Mercury and cadmium trigger expression of the copper importer Ctr1B, which enables Drosophila to thrive on heavy metal-loaded food

Organisms from insects to mammals respond to heavy metal load (copper, zinc, cadmium, and mercury) by activating the metal-responsive transcription factor 1 (MTF-1). MTF-1 binds to short DNA sequence motifs, termed metal response elements, and boosts transcription of a number of genes, notably those for metallothioneins. In Drosophila, MTF-1 somewhat counter-intuitively also activates transcription of a copper importer gene (Ctr1B) in response to copper starvation. Here, we report that mutant flies lacking Ctr1B are extremely sensitive to cadmium and mercury treatment, but can be rescued by excess copper in the food. We thus propose that copper, by competing for binding sites on cellular proteins, alleviates the toxic effects of mercury and cadmium. Such a scenario also explains a seemingly fortuitous metal response, namely, that cadmium and mercury strongly induce the expression of a Ctr1B reporter gene. Thus, the transcription enhancer/promoter region of the Ctr1B copper importer gene is subject to three modes of regulation. All of them depend on MTF-1 and all make biological sense, namely, (i) induction by copper starvation, (ii) repression by copper abundance, and (iii), as shown here, induction by cadmium or mercury at normal copper suppl

Impaired DNA double-strand break repair contributes to chemoresistance in HIF-1α-deficient mouse embryonic fibroblasts

A mismatch between metabolic demand and oxygen delivery leads to microenvironmental changes in solid tumors. The resulting tumor hypoxia is associated with malignant progression, therapy resistance and poor prognosis. However, the molecular mechanisms underlying therapy resistance in hypoxic tumors are not fully understood. The hypoxia-inducible factor (HIF) is a master transcriptional activator of oxygen-regulated gene expression. Transformed mouse embryonic fibroblasts (MEFs) derived from HIF-1α-deficient mice are a popular model to study HIF function in tumor progression. We previously found increased chemotherapy and irradiation susceptibility in the absence of HIF-1α. Here, we show by single-cell electrophoresis, histone 2AX phosphorylation and nuclear foci formation of γH2AX and 53BP1, that the number of DNA double-strand breaks (DSB) is increased in untreated and etoposide-treated HIF-deficient MEFs. In etoposide-treated cells, cell cycle control and p53-dependent gene expression were not affected by the absence of HIF-1α. Using a candidate gene approach to screen 17 genes involved in DNA repair, messenger RNA (mRNA) and protein of three members of the DNA-dependent protein kinase complex were found to be decreased in HIF-deficient MEFs. Of note, residual HIF-1α protein in cancer cells with a partial HIF-1α mRNA knockdown was sufficient to confer chemoresistance. In summary, these data establish a novel molecular link between HIF and DNA DSB repair. We suggest that selection of early, non-hypoxic tumor cells expressing low levels of HIF-1α might contribute to HIF-dependent tumor therapy resistanc

Copper sensing function of Drosophila metal-responsive transcription factor-1 is mediated by a tetranuclear Cu(I) cluster

Drosophila melanogaster MTF-1 (dMTF-1) is a copper-responsive transcriptional activator that mediates resistance to Cu, as well as Zn and Cd. Here, we characterize a novel cysteine-rich domain which is crucial for sensing excess intracellular copper by dMTF-1. Transgenic flies expressing mutant dMTF-1 containing alanine substitutions of two, four or six cysteine residues within the sequence 547CNCTNCKCDQTKSCHGGDC565 are significantly or completely impaired in their ability to protect flies from copper toxicity and fail to up-regulate MtnA (metallothionein) expression in response to excess Cu. In contrast, these flies exhibit wild-type survival in response to copper deprivation thus revealing that the cysteine cluster domain is required only for sensing Cu load by dMTF-1. Parallel studies show that the isolated cysteine cluster domain is required to protect a copper-sensitive S. cerevisiae ace1Δ strain from copper toxicity. Cu(I) ligation by a Cys-rich domain peptide fragment drives the cooperative assembly of a polydentate [Cu4-S6] cage structure, characterized by a core of trigonally S3 coordinated Cu(I) ions bound by bridging thiolate ligands. While reminiscent of Cu4-L6 (L = ligand) tetranuclear clusters in copper regulatory transcription factors of yeast, the absence of significant sequence homology is consistent with convergent evolution of a sensing strategy particularly well suited for Cu(I

Copper sensing function of Drosophila metal-responsive transcription factor-1 is mediated by a tetranuclear Cu(I) cluster

Drosophila melanogaster MTF-1 (dMTF-1) is a copper-responsive transcriptional activator that mediates resistance to Cu, as well as Zn and Cd. Here, we characterize a novel cysteine-rich domain which is crucial for sensing excess intracellular copper by dMTF-1. Transgenic flies expressing mutant dMTF-1 containing alanine substitutions of two, four or six cysteine residues within the sequence 547CNCTNCKCDQTKSCHGGDC565 are significantly or completely impaired in their ability to protect flies from copper toxicity and fail to up-regulate MtnA (metallothionein) expression in response to excess Cu. In contrast, these flies exhibit wild-type survival in response to copper deprivation thus revealing that the cysteine cluster domain is required only for sensing Cu load by dMTF-1. Parallel studies show that the isolated cysteine cluster domain is required to protect a copper-sensitive S. cerevisiae ace1Δ strain from copper toxicity. Cu(I) ligation by a Cys-rich domain peptide fragment drives the cooperative assembly of a polydentate [Cu4-S6] cage structure, characterized by a core of trigonally S3 coordinated Cu(I) ions bound by bridging thiolate ligands. While reminiscent of Cu4-L6 (L = ligand) tetranuclear clusters in copper regulatory transcription factors of yeast, the absence of significant sequence homology is consistent with convergent evolution of a sensing strategy particularly well suited for Cu(I)

Partners in Crime: Fledgling Tumors Hijack Inflammation

While inflammation is a normal physiological response after tissue injury, the chemicals/mediators that are released by the damaged tissue can be toxic to the cells. This underlying inflammation increases the likelihood of cellular DNA damage and aberrant cell growth (Kiraly and others 2015). In this scenario, inflammation functions as a ‘‘behind-curtain factor’’ for many disorders. Cancer has long been known to be closely tethered to inflammation. Widespread evidence shows that inflammatory diseases such as colitis, pancreatitis, and hepatitis make their respective organs highly susceptible to eventual cancer development (Shalapour and Karin 2015). However, other studies have shown that in due course, a growing cancer starts recruiting and relying on various mediators of inflammation to promote angiogenesis, further proliferating, metastasizing, and subverting the innate and adaptive immune response (Chan and others 2012). While we continue to ponder the chicken or egg scenario of how cancer and inflammation are related, the critical role inflammation plays in cancer progression cannot be denied. For example, some studies demonstrate that antiinflammatory drugs such as aspirin not only act by reducing inflammation-related disorders, but also decrease the risk of colon cancer and gastrointestinal cancers. In colon cancer, aspirin specifically decreased the incidence of polyps, including advanced polyps, which are the precursors to colorectal cancer (Husain and others 2002; Wang and others 2018). However, experimental data from other cancers, including breast cancer, are not very promising, largely due to the differences in the inflamed tumor microenvironment (Strasser-Weippl and others 2018). Therefore, understanding the intricate cellular signaling pathways and the players involved in the smoldering inflammatory tumor microenvironment is paramount to identifying new strategies for the better management of cancers. In this special issue, several leaders in the field discuss the latest developments in our understanding of cancer-associated inflammation, with a focus on the role of inflammatory cytokines and interferons. Emphasis is also placed on identifying opportunities and developing road maps for novel treatment approaches to combine immunotherapies with direct modulation of cytokine levels in the host

Caspase-3: Its potential involvement in Cr(III)-induced apoptosis of lymphocytes

In this study, we have examined the role of caspase-3 in apoptosis of lymphocytes induced by the chromium(III) complexes viz. tris-(1,10-phenanthroline)chromium(III) chloride (Cr(III)-phen) and trans-diaqua[1,3-bis(salicylideneamino)propane-chromium(III)] perchlorate (Cr(III)-salprn). Evidence for caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage in lymphocytes exposed to Cr(III) complexes is revealed through Western blotting analysis. Blocking the activity of caspase-3 with z-DEVD-fmk, prevents apoptosis as evidenced through [3H]-thymidine incorporation, DNA fragmentation assay and measurement of sub-G1 cells by flow cytometry. Pretreatment of lymphocytes with free radical scavengers completely attenuates the activity of caspase-3 suggesting that reactive oxygen species (ROS) are upstream activators of caspase-3. Preincubation of lymphocytes with PP2, a selective Src-family tyrosine kinase inhibitor, abolishes the activation of caspase-3 indicating that Src-family tyrosine kinases viz. p56lck, p59fyn and p53/56lyn are mediators of caspase-3 activation during Cr(III) exposure. Collectively, our findings support a plausible mechanism in which Cr(III) mediates ROS generation that precedes the up-regulation of p56lck, p59fyn and p53/56lyn which eventually activates caspase-3 to promote apoptotic cell death of lymphocytes. To our knowledge, this is the first report suggesting the importance of Src-family tyrosine kinases for the activation of caspase-3 in metal-induced apoptotic cell death

Chromium(III)-induced apoptosis of lymphocytes: death decision by ROS and Src-family tyrosine kinases

Apoptosis is an active process induced by a variety of physiological and external stimuli, in which elimination of damaged cells are effected through a genetically controlled process. In this study, we have examined the mechanism of chromium(III) [Cr(III)]-induced cytotoxicity with respect to its relationship to oxidative stress. Morphology, flow cytometry, and DNA fragmentation studies show that tris-(1,10-phenanthroline)chromium(III) [Cr(III)-phen], tris-(2,2'-bipyridyl)chromium(III) [Cr(III)-bpy], trans-diaqua[1,2-bis(salicylideneamino)ethanechromium(III)] [Cr(III)-salen], and trans-diaqua[1,3-bis(salicylideneamino)propanechromium(III)] [Cr(III)-salprn] induced apoptosis of lymphocytes. Pentaammineaquachromium(III) [Cr(III)-hpa] does not induce apoptosis. Apoptosis induced by these complexes involves the generation of reactive oxygen species (ROS) as seen by increased fluorescence of dichloroflourescein (DCF) observed through flow cytometry. Pretreatment of lymphocytes with antioxidants completely abrogate apoptosis. Cr(III) treatment also increased the expression and activation of Src-family tyrosine kinases viz. p56lck, p59fyn, and p53/56lyn, as seen by immunoblotting and immune complex kinase assay. PP2, a selective Src-family tyrosine kinase inhibitor, abolishes apoptosis, indicating that Src-family tyrosine kinases are directly involved in eliciting apoptosis. Interestingly, a one-to-one correlation between the expression of Src-family tyrosine kinases and ROS is observed, since antioxidants pretreatment inhibits the expression and the activation of these kinases. These results further indicate that Cr(III)-induced apoptosis is mediated through production of ROS, which in turn activates the Src-family tyrosine kinases. The increased activation of Src-family tyrosine kinases may be a mechanism involved in apoptosis of lymphocytes elicited by various other physiological stimuli that exploit ROS as a second messenger

Hydroxopentaamminechromium(III) promoted phosphorylation of bovine serum albumin: its potential implications in understanding biotoxicity of chromium

Evidence for chromium(III) induced phosphorylation of a biomarker protein bovine serum albumin (BSA) is presented. Radiolabelled adenosine 5'-triphosphate (ATP) was reacted with BSA in the presence of various Cr(III) salts. While [Cr(NH<SUB>3</SUB>)<SUB>5</SUB>(H<SUB>2</SUB>O)]<SUP>3+</SUP> brought about phosphorylation of BSA, several Cr(III) complexes, viz. [Cr(bpy)<SUB>3</SUB>]<SUP>3+</SUP>, [Cr(phen)<SUB>3</SUB>]<SUP>3+</SUP>, [Cr(en)<SUB>3</SUB>]<SUP>3+</SUP>, [Cr(salen)(H<SUB>2</SUB>O)<SUB>2</SUB>]<SUP>+</SUP> and [Cr(salprn)(H<SUB>2</SUB>O)<SUB>2</SUB>]<SUP>+</SUP>, did not phosphorylate BSA. The Cr(III) mediated the transfer of γ- and α-phosphates but not the adenine and the sugar moieties of the ATP molecule to BSA. The observed stoichiometry was 0.75 mol P<SUB>i</SUB> to mol BSA for the γ-phosphate and 0.5 mol P<SUB>i</SUB> to mol BSA for the α-phosphate of ATP. The presence of serine phosphate and threonine phosphate was detected in the hydrolysate of phosphorylated BSA by means of comparison of R<SUB>f</SUB> values with authentic samples of phosphoserine and phosphothreonine after chromatographic separation and autoradiography. [Cr(NH<SUB>3</SUB>)<SUB>5</SUB>(H<SUB>2</SUB>O)]<SUP>3+</SUP> at pH 7.4 is known to exist as the conjugate base [Cr(NH<SUB>3</SUB>)<SUB>5</SUB>(OH)]<SUP>2+</SUP> and is capable of ligand substitution involving metal-oxygen bond retention. Such anation reaction of [Cr(NH<SUB>3</SUB>)<SUB>5</SUB>(OH)]<SUP>2+</SUP> with ATP subsequently leads to the esterification of alcoholic hydroxyl groups of serine and threonine of BSA. Possible consequences of chromium(III) induced in vivo phosphorylation of proteins are discussed

The Experts Speak: A New Feature in the JICR

This month we launch a new feature in the JICR: The Experts Speak. This feature is designed to provide first-hand insight from topical experts in the fields of interferon, cytokines, innate immunity, and adaptive immune actions of cytokine biology in disease. We start by asking experts to comment on interferon, cytokines, and SARS-CoV-2

Apoptosis of lymphocytes in the presence of Cr(V) complexes: role in Cr(VI)-induced toxicity

Cr(VI) compounds have been declared as a potent occupational carcinogen by IARC (1990) through epidemiological studies among workers in chrome plating, stainless-steel, and pigment industries. Studies relating to the role of intermediate oxidation states such as Cr(V) and Cr(IV) in Cr(VI)-induced carcinogenicity are gaining importance. In this study, issues relating to toxicity elicited by Cr(V) have been addressed and comparisons made with those relating to Cr(VI) employing human peripheral blood lymphocytes. Lymphocytes have been isolated from heparinized blood by Ficoll-Hypaque density gradient centrifugation and exposed to Cr(V) complexes viz. sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V), Na[Cr<SUP>ν</SUP>O(ehba)<SUB>2</SUB>], 1 and sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V), Na[Cr<SUP>ν</SUP>O(hmba)<SUB>2</SUB>], 2 and Cr(VI). The phytohemagglutinin (PHA)-induced proliferation of lymphocytes has been found to be inhibited by the two complexes of Cr(V) and chromate Cr(VI) in a time-and concentration-dependent manner. Viability of cells decreases in the presence of Cr(V). Apoptosis appears to be the mode of cell death in the presence of both Cr(V) and Cr(VI). Pretreatment of cells with antioxidants before exposure to chromium(V) complexes reverse apoptosis partially. Possibility for the formation and implication of reactive oxygen species in Cr(V)-induced apoptosis of human lymphocyte cells has been indicated in this investigation. The intermediates of Cr(V) and radical species in the biotoxic pathways elicited by Cr(VI) seems feasible