Faculty Bibliography

Hexavalent chromium [Cr(VI)] is a human carcinogen that results in the generation of reactive oxygen species (ROS) and a variety of DNA lesions leading to cell death. Epigallocatechin-3-gallate (EGCG), the major polyphenol present in green tea, possesses potent antioxidative activity capable of protecting normal cells from various stimuli-induced oxidative stress and cell death. Here we demonstrated that co-treatment with EGCG protected human normal bronchial epithelial BEAS-2B cells from Cr(VI)-induced cell death in a dose-dependent manner. Cr(VI) induces apoptosis as the primary mode of cell death. Co-treatment of BEAS-2B cells with EGCG dose-dependently suppressed Cr(VI)-induced apoptosis. Fluorescence microscopic analyses and quantitative measurement revealed that EGCG significantly decreased intracellular levels of ROS induced by Cr(VI) exposure. Using a well-established K(+)/SDS precipitation assay, we further showed that EGCG was able to dose-dependently reduce DNA-protein cross-links (DPC), lesions that could be partially attributed to Cr(VI)-induced oxidative stress. Finally, analyses of Affymetrix microarray containing 28,869 well-annotated genes revealed that, among the 3412 genes changed more than 1.5-fold by Cr(VI) treatment, changes of 2404 genes (70%) were inhibited by pretreatment of EGCG. Real-time PCR confirmed the induction of 3 genes involved in cell death and apoptosis by Cr(VI), which was eliminated by EGCG. In contrast, Cr(VI) reduced the expression of 3 genes related to cellular defense, and this reduction was inhibited by EGCG. Our results indicate that EGCG protects BEAS-2B cells from Cr(VI)-induced cytotoxicity presumably by scavenging ROS and modulating a subset of genes. EGCG, therefore, might serve as a potential chemopreventive agent against Cr(VI) carcinogenesis

Epigenetic dysregulation is being increasingly recognized as a hallmark of cancer. Post-translational modifications of histones, in particular, are known to play important roles gene expression alterations in cancer development and progression. Given their key involvement in the various stages of carcinogenesis, histone modifications are also being explored as potential biomarkers of disease progression and prognosis. This review will therefore discuss the role of histone modifications in cancer biology and will explore their prognostic potential.

A major target of Ni ions in cells is the dioxygenase family of enzymes. Since Fe binding is required for catalytic activity and Ni ions readily displace the Fe from these enzymes, Ni exposure results in an inactive enzyme. ABH2 is an oxidative demethylase that is inhibited by Ni ions. The Kd for nickel binding is about three times better than the Fe binding to this enzyme. In addition, using a mutant ABH2 where a histidine has been replaced by an aspartic acid and the wild-type enzyme, Ni ions were found to bind more to the wild-type enzyme when both were transfected into cultured cells. Nickel ions were found to induce global increases in H3K9 dime and H3K4 trime which were mapped in the genome with Chip- Seq and correlated with gene expression using RNA-Seq. We have been studying histone tail modifications in human peripheral blood lymphocytes from an unexposed and exposed cohort of nickel refinery workers in China. We will report these results at the meeting

Human ABH2 repairs DNA lesions by using an Fe(II)- and alphaKG-dependent oxidative demethylation mechanism. The structure of the active site features the facial triad of protein ligands consisting of the side chains of two histidine residues and one aspartate residue that is common to many non-heme Fe(II) oxygenases. X-ray absorption spectroscopy (XAS) of metallated (Fe and Ni) samples of ABH2 was used to investigate the mechanism of ABH2 and its inhibition by Ni(II) ions. The data are consistent with a sequential mechanism that features a five-coordinate metal center in the presence and absence of the alpha-ketoglutarate cofactor. This aspect is not altered in the Ni(II)-substituted enzyme, and both metals are shown to bind the cofactor. When the substrate is bound to the native Fe(II) complex with alpha-ketoglutarate bound, a five-coordinate Fe(II) center is retained that features an open coordination position for O(2) binding. However, in the case of the Ni(II)-substituted enzyme, the complex that forms in the presence of the cofactor and substrate is six-coordinate and, therefore, features no open coordination site for oxygen activation at the metal

Occupational exposure to nickel compounds has been associated with lung and nasal cancers. We have previously shown that exposure of the human lung adenocarcinoma A549 cells to NiCl(2) for 24 hr significantly increased global levels of trimethylated H3K4 (H3K4me3), a transcriptional activating mark that maps to the promoters of transcribed genes. To further understand the potential epigenetic mechanism(s) underlying nickel carcinogenesis, we performed genome-wide mapping of H3K4me3 by chromatin immunoprecipitation and direct genome sequencing (ChIP-seq) and correlated with transcriptome genome-wide mapping of RNA transcripts by massive parallel sequencing of cDNA (RNA-seq). The effect of NiCl(2) treatment on H3K4me3 peaks within 5,000 bp of transcription start sites (TSSs) on a set of genes highly induced by nickel in both A549 cells and human peripheral blood mononuclear cells were analyzed. Nickel exposure increased the level of H3K4 trimethylation in both the promoters and coding regions of several genes including CA9 and NDRG1 that were increased in expression in A549 cells. We have also compared the extent of the H3K4 trimethylation in the absence and presence of formaldehyde crosslinking and observed that crosslinking of chromatin was required to observe H3K4 trimethylation in the coding regions immediately downstream of TSSs of some nickel-induced genes including ADM and IGFBP3. This is the first genome-wide mapping of trimethylated H3K4 in the promoter and coding regions of genes induced after exposure to NiCl(2). This study may provide insights into the epigenetic mechanism(s) underlying the carcinogenicity of nickel compounds

BACKGROUND: Hexavalent chromium [Cr(VI)] is a potent human carcinogen. Occupational exposure has been associated with increased risk of respiratory cancer. Multiple mechanisms have been shown to contribute to Cr(VI) induced carcinogenesis, including DNA damage, genomic instability, and epigenetic modulation, however, the molecular mechanism and downstream genes mediating chromium's carcinogenicity remain to be elucidated. METHODS/RESULTS: We established chromate transformed cell lines by chronic exposure of normal human bronchial epithelial BEAS-2B cells to low doses of Cr(VI) followed by anchorage-independent growth. These transformed cell lines not only exhibited consistent morphological changes but also acquired altered and distinct gene expression patterns compared with normal BEAS-2B cells and control cell lines (untreated) that arose spontaneously in soft agar. Interestingly, the gene expression profiles of six Cr(VI) transformed cell lines were remarkably similar to each other yet differed significantly from that of either control cell lines or normal BEAS-2B cells. A total of 409 differentially expressed genes were identified in Cr(VI) transformed cells compared to control cells. Genes related to cell-to-cell junction were upregulated in all Cr(VI) transformed cells, while genes associated with the interaction between cells and their extracellular matrices were down-regulated. Additionally, expression of genes involved in cell proliferation and apoptosis were also changed. CONCLUSION: This study is the first to report gene expression profiling of Cr(VI) transformed cells. The gene expression changes across individual chromate exposed clones were remarkably similar to each other but differed significantly from the gene expression found in anchorage-independent clones that arose spontaneously. Our analysis identified many novel gene expression changes that may contribute to chromate induced cell transformation, and collectively this type of information will provide a better understanding of the mechanism underlying chromate carcinogenicity

Non-heme Fe enzymes catalyze a wide range of oxidations involving 02. We have used a combination ofspectroscopic techniques to investigate the structure and function of three different kinds of mononuclear non-heme Fe enzymes -ABH2 (a DNA demethylase), histone demethylases (JMJD2A, JMJD2C and JMJD2D), and rat liver cysteine dioxygenase (CDO). The demethylases have the 2-His-1-carboxylate Fe-binding motif, while CDO has a triad of histidines. It has been shown that Ni substitution causes inactivation ofABH2 that leads to transcriptional silencing. X-ray absorption spectroscopy (XAS) has been used to investigate the active site ofABH2 in presence of Fe and Ni. XAS shows that in the presence of both cofactor and substrate Fe is 5-coordinate (leaving an empty coordination site for 02 activation) but Ni is 6-coordinate (no empty coordination site for 02 binding). CDO catalyzes the conversion of cysteine to cysteine sulfinic acid (CSA). XAS of rat liver CDO reconstituted with Fe(ll) shows the presence of a 5- coordinate Fe site both in presence and absence of cysteine. Ligation of cysteine is confirmed by the presence of a S- donor. Mossbauer spectroscopy of rat liver CDO reconstituted with 57Fe shows that without substrate CDO contains mostly Fe(lll), while in the presence ofsubstrate CDO contains mostly Fe(ll). Posttranslational modifications of histone tails regulate chromatin structure and gene expression. It has already been shown that Ni substitution causes inactivation of JMJD2A. It has also been shown that JMJD2A is a H3-K9me3- and H3K36me3-specific demethylase. However, little is known about the basis ofsubstrate selectivity by the JMJD2 family of histone demethylases. We will report on XAS experiments involving the JMJD2 family of histone demethylases. The results will be compared with results obtained from ABH2 and from CDO. These XAS experiments, along with Mossbauer and MCD studies will allow us to compare metal site structures, substrate selectivity and reaction mechanism among different types of nonheme Fe enzymes. (Figure presented)

Liprin-alpha4 was strongly induced following nickel (II) chloride exposure in a variety of cell types including BEAS-2B, A549, BEP2D and BL41 cells. Liprin-alpha4, a member of the Liprin alpha family, has seven isoforms but only three of these variants were detected in BEAS-2B cells (004, 201 and 202). The level of Liprin-alpha4 variants 201 and 004 were highly increased in BEAS-2B cells in response to nickel. We showed that Liprin-alpha4 bound directly to the cytoplasmic region of RPTP-LAR (receptor protein tyrosine phosphatase-leukocyte antigen-related receptor F). The cytoplasmic region of RPTP-LAR contains two phosphatase domains but only the first domain shows activity. The second domain interacts with other proteins. The phosphatase activity was increased both following nickel treatment and also in the presence of nickel ions in cell extracts. Liprin-alpha4 knock-down lines with decreased expression of Liprin-alpha4 variants 004 and 201 exhibited greater nickel toxicity compared to controls. The RPTP-LAR phosphatase activity was only slightly increased in a Liprin-alpha4 knock-down line. Liprin-alpha4 appeared necessary for the nickel induced tyrosine phosphatase activity. The presence of Liprin-alpha4 and nickel increased tyrosine phosphatase activity that reduced the global levels of tyrosine phosphorylation in the cell