expression at transcriptional level, whereas the introduction of p27 brought back COX-2 expression to a level similar to that of p27+/+ cells, suggesting that p27 exhibits an inhibitory effect on COX-2 expression. Further studies identified that p27 inhibition of COX-2 expression was specifically due to phosphorylation of transcription factor cAMP response element binding (CREB) phosphorylation mediated by p38β and p38δ. These results demonstrate a novel mechanism underlying tumor suppression effect of p27 and will contribute to the understanding of the overall mechanism of p27 tumor suppression in a CDK-independent manner.

In the present study, we examined the effects of CoCl(2) on multiple histone modifications at the global level. We found that in both human lung carcinoma A549 cells and human bronchial epithelial Beas-2B cells, exposure to CoCl(2) (>/=200 muM) for 24 h increased H3K4me3, H3K9me2, H3K9me3, H3K27me3, H3K36me3, uH2A and uH2B but decreased acetylation at histone H4 (AcH4). Further investigation demonstrated that in A549 cells, the increase in H3K4me3 and H3K27me3 by cobalt ions exposure was probably through enhancing histone methylation processes, as methionine-deficient medium blocked the induction of H3K4me3 and H3K27me3 by cobalt ions, whereas cobalt ions increased H3K9me3 and H3K36me3 by directly inhibiting JMJD2A demethylase activity in vitro, which was probably due to the competition of cobalt ions with iron for binding to the active site of JMJD2A. Furthermore, in vitro ubiquitination and deubiquitination assays revealed that the cobalt-induced histone H2A and H2B ubiquitination is the result of inhibition of deubiquitinating enzyme activity. Microarray data showed that exposed to 200 microM of CoCl(2) for 24 h, A549 cells not only increased but also decreased expression of hundreds of genes involved in different cellular functions, including tumorigenesis. This study is the first to demonstrate that cobalt ions altered epigenetic homeostasis in cells. It also sheds light on the possible mechanisms involved in cobalt-induced alteration of histone modifications, which may lead to altered programs of gene expression and carcinogenesis since cobalt at higher concentrations is a known carcinogen.

Nickel (Ni) compounds are known carcinogens but underlying mechanisms are not clear. Epigenetic changes are likely to play an important role in nickel ion carcinogenesis. Previous studies have shown epigenetic effects of nickel ions, including the loss of histone acetylation and a pronounced increase in dimethylated H3K9 in nickel-exposed cells. In this study, we demonstrated that both water-soluble and insoluble nickel compounds induce histone ubiquitination (uH2A and uH2B) in a variety of cell lines. Investigations of the mechanism by which nickel increases histone ubiquitination in cells reveal that nickel does not affect cellular levels of the substrates of this modification, i.e., ubiquitin, histones, and other non-histone ubiquitinated proteins. In vitro ubiquitination and deubiquitination assays have been developed to further investigate possible effects of nickel on enzymes responsible for histone ubiquitination. Results from the in vitro assays demonstrate that the presence of nickel did not affect the levels of ubiquitinated histones in the ubiquitinating assay. Instead, the addition of nickel significantly prevents loss of uH2A and uH2B in the deubiquitinating assay, suggesting that nickel-induced histone ubiquitination is the result of inhibition of (a) putative deubiquitinating enzyme(s). Additional supporting evidence comes from the comparison of the response to nickel ions with a known deubiquitinating enzyme inhibitor, iodoacetamide (IAA). This study is the first to demonstrate such effects of nickel ions on histone ubiquitination. It also sheds light on the possible mechanisms involved in altering the steady state of this modification. The study provides further evidence that supports the notion that nickel ions alter epigenetic homeostasis in cells, which may lead to altered programs of gene expression and carcinogenesis.

Nickel (Ni) is a known carcinogen, although the mechanism of its carcinogenicity is not clear. Here, we provide evidence that Ni can induce phosphorylation of histone H3 at its serine 10 residue in a c-jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK)-dependent manner. Ni induces the phosphorylation of JNK, with no effect on the phosphorylation states of the extracellular signal-regulated kinase (ERK) or p38 mitogen-activated protein kinases. An inhibitor of JNK eliminated the Ni-initiated JNK-mediated induction of histone H3 phosphorylation at serine 10, whereas inhibitors specific for ERK or p38 kinases had no effect on the phosphorylation levels of histone H3 at serine 10 (P-H3S10) in Ni-treated cells. A complete loss of Ni ion-induced phosphorylation of H3S10 was observed when JNK was specifically knocked down with RNAi. These results are the first to show the specific JNK-mediated phosphorylation of histone H3 at its serine 10 residue. We show that addition of Ni to an in vitro P-H3S10 dephosphorylation reaction does not change the loss of phosphorylation in the reaction, supporting the notion that Ni causes H3S10 phosphorylation via the JNK/SAPK pathway. It is likely that modification of H3S10 is one of a growing number of epigenetic changes believed to be involved in the carcinogenesis caused by Ni.

The carcinogenic activity of various nickel (Ni) compounds is likely dependent upon their ability to enter cells and elevate intracellular levels of Ni ions. Water-insoluble Ni compounds such as NiS and Ni(3)S(2) were shown in vitro to enter cells by phagocytosis and potently induce tumors in experimental animals at the site of exposure. These water-insoluble nickel compounds are generally considered to be more potent carcinogens than the water-soluble forms. However, recent in vitro studies have shown similar effects for insoluble and soluble Ni compounds. Using a dye that fluoresces when intracellular Ni ion binds to it, we showed that both soluble and insoluble Ni compounds were able to elevate the levels of Ni ions in the cytoplasmic and nuclear compartments. However, when the source of Ni ions was removed from the culture dish, the intracellular Ni ions derived from soluble Ni compound were lost from the cells at a significantly faster rate than those derived from the insoluble Ni compound. Within 10 h after NiCl(2) removal from the culture medium, Ni ions disappeared from the nucleus and were not detected in the cells by 16 h, while insoluble Ni(3)S(2) yielded Ni ions that persisted in the nucleus after 16 h and were detected in the cytoplasm even after 24 h following Ni removal. These effects are discussed in terms of whole body exposure to water-soluble and -insoluble Ni compounds and consistency with animal carcinogenicity studies.

N-myc downstream-regulated gene 1 (NDRG1) is an intracellular protein that is induced under a wide variety of stress and cell growth-regulatory conditions. NDRG1 is up-regulated by cell differentiation signals in various cancer cell lines and suppresses tumor metastasis. Despite its specific role in the molecular cause of Charcot-Marie-Tooth type 4D disease, there has been more interest in the gene as a marker of tumor progression and enhancer of cellular differentiation. Because it is strongly up-regulated under hypoxic conditions, and this condition is prevalent in solid tumors, its regulation is somewhat complex, governed by hypoxia-inducible factor 1 alpha (HIF-1alpha)- and p53-dependent pathways, as well as its namesake, neuroblastoma-derived myelocytomatosis, and probably many other factors, at the transcriptional and translational levels, and through mRNA stability. We survey the data for clues to the NDRG1 gene's mechanism and for indications that the NDRG1 gene may be an efficient diagnostic tool and therapy in many types of cancers.

This chapter will summarize some of the molecular responses exhibited by cells that come into contact with toxic metals. We will consider the transport of toxic metals into cells and how this interferes with the transport of essential metals. Toxic metals also interfere with the intracellular action of essential metals and may cause toxicity and cancer by this mechanism. Some metals have very specialized effects on enzymes, and there are proteins that can bind toxic metals such as metallothionein, which will also be discussed. Some metals are also slightly mutagenic and genotoxic, and these effects will be discussed. Because most metals that are carcinogenic, such as nickel (Ni), arsenic (As), beryllium (Be), and cadmium (Cd), with the exception of chromium (Cr), do not interact with DNA and are not mutagenic, we discuss other mechanisms such as epigenetic effects to account for their carcinogenic activity, as well as how they affect the expression of genes. Metals also interfere with cell signaling, and, in particular, we will discuss the hypoxic signaling pathway as well as those involving PI3K, AKT, ROS, MAPK, NF kappa B, NFAT, and AP1. Finally, the basis of the interaction of toxic metals with all cellular constituents involves their coordination with biological ligands, which will be addressed throughout the chapter.

Adaptation to low oxygen tension (hypoxia) in cells and tissues leads to the transcriptional induction of a series of genes that participate in angiogenesis, iron metabolism, glucose metabolism, and cell proliferation/survival. The primary factor mediating this response is the hypoxia-inducible factor-1 (HIF-1), an oxygen-sensitive transcriptional activator. HIF-1 consists of a constitutively expressed subunit HIF-1β and an oxygen-regulated subunit HIF-1α (or its paralogs HIF-2α and HIF-3α). The stability and activity of the α subunit of HIF are regulated by its post-translational modifications such as hydroxylation, ubiquitination, acetylation, and phosphorylation. In normoxia, hydroxylation of two proline residues and acetylation of a lysine residue at the oxygen-dependent degradation domain (ODDD) of HIF-1α trigger its association with pVHL E3 ligase complex, leading to HIF-1α degradation via ubiquitin-proteasome pathway. In hypoxia, the HIF-1α subunit becomes stable and interacts with coactivators such as cAMP response element-binding protein binding protein/p300 and regulates the expression of target genes. Overexpression of HIF-1 has been found in various cancers, and targeting HIF-1 could represent a novel approach to cancer therapy.

Occupational exposure to crystalline silica has been associated with progressive pulmonary silicosis and lung cancer, but the underlying molecular mechanisms are not well understood. Previous studies have shown that crystalline silica exposure can generate reactive oxygen species (ROS) and induce the expression of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) in cells. TNF-alpha is believed to be critical in the development of silica-related diseases. Thus it will be of significance to understand the mechanisms of TNF-alpha induction by silica exposure. Given the fact that the transcription factor nuclear factor of activated T cells (NFAT) plays an important role in the regulation of TNF-alpha and can also be activated by ROS, in this study we investigated the potential role of ROS in silica-induced NFAT activity as well as TNF-alpha expression in Cl41 cells. The results showed that exposure of cells to silica led to NFAT transactivation and TNF-alpha induction, where superoxide anion radical (O(2)(-).), but not H(2)O(2), was involved. The knockdown of NFAT3 by its specific small interfering RNA significantly attenuated the silica-induced TNF-alpha transcription. This study demonstrated that silica was able to activate NFAT in an O(2)(-).-dependent manner, which was required for TNF-alpha induction.

Although it has been well established that insoluble nickel compounds are potent carcinogens and soluble nickel compounds are less potent, the mechanisms remain unclear. Nickel compounds are weakly mutagenic, but cause epigenetic effects in cells. Previous studies have shown that insoluble nickel compounds enter cells by phagocytosis and silence gene expression, but the entry of soluble nickel compounds and their effects on gene silencing have not been well studied. Here, we have demonstrated, using a dye that fluoresces when nickel ions bind, that soluble nickel compounds were taken up by cells. Nickel ions localized initially in the cytoplasm, but later entered the nucleus and eventually silenced a transgene. In addition, we described three major changes in histone modification of cells exposed to soluble nickel compounds: (i) loss of acetylation of H2A, H2B, H3 and H4; (ii) increases of H3K9 dimethylation; and (iii) substantial increases of the ubiquitination of H2A and H2B. These effects were observed at nickel exposure conditions that had minimum effects on cell cytotoxicity. Moreover, we demonstrated that nickel-induced transgene silencing was associated with similar changes of histone modifications in their nuclesomes. This study is the first to show that nickel compounds increase histone ubiquitination in cells. These new findings will further our understanding of the epigenetic mechanisms of nickel-mediated carcinogenesis.

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.

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.

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-1a, 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-1 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-1a 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‐κB (NFκB) 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‐κB, whereas B[a]P showed only marginal effect on AP‐1 activation, and B[a]P and CDE had no effect on NF‐κB 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 (IκBα) 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‐κB in tumor promotion, we speculated the activation of AP‐1 and NF‐κB 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. © 2004 Wiley‐Liss, Inc.

The p70 S6 kinase (p70S6k) is a Ser/Thr kinase that plays an important role in cell growth, transformation, and the transition of the cell cycle in mammalian cells. Because UV radiation has been reported to induce activation of p70S6k, which is believed to play some role in the carcinogenic effects of sun exposure, the present study investigated the signaling path- ways involved in this activation induced by UV radiation in mouse epi- dermal JB6 Cl41 cells. Exposure of cells to UV radiation led to marked increases in p70S6k activity and phosphorylation at Thr389 and Thr421/ Ser424. UV radiation also generated reactive oxygen species as measured by electron spin resonance and by H2O2 and O2 . fluorescence staining assays in JB6 Cl 41 cells. The scavenging of UV-generated H2O2 by N-acety-L-cyteine (a general antioxidant) or catalase (a specific H 2O2 scavenger) inhibited p70S6k phosphorylation at Thr389 and Thr421/Ser424, whereas pretreatment of cells with sodium formate (an OH radical scav- enger) or superoxide dismutase (an O2 . radical scavenger) did not show any inhibitory effects. Importantly, UV-induced increases in p70S6k phos- phorylation at Thr389 and Thr421/Ser424 were dramatically inhibited by pretreatment of cells with rapamycin, LY294002, or PD98059, whereas overexpression of dominant-negative mutants of PKC/ and Akt1 did not inhibit p70S6k phosphorylation at Thr389 and Thr421/Ser424. These results demonstrated that H2O2, phosphatidylinositol 3-kinase, and mammalian target of rapamycin were important players for UV-induced p70S6k phos- phorylation at Thr389 and Thr421/Ser424, whereas Akt and atypical protein kinase C were not involved in this activation. The role of H2O2 in p70 S6k phosphorylation at Thr389 and Thr421/Ser424 was further supported by the findings that treatment of cells with H2O2 also caused p70 S6k phosphoryl- ation at Thr389 and Thr421/Ser424.

Arsenic is a metalloid compound that is widely distributed in the environment. Human exposure of this compound has been associated with increased cancer incidence. Although the exact mechanisms remain to be investigated, numerous carcinogenic pathways have been proposed. Potential carcinogenic actions for arsenic include oxidative stress, genotoxic damage, DNA repair inhibition, epigenetic events, and activation of certain signal transduction pathways leading to abberrant gene expression. In this article, we summarize current knowledge on the molecular mechanisms of arsenic carcinogenesis with an emphasis on ROS and signal transduction pathways.