A profile of 8-oxo-dGTPase activities in the NCI-60 human cancer panel: Meta-analytic insight into the regulation and role of MTH1 (NUDT1) gene expression in carcinogenesis.

We measured the specific 8-oxo-dGTPase activity profile of the NCI-60 panel of malignant cell lines, and MTH1 protein levels in a subset of 16 lines. Their 8-oxo-dGTPase activity was compared to twelve publicly accessible MTH1 mRNA expression data bases and their cross-consistency was analyzed. 8-oxo-dGTPase and MTH1 protein levels in these cell lines are generally, but not always, mainly determined by MTH1 mRNA expression levels. The aneuploidy number of MTH1 gene copies only slightly affects its mRNA expression levels. By using the data mining platforms Compare and CellMiner, our 8-oxo-dGTPase profile was compared to five global gene expression datasets to identify genes whose expression levels are directly or inversely associated with 8-oxo-dGTPase. We analyzed effects of SNP within MTH1 on MTH1 mRNA level and enzyme activity. Similar association analysis was performed for five microRNA expression datasets. We identified several proteins and microRNA which might be involved in the regulation of MTH1 expression and we discuss potential mechanisms. Comparison of chemical and natural products sensitivities of the NCI-60 panel suggests seven compounds which are directly or inversely associated with 8-oxo-dGTPase. We provide an integrated picture of MTH1 expression combined from eleven consistent MTH1 mRNA and our 8-oxo-dGTPase activity NCI-60 profiles.

Molecular and organismal changes in offspring of male mice treated with chemical stressors.

Both gene methylation changes and genetic instability have been noted in offspring of male rodents exposed to radiation or chemicals, but few specific gene targets have been established. Previously, we identified the gene for ribosomal RNA, rDNA, as showing methylation change in sperm of mice treated with the preconceptional carcinogen, chromium(III) chloride. rDNA is a critical cell growth regulator. Here, we investigated the effects of paternal treatments on rDNA in offspring tissue. A total of 93 litters and 758 offspring were obtained, permitting rigorous mixed-effects models statistical analysis of the results. We show that the offspring of male mice treated with Cr(III) presented increased methylation in a promoter sequence of the rDNA gene, specifically in lung. Furthermore polymorphic variants of the multi-copy rDNA genes displayed altered frequencies indicative of structural changes, as a function of both tissue type and paternal treatments. Organismal effects also occurred: some groups of offspring of male mice treated with either Cr(III) or its vehicle, acidic saline, compared with those of untreated mice, had altered average body and liver weights and levels of serum glucose and leptin. Males treated directly with Cr(III) or acidic saline presented serum hormone changes consistent with a stress response. These results establish for the first time epigenetic and genetic instability effects in a gene of central physiological importance, in offspring of male mice exposed preconceptionally to chemicals, possibly related to a stress response in these males.

Effects of ascorbic acid on carcinogenicity and acute toxicity of nickel subsulfide, and on tumor transplants growth in gulonolactone oxidase knock-out mice and wild-type C57BL mice.

The aim of this study was to test a hypothesis that ascorbate depletion could enhance carcinogenicity and acute toxicity of nickel. Homozygous L-gulono--lactone oxidase gene knock-out mice (Gulo-/- mice) unable to produce ascorbate and wild-type C57BL mice (WT mice) were injected intramuscularly with carcinogenic nickel subsulfide (Ni3S2), and observed for the development of injection site tumors for 57 weeks. Small pieces of one of the induced tumors were transplanted subcutaneously into separate groups of Gulo-/- and WT mice and the growth of these tumors was measured for up to 3 months. The two strains of mice differed significantly with regard to (1) Ni3S2 carcinogenesis: Gulo-/- mice were 40% more susceptible than WT mice; and (2) transplanted tumors development: Gulo-/- mice were more receptive to tumor growth than WT mice, but only in terms of a much shorter tumor latency; later in the exponential phase of growth, the growth rates were the same. And, with adequate ascorbate supplementation, the two strains were equally susceptible to acute toxicity of Ni3S2. Statistically significant effects of dietary ascorbate dosing levels were the following: (1) reduction in ascorbate supplementation increased acute toxicity of Ni3S2 in Gulo-/- mice; (2) ascorbate supplementation extended the latency of transplanted tumors in WT mice. In conclusion, the lack of endogenous ascorbate synthesis makes Gulo-/- mice more susceptible to Ni3S2 carcinogenesis. Dietary ascorbate tends to attenuate acute toxicity of Ni3S2 and to extend the latency of transplanted tumors. The latter effects may be of practical importance to humans and thus deserve further studies.

Ontogeny-driven rDNA rearrangement, methylation, and transcription, and paternal influence.

Gene rearrangement occurs during development in some cell types and this genome dynamics is modulated by intrinsic and extrinsic factors, including growth stimulants and nutrients. This raises a possibility that such structural change in the genome and its subsequent epigenetic modifications may also take place during mammalian ontogeny, a process undergoing finely orchestrated cell division and differentiation. We tested this hypothesis by comparing single nucleotide polymorphism-defined haplotype frequencies and DNA methylation of the rDNA multicopy gene between two mouse ontogenic stages and among three adult tissues of individual mice. Possible influences to the genetic and epigenetic dynamics by paternal exposures were also examined for Cr(III) and acid saline extrinsic factors. Variables derived from litters, individuals, and duplicate assays in large mouse populations were examined using linear mixed-effects model. We report here that active rDNA rearrangement, represented by changes of haplotype frequencies, arises during ontogenic progression from day 8 embryos to 6-week adult mice as well as in different tissue lineages and is modifiable by paternal exposures. The rDNA methylation levels were also altered in concordance with this ontogenic progression and were associated with rDNA haplotypes. Sperm showed highest level of methylation, followed by lungs and livers, and preferentially selected haplotypes that are positively associated with methylation. Livers, maintaining lower levels of rDNA methylation compared with lungs, expressed more rRNA transcript. In vitro transcription demonstrated haplotype-dependent rRNA expression. Thus, the genome is also dynamic during mammalian ontogeny and its rearrangement may trigger epigenetic changes and subsequent transcriptional controls, that are further influenced by paternal exposures.

Genotoxicity of metal ions: chemical insights.

The purpose of this review is to provide a reader with a brief account of current results and views in the area of genotoxicity of metal ions, with a special attention to underlying chemical mechanisms. The text is divided into six sections. Following a general introduction in Section 1, Section 2 describes main molecular mechanisms of formation of genotoxic lesions: hydrolysis, alkylation, and radical reactions of nucleobases and the phosphosugar DNA backbone. The basics of cellular repair of DNA lesions are also shortly presented. This section serves as a background source for Sections 3, 4, and 5. Section 3 covers the main mechanisms of metal ion genotoxicity, followed by Section 4, which describes genotoxicity of individual metals; i.e., of the confirmed carcinogens As, Be, Cd, Cr, and Ni, as well as of the suspected carcinogens Co, Cu, Fe, Pb, Pt, and 238U (also known as depleted uranium). The genotoxicity of exposures to metal mixtures is also discussed. Section 5 provides a critical overview of methodologies used for studying mutagenicity and carcinogenicity of metals; the final Section 6 summarizes the current state and future perspectives of research in genotoxic mechanisms of metal ions.

Truncation of histone H2A's C-terminal tail, as is typical for Ni(II)-assisted specific peptide bond hydrolysis, has gene expression altering effects.

Nickel(II), capable of transforming cells and causing tumors in humans and animals, has been previously shown by us to mediate hydrolytic truncation of histone H2A's C-terminal tail by 8 amino acids in both cell-free and cell culture systems. Since H2A's C-tail is involved in maintaining chromatin structure, such truncation might alter this structure and affect gene expression. To test the latter possibility, we transfected cultured T-REx 293 human embryonic kidney cells with plasmids expressing either wild type (wt) or truncated (q) histone H2A proteins, which were either untagged or N-terminally tagged with fluorescent proteins. Each histone variant was found to be incorporated into chromatin at 24 and 48 hr post-transfection. Cells transfected with the untagged plasmids were tested for gene expression by microarray and real-time PCR. Evaluation of the results for over 21,000 genes using the multidimensional scaling and hierarchical clustering methods revealed significant differences in expression of numerous genes between the q-H2A and wt-H2A transfectants. Many of the differentially expressed genes, including BAZ2A, CLDN18, CYP51A1, GFR, GIPC2, HMGB1, IRF7, JAK3, PSIP1, and VEGF, are cancer-related genes. The results thus demonstrate the potential of q-H2A to contribute to the process of carcinogenesis through epigenetic mechanisms.

Up-regulation of 8-oxo-dGTPase activity of MTH1 protein in the brain, testes and kidneys of mice exposed to (137)Cs gamma radiation.

Abstract Mammalian MTH1 protein is an antimutagenic (2'-deoxy)ribonucleoside 5'-triphosphate pyrophosphohydrolase that prevents the incorporation of oxidatively modified nucleotides into nucleic acids. It decomposes most specifically the miscoding products of oxidative damage to purine nucleic acid precursors (e.g. 8-oxo-dGTP, 2-oxo-dATP, 2-oxo-ATP, 8-oxo-GTP) that may cause point mutations or transcription errors when incorporated into DNA and RNA, respectively. The increased expression of MTH1 mRNA and MTH1 protein was previously proposed as a molecular marker of oxidative stress. Therefore, we hypothesized that increased 8-oxo-dGTPase activity of MTH1 protein in mouse organs could serve as a dose-dependent marker of exposure to ionizing radiation, which is known to induce oxidative stress. To test our hypothesis, we measured 8-oxo-dGTPase activity in six organs of male BL6 mice after exposure to 0, 10, 25 and 50 cGy and 1 Gy of (137)Cs gamma radiation given as a single whole-body dose (1 Gy/min). The mice were killed 4, 8 and 24 h after irradiation. A statistically significant induction of 8-oxo-dGTPase was found in brains, testes and kidneys but not in lungs, hearts or livers. Brains, which demonstrated the highest (4.3-fold) increase of 8-oxo-dGTPase activity, were shown to express approximately 50% higher levels of MTH1 protein. However, due to the lack of a simple positive correlation between the dose and the observed 8-oxo-dGTPase activity in brain, testes and kidneys, we conclude that measurements of 8-oxo-dGTPase activity in these organs may serve as a rough indicator rather than a quantifiable marker of radiation-induced oxidative stress.

Metal ions-stimulated iron oxidation in hydroxylases facilitates stabilization of HIF-1 alpha protein.

The exposure of cells to several metal ions stabilizes HIF-1 alpha protein. However, the molecular mechanisms are not completely understood. They may involve inhibition of hydroxylation by either substitution of iron by metal ions or by iron oxidation in the hydroxylases. Here we provide evidence supporting the latter mechanism. We show that HIF-1 alpha stabilization in human lung epithelial cells occurred following exposure to various metal and metalloid ions, including those that cannot substitute for iron in the hydroxylases. In each case addition of the reducing agent ascorbic acid (AA)* abolished HIF-1 alpha protein stabilization. To better understand the role of iron oxidation in hydroxylase inhibition and to define the role of AA in the enzyme recovery we applied molecular modeling techniques. Our results indicate that the energy required for iron substitution by Ni(II) in the enzyme is high and unlikely to be achieved in a biological system. Additionally, computer modeling allowed us to identify a tridentate coordination of AA with the enzyme-bound iron, which explains the specific demand for AA as the iron reductant. Thus, the stabilization of HIF-1 alpha by numerous metal ions that cannot substitute for iron in the enzyme, the alleviation of this effect by AA, and our computer modeling data support the hypothesis of iron oxidation in the hydroxylases following exposure to metal ions.

Marked liver tumorigenesis by Helicobacter hepaticus requires perinatal exposure.

BACKGROUND: Although severe hepatitis and liver tumors occur in a high percentage of A/J male mice naturally infected with Helicobacter hepaticus, these effects have not been observed after injection of adult mice with the bacteria. OBJECTIVES: We tested the hypothesis that perinatal exposure to the bacteria is required for liver tumorigenesis. METHODS: A/J female mice were infected by intragastric (ig) or intraperitoneal (ip) treatment with 1.5 x 10(8) H. hepaticus before pregnancy. We examined offspring at progressive time intervals, including some kept until natural death in old age. A/J, BALB/c, and C57BL/6 weanling male mice were similarly treated ig with the bacteria and observed for up to 2 years. RESULTS: After ip bacterial infection of A/J females, 41% of their male offspring developed hepatitis and 33% had hepatocellular tumors, including 18% with hepatocellular carcinoma. Treatment by the ig route resulted in a similar incidence of hepatitis in offspring (35%) but fewer total liver tumors (8%) and carcinomas (4%). By contrast, ig instillation of H. hepaticus in weanling A/J, C57BL/6, or BALB/c mice resulted in low incidence of hepatitis (0-20%) and few liver tumors, despite presence of bacteria confirmed in feces. CONCLUSIONS: Results indicate that a high incidence of liver tumors in mice infected with H. hepaticus requires perinatal exposure. Contributing perinatal factors could include known high sensitivity of neonatal liver to tumor initiation, and/or modulation of immune response to the bacterium or its toxins. Mechanisms of human perinatal sensitivity to such phenomena can be studied with this model.

Regulation of hypoxia-inducible genes by ETS1 transcription factor.

Hypoxia-inducible factor (HIF-1) regulates the expression of genes that facilitate tumor cell survival by making them more resistant to therapeutic intervention. Recent evidence suggests that the activation of other transcription factors, in cooperation with HIF-1 or acting alone, is involved in the upregulation of hypoxia-inducible genes. Here we report that high cell density, a condition that might mimic the physiologic situation in growing tumor and most probably representing nutritional starvation, upregulates hypoxia-inducible genes. This upregulation can occur in HIF-independent manner since hypoxia-inducible genes carbonic anhydrase 9 (CA9), lysyloxidase like 2 (LOXL2) and n-myc-down regulated 1 (NDRG1)/calcium activated protein (Cap43) can be upregulated by increased cell density under both normoxic and hypoxic conditions in both HIF-1 alpha-proficient and -deficient mouse fibroblasts. Moreover, cell density upregulates the same genes in 1HAEo- and A549 human lung epithelial cells. Searching for other transcription factors involved in the regulation of hypoxia-inducible genes by cell density, we focused our attention on ETS1. As reported previously, members of v-ets erythroblastosis virus E26 oncogene homolog (ETS) family transcription factors participate in the upregulation of hypoxia-inducible genes. Here, we provide evidence that ETS1 protein is upregulated at high cell density in both human and mouse cells. The involvement of ETS1 in the upregulation of hypoxia-inducible genes was further confirmed in a luciferase reporter assay using cotransfection of ETS1 expression vector with NDRG1/Cap43 promoter construct. The downregulation of ETS1 expression with small interfering RNA (siRNA) inhibited the upregulation of CA9 and NDRG1/Cap43 caused by increased cell density. Collectively, our data indicate the involvement of ETS1 along with HIF-1 in regulating hypoxia-inducible genes.

DNA damage, superoxide, and mutant K-ras in human lung adenocarcinoma cells.

DNA single-strand breaks (quantitative comet assay) were assessed to indicate ongoing genetic instability in a panel of human lung adenocarcinoma cell lines. Of these, 19/20 showed more DNA damage than a nontransformed cell line from human peripheral lung epithelium, HPL1D. DNA damage was significantly greater in those derived from pleural effusates vs those from lymph node metastases. DNA strand breaks correlated positively with superoxide (nitroblue tetrazolium reduction assay), and negatively with amount of OGG1, a repair enzyme for oxidative DNA damage. Levels of CuZn superoxide dismutase varied moderately among the lines and did not correlate with other parameters. A role for mutant K-ras through generation of reactive oxygen species was examined. Cells with mutant K-ras had significantly lower amounts of manganese superoxide dismutase (MnSOD) vs those with wild-type K-ras, but MnSOD protein correlated positively with superoxide levels. In a subset of cell lines with similar levels of MnSOD, comparable to those in HPL1D cells, K-ras activity correlated positively with levels of both superoxide and DNA strand breaks. These results suggest that persistent DNA damage in some lung adenocarcinoma cells may be caused by superoxide resulting from mutant K-ras activity, and that OGG1 is important for prevention of this damage.

The role of ascorbate in the modulation of HIF-1alpha protein and HIF-dependent transcription by chromium(VI) and nickel(II).

Molecular oxygen is involved in hydroxylation and subsequent degradation of HIF-1alpha, a subunit of HIF-1 transcription factor; therefore oxygen shortage (hypoxia) stabilizes this protein. However, HIF-1alpha can also be stabilized by transition metal ions in the presence of oxygen, suggesting that a different mechanism is involved in metal-induced hypoxic stress. Recently, we showed that the depletion of intracellular ascorbate by metals may lead to the inhibition of hydroxylases. Because nickel(II) has similarity to iron(II), an alternative hypothesis suggests that iron substitution for nickel in the enzyme inhibits hydroxylase activity. Here we investigated the induction of HIF-1 by another metal, chromium, which cannot replace iron in the enzyme. We show that chromium(VI), but not chromium(III), can oxidize ascorbate both in cells and in a cell-free system. In agreement with these data chromium(VI) stabilizes HIF-1alpha protein in cells only until it is reduced to chromium(III). In contrast, nickel(II) was found to be a catalyst, which facilitated continuous oxidation of ascorbate by ambient oxygen. These data correlate with extended stabilization of HIF-1alpha after acute exposure to nickel(II). The HIF-1-dependent reporter assays revealed that 20-24 h was required to fully develop the HIF-1 transcriptional response, and the acute exposure to nickel(II), but not chromium(VI), meets this requirement. However, repeated (chronic) exposure to chromium(VI) can also lead to extended stabilization of HIF-1alpha. Thus, the obtained data emphasize the important role of ascorbate in regulation of HIF-1 transcriptional activity in metal-exposed human lung cells.

Effects of selenium supplementation on expression of glutathione peroxidase isoforms in cultured human lung adenocarcinoma cell lines.

Selenium is an essential nutrient, a component of several anti-oxidant enzymes, and a possible factor in cancer risk, including lung cancer. We determined the subtoxic range of selenium concentration (as sodium selenite) required to increase and maintain the expression of anti-oxidant selenoproteins gluthathione peroxidases GPX1 and GPX4 at a constant level in cultures of human lung adenocarcinoma cell lines (H460, H1703 and H1944) and in HPL1D, a non-transformed lung epithelial cell line. Selenium dose-dependently increased GPX1 protein expression 1.8-fold in HPL1D cells and approximately 40-fold in H460 and H1944 cancer cells, with maximum effects at 20-40 nM. GPX4 protein was also increased, but more so in HPL1D (five-fold) than in H460 or H1944 cells (two- to three-fold). GPX1 mRNA showed similar patterns but differences of lesser magnitude. GPX1 protein and activity level was not consistently detectable in H1703 cells, with or without Se supplementation; its mRNA was present but very low. GPX4 protein level was also low in H1703 cells, but was markedly increased by selenium supplementation (48-fold). These results confirm a role for selenium in risk of lung cancer and the independent regulation of GPX1 and GPX4. Characterization of individual tumors with regard to GPX1 and GPX4 levels and regulation might be useful for interpretation of clinical studies on effects of selenium in lung cancer risk.

Ni(II) affects ubiquitination of core histones H2B and H2A.

The molecular mechanisms of nickel-induced malignant cell transformation include effects altering the structure and covalent modifications of core histones. Previously, we found that exposure of cells to Ni(II) resulted in truncation of histones H2A and H2B and thus elimination of some modification sites. Here, we investigated the effect of Ni(II) on one such modification, ubiquitination, of histones H2B and H2A in nuclei of cultured 1HAEo- and HPL1D human lung cells. After 1-5 days of exposure, Ni(II) up to 0.25 mM stimulated mono-ubiquitination of both histones, while at higher concentrations a suppression was found. Di-ubiquitination of H2A was not affected except for a drop after 5 days at 0.5 mM Ni(II). The decrease in mono-ubiquitination coincided with the appearance of truncated H2B that lacks the K120 ubiquitination site. However, prevention of truncation did not avert the decrease of H2B ubiquitination, indicating mechanistic independence of these effects. The changes in H2B ubiquitination did not fully coincide with concurrent changes in the nuclear levels of the ubiquitin-conjugating enzymes Rad6 and UbcH6. Overall, our results suggest that dysregulation of H2B ubiquitination is a part of Ni(II) adverse effects on gene expression and DNA repair which may assist in cell transformation.

Quantum chemical modeling of reaction mechanism for 2-oxoglutarate dependent enzymes: effect of substitution of iron by nickel and cobalt.

Enzymatic hydroxylation reactions carried out by 2-oxoglutarate (2OG) dependent iron-containing oxygenases were recently implicated in oxygen sensing. In addition to oxygen depletion, two metals, cobalt and nickel, are capable of inducing hypoxic stress in cells by inhibiting oxygenase activity. Two possible scenarios have been proposed for the explanation of the hypoxic effects of cobalt and nickel: oxidation of enzyme-bound iron following cobalt or nickel exposure, and substitution of iron by cobalt or nickel. Here, by using density functional theory calculations, we modeled the reaction route from the reaction components to the high-spin metal-oxide intermediate in the activation of oxygen molecule by 2OG-dependent enzymes for three metal ions Fe(II), Ni(II), and Co(II) in the active site. An initial molecular model was constructed based on the crystal structure of iron-containing asparaginyl hydroxylase (FIH-1). Nickel- and cobalt-containing enzymes were modeled by a consequent replacement of the iron in the active center. The energy profiles connecting stationary points on the potential surfaces were computed by using the intrinsic reaction coordinate (IRC) technique from the located transition states. The results of calculations show that the substitution of iron by nickel or cobalt modifies the reaction energy profile; however, qualitatively, the reaction mechanism remains essentially the same. Thus, we would postulate that if the iron ion in the active site were substitutable by nickel and/or cobalt ions enzyme activity would be considerably altered due to high activation barriers.

Hypersusceptibility to cisplatin carcinogenicity in metallothionein-I/II double knockout mice: production of hepatocellular carcinoma at clinically relevant doses.

Metallothionein (MT) is a high-affinity metal binding protein thought to mitigate the toxicity of various metals. Cisplatin is a widely used cancer chemotherapeutic that is a rodent carcinogen and may have carcinogenic potential in humans. MT seems to reduce cisplatin toxicity by binding the metal compound but how MT deficiency might impact the carcinogenic effects of cisplatin is unknown. Thus, groups (n = 25) of male MT-I/II double knockout (MT-null) or MT wild-type (WT) mice were exposed to a single treatment of cisplatin (5 or 10 mg/kg, i.p.), or left untreated (control) and observed over the next 104 weeks. The doses of cisplatin used equate to only a fraction of the total dose used typically in clinical settings. In cisplatin-treated MT-null mice, a dose-related increase in hepatocellular carcinoma (HCC) occurred (control, 0%; 5 mg/kg, 17%; 10 mg/kg, 36%) that was not seen in WT mice. Similarly, liver carcinoma multiplicity (HCC/liver) was increased markedly by cisplatin but only in MT-null mice, indicating the formation of multiple primaries in MT deficient mice. Harderian gland carcinoma incidence was also increased by cisplatin treatment in MT-null mice but not WT mice. Our results indicate that MT-null mice are hypersusceptible to the hepatocarcinogenic effects of cisplatin, and poor MT expression may be a predisposing factor for cisplatin-induced secondary tumors after chemotherapy.

Ascorbate depletion mediates up-regulation of hypoxia-associated proteins by cell density and nickel.

Exposure of human lung cells to carcinogenic nickel compounds in the presence of oxygen up-regulated carbonic anhydrase IX (CA IX) and NDRG1/Cap43, both known as intrinsic hypoxia markers and cancer-associated genes. This suggests that factors other than a shortage of oxygen may be involved in this induction. Both proteins can also be induced in the presence of oxygen by culturing these cells to a high density without medium change. The intracellular ascorbate measurements revealed its rapid depletion in both metal- and density-exposed cells. Nickel exposure caused strong activation of HIF-1alpha and HIF-2alpha proteins, underscoring activation of HIF-1-dependent transcription. In contrast, cell density-dependent transcription was characterized by minor induction of HIF-1alpha or HIF-2alpha. Moreover, the up-regulation of NDRG1/Cap43 in HIF-1alpha deficient fibroblasts suggested the involvement of different transcription factor(s). The repletion of intracellular ascorbate reversed the induction of CA IX and NDRG1/Cap43 caused by cell density or nickel exposure. Thus, the loss of intracellular ascorbate triggered the induction of both tumor markers by two different conditions in the presence of oxygen. Ascorbate is delivered to lung cells via the SVCT2 ascorbate transporter, which was found to be sensitive to nickel or cell density. Collectively these findings establish the importance of intracellular ascorbate levels for the regulation of expression of CA IX and NDRG1/Cap43. We suggest, that, in addition to low oxygenation, insufficient supply of ascorbate or its excessive oxidation in tumors, can contribute to the induction of hypoxia-associated proteins via both HIF-dependent and independent mechanisms.

Truncation, deamidation, and oxidation of histone H2B in cells cultured with nickel(II).

Molecular mechanisms of nickel-induced carcinogenesis include interactions of Ni(II) cations with histones. Previously, we demonstrated in vitro and in cells that Ni(II) cleaved off the -SHHKAKGK C-terminal motif of histone H2A. In the present study, Western blotting of histones isolated from rat and human cell lines, cultured for 3-5 days with 0.05-0.5 mM Ni(II), revealed time- and dose-dependent appearance of a new band of histone H2B. This effect was also induced by Co(II), but not by Cu(II), Cd(II), and Zn(II). Mass spectrometry and amino acid sequencing of proteins from the new band allowed for identification of two derivatives of the major variant of histone H2B. The larger protein was histone H2B lacking 16 N-terminal amino acids. The smaller one was histone H2B which, in addition to being shortened at the N-terminus, had nine amino acids deleted from its C-terminus. At both termini, the truncation occurred between lysine and alanine in the two identical -KAVTK- repeats of histone H2B. Also, the truncated H2B proteins had their Q22 residues deamidated and M59 and M62 residues oxidized to sulfoxides, a signature of oxidative stress. The truncation did not concur with apoptosis. Its mechanism involved activation by Ni(II) treatment of specific nuclear proteolytic enzymes belonging to the calpain family. The terminal tails of core histones participate in structuring chromatin and regulating gene expression. Therefore, the observed truncation and other modifications of histone H2B may assist in Ni(II) carcinogenesis through epigenetic mechanisms.

Inhibition of core histones acetylation by carcinogenic nickel(II).

Nickel, a well-established human carcinogen, was shown to decrease acetylation of histones H4 and H3 in cultured cells. Such a decrease is expected to suppress gene expression. However, nickel is known to not only suppress but also enhance the expression of many genes. So, perhaps, nickel can alter histone acetylation in a more complex way? In a first step of testing this presumption, we examined acetylation status of histones H2A, H2B, H3 and H4, in human (HAE) and rat (NRK) cells exposed to nickel(II) under various conditions. In both cell lines, acetylation of all four histones was down-regulated by nickel(II) in a concentration- and time-dependent manner. Acetylation of histone H2B was suppressed to greater extent than that of the others, with histone H3 being relatively least affected. The analysis of acetylation status of each of the four lysine sites at the N-terminal tail of histone H2B revealed decreases consistent with those observed in the total acetylation patterns, with the K12 and K20 residues being markedly more affected than K5 and K15 residues. Thus, the decrease in acetylation was to some degree site specific. In NRK cells, the observed uniform down-regulation of histone acetylation was consistent with a marked suppression of global gene transcription measured as [3H]-uridine incorporation into mRNA. However, in HAE cells, global RNA expression was transiently increased (in 24 h) before dropping below control after longer exposure (3 days). In conclusion, the effects of Ni(II) on histone acetylation are inhibitory, with their extent depending on the dose and exposure time. This uniform inhibition, however, is not consistently reflected in global RNA expression that in HAE cells may include both increase and decrease of the expression, clearly indicating the involvement of factors other than histone acetylation. The observed effects may contribute to neoplastic transformation of Ni(II)-exposed cells.

Metallothionein-I/II double knockout mice are no more sensitive to the carcinogenic effects of nickel subsulfide than wild-type mice.

Metallothionein (MT) is a high-affinity metal-binding protein thought to mitigate the toxicity of various metals. MT may limit the toxicity of a metal by direct binding or through action as an antioxidant for metals that generate reactive oxygen species. Nickel compounds have carcinogenic potential in humans and animals, possibly by production of oxidative stress. The impact of MT deficiency on the carcinogenic effects of nickel is unknown. Thus, groups (n=25) of male MT-I/II double knockout (MT-null) or MT wild-type (WT) mice were exposed to a single treatment of nickel (0.5 or 1.0 mg Ni3S2/site, intramuscularly, [i.m.], into both hind legs), or left untreated (control) and observed over the next 104 weeks. There were no differences in the incidence of spontaneous tumors in MT-null and WT mice. Nickel induced injection site fibrosarcomas in a dose-related fashion to a similar extent in both WT and MT-null mice. Nickel-treatment had no effect on total lung tumor incidence, although some phenotypic-specific differences occurred in the proportion of benign and malignant pulmonary tumors. Overall, MT-null mice appear no more sensitive to the carcinogenic effects of nickel than WT mice. Thus, poor MT production does not appear to be a predisposing factor for nickel carcinogenesis.