Transgenerational epigenetic self-memory of Dio3 dosage is associated with Meg3 methylation and altered growth trajectories and neonatal hormones.

Intergenerational and transgenerational epigenetic effects resulting from conditions in previous generations can contribute to environmental adaptation as well as disease susceptibility. Previous studies in rodent and human models have shown that abnormal developmental exposure to thyroid hormone affects endocrine function and thyroid hormone sensitivity in later generations. Since the imprinted type 3 deiodinase gene (Dio3) regulates sensitivity to thyroid hormones, we hypothesize its epigenetic regulation is altered in descendants of thyroid hormone overexposed individuals. Using DIO3-deficient mice as a model of developmental thyrotoxicosis, we investigated Dio3 total and allelic expression and growth and endocrine phenotypes in descendants. We observed that male and female developmental overexposure to thyroid hormone altered total and allelic Dio3 expression in genetically intact descendants in a tissue-specific manner. This was associated with abnormal growth and neonatal levels of thyroid hormone and leptin. Descendant mice also exhibited molecular abnormalities in the Dlk1-Dio3 imprinted domain, including increased methylation in Meg3 and altered foetal brain expression of other genes of the Dlk1-Dio3 imprinted domain. These molecular abnormalities were also observed in the tissues and germ line of DIO3-deficient ancestors originally overexposed to thyroid hormone in utero. Our results provide a novel paradigm of epigenetic self-memory by which Dio3 gene dosage in a given individual, and its dependent developmental exposure to thyroid hormone, influences its own expression in future generations. This mechanism of epigenetic self-correction of Dio3 expression in each generation may be instrumental in descendants for their adaptive programming of developmental growth and adult endocrine function.

Thyroid hormone influences brain gene expression programs and behaviors in later generations by altering germ line epigenetic information.

Genetic factors do not fully account for the relatively high heritability of neurodevelopmental conditions, suggesting that non-genetic heritable factors contribute to their etiology. To evaluate the potential contribution of aberrant thyroid hormone status to the epigenetic inheritance of neurological phenotypes, we examined genetically normal F2 generation descendants of mice that were developmentally overexposed to thyroid hormone due to a Dio3 mutation. Hypothalamic gene expression profiling in postnatal day 15 F2 descendants on the paternal lineage of ancestral male and female T3-overexposed mice revealed, respectively, 1089 and 1549 differentially expressed genes. A large number of them, 675 genes, were common to both sets, suggesting comparable epigenetic effects of thyroid hormone on both the male and female ancestral germ lines. Oligodendrocyte- and neuron-specific genes were strongly overrepresented among genes showing, respectively, increased and decreased expression. Altered gene expression extended to other brain regions and was associated in adulthood with decreased anxiety-like behavior, increased marble burying and reduced physical activity. The sperm of T3-overexposed male ancestors revealed significant hypomethylation of CpG islands associated with the promoters of genes involved in the early development of the central nervous system. Some of them were candidates for neurodevelopmental disorders in humans including Nrg3, Nrxn1, Gabrb3, Gabra5, Apba2, Grik3, Reln, Nsd1, Pcdh8, En1, and Elavl2. Thus, developmental levels of thyroid hormone influence the epigenetic information of the germ line, disproportionately affecting genes with critical roles in early brain development, and leading in future generations to disease-relevant alterations in postnatal brain gene expression and adult behavior.

Spermatogonial Type 3 Deiodinase Regulates Thyroid Hormone Target Genes in Developing Testicular Somatic Cells.

Premature overexposure to thyroid hormone causes profound effects on testis growth, spermatogenesis, and male fertility. We used genetic mouse models of type 3 deiodinase (DIO3) deficiency to determine the genetic programs affected by premature thyroid hormone action and to define the role of DIO3 in regulating thyroid hormone economy in testicular cells. Gene expression profiling in the neonatal testis of DIO3-deficient mice identified 5699 differentially expressed genes. Upregulated and downregulated genes were, respectively, involved according to DAVID analysis with cell differentiation and proliferation. They included anti-Müllerian hormone and genes involved in the formation of the blood-testis barrier, which are specific to Sertoli cells (SCs). They also included steroidogenic genes, which are specific to Leydig cells. Comparison with published data sets of genes enriched in SCs and spermatogonia, and responsive to retinoic acid (RA), identified a subset of genes that were regulated similarly by RA and thyroid hormone. This subset of genes showed an expression bias, as they were downregulated when enriched in spermatogonia and upregulated when enriched in SCs. Furthermore, using a genetic approach, we found that DIO3 is not expressed in SCs, but spermatogonia-specific inactivation of DIO3 led to impaired testis growth, reduced SC number, decreased cell proliferation and, especially during neonatal development, altered gene expression specific to somatic cells. These findings indicate that spermatogonial DIO3 protects testicular cells from untimely thyroid hormone signaling and demonstrate a mechanism of cross-talk between somatic and germ cells in the neonatal testis that involves the regulation of thyroid hormone availability and action.

The Thyroid Hormone Inactivating Type 3 Deiodinase Is Essential for Optimal Neutrophil Function: Observations From Three Species.

Neutrophils are essential effector cells of the innate immune system that have recently been recognized as thyroid hormone (TH) target cells. Cellular TH bioavailability is regulated by the deiodinase enzymes, which can activate or inactivate TH. We have previously shown that the TH inactivating enzyme type 3 deiodinase (D3) is present in neutrophils. Furthermore, D3 knockout (D3KO) mice show impaired bacterial killing upon infection. We hypothesized that D3 plays a role in neutrophil function during infection by actively regulating local TH availability. We measured TH concentrations in cerebrospinal fluid (CSF) from patients with bacterial meningitis and controls. Bacterial meningitis resulted in marked changes in CSF TH levels, characterized by a strong increase of thyroxine and reverse-triiodothyronine concentrations. This altered TH profile was consistent with elevated D3 activity in infiltrating neutrophils at the site of infection. D3 knockdown in zebrafish embryos with pneumococcal meningitis resulted in increased mortality and reduced neutrophil infiltration during infection. Finally, stimulated neutrophils from female D3KO mice exhibited impaired NADPH-oxidase activity, an important component of the neutrophil bacterial killing machinery. These consistent findings across experimental models strongly support a critical role for reduced intracellular TH concentrations in neutrophil function during infection, for which the TH inactivating enzyme D3 appears essential.

Novel roles for podocalyxin in regulating stress myelopoiesis, Rap1a, and neutrophil migration.

Podocalyxin (Podxl) is a CD34 orthologue and cell surface sialomucin reported to have roles in renal podocyte diaphragm slit development; vascular cell integrity; and the progression of blood, breast, and prostate cancers. Roles for Podxl during nonmalignant hematopoiesis, however, are largely undefined. We have developed a Vav-Cre Podxl knockout (KO) mouse model, and report on novel roles for Podxl in governing stress myelopoiesis. At steady state, Podxl expression among hematopoietic progenitor cells was low level but was induced by granulocyte colony-stimulating factor (G-CSF) in myeloid progenitors and by thrombopoietin in human stem cells. In keeping with low-level Podxl expression at steady state, Vav-Cre deletion of Podxl did not markedly alter peripheral blood cell levels. A G-CSF challenge in Podxl-KO mice, in contrast, hyperelevated peripheral blood neutrophil and monocyte levels. Podxl-KO also substantially heightened neutrophil levels after 5-fluorouracil myeloablation. These loss-of-function phenotypes were selective, and Podxl-KO did not alter lymphocyte, basophil, or eosinophil levels. Within bone marrow (and after G-CSF challenge), Podxl deletion moderately decreased colony forming units-granulocytes, eyrthrocytes, monocyte/macrophages, megakaryocytes and CD16/32posCD11bpos progenitors but did not affect Gr-1pos cell populations. Notably, Podxl-KO did significantly heighten peripheral blood neutrophil migration capacities. To interrogate Podxl's action mechanisms, a co-immunoprecipitation plus liquid chromatography-mass spectrometry approach was applied using hematopoietic progenitors from G-CSF-challenged mice. Rap1a, a Ras-related small GTPase, was a predominant co-retrieved Podxl partner. In bone marrow human progenitor cells, Podxl-KO led to heightened G-CSF activation of Rap1aGTP, and Rap1aGTP inhibition attenuated Podxl-KO neutrophil migration. Studies have revealed novel roles for Podxl as an important modulator of neutrophil and monocyte formation and of Rap1a activation during stress hematopoiesis.

Human NUMB6 Induces Epithelial-Mesenchymal Transition and Enhances Breast Cancer Cells Migration and Invasion.

Mammalian NUMB is alternatively spliced generating four isoforms NUMB1-NUMB4 that can function as tumor suppressors. NUMB1-NUMB4 proteins, which normally determine how different cell types develop, are reduced in 21% of primary breast tumors. Our previous work has, however, indicated that two novel NUMB isoforms, NUMB5 and NUMB6 have the pro-oncogenic functions. Herein, we address a novel function of human NUMB isoform 6 (NUMB6) in promoting cancer cell migration and invasion. We found that NUMB6 induced expression of embryonic transcription factor Slug, which in turn actively repressed E-cadherin, prompting cells to undergo epithelial-mesenchymal transition (EMT). Low-metastatic breast cancer cells DB-7 stably expressing NUMB6, lost their epithelial phenotype, exhibited migratory and pro-invasive behavior, and ultimately elevated expression of mesenchymal markers. Among these markers, increased vimentin, ß-catenin, and fibronectin expression elicited metalloproteinase 9 (MMP9) production. Our results revealed that NUMB6-DB-7 cells have significantly increased level of Akt1 and Akt2 phosphorylation. Therefore, antagonizing Akt signaling using a chemical inhibitor LY294002, we found that NUMB6-induced Slug expression was reduced, and ultimately accompanied with decreased cell migration and invasion. In summary, this study identified a novel molecular determinant of breast cancer progression, uncovering a potential oncogenic role for the NUMB6 protein in cancer cell migration and invasion, coupled to the maintenance of mesenchymal-like cells. J. Cell. Biochem. 118: 237-251, 2017. © 2016 Wiley Periodicals, Inc.

The Type 3 Deiodinase Is a Critical Determinant of Appropriate Thyroid Hormone Action in the Developing Testis.

Timely and appropriate levels of thyroid hormone (TH) signaling are necessary to ensure normal developmental outcomes in many tissues. Studies using pharmacological models of altered TH status have revealed an influence of these hormones on testis development and size, but little is known about the role of endogenous determinants of TH action in the developing male gonads. Using a genetic approach, we demonstrate that the type 3 deiodinase (D3), which inactivates TH and protects developing tissues from undue TH action, is a key factor. D3 is highly expressed in the developing testis, and D3-deficient (D3KO) mice exhibit thyrotoxicosis and cell proliferation arrest in the neonatal testis, resulting in an approximately 75% reduction in testis size. This is accompanied by larger seminiferous tubules, impaired spermatogenesis, and a hormonal profile indicative of primary hypogonadism. A deficiency in the TH receptor-α fully normalizes testis size and adult testis gene expression in D3KO mice, indicating that the effects of D3 deficiency are mediated through this type of receptor. Similarly, genetic deficiencies in the D2 or in the monocarboxylate transporter 8 partially rescue the abnormalities in testis size and gonadal axis gene expression featured in the D3KO mice. Our study highlights the testis as an important tissue in which determinants of TH action coordinately converge to ensure normal development and identifies D3 as a critical factor in testis development and in testicular protection from thyrotoxicosis.

A small peptide modeled after the NRAGE repeat domain inhibits XIAP-TAB1-TAK1 signaling for NF-κB activation and apoptosis in P19 cells.

In normal growth and development, apoptosis is necessary to shape the central nervous system and to eliminate excess neurons which are not required for innervation. In some diseases, however, apoptosis can be either overactive as in some neurodegenerative disorders or severely attenuated as in the spread of certain cancers. Bone morphogenetic proteins (BMPs) transmit signals for regulating cell growth, differentiation, and apoptosis. Responding to BMP receptors stimulated from BMP ligands, neurotrophin receptor-mediated MAGE homolog (NRAGE) binds and functions with the XIAP-TAK1-TAB1 complex to activate p38(MAPK) and induces apoptosis in cortical neural progenitors. NRAGE contains a unique repeat domain that is only found in human, mouse, and rat homologs that we theorize is pivotal in its BMP MAPK role. Previously, we showed that deletion of the repeat domain inhibits apoptosis, p38(MAPK) phosphorylation, and caspase-3 cleavage in P19 neural progenitor cells. We also showed that the XIAP-TAB1-TAK1 complex is dependent on NRAGE for IKK-α/ß phosphorylation and NF-κB activation. XIAP is a major inhibitor of caspases, the main executioners of apoptosis. Although it has been shown previously that NRAGE binds to the RING domain of XIAP, it has not been determined which NRAGE domain binds to XIAP. Here, we used fluorescence resonance energy transfer (FRET) to determine that there is a strong likelihood of a direct interaction between NRAGE and XIAP occurring at NRAGE's unique repeat domain which we also attribute to be the domain responsible for downstream signaling of NF-κB and activating IKK subunits. From these results, we designed a small peptide modeled after the NRAGE repeat domain which we have determined inhibits NF-κB activation and apoptosis in P19 cells. These intriguing results illustrate that the paradigm of the NRAGE repeat domain may hold promising therapeutic strategies in developing pharmaceutical solutions for combating harmful diseases involving excessive downstream BMP signaling, including apoptosis.

Two novel human NUMB isoforms provide a potential link between development and cancer.

We previously identified four functionally distinct human NUMB isoforms. Here, we report the identification of two additional isoforms and propose a link between the expression of these isoforms and cancer. These novel isoforms, NUMB5 and NUMB6, lack exon 10 and are expressed in cells known for polarity and migratory behavior, such as human amniotic fluid cells, glioblastoma and metastatic tumor cells. RT-PCR and luciferase assays demonstrate that NUMB5 and NUMB6 are less antagonistic to NOTCH signaling than other NUMB isoforms. Immunocytochemistry analyses show that NUMB5 and NUMB6 interact and complex with CDC42, vimentin and the CDC42 regulator IQGAP1 (IQ (motif) GTPase activating protein 1). Furthermore, the ectopic expression of NUMB5 and NUMB6 induces the formation of lamellipodia (NUMB5) and filopodia (NUMB6) in a CDC42- and RAC1-dependent manner. These results are complemented by in vitro and in vivo studies, demonstrating that NUMB5 and NUMB6 alter the migratory behavior of cells. Together, these novel isoforms may play a role in further understanding the NUMB function in development and cancer.

A role for NRAGE in NF-kappaB activation through the non-canonical BMP pathway.

BACKGROUND: Previous studies have linked neurotrophin receptor-interacting MAGE protein to the bone morphogenic protein signaling pathway and its effect on p38 mediated apoptosis of neural progenitor cells via the XIAP-Tak1-Tab1 complex. Its effect on NF-kappaB has yet to be explored. RESULTS: Herein we report that NRAGE, via the same XIAP-Tak1-Tab1 complex, is required for the phosphorylation of IKK -alpha/beta and subsequent transcriptional activation of the p65 subunit of NF-kappaB. Ablation of endogenous NRAGE by siRNA inhibited NF-kappaB pathway activation, while ablation of Tak1 and Tab1 by morpholino inhibited overexpression of NRAGE from activating NF-kappaB. Finally, cytokine profiling of an NRAGE over-expressing stable line revealed the expression of macrophage migration inhibitory factor. CONCLUSION: Modulation of NRAGE expression revealed novel roles in regulating NF-kappaB activity in the non-canonical bone morphogenic protein signaling pathway. The expression of macrophage migration inhibitory factor by bone morphogenic protein -4 reveals novel crosstalk between an immune cytokine and a developmental pathway.

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.

NRAGE: a potential rheostat during branching morphogenesis.

Branching morphogenesis is a developmental process characteristic of many organ systems. Specifically, during renal branching morphogenesis, its been postulated that the final number of nephrons formed is one key clinical factor in the development of hypertension in adulthood. As it has been established that BMPs regulate, in part, renal activity of p38 MAP kinase (p38(MAPK)) and it has demonstrated that the cytoplasmic protein Neurotrophin Receptor MAGE homologue (NRAGE) augments p38(MAPK) activation, it was hypothesized that a decrease in the expression of NRAGE during renal branching would result in decreased branching of the UB that correlated with changes in p38(MAPK) activation. To verify this, the expression of NRAGE was reduced in ex vivo kidney explants cultures using antisense morpholino. Morpholino treated ex vivo kidney explants expression were severely stunted in branching, a trait that was rescued with the addition of exogenous GDNF. Renal explants also demonstrated a precipitous drop in p38(MAPK) activation that too was reversed in the presence of recombinant GDNF. RNA profiling of NRAGE diminished ex vivo kidney explants resulted in altered expression of GDNF, Ret, BMP7 and BMPRIb mRNAs. Our results suggested that in early kidney development NRAGE might have multiple roles during renal branching morphogenesis through association with both the BMP and GDNF signaling pathways.

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.

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.

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.

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.

Microarray analysis of altered gene expression in murine fibroblasts transformed by nickel(II) to nickel(II)-resistant malignant phenotype.

B200 cells are Ni(II)-transformed mouse BALB/c-3T3 fibroblasts displaying a malignant phenotype and increased resistance to Ni(II) toxicity. In an attempt to find genes whose expression has been altered by the transformation, the Atlas Mouse Stress/Toxicology cDNA Expression Array (Clontech Laboratories, Inc., Palo Alto, CA) was used to analyze the levels of gene expression in both parental and Ni(II)-transformed cells. Comparison of the results revealed a significant up- or downregulation of the expression of 62 of the 588 genes present in the array (approximately 10.5%) in B200 cells. These genes were assigned to different functional groups, including transcription factors and oncogenes (9/14; fractions in parentheses denote the number of up-regulated versus the total number of genes assigned to this group), stress and DNA damage response genes (11/12), growth factors and hormone receptors (6/9), metabolism (7/7), cell adhesion (2/7), cell cycle (3/6), apoptosis (3/4), and cell proliferation (2/3). Among those genes, overexpression of beta-catenin and its downstream targets c-myc and cyclin D1, together with upregulated cyclin G, points at the malignant character of B200 cells. While the increased expression of glutathione (GSH) synthetase, glutathione-S-transferase A4 (GSTA4), and glutathione-S-transferase theta (GSTT), together with high level of several genes responding to oxidative stress, suggests the enforcement of antioxidant defenses in Ni-transformed cells.

The octapeptidic end of the C-terminal tail of histone H2A is cleaved off in cells exposed to carcinogenic nickel(II).

We have demonstrated previously that Ni(II) binds to the C-terminal -TESHHKAKGK motif of isolated bovine histone H2A. At physiological pH, the bound Ni(II) assists in hydrolysis of the E-S peptide bond in this motif that results in a cleavage of the terminal octapeptide SHHKAKGK off the histone's C-tail. To test if the hydrolysis could also occur in living cells, we cultured CHO (Chinese hamster ovary), NRK-52 (rat renal tubular epithelium), and HPL1D (human lung epithelium) cells with 0.1-1 mM Ni(II) for 3-7 days. As found by gel electrophoresis, Western blotting, and liquid chromatography/mass spectrometry, histones extracted from the cells contained a new fraction of histone H2A lacking the terminal octapeptide (q-H2A). The abundance of q-H2A increased with Ni(II) concentration and exposure time. It can be anticipated that the truncation of histone H2A may alter chromatin structure and affect gene expression. The present results provide evidence for novel mechanisms of epigenetic effects of Ni(II) that may be involved in nickel toxicity and carcinogenesis.

The role of chromatin damage in nickel-induced carcinogenesis. A review of recent developments.

Over the last years, we have been testing a hypothesis that molecular mechanisms of nickel-induced carcinogenesis include interactions of this metal with major chromatin components; DNA, histones, and protamines. Our investigations using synthetic peptide models have resulted in identification of nickel-binding sites in core histones H3 and H2A and in protamine P2. These are: the internal -Cys110-AIH- motif in histone H3: the C-terminal-E121-SHHKAKGK "tail" motif in histone H2A; and the N-terminal RTH- motif in protamine P2. Ni(II) bound to the H3 and P2 motifs enhances oxidative DNA base damage by H2O2. In contrast, Ni(II) complex with the H2A "tail" is not redox active. However, at pH 7.4, it undergoes hydrolysis yielding a new complex, Ni(II)-SHHKAKGK, reactive with H2O2 and capable of mediating DNA oxidation. The "tail" cutting of H2A has also been observed in cells cultured with Ni(II). In Ni(II) complex with the protamine P2 peptides, H2O2 causes degradation of the metal-binding His3 and the distant Tyr8 residues. This site-specificity results from a long-range structuring effect of Ni(II) on its protamine ligand. In conclusion, Ni(II) binding to some chromatin proteins in somatic and sperm cells may result in oxidative and structural damage to the proteins and DNA. These effects may alter the fidelity of DNA replication and gene expression and thus facilitate carcinogenesis, including paternally-mediated cancer in the progeny.