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ARID1A is a tumor suppressor gene that belongs to the switch/sucrose non-fermentable chromatin remodeling gene family. It is mutated in many types of human cancer with the highest frequency in endometrium-related ovarian and uterine neoplasms including ovarian clear cell, ovarian endometrioid, and uterine endometrioid carcinomas. We have previously reported that mutations in the promoter of human telomerase reverse transcriptase (TERT) rarely co-occur with the loss of ARID1A protein expression, suggesting a potential role of ARID1A in telomere biology. In this study, we demonstrate that ARID1A negatively regulates TERT transcriptional regulation and activity via binding to the regulatory element of TERT and promotes a repressive histone mode. Induction of ARID1A expression was associated with increased occupancy of SIN3A and H3K9me3, known transcription repressor and histone repressor marks, respectively. Thus, loss of ARID1A protein expression caused by inactivating mutations reactivates TERT transcriptional activity and confers a survival advantage of tumor cells by maintaining their telomeres., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Iron is critical for cellular proliferation and its depletion leads to a suppression of both DNA synthesis and global translation. These observations suggest that iron depletion may trigger a cellular "stress response". A canonical response of cells to stress is the formation of stress granules, which are dynamic cytoplasmic aggregates containing stalled pre-initiation complexes that function as mRNA triage centers. By differentially prioritizing mRNA translation, stress granules allow for the continued and selective translation of stress response proteins. Although the multi-subunit eukaryotic initiation factor 3 (eIF3) is required for translation initiation, its largest subunit, eIF3a, may not be essential for this activity. Instead, eIF3a is a vital constituent of stress granules and appears to act, in part, by differentially regulating specific mRNAs during iron depletion. Considering this, we investigated eIF3a's role in modulating iron-regulated genes/proteins that are critically involved in proliferation and metastasis. In this study, eIF3a was down-regulated and recruited into stress granules by iron depletion as well as by the classical stress-inducers, hypoxia and tunicamycin. Iron depletion also increased expression of the metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), and a known downstream repressed target of eIF3a, namely the cyclin-dependent kinase inhibitor, p27(kip1). To determine if eIF3a regulates NDRG1 expression, eIF3a was inducibly over-expressed or ablated. Importantly, eIF3a positively regulated NDRG1 expression and negatively regulated p27(kip1) expression during iron depletion. This activity of eIF3a could be due to its recruitment to stress granules and/or its ability to differentially regulate mRNA translation during cellular stress. Additionally, eIF3a positively regulated proliferation, but negatively regulated cell motility and invasion, which may be due to the eIF3a-dependent changes in expression of NDRG1 and p27(kip1) observed under these conditions.

There is no effective treatment for the cardiomyopathy of the most common autosomal recessive ataxia, Friedreich ataxia (FA). This disease is due to decreased expression of the mitochondrial protein, frataxin, which leads to alterations in mitochondrial iron (Fe) metabolism. The identification of potentially toxic mitochondrial Fe deposits in FA suggests Fe plays a role in its pathogenesis. Studies using the muscle creatine kinase (MCK) conditional frataxin knockout mouse that mirrors the disease have demonstrated frataxin deletion alters cardiac Fe metabolism. Indeed, there are pronounced changes in Fe trafficking away from the cytosol to the mitochondrion, leading to a cytosolic Fe deficiency. Considering Fe deficiency can induce apoptosis and cell death, we examined the effect of dietary Fe supplementation, which led to body Fe loading and limited the cardiac hypertrophy in MCK mutants. Furthermore, this study indicates a unique effect of heart and skeletal muscle-specific frataxin deletion on systemic Fe metabolism. Namely, frataxin deletion induces a signaling mechanism to increase systemic Fe levels and Fe loading in tissues where frataxin expression is intact (i.e., liver, kidney, and spleen). Examining the mutant heart, native size-exclusion chromatography, transmission electron microscopy, Mössbauer spectroscopy, and magnetic susceptibility measurements demonstrated that in the absence of frataxin, mitochondria contained biomineral Fe aggregates, which were distinctly different from isolated mammalian ferritin molecules. These mitochondrial aggregates of Fe, phosphorus, and sulfur, probably contribute to the oxidative stress and pathology observed in the absence of frataxin.

Background: Melanotransferrin was discovered in the 1980s as one of the first melanoma tumour antigens. The molecule is a transferrin homologue that is found predominantly bound to the cell membrane by a glycosyl-phosphatidylinositol anchor. MTf was described as an oncofoetal antigen expressed in only small quantities in normal tissues, but in much larger amounts in neoplastic cells. Several diseases are associated with expression of melanotransferrin, including melanoma and Alzheimer's disease, although the significance of the protein to the pathogenesis of these conditions remains unclear., Scope of Review: In this review, we discuss the roles of melanotransferrin in physiological and pathological processes and its potential use as an immunotherapy., Major Conclusions: Although the exact biological functions of melanotransferrin remain elusive, a growing number of roles have been attributed to the protein, including iron transport/metabolism, angiogenesis, proliferation, cellular migration and tumourigenesis., General Significance: The high expression of melanotransferrin in several disease states, particularly malignant melanoma, remains intriguing and may have clinical significance. Further studies on the biology of this protein may provide new insights as well as potential therapeutic avenues for cancer treatment. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Siderocalin (also lipocalin 2, NGAL or 24p3) binds iron as complexes with specific siderophores, which are low molecular weight, ferric ion-specific chelators. In innate immunity, siderocalin slows the growth of infecting bacteria by sequestering bacterial ferric siderophores. Siderocalin also binds simple catechols, which can serve as siderophores in the damaged urinary tract. Siderocalin has also been proposed to alter cellular iron trafficking, for instance, driving apoptosis through iron efflux via BOCT. An endogenous siderophore composed of gentisic acid (2,5-dihydroxybenzoic acid) substituents was proposed to mediate cellular efflux. However, binding studies reported herein contradict the proposal that gentisic acid forms high-affinity ternary complexes with siderocalin and iron, or that gentisic acid can serve as an endogenous siderophore at neutral pH. We also demonstrate that siderocalin does not induce cellular iron efflux or stimulate apoptosis, questioning the role siderocalin plays in modulating iron metabolism.

Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone possesses potent and selective antitumor activity. Its cytotoxicity has been attributed to iron chelation leading to inhibition of the iron-containing enzyme ribonucleotide reductase (RR). Thiosemicarbazone iron complexes have been shown to be redox-active, although their effect on cellular antioxidant systems is unclear. Using a variety of antioxidants, we found that only N-acetylcysteine significantly inhibited thiosemicarbazone-induced antiproliferative activity. Thus, we examined the effects of thiosemicarbazones on major thiol-containing systems considering their key involvement in providing reducing equivalents for RR. Thiosemicarbazones significantly (p < 0.001) elevated oxidized trimeric thioredoxin levels to 213 ± 5% (n = 3) of the control. This was most likely due to a significant (p < 0.01) decrease in thioredoxin reductase activity to 65 ± 6% (n = 4) of the control. We were surprised to find that the non-redox-active chelator desferrioxamine increased thioredoxin oxidation to a lower extent (152 ± 9%; n = 3) and inhibited thioredoxin reductase activity (62 ± 5%; n = 4), but at a 10-fold higher concentration than thiosemicarbazones. In contrast, only the thiosemicarbazones significantly (p < 0.05) reduced the glutathione/oxidized-glutathione ratio and the activity of glutaredoxin that requires glutathione as a reductant. All chelators significantly decreased RR activity, whereas the NADPH/NADP(total) ratio was not reduced. This was important to consider because NADPH is required for thiol reduction. Thus, thiosemicarbazones could have an additional mechanism of RR inhibition via their effects on major thiol-containing systems.

Regulation of gene expression is a fundamental step in cellular physiology as abnormalities in this process may lead to de-regulated growth and cancer. Translation of mRNA is mainly regulated at the rate-limiting initiation step, where many eukaryotic initiation factors (eIFs) are involved. The largest and most complex initiation factor is eIF3 which plays a role in translational regulation, cell growth and cancer. The largest subunit of eIF3 is eIF3a, although it is not required for the general function of eIF3 in translation initiation. However, eIF3a may play a role as a regulator of a subset of mRNAs and has been demonstrated to regulate the expression of p27(kip1), tyrosinated α-tubulin and ribonucleotide reductase M2 subunit. These molecules have a pivotal role in the regulation of the cell cycle. Moreover, the eIF3a mRNA is ubiquitously expressed in all tissues at different levels and is found elevated in a number of cancer types. eIF3a can modulate the cell cycle and may be a translational regulator for proteins important for entrance into S phase. The expression of eIF3a is decreased in differentiated cells in culture and the suppression of eIF3a expression can reverse the malignant phenotype and change the sensitivity of cells to cell cycle modulators. However, the role of eIF3a in cancer is still unclear. In fact, some studies have identified eIF3a to be involved in cancer development, while other results indicate that it could provide protection against evolution into higher malignancy. Together, these findings highlight the "tricky" and interesting nature of eIF3a., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The mitochondrion is well known for its key role in energy transduction. However, it is less well appreciated that it is also a focal point of iron metabolism. Iron is needed not only for heme and iron sulfur cluster (ISC)-containing proteins involved in electron transport and oxidative phosphorylation, but also for a wide variety of cytoplasmic and nuclear functions, including DNA synthesis. The mitochondrial pathways involved in the generation of both heme and ISCs have been characterized to some extent. However, little is known concerning the regulation of iron uptake by the mitochondrion and how this is coordinated with iron metabolism in the cytosol and other organelles (e.g., lysosomes). In this article, we discuss the burgeoning field of mitochondrial iron metabolism and trafficking that has recently been stimulated by the discovery of proteins involved in mitochondrial iron storage (mitochondrial ferritin) and transport (mitoferrin-1 and -2). In addition, recent work examining mitochondrial diseases (e.g., Friedreich's ataxia) has established that communication exists between iron metabolism in the mitochondrion and the cytosol. This finding has revealed the ability of the mitochondrion to modulate whole-cell iron-processing to satisfy its own requirements for the crucial processes of heme and ISC synthesis. Knowledge of mitochondrial iron-processing pathways and the interaction between organelles and the cytosol could revolutionize the investigation of iron metabolism.

Iron deficiency affects 500 million people, yet the molecular role of iron in gene expression remains poorly characterized. In addition, the alterations in global gene expression after iron chelation remain unclear and are important to assess for understanding the molecular pathology of iron deficiency and the biological effects of chelators. Considering this, we assessed the effect on whole genome gene expression of two iron chelators (desferrioxamine and 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone) that have markedly different permeability properties. Sixteen genes were significantly regulated by both ligands, whereas a further 50 genes were significantly regulated by either compound. Apart from iron-mediated regulation of expression via hypoxia inducible factor-1 alpha, it was noteworthy that the transcription factor p53 was also involved in iron-regulated gene expression. Examining 16 genes regulated by both chelators in normal and neoplastic cells, five genes (APP, GDF15, CITED2, EGR1, and PNRC1) were significantly differentially expressed between the cell types. In view of their functions in tumor suppression, proliferation, and apoptosis, these findings are important for understanding the selective antiproliferative effects of chelators against neoplastic cells. Most of the genes identified have not been described previously to be iron-regulated and are important for understanding the molecular and cellular effects of iron depletion.

We used the muscle creatine kinase (MCK) conditional frataxin knockout mouse to elucidate how frataxin deficiency alters iron metabolism. This is of significance because frataxin deficiency leads to Friedreich's ataxia, a disease marked by neurologic and cardiologic degeneration. Using cardiac tissues, we demonstrate that frataxin deficiency leads to down-regulation of key molecules involved in 3 mitochondrial utilization pathways: iron-sulfur cluster (ISC) synthesis (iron-sulfur cluster scaffold protein1/2 and the cysteine desulferase Nfs1), mitochondrial iron storage (mitochondrial ferritin), and heme synthesis (5-aminolevulinate dehydratase, coproporphyrinogen oxidase, hydroxymethylbilane synthase, uroporphyrinogen III synthase, and ferrochelatase). This marked decrease in mitochondrial iron utilization and resultant reduced release of heme and ISC from the mitochondrion could contribute to the excessive mitochondrial iron observed. This effect is compounded by increased iron availability for mitochondrial uptake through (i) transferrin receptor1 up-regulation, increasing iron uptake from transferrin; (ii) decreased ferroportin1 expression, limiting iron export; (iii) increased expression of the heme catabolism enzyme heme oxygenase1 and down-regulation of ferritin-H and -L, both likely leading to increased "free iron" for mitochondrial uptake; and (iv) increased expression of the mammalian exocyst protein Sec15l1 and the mitochondrial iron importer mitoferrin-2 (Mfrn2), which facilitate cellular iron uptake and mitochondrial iron influx, respectively. Our results enable the construction of a model explaining the cytosolic iron deficiency and mitochondrial iron loading in the absence of frataxin, which is important for understanding the pathogenesis of Friedreich's ataxia.

Melanotransferrin (MTf) is a transferrin homologue that binds iron (Fe) through a high affinity Fe-binding site. MTf has been implicated in diverse processes, e.g., iron metabolism, plasminogen activation, eosinophil differentiation and cancer cell migration, proliferation and tumourigenesis. Our previous studies using a knockout mouse demonstrated that MTf does not have an essential function in Fe metabolism (E.O. Sekyere, L.L. Dunn, Y.S. Rahmanto, D.R. Richardson, Role of melanotransferrin in iron metabolism: studies using targeted gene disruption in vivo, Blood 107 (2006) 2599-2601). However, it does play a role in melanoma cell proliferation and tumourigenesis. In this investigation, we report generation and characterization of transgenic mice bearing the MTf gene (MTf(Tg)) produced via lentiviral delivery. In MTf(Tg) mice, MTf mRNA and protein were hyper-expressed in tissues compared to control mice. These animals exhibited no gross morphological, histological, nor Fe status changes. The MTf(Tg) mice were also born in accordance with classical Mendelian ratios. However, hyper-expression of MTf leads to a mild, but significant decrease in erythrocyte count. This animal provides a novel MTf hyper-expression transgenic model for further investigating the biological function(s) of MTf.

There is no effective treatment for the cardiomyopathy of the most common autosomal recessive ataxia, Friedreich's ataxia (FA). The identification of potentially toxic mitochondrial (MIT) iron (Fe) deposits in FA suggests that Fe plays a role in its pathogenesis. This study used the muscle creatine kinase conditional frataxin (Fxn) knockout (mutant) mouse model that reproduces the classical traits associated with cardiomyopathy in FA. We examined the mechanisms responsible for the increased cardiac MIT Fe loading in mutants. Moreover, we explored the effect of Fe chelation on the pathogenesis of the cardiomyopathy. Our investigation showed that increased MIT Fe in the myocardium of mutants was due to marked transferrin Fe uptake, which was the result of enhanced transferrin receptor 1 expression. In contrast to the mitochondrion, cytosolic ferritin expression and the proportion of cytosolic Fe were decreased in mutant mice, indicating cytosolic Fe deprivation and markedly increased MIT Fe targeting. These studies demonstrated that loss of Fxn alters cardiac Fe metabolism due to pronounced changes in Fe trafficking away from the cytosol to the mitochondrion. Further work showed that combining the MIT-permeable ligand pyridoxal isonicotinoyl hydrazone with the hydrophilic chelator desferrioxamine prevented cardiac Fe loading and limited cardiac hypertrophy in mutants but did not lead to overt cardiac Fe depletion or toxicity. Fe chelation did not prevent decreased succinate dehydrogenase expression in the mutants or loss of cardiac function. In summary, we show that loss of Fxn markedly alters cellular Fe trafficking and that Fe chelation limits myocardial hypertrophy in the mutant.

Melanotransferrin (MTf) is a member of the transferrin (Tf) family of iron (Fe)-binding proteins that was first identified as a cell-surface marker of melanoma. Although MTf has a high-affinity Fe-binding site that is practically identical to that of serum Tf, the protein does not play an essential role in Fe homeostasis and its precise molecular function remains unclear. A Zn(II)-binding motif, distinct from the Fe-binding site, has been proposed in human MTf based on computer modelling studies. However, little is known concerning the interaction of its proposed binding site(s) with metals and the consequences in terms of MTf conformation. For the first time, biochemical and spectroscopic techniques have been used in this study to characterise metal ion-binding to recombinant MTf. Initially, the binding of Fe to MTf was examined using 6M urea gel electrophoresis. Although four different iron-loaded forms were observed with serum Tf, only two forms were found with MTf, the apo-form and the N-monoferric holo-protein, suggesting a single high-affinity site. The presence of a single Fe(III)-binding site was also supported by EPR results which indicated that the Fe(III)-binding characteristics of MTf were unique, but somewhat comparable to the N-lobes of human serum Tf and chicken ovo-Tf. Circular dichroism (CD) analysis indicated that, as for Tf, no changes in secondary structure could be observed upon Fe(III)-binding. The ability of MTf to bind Zn(II) was also investigated using CD which demonstrated that the single high-affinity Fe-binding site was distinct from a potential Zn(II)-binding site.

Iron is an essential element for metabolic processes intrinsic to life, and yet the properties that make iron a necessity also make it potentially deleterious. To avoid harm, iron homeostasis is achieved through iron transport, storage and regulatory proteins. The functions of some of these molecules are well described, for example transferrin and transferrin receptor-1, whereas the roles of others, such as the transferrin homolog melanotransferrin, remain unclear. The past decade has seen the identification of new molecules involved in iron metabolism, such as divalent metal transporter-1, ferroportin-1, hepcidin, hemojuvelin and heme carrier protein-1. Here, we focus on these intriguing new molecules and the insights gained from them into cellular iron uptake and the regulation of iron metabolism.

Gallium (Ga) shows significant antitumor activity by markedly interfering with iron (Fe) metabolism, and (67)Ga is used as a radio-imaging agent for cancer detection. Therefore, the mechanisms involved in (67)Ga uptake, metabolism, and resistance are critical to understand. The development of tumor lines that are gallium-resistant suggests (67)Ga uptake may be different in these cells, providing an opportunity for understanding intracellular (67)Ga and (59)Fe transport and gallium resistance. In this study, gallium-resistant cells were used to assess (67)Ga and (59)Fe uptake using native polyacrylamide gel electrophoresis autoradiography. In contrast to the common view that (67)Ga and (59)Fe use the same uptake pathways, we show that the trafficking of these two metal ions is different in cells either resistant (R) or sensitive (S) to gallium. Indeed, in contrast to (59)Fe, little (67)Ga is incorporated into ferritin, with most present as a labile (67)Ga pool. We also report unique changes in (67)Ga and (59)Fe trafficking between R and S cells. In particular, in R cells, there was a distinct transferrin-transferrin receptor 1-hemochromatosis protein (HFE) complex (band B) not observed in S cells. Furthermore, because HFE regulates iron and gallium uptake, the two Tf-TfR1-HFE complexes in R cells may be involved in reduced (67)Ga and (59)Fe uptake compared with S cells. In S cells, a novel iron-binding intermediate (band D) was identified that was not present in R cells and may be a "sensitivity factor" to gallium. In contrast to the general view that (67)Ga and (59)Fe use the same or similar uptake pathways, we show that their distribution and trafficking is markedly different in R and S cells.

Melanotransferrin (MTf) or tumor antigen p97 is a transferrin homolog that binds one iron (Fe) atom and has been suggested to play roles in a variety of processes, including Fe metabolism, eosinophil differentiation, and plasminogen activation. Considering the vital role of Fe in many metabolic pathways, such as DNA and heme synthesis, it is important to understand the function of MTf. To define this, a MTf knockout (MTf-/-) mouse was generated through targeted disruption of the MTf gene. The MTf-/- mice were viable and fertile and developed normally, with no morphologic or histologic abnormalities. Assessment of Fe indices, tissue Fe levels, hematology, and serum chemistry parameters demonstrated no differences between MTf-/- and wild-type (MTf+/+) mice, suggesting MTf was not essential for Fe metabolism.