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Polo-like kinase 3 (Plk3) plays an important role in the regulation of cell cycle progression and stress responses. Plk3 also has a tumor-suppressing activity as aging PLK3-null mice develop tumors in multiple organs. The growth of highly vascularized tumors in PLK3-null mice suggests a role for Plk3 in angiogenesis and cellular responses to hypoxia. By studying primary isogenic murine embryonic fibroblasts, we tested the hypothesis that Plk3 functions as a component in the hypoxia signaling pathway. PLK3(-/-) murine embryonic fibroblasts contained an enhanced level of HIF-1α under hypoxic conditions. Immunoprecipitation and pulldown analyses revealed that Plk3 physically interacted with HIF-1α under hypoxia. Purified recombinant Plk3, but not a kinase-defective mutant, phosphorylated HIF-1α in vitro, resulting in a major mobility shift. Mass spectrometry identified two unique serine residues that were phosphorylated by Plk3. Moreover, ectopic expression followed by cycloheximide or pulse-chase treatment demonstrated that phospho-mutants exhibited a much longer half-life than the wild-type counterpart, strongly suggesting that Plk3 directly regulates HIF-1α stability in vivo. Combined, our study identifies Plk3 as a new and essential player in the regulation of the hypoxia signaling pathway.

Epigenetic silencing of tumor suppressor genes commonly occurs in human cancers via increasing DNA methylation and repressive histone modifications at gene promoters. However, little is known about how pathogenic environmental factors contribute to cancer development by affecting epigenetic regulatory mechanisms. Previously, we reported that both hypoxia and nickel (an environmental carcinogen) increased global histone H3 lysine 9 methylation in cells through inhibiting a novel class of iron- and α-ketoglutarate-dependent histone demethylases. Here, we investigated whether inhibition of histone demethylase JMJD1A by hypoxia and nickel could lead to repression/silencing of JMJD1A-targeted gene(s). By using Affymetrix GeneChip and ChIP-on-chip technologies, we identified Spry2 gene, a key regulator of receptor tyrosine kinase/extracellular signal-regulated kinase (ERK) signaling, as one of the JMJD1A-targeted genes in human bronchial epithelial BEAS-2B cells. Both hypoxia and nickel exposure increased the level of H3K9me2 at the Spry2 promoter by inhibiting JMJD1A, which probably led to a decreased expression of Spry2 in BEAS-2B cells. Repression of Spry2 potentiated the nickel-induced ERK phosphorylation, and forced expression of Spry2 in BEAS-2B cells decreased the nickel-induced ERK phosphorylation and significantly suppressed nickel-induced anchorage-independent growth. Taken together, our results suggest that histone demethylases could be targets of environmental carcinogens and their inhibition may lead to altered gene expression and eventually carcinogenesis.

Breast cancer is the most prevalent cancer among women worldwide, and is the second leading cause of cancer mortality among women. Triple-negative breast cancers (TNBCs; ER-/PR-/HER2-) are highly aggressive tumors with poor prognosis. Due to lack of these specific receptors, TNBCs cannot be treated with hormone therapies or anti-HER2 targeted therapies. Chemotherapy, combined with surgery and radiation therapy, is the primary strategy for treating TNBCs. Thus, discovery and evaluation of new alternative medications targeting triple-negative breast cancer is of tremendous importance for reducing breast cancer mortality. Isorhapontigenin (ISO), a new derivative of stilbene isolated from Chinese herb Gnetum Cleistostachyun, exhibits a strong inhibitory effect on human bladder cancers. However, its anticancer activity with respect to breast cancer remains unclear. In the present study, we investigated the potential anticancer effect of ISO on TNBCs. ISO treatment inhibited the proliferation and anchorage-independent growth of 4T1 cells, a murine triple-negative breast cancer cell line, in a dose-dependent manner. In addition, the results from wound healing and Transwell invasion assay indicated significantly reduced cell migration and invasion in ISO-treated cells. Moreover, we performed the whole transcriptome analysis using RNA-seq to elucidate the molecules and pathways that mediate ISO's anticancer activity. A total of 1972 differentially expressed genes were identified in ISO-treated cells. IPA analysis revealed that the top canonical pathway altered by ISO is NRF2-mediated pathway. ISO-induced NRF2 activation was further confirmed by nuclear translocation of NRF2 protein and upregulation of NRF2 downstream target genes (GSTA1, GSTM1, HMOX1, and NQO1). In summary, our study is the first to demonstrate that ISO inhibits breast cancer cell growth and invasion, in part, by activating NRF2-mediated signaling pathway. Our results provide a novel mechanism for ISO-induced anticancer effect, and strongly support ISO as a promising therapeutic agent for triple-negative breast cancers. Citation Format: Yusha Zhu, Anthony Murphy, Thomas Kluz, Bo Li, Max Costa, Chuanshu Huang, Hong Sun. Isorhapontigenin inhibits triple-negative breast cancer via activating NRF2-mediated pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5857.

Naturally occurring inorganic arsenic has been identified as a prominent causal agent in lung, bladder, liver, and prostate cancers. Although arsenic (As) has long been known to induce carcinogenicity via epigenetic mechanisms, alteration in histone gene expression has not been explored. The replication-dependent histone genes (also known as canonical histone genes) are the only genes found in multicellular organisms whose messenger RNA (mRNA) does not terminate with a poly(A) tail at the 3' end. Instead, they contain a conserved 26-nucleotide sequence that forms a stem-loop structure, and serves as the binding site for the Stem-loop binding protein (SLBP). SLBP is critical for canonical histone pre-mRNA processing and histone mRNA translation. Our previous studies demonstrated that arsenic exposure increases polyadenylation of histone H3.1 mRNA (a canonical histone H3) and its presence outside of S phase of the cell cycle due to the loss of SLBP protein and mRNA. Here we report that ectopic expression of polyadenylated H3.1 mRNA induces cell transformation and that the transformed cells are able to form tumors in athymic nude mice. These carcinogenic effects are dependent on the level of polyadenylation of canonical H3.1 mRNA. Notably, transfection of H3.1 cDNA with a stem-loop sequence in the front of the poly(A) signal generates less polyadenylated H3.1 mRNA and thereby attenuates cell transformation. Furthermore, the increase in polyadenylated H3.1 mRNA is able to antagonize histone H3.3 (a non-canonical variant of H3) in the promoter of active genes and perturb expression of cancer-related genes. In addition transfection of H3.1 with a poly(A) tail causes mitotic block in the cell cycle as well as chromosome instability. Furthermore, acute arsenic exposure by inhalation in mice results in the loss of SLBP and gain of polyadenylated H3.1 mRNA in lung tissues. These results suggest that the polyadenylation of canonical histone mRNAs induce by Arsenic is very disruptive to cellular genomic integrity and contributes to development of human cancers. Citation Format: Qiao Yi Chen, Danqi Chen, Thomas Kluz, Feng Wu, Yusha Zhu, Thomas Des Marais, Jinquan Li, Xiaoru Zhang, Ashley Jordan, Hong Sun, Chunyuan Jin, Max Costa. Polyadenyaltion of canonical histone mRNA: A potential mechanism of arsenic-induced carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 356.

This work compares the performances of two Golomb family entropy coders applied to a video codec system named FHVC (fast hadamard video codec). The entropy coders considered have different operation modes and specific adaptation strategies. The work also presents a new 3D-transform coefficient scan order developed for the FHVC. This scan process is based on the multiplication of the three-dimensional sequency numbers of each coefficient. The FHVC (which is also described in this work) is a fast embedded color video codec developed to be implemented in a video set-top box used in a fiber optics network. The focus is on more reduced execution times, and not on higher compression rates. Low computational complexity and use of meager computational resources are also required. All the multiplications and divisions operations are performed by binary shifts and the system is implemented exclusively with 16-bit integer arithmetic. Even with these constraints, good distortion versus bit-rate results were achieved. The Hadamard transform is used in a three-dimensional fashion, in order to reduce spatial and temporal correlation and to avoid costly motion estimation and compensation techniques. The proposed scan procedure allows the transform coefficient reading in an idealistic ''decreasing in the average'' order. After the scan procedure, the encoding of the bit sequence of the 3D-Hadamard coefficients is carried out, bit-plane-by-bit-plane, with an adaptive Golomb run-length entropy coder, which produces a fully embedded output bitstream. Two entropy coders were considered. The first one uses an empirical, but fast and efficient, adaptation strategy that shows good results on non-stationary data. The second one has an adaptation strategy that is nearly optimum, in a maximum-likelihood sense, for independent Bernoulli identically distributed data.

Animal studies and small clinical trials have shown that taurine (2-aminoethanesulphonic acid), a sulphur-containing molecule mainly obtained from the diet in human subjects, has a variety of biological actions that are related to atherosclerosis and cardiovascular functions. However, epidemiological studies of taurine and CHD risk are lacking. We evaluated whether a single measurement of serum taurine could serve as an estimate for long-term serum levels. Serum taurine was measured using HPLC in three annual samples from thirty postmenopausal women selected from the New York University Women's Health Study. Overall, serum taurine values ranged from 62·8 to 245·3 nmol/ml, with a mean of 140 nmol/ml. The intraclass correlation coefficient of a single measurement of serum taurine was 0·48 (95 % CI 0·26, 0·68), which can be improved to 0·65 by using the mean of two annual measurements. The CV was 7 %. These results indicate that the mean of two or more annual measurements of serum taurine is a sufficiently reliable measure of long-term serum levels that can be used in epidemiological studies.

In recent years, research efforts have been centered on the functional roles of special AT-rich sequence-binding protein (SATB2) in cancer development. Existing studies differ in the types of tumor tissues and cell lines used, resulting in mixed results, which hinder the clear understanding of whether SATB2 acts as a tumor suppressor or promoter. Literature search for this review consisted of a basic search on PubMed using keywords "SATB2" and "special AT-rich sequence-binding protein 2." Each article was then selected for further examination based on relevance of the title. In consideration to possible missing data from a primary PubMed search, after coding for relevant information, articles listed in the references section were filtered for further review. The current literature suggests that SATB2 can act both as a tumor suppressor and as a promoter since it can be regulated by multiple factors and is able to target different downstream genes in various types of cancer cell lines as well as tissues. Future studies should focus on its contradictory roles in different types of tumors. This paper provides a comprehensive review of currently available research on the role of SATB2 in different cancer cells and tissues and may provide some insight into the contradictory roles of SATB2 in cancer development.

Hypoxia induced factor-1α (HIF-1α) is a master transcription factor that is critical for the regulation of various cellular functions. HIF-1α is rapidly degraded via a von Hippel-Lindau tumor suppressor gene product (VHL)-mediated ubiquitin-proteasome pathway in normoxia conditions. Most recent studies reveal that heat-shock proteins (Hsp) can regulate HIF-1α protein degradation via a VHL-independent pathway. Here we demonstrate that c-Jun N-terminal kinase 1 (JNK1) is required for such an Hsp-dependent HIF-1α regulation. HIF-1α stabilization was impaired in JNK1−/− cells, and could be rescued by reconstitutional expression of JNK1 in either hypoxia or chemical-mimicked hypoxia conditions. This phenomena was confirmed in different cell lines using the shRNA-JNK1 knockdown approach. Accordingly, HIF-1-dependent transcriptional activity and its downstream gene’s expression were dramatically reduced in JNK1-deficient cells. We further found that in JNK1−/− cells the low expression level of Hsp90/Hsp70 proteins affected the protective roles of these chaperones in maintaining newly synthesized HIF-1α stabilization, and forced expression of Hsp90 or Hsp70 in JNK1−/− cells showed a notable increase in HIF-1α stability compared with that of parental cells. Furthermore, our studies found that defective HDAC6 expression and subsequently increased Hsp90 acetylation could account for the reduction of Hsp90 chaperon activity in JNK1−/− cells. Taken together, our studies provide strong evidence for a novel function of JNK1 in regulating VHL-independent HIF-1α degradation.

Higher order chromosome structures are the hallmark of mitotic and meiotic cells. Chromatin condensation and compaction are essential for rapid chromosome congression and accurate chromosome segregation during cell division. The core histones possess tails at their amino-termini. These tails, which extend from the surface of the nucleosomes, are highly dynamic and subject to an extensive array of covalent modifications. Modified histone tails play an important role, not only in the folding of nucleosomal arrays into higher order chromatin structures but also in gene regulation. The combination of these distinct covalent modifications of histones constitutes "the histone code" that regulates various cellular processes, including mitotic and meiotic progression.

We have used Saccharomyces cerevisiae to identify toxicologically important proteins and pathways involved in arsenic-induced toxicity and carcinogenicity in humans. We performed a systemic screen of the complete set of 4733 haploid S. cerevisiae single-gene-deletion mutants to identify those that have decreased or increased growth, relative to wild type, after exposure to sodium arsenite (NaAsO2). IC50 values for all mutants were determined to further validate our results. Ultimately we identified 248 mutants sensitive to arsenite and 5 mutants resistant to arsenite exposure. We analyzed the proteins corresponding to arsenite-sensitive mutants and determined that they belonged to functional categories that include protein binding, phosphate metabolism, vacuolar/lysosomal transport, protein targeting, sorting, and translocation, cell growth/morphogenesis, cell polarity and filament formation. Furthermore, these data were mapped onto a protein interactome to identify arsenite-toxicity-modulating networks. These networks are associated with the cytoskeleton, ubiquitination, histone acetylation and the MAPK signaling pathway. Our studies have potential implications for understanding toxicity and carcinogenesis in arsenic-induced human conditions, such as cancer and aging.

Global acetylation of histone H4 is a mark of gene transcriptional activation. The c-Myc transcription factor binds to specific DNA sites in cellular chromatin and induces the acetylation of histone H4. In this study, hypoxia (1% Oxygen) induced a decrease in both global acetylated histone H4 (AcH4) and c-Myc in human lung carcinoma A549 cells and in human bronchial epithelial Beas-2B cells, The decline was more striking in A549 cells compared to Beas2-B cells, when cells were exposed to hypoxic stress for 24 h. Further studies showed that these alterations of global AcH4 can be attributed to the decrease in c-Myc protein levels. While hypoxia-induced gene activation is known to be mediated by Hypoxia Response Elements (HRE), the mechanism for down-regulation of genes by hypoxia is not known. The decrease in c-Myc protein levels induced by hypoxia may contribute to hypoxia-induced gene repression.

Hexavalent chromium [Cr(VI)] is a mutagen and carcinogen, and occupational exposure can lead to lung cancers and other adverse health effects. Genetic changes resulting from DNA damage have been proposed as an important mechanism that mediates chromate's carcinogenicity. Here we show that chromate exposure of human lung A549 cells increased global levels of di- and tri-methylated histone H3 lysine 9 (H3K9) and lysine 4 (H3K4) but decreased the levels of tri-methylated histone H3 lysine 27 (H3K27) and di-methylated histone H3 arginine 2 (H3R2). Most interestingly, H3K9 dimethylation was enriched in the human MLH1 gene promoter following chromate exposure and this was correlated with decreased MLH1 mRNA expression. Chromate exposure increased the protein as well as mRNA levels of G9a a histone methyltransferase that specifically methylates H3K9. This Cr(VI)-induced increase in G9a may account for the global elevation of H3K9 dimethylation. Furthermore, supplementation with ascorbate, the primary reductant of Cr(VI) and also an essential cofactor for the histone demethylase activity, partially reversed the H3K9 dimethylation induced by chromate. Thus our studies suggest that Cr(VI) may target histone methyltransferases and demethylases, which in turn affect both global and gene promoter specific histone methylation, leading to the silencing of specific tumor suppressor genes such as MLH1.

Occupational exposure to nickel (Ni), chromium (Cr), and arsenic (As) containing compounds has been associated with lung cancer and other adverse health effects. Their carcinogenic properties may be attributable in part, to activation and/or repression of gene expression induced by changes in the DNA methylation status and histone tail post-translational modifications. Here we show that individual treatment with nickel, chromate, and arsenite all affect the gene activating mark H3K4 methylation. We found that nickel (1 mM), chromate (10 microM), and arsenite (1 microM) significantly increase tri-methyl H3K4 after 24 h exposure in human lung carcinoma A549 cells. Seven days of exposure to lower levels of nickel (50 and 100 microM), chromate (0.5 and 1 microM) or arsenite (0.1, 0.5 and 1 microM) also increased tri-methylated H3K4 in A549 cells. This mark still remained elevated and inherited through cell division 7 days following removal of 1 microM arsenite. We also demonstrate by dual staining immunofluorescence microscopy that both H3K4 tri-methyl and H3K9 di-methyl marks increase globally after 24 h exposure to each metal treatment in A549 cells. However, the tri-methyl H3K4 and di-methyl H3K9 marks localize in different regions in the nucleus of the cell. Thus, our study provides further evidence that a mechanism(s) of carcinogenicity of nickel, chromate, and arsenite metal compounds may involve alterations of various histone tail modifications that may in turn affect the expression of genes that may cause transformation.

Nickel compounds are carcinogenic to humans and have been shown to alter epigenetic homeostasis. The c-Myc protein controls 15 % of human genes and it has been shown that fluctuations of c-Myc protein alter global epigenetic marks. Therefore, the regulation of c-Myc by nickel ions in immortalized but not tumorigenic human bronchial epithelial Beas-2B cells was examined in this study. It was found that c-Myc protein expression was increased by nickel ions in non-tumorigenic Beas-2B and human keratinocyte HaCaT cells. The results also indicated that nickel ions induced apoptosis in Beas-2B cells. Knockout of c-Myc and its restoration in a rat cell system confirmed the essential role of c-Myc in nickel ions induced apoptosis. Further studies in Beas-2B cells showed that nickel ion increased the c-Myc mRNA level and c-Myc promoter activity, but did not increase c-Myc mRNA and protein stability. Moreover, nickel ion upregulated c-Myc in Beas-2B cells through the MEK/ERK pathway. Collectively, the results demonstrate that c-Myc induction by nickel ions occurs via an ERK-dependent pathway and plays a crucial role in nickel-induced apoptosis in Beas-2B cells.

It is well-known that insoluble nickel compounds possess much more potent carcinogenic activities as compared with soluble nickel compounds. Although it is assumed that the different entry and clearance rate are responsible for the difference, the mechanisms underlying the different carcinogenic activities are still not well understood yet. In the present study, we found that exposure to soluble, but not insoluble nickel compounds, caused a significant inhibition of cell growth and G1/G0 cell cycle arrest, which was concomitant with a marked down-regulation of cylin D1, an essential nuclear protein for controlling G1/S transition, while both soluble and insoluble nickel compounds showed similar effects on NFkappaB activation, HIF-1alpha protein accumulation and TNF-alpha transcription and CAP43 protein expression at same doses range. The down-regulation of cyclin D1 is due to protein degradation rather than inhibition of transcription, because the nickel compounds treatment did not change cyclin D1 mRNA level, while MG132, the proteasome inhibitor, can rescue the degradation of cyclin D1 caused by soluble nickel compound. Moreover, the soluble nickel-induced cyclin D1 degradation is dependent on its Thr286 residue and requires IKKalpha, but not HIF-1alpha, which are both reported to be involved in cyclin D1 down-regulation. Taken together, we demonstrate that soluble, but not insoluble nickel compound, is able to cause cyclin D1 degradation and a cell growth arrest in an IKKalpha-dependent manner. Given the role of cyclin D1 and cell proliferation in carcinogenesis, we anticipate that the different effects of soluble and insoluble nickel compounds on cyclin D1 degradation and cell growth arrest may at least partially account for their different carcinogenic activities.

Nickel compounds are important occupational and environmental pollutants. Chronic exposure to these pollutants has been connected with increased risks of respiratory cancers and cardiovascular diseases. However, it is still not clear what are the specific molecular targets for nickel toxicity and carcinogenicity. Here, we propose that the iron- and 2-oxoglutarate-dependent dioxygenase family enzymes are important intracellular targets that mediate the toxicity and carcinogenicity of nickel. In support of this hypothesis, our data show that three different classes of enzymes in this iron- and 2-oxoglutarate-dependent dioxygenase family, including HIF-prolyl hydroxylase PHD2, histone demethylase JHDM2A/JMJD1A, and DNA repair enzyme ABH3, are all highly sensitive to nickel inhibition. Inactivation of these enzymes accounts for a number of deleterious effects caused by nickel in cells, namely hypoxia-mimic stress and aberrant epigenetic changes. Future studies on nickel’s effects on these iron- and 2-oxoglutarate-dependent dioxygenases would deepen our understanding on nickel toxicity and carcinogenicity.

Post-translational modifications of histones play a critical role in regulating genome structures and integrity. We have focused on the regulatory relationship between covalent modifications of histone H3 lysine 9 (H3K9) and H3S10 during the cell cycle. Immunofluorescence microscopy revealed that H3S10 phosphorylation in HeLa, A549, and HCT116 cells was high during prophase, prometaphase, and metaphase, whereas H3K9 monomethylation (H3K9me1) and dimethylation (H3K9me2), but not H3K9 trimethylation (H3K9me3), were significantly suppressed. When H3S10 phosphorylation started to diminish during anaphase, H3K9me1 and H3K9me2 signals reemerged. Western blot analyses confirmed that mitotic histones, extracted in an SDS-containing buffer, had little H3K9me1 and H3K9me2 signals but abundant H3K9me3 signals. However, when mitotic histones were extracted in the same buffer without SDS, the difference in H3K9me1 and H3K9me2 signals between interphase and mitotic cells disappeared. Removal of H3S10 phosphorylation by pretreatment with lambda-phosphatase unmasked mitotic H3K9me1 and H3K9me2 signals detected by both fluorescence microscopy and Western blotting. Further, H3S10 phosphorylation completely blocked methylation of H3K9 but not demethylation of the same residue in vitro. Given that several conserved motifs consisting of a Lys residue immediately followed by a Ser residue are present in histone tails, our studies reveal a potential new mechanism by which phosphorylation not only regulates selective access of methylated lysines by cellular factors but also serves to preserve methylation patterns and epigenetic programs during cell division.

Arsenic (As) is a well-characterized human carcinogen but is generally not mutagenic. The evidence that As induces both loss of global DNA methylation and gene promoter DNA hypermethylation has suggested that epigenetic mechanisms may play an important role in As-induced carcinogenesis. In the present study, we examined the change in histone methylation by As exposure. In human lung carcinoma A549 cells, exposure to inorganic trivalent As (arsenite) increased H3K9 dimethylation (H3K9me2) and decreased H3K27 trimethylation (H3K27me3), both of which represent gene silencing marks, while increasing the global levels of the H3K4 trimethylation (H3K4me3), a gene-activating mark. The increase in H3K9me2 was mediated by an increase in the histone methyltransferase G9a protein and messenger RNA levels. We also observed strikingly significant altered histone modifications induced by very low-dose (0.1 microM) arsenite. Taken together, these results suggest a potential mechanism by which As induces carcinogenesis through the alteration of specific histone methylations that represent both gene silencing and activating marks. Furthermore, these marks are known to affect DNA methylation, and it is likely that arsenic's effect is not limited to histone modifications alone, but extends, perhaps by them, to DNA methylations as well. Future studies in our laboratory will address the genomic location of these silencing and activating marks using ChIP-on-chip technology.

Polo-like kinase 3 (Plk3) is an important mediator of the cellular responses to genotoxic stresses. In this study, we examined the physiologic function of Plk3 by generating Plk3-deficient mice. Plk3−/− mice displayed an increase in weight and developed tumors in various organs at advanced age. Many tumors in Plk3−/− mice were large in size, exhibiting enhanced angiogenesis. Plk3−/− mouse embryonic fibroblasts were hypersensitive to the induction of hypoxia-inducible factor-1α (HIF-1α) under hypoxic conditions or by nickel and cobalt ion treatments. Ectopic expression of the Plk3-kinase domain (Plk3-KD), but not its Polo-box domain or a Plk3-KD mutant, suppressed the nuclear accumulation of HIF-1α induced by nickel or cobalt ions. Moreover, hypoxia-induced HIF-1α expression was tightly associated with a significant down-regulation of Plk3 expression in HeLa cells. Given the importance of HIF-1α in mediating the activation of the “survival machinery” in cancer cells, these studies strongly suggest that enhanced tumorigenesis in Plk3-null mice is at least partially mediated by a deregulated HIF-1 pathway. [Cancer Res 2008;68(11):4077–85]

Oxygen levels range from 2% to 9% in vivo. Atmospheric O2 levels (21%) are known to induce cell proliferation defects and cellular senescence in primary cell cultures. However, the mechanistic basis of the deleterious effects of higher O2 levels is not fully understood. On the other hand, immortalized cells including cancer cell lines, which evade cellular senescence are normally cultured at 21% O2 and the effects of higher O2 on these cells are understudied. Here, we addressed this problem by culturing immortalized human bronchial epithelial (BEAS-2B) cells at ambient atmospheric, 21% O2 and lower, 10% O2. Our results show increased inflammatory response at 21% O2 but not at 10% O2. We found higher RelA binding at the NF-κB1/RelA target gene promoters as well as upregulation of several pro-inflammatory cytokines in cells cultured at 21% O2. RelA knockdown prevented the upregulation of the pro-inflammatory cytokines at 21% O2, suggesting NF-κB1/RelA as a major mediator of inflammatory response in cells cultured at 21% O2. Interestingly, unlike the 21% O2 cultured cells, exposure of 10% O2 cultured cells to H2O2 did not elicit inflammatory response, suggesting increased ability to tolerate oxidative stress in cells cultured at lower O2 levels.

Distributed Raman amplifiers (DRAs) have gained interest for elastic optical networking (EON) because they allow, in some configurations, to increase the amplification bandwidth, to enhance the gain flatness and to decrease the noise insertion compared to conventional erbium doped fiber amplifiers (EDFAs). However, there are operational issues in the deployment of DRAs because the transmission fiber itself serves as the gain medium. The characteristics of this fiber, that are not always perfectly known in field installations, have an influence on the Raman gain used for the signal amplification. As a consequence, the pump power of the DRAs must be adjusted to adapt to the actual fiber properties to provide accurate automatic gain control. In this paper, we present a field calibration procedure for distributed counter-propagating Raman amplifiers to enhance their automatic gain control. We demonstrate the performance of this calibration procedure in simulations for transmission fibers of different attenuation profiles.

Cadmium (Cd) is a toxic and carcinogenic metal naturally occurring in the earth’s crust. A common route of human exposure is via diet and cadmium accumulates in the liver. The effects of Cd exposure on gene expression in human hepatocellular carcinoma (HepG2) cells were examined in this study. HepG2 cells were acutely-treated with 0.1, 0.5, or 1.0 μM Cd for 24 hours; or chronically-treated with 0.01, 0.05, or 0.1 μM Cd for three weeks and gene expression analysis was performed using Affymetrix GeneChip® Human Gene 1.0 ST Arrays. Acute and chronic exposures significantly altered the expression of 333 and 181 genes, respectively. The genes most upregulated by acute exposure included several metallothioneins. Downregulated genes included the monooxygenase CYP3A7, involved in drug and lipid metabolism. In contrast, CYP3A7 was upregulated by chronic Cd exposure, as was DNAJB9, an anti-apoptotic J protein. Genes downregulated following chronic exposure included the transcriptional regulator early growth response protein 1. Ingenuity Pathway Analysis revealed that the top networks altered by acute exposure were lipid metabolism, small molecule biosynthesis, and cell morphology, organization, and development; while top networks altered by chronic exposure were organ morphology, cell cycle, cell signaling, and renal and urological diseases/cancer. Many of the dysregulated genes play important roles in cellular growth, proliferation, and apoptosis, and may be involved in carcinogenesis. In addition to gene expression changes, HepG2 cells treated with cadmium for 24 hours indicated a reduction in global levels of histone methylation and acetylation that persisted 72 hours post-treatment.

Long Term Evolution (LTE) is the standard developed by the 3rd Generation Partnership Project (3GPP) to address the ITU's 4G requirements. For the first time, we have technology convergence in a generation of cellular communication systems. LTE promises data transmission of about 100 Mbps and its advanced version (LTE-A) goes even further with more than 1 Gbps in the downlink channel. Such very high data rates will only be achievable with appropriate network resource optimizations. Accordingly, this paper presents a flexible and adaptive scheme of multi-user scheduling considering data traffic prioritization in the LTE downlink channel. The proposed scheduling algorithm uses Proportional Fairness and Analytic Hierarchy Process (AHP) that allows to aggregate performance criteria and to improve resource allocation for users based on the general traffic.

Future flexible transceivers will be able to adjust modulation format, symbol rate and FEC scheme according to the channel and network conditions. This paper uses a combination of theoretical derivations and experimental results to recommend the best set of parameters to satisfy a specific demand to be routed along the network. In particular, for a given required OSNR (which is related to reach) and desired net bit rate, and assuming a capacity-achieving FEC scheme, the paper provides the set of modulation format, symbol rate and code rate which requires the minimum bandwidth, while offering an arbitrarily low bit error rate. We observed that optimum operating points required FEC schemes with overheads ranging from almost 0 to 100%, which is an important guideline for future flexible optical communications systems.

Metals such as arsenic, cadmium, beryllium, and nickel are known human carcinogens; however, other transition metals, such as tungsten (W), remain relatively uninvestigated with regard to their potential carcinogenic activity. Tungsten production for industrial and military applications has almost doubled over the past decade and continues to increase. Here, for the first time, we demonstrate tungsten's ability to induce carcinogenic related endpoints including cell transformation, increased migration, xenograft growth in nude mice, and the activation of multiple cancer-related pathways in transformed clones as determined by RNA sequencing. Human bronchial epithelial cell line (Beas-2B) exposed to tungsten developed carcinogenic properties. In a soft agar assay, tungsten-treated cells formed more colonies than controls and the tungsten-transformed clones formed tumors in nude mice. RNA-sequencing data revealed that the tungsten-transformed clones altered the expression of many cancer-associated genes when compared to control clones. Genes involved in lung cancer, leukemia, and general cancer genes were deregulated by tungsten. Taken together, our data show the carcinogenic potential of tungsten. Further tests are needed, including in vivo and human studies, in order to validate tungsten as a carcinogen to humans.

Long Term Evolution (LTE), a standard developed by the 3rd Generation Partnership Project (3GPP), is currently the 4G mainstream technology, providing technology convergence for this generation of cellular communication systems. LTE networks promise data transmission rates of 100 Mbps in the downlink channel. For this high throughput it needs to optimize the use of network resources. LTE uses scheduling techniques for radio resource allocation based on communication channel conditions. This paper presents a heuristic customizable algorithm for multi-user data scheduling in the LTE downlink channel that can prioritize selected types of services. The proposed scheduling scheme is a modified version of the proportional fairness scheduler that uses Analytic Hierarchy Process (AHP) techniques. This allows to aggregate performance criteria to improve resource allocation based on general traffic conditions and a set of predefined service priorities. The approach for this customization is to assign weights to certain parameters used in the resource allocation process, thus modifying the relative importance of these parameters and creating a hierarchy of criteria. Criteria for different types of data are added through comparison matrices. A comparison matrix quantifies the judgment of the decision maker with respect to the relative importance of different criteria. Thus the scheduler can be made flexible and adaptive to different traffic scenarios. Examples are given where video traffic is prioritized.

Almost-Gaussian (AG) and almost-lossless (AL) properties are used to derive almost trivial proofs of almost-achievable corner points of the capacity region of Gaussian interference channels. For the missing corner point, the proof is almost complete.

Arsenic contamination of drinking water occurs globally and is associated with numerous diseases including skin, lung and bladder cancers, and cardiovascular disease. Recent research indicates that arsenic may be an endocrine disruptor. This study was conducted to evaluate the nature of gene expression changes among males and females exposed to arsenic contaminated water in Bangladesh at high and low doses. Twenty-nine (55% male) Bangladeshi adults with water arsenic exposure ranging from 50 to 1000 μg/L were selected from the Folic Acid Creatinine Trial. RNA was extracted from peripheral blood mononuclear cells for gene expression profiling using Affymetrix 1.0 ST arrays. Differentially expressed genes were assessed between high and low exposure groups for males and females separately and findings were validated using quantitative real-time PCR. There were 534 and 645 differentially expressed genes (p

In 1990, the International Agency for Cancer Research (IARC) concluded that nickel compounds were human carcinogens; prior to that time, the majority of in vivo animal data and in vitro genetic toxicology data pointed to insoluble particulate nickel species as the most carcinogenic compounds. However, in following the two decades, experimental data have shown that water-soluble nickel compounds also invoke adverse effects. The IARC affirmed the Group 1 carcinogenicity of soluble and insoluble nickel in 2009 (IARC, 2012). Upon reconsideration of previous experimental studies with shorter than 24-48 h acute exposures to insoluble nickel compounds in vitro, it is now thought that those actual nickel exposures were in fact of much longer duration than previously thought, owing to the unlikelihood of adherent nickel particle removal from the cell surfaces. Nevertheless, whereas accumulating studies of molecular and epigenetic pathways reviewed in this chapter show that the soluble NiCl2 are genotoxic and epigenotoxic, especially when cells are chronically exposed for long durations of up to several weeks to allow sufficient nickel uptake, there is still some debate within the field on this topic. This chapter briefly reviews some of the most recent work on nickel uptake, metabolism, environmental and occupational exposures, and human disease epidemiology. Recent reviews on nickel, including those of Munoz and Costa (2012) and Arita and Costa (2009), provide information on further resources. The newest information on the molecular signaling and epigenetic mechanisms of nickel’s action in biological systems is briefly summarized herein and more in-depth knowledge can be gained from a recent review (Chervona et al., 2012), as well as from Chapter 9 of this handbook, “Selected Molecular Mechanisms of Metal Toxicity and Carcinogenicity.” Furthermore, additional details on the carcinogenicity of nickel compounds can be gleaned from Chapter 18 of this handbook, “Carcinogenicity of Metal Compounds.”

Chromium (Cr) is found in nature primarily as chromite ore with Cr in the trivalent [Cr(III)] form. This ore is used for manufacturing monochromates, dichromates, chromic acid, and Cr pigments, as well as Cr metal. Chromium chemicals are of great significance to the health of workers. The uptake of Cr via the airways and the digestive tract is much quicker in the hexavalent [Cr(VI)] than in the Cr(III) state. Chromium is found in all human organs, in both adults and newborns. Cr concentrations are generally highest in lung tissue, where the concentration tends to increase with age, most likely as a result of inhalation and retention of Cr compounds with low water solubility. Chromium is excreted via both urine and feces, but predominantly via the urine. Chromium excretion in rats has been reported to take place exponentially and with half-times of 1.5, 5.9, and 83.4 days. Trivalent Cr appears to be a nutritional supplement for humans and animals, and may play a role in the in metabolism of glucose. However, the mechanism remains to be further elucidated. The daily diet is the main source of Cr in humans and minute amounts of the intake come from drinking water and inhalation. There is recent concern about the potential toxicity of supplementation with Cr(III). Exposure to Cr(VI) compounds is the main source of acute and long-term untoward effects of Cr. Allergic reaction of the skin is the most frequently observed adverse effect of Cr, and Cr(VI)-induced ulceration of the nasal septum mucosa and of the skin are still frequently seen in developing countries. Long-term inhalation exposure to various Cr(VI) compounds has been shown to result in a high risk of carcinomas of the respiratory organs. When inhaled, some of the Cr(VI) compounds appear to be among the most potent human carcinogens. Many animal experiments involving instillation and injection of different Cr compounds have resulted in local cancers in the skin and muscle tissue. Compounds of Cr(VI) are shown to induce epigenetic changes, mutations, chromosomal aberrations, and DNA damage in the form of single-strand breaks and both DNA-protein and DNA-DNA crosslinks. The end reductant product of Cr(VI) in the cell is Cr(III), which is thought to be responsible for much of the DNA damage and mutations induced by Cr(VI). The most significant exposure to Cr(III) compounds in the general population is via food, but no untoward effects of Cr have been reported based on Cr in food. The manufacture, exposure, and analysis of Cr compounds have been reviewed by the International Agency for Research on Cancer (IARC, 1990) and they have been classified as carcinogenic to humans. Toxic and biological effects of Cr compounds are also covered in a number of reviews (e.g. WHO, 2013).

We consider lower and upper bounds on the difference of differential entropies of a Gaussian random vector and an approximately Gaussian random vector after they are “smoothed” by an arbitrarily distributed random vector of finite power. These bounds are important to establish the optimality of the corner points in the capacity region of Gaussian interference channels. A problematic issue in a previous attempt to establish these bounds was detected in 2004 and the mentioned corner points have since been dubbed “the missing corner points”. The importance of the given bounds comes from the fact that they induce Fano-type inequalities for the Gaussian interference channel. Usual Fano inequalities are based on a communication requirement. In this case, the new inequalities are derived from a non-disturbance constraint. The upper bound on the difference of differential entropies is established by the data processing inequality (DPI). For the lower bound, we do not have a complete proof, but we present an argument based on continuity and the DPI.

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.

Activation of the signaling pathways leading to gene expression regulation is critical in the carcinogenic effects of nickel exposure. In the present study, we found nickel exposure could induce cyclooxygenase-2 (COX-2) expression at transcriptional and protein levels in both human bronchoepithelial cells (Beas-2B) and murine embryonic fibroblasts (MEFs). We further provided direct evidence for the specific involvement of the JNK1 signaling pathway in the COX-2 induction using specific gene knockout approaches. Our results demonstrated that COX-2 induction by nickel was impaired in JNK1(-/-) MEFs, but not in JNK2(-/-) MEFs. Moreover, re-constitutional expression of JNK1 restored COX-2 induction, confirming the specific requirement of JNK1 in COX-2 induction. Further investigation revealed that JNK1 mediated the nickel-induced COX-2 expression in a c-Jun/AP-1-dependent manner. Ectopic expression of TAM67, a c-Jun dominant negative mutant, also suppressed the COX-2 induction. Our results demonstrate that the JNK1/c-Jun/AP-1 pathway, but not the JNK2 pathway, plays a critical role in nickel-induced COX-2 expression.

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-1beta and an oxygen-regulated subunit HIF-1alpha (or its paralogs HIF-2alpha and HIF-3alpha). The stability and activity of the alpha 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-1alpha trigger its association with pVHL E3 ligase complex, leading to HIF-1alpha degradation via ubiquitin-proteasome pathway. In hypoxia, the HIF-1alpha 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.

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.

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.

DMT1-Divalent Metal (Ion) Transporter 1 or SLC11A2/DCT1/Nramp2 - transports Fe2+ into the duodenum and out of the endosome during the transferrin cycle. DMTI also is important in non-transferrin bound iron uptake. It plays similar roles in Mn2+ trafficking. Voltage clamping showed that six other metals evoked currents, but it is unclear if these metals are substrates for DMT1. This report summarizes progress on which metals DMT1 transports, focusing on results from the authors' labs. We recently cloned 1A/+IRE and 2/-IRE DMT1 isoforms to generate HEK293 cell lines that express them in a tetracycline-inducible fashion, then compared induced expression to uninduced expression and to endogenous DMT1 expression. Induced expression increases approximately 50x over endogenous expression and approximately 10x over uninduced levels. Fe2+, Mn2+, Ni2+ and Cu1+ or Cu2+ are transported. We also explored competition between metal ions using this system because incorporation essentially represents DMT1 transport and find this order for transport affinity: Mn>?Cd>?Fe>Pb-Co-Ni>Zn. The effects of decreased DMT1 also could be examined. The Belgrade rat has diminished DMT1 function and thus provides ways of testing. A series of DNA constructs that generate siRNAs specific for DMT1 or certain DMT1 isoforms yield another way to test DMT1-based transport.

Chromium is a human carcinogen primarily by inhalation exposure in occupational settings. Although lung cancer has been established as a consequence of hexavalent chromium exposure in smokers and nonsmokers, some cancers of other tissues of the gastrointestinal and central nervous systems have also been noted. Except for a few reports from China, little is known about the health risks of environmental exposures to chromium. Likewise, there has been a lack of epidemiological studies of human exposure to hexavalent Cr by drinking water or ingestion, and it has been suggested that humans can perhaps tolerate hexavalent Cr at higher levels than the current drinking water standard of 50 ppb. This review highlights the most recent data on the induction of skin tumors in mice by chronic drinking-water exposure to hexavalent chromium in combination with solar ultraviolet light. This experimental system represents an important new animal model for chromate-induced cancers by ingestion of drinking water, and it suggests by extrapolation that chromate can likely be considered a human carcinogen by ingestion as well. The potential use of this animal model for future risk assessment is discussed.

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.

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.

Hexavalent chromium [Cr(VI)] is a known etiological factor in human lung cancer. Cr(VI) exposure-related lung cancer has a high mutation incidence in the p53 gene. Upon intake in human cells Cr(VI) is reduced to Cr(III), which is able to conjugate with amino acids and consequently form either binary Cr(III)-DNA or ternary Cr(III)-amino acid-DNA adducts. Both binary and ternary Cr(III)-DNA adducts are mutagenic. We have found that the Escherichia coli nucleotide excision enzyme UvrABC nuclease is able to incise Cr(III)- and Cr(III)-histidine-modified plasmid DNA and the extent of incision is proportional to the amount of Cr(III)-DNA adducts in the plasmid. In order to determine the role of Cr(III)-DNA adducts in the mutagenesis of the p53 gene in human cancer using the UvrABC nuclease incision method, we have mapped the Cr(III)-DNA distribution in PCR DNA fragments amplified from exons 5, 7 and 8 of the p53 gene. We have found that the sequence specificities of Cr(III)-DNA and Cr(III)-histidine-DNA adducts in the p53 gene sequence are identical and that both types of adducts are preferentially formed at -NGG- sequences, including codons 245, 248 and 249, the mutational hotspots in human lung cancer. It has been found that Cr(III)-DNA adducts induce mainly G to T mutations. Therefore, these results suggest that Cr(III)-DNA adduct formation contributes to the p53 gene mutations in lung carcinogenesis.

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.

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.

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.

Nickel (Ni) carcinogenesis is thought to involve gene chip silencing by epigenetic mechanisms. Serpina3g, a member of the mouse serpin family, was among the most down-regulated genes (32-fold) in response to Ni exposure of mouse cells based on the Affymetrix gene chip. Serpina3g down-regulation was controlled by a hypoxia inducible factor (HIF) mechanism. The exposure of cells to cobalt (Co), hypoxia, the iron chelator deferoxamine, and the proline hydroxylase inhibitor dimethyloxalylglycine (DMOG) also down-regulated serpina3g transcription to similar extents as soluble Ni exposure. These results support the mounting experimental evidence that water-soluble Ni compounds have a predominant effect on hypoxia signaling because of their ability to interfere with Fe homeostasis in the cell. Trichostatin A (TSA) and 5-azacytidine (5-AzaC) reactivated the Ni-silenced serpina3g gene, indicating that its silencing by Ni involved either a direct or indirect epigenetic mechanism. Analysis of the chromatin state of the serpina3g promoter by the ChIP assay revealed that exposure of mouse fibroblast cells to Ni resulted in the methylation of H3 lysine 9 within its promoter, as well as a decrease in the phosphorylation of serine 10 of H3 and a marked decrease in the acetylation of H3 and H4. Serpina3g gene expression returned to basal levels following Ni removal, suggesting that the observed silencing was a dynamic and reversible process.

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.

Vanadium is a metal widely distributed in the environment. Although vanadate-containing compounds exert potent toxic effects on a wide variety of biological systems, the mechanisms by which vanadate mediates adverse effects are not well understood. The present study investigated the vanadate-induced phosphorylation of Akt and p70S6K, two kinases known to be vital for cell survival, growth, transformation, and transition of the cell cycle in mammals. Exposure of mouse epidermal JB6 cells to vanadium led to phosphorylation of Akt and p70S6K in a time- and dose-dependent manner. Vanadium exposure also caused translocation of atypical isoforms of PKC (lambda, zeta) from the cytosol to the membrane, but had no effect on PKCalpha translocation, suggesting that the atypical PKCs (aPKC) were specifically involved in vanadium-induced cellular response. Importantly, overexpression of a dominant negative mutant PKClambda blocked Akt phosphorylation at Ser473 and Thr308, whereas it did not inhibit p70S6k phosphorylation at Thr389 and Thr421/Ser424, suggesting that aPKC activation is specifically involved in vanadium-induced activation of Akt, but not in activation of p70S6k. Furthermore, vanadium-induced p70S6k phosphorylation at Thr389 and Thr421/Ser424 and Akt phosphorylation at Thr308 occurred through a PI-3K-dependent pathway because a PI-3K dominant negative mutant inhibited induction as compared with vector control cells. These results indicate that there was a differential role of aPKC in vanadate-induced phosphorylation of Akt and p70S6k, suggesting that signal transduction pathways leading to the activation of Akt and p70S6k were different.

The carcinogenic process initiated by nongenotoxic carcinogens involves modulation of gene expression. Nickel compounds have low mutagenic activity, but are highly carcinogenic. In vitro both mouse and human cells can be efficiently transformed by soluble and insoluble nickel compounds to anchorage-independent growth. Because previous studies have shown that carcinogenic nickel compounds silence genes by inhibiting histone acetylation and enhancing DNA methylation, we investigated the effect of enhancing histone acetylation on cell transformation. The exposure of nickel-transformed cells to the histone deacetylase inhibitor trichostatin A (TSA) resulted in the appearance of significant number of revertants measured by their inability to grow in soft agar. Using the Affymetrix GeneChip we found that the level of expression of a significant number of genes was changed (suppressed or upregulated) in nickel-transformed clones but returned to a normal level in revertants obtained following TSA treatment. Moreover, we found that treatment of cells with TSA inhibited the ability of nickel to transform mouse PW cells to anchorage-independent growth. Treatment with TSA also inhibited the ability of nickel to transform human HOS cells, although to a lesser extent. In contrast, treatment with TSA was not able to revert established cancer cell lines as readily as the nickel-transformed cells. These data indicated that modulation of gene expression is important for nickel-induced transformation.

We have previously reported that the gpt transgene in G12 Chinese hamster cells could be silenced by water-insoluble nickel compounds nickel sulfide (NiS) or nickel subsulfide (Ni(3)S(2)) and showed that the transgene was silenced by de novo DNA methylation and chromatin condensation. To further understand the nature of this silencing, we used the chromatin immunoprecipitation assay to elucidate the chromatin structure in nickel-induced silenced G12 clones. We also analyzed the effects of the DNA methyltransferase inhibitor 5-azacytidine (5-AzaC) and a histone deacetylase inhibitor trichostatin A (TSA) on histone H3 and H4 acetylation and gpt gene expression in selected nickel-silenced clones. We observed that both histone H3 and H4 were hypoacetylated and a methyl DNA-binding protein MeCP2 was targeted to the gpt gene locus, resulting in a localized inactive chromatin configuration in nickel-silenced cell clones. The histone H3K9 was also found methylated in three of four nickel- silenced cell clones, whereas the histone H3K9 was deacetylated in all four cell clones, indicating that the H3K9 methylation was involved in nickel-induced gene silencing. The acetylation of the gpt gene could be increased by a combination of 5-AzaC and TSA treatment, but not by either 5-AzaC or TSA alone. The gpt transcript was studied by either Northern blot or by semiquantitative RT-PCR following treatment of the silenced clones with TSA or 5-AzaC. An increase in gpt mRNA could be detected by RT-PCR in the clones that regained acetylation of H3 and H4. These data show that gene silencing induced by nickel in the gpt transgenic cell line involved a loss of histone acetylation and an activation of histone methylation. Both H4 and H3 histone acetylation were lost in the silenced clones and these clones exhibited an increase in the methylation of the lysine 9 in histone H3.