Metal-regulatory Transcription Factor Research Articles

Effects of Cu 2O nanoparticle and CuCl 2 on zebrafish larvae and a liver cell-line

The extensive uses of nanomaterials have caused many concerns of their potential hazards to the aquatic environments. As partial dissolution of metal nanoparticles may occur, it is important to study the toxic effects of nanoparticles and determine the no observable effect levels (NOELs) and lowest observable effect levels (LOELs) of these materials in water by using biomarker genes’ expression in zebrafish ( Danio rerio). In this study, the toxic effects of Cu 2O nanoparticle (NP) on zebrafish larvae and zebrafish liver cell-line (ZFL) were evaluated by determining their 96 h LC50 values (zebrafish larvae: 242.4 ppb; ZFL: 110 ppm), which was less toxic than CuCl 2 (zebrafish larvae: 85.73 ppb; ZFL: 23.04 ppm). However, zebrafish larvae are sensitive to both Cu 2O NP and CuCl 2. We also examined the effects of elevated Cu 2O NP and CuCl 2 on the expression of several copper related genes in zebrafish larvae by using real-time quantitative polymerase chain reaction. It was found that Cu 2O NP and CuCl 2 induced the mRNA levels of metallothionein (MT), Cu/Zn superoxide dismutase (Cu/Zn SOD), metal regulatory transcription factor 1 (MTF1) and copper transporters, ATP7A and 7B, but down-regulated the mRNA levels of glutathione sulfur transferase (GST). Interestingly, the inductions of MT, ATP7A and ATP7B in the Cu 2O NP exposure groups were much higher than that of the CuCl 2 exposure groups, and resulted in higher copper accumulation in the Cu 2O NP exposure groups. Furthermore, as determined by using MT, ATP7A and ATP7B gene expression, the NOELs of CuCl 2 and Cu 2O NP were 11 ppb and 30 ppb whereas the LOELs of CuCl 2 and Cu 2O NP were 43 ppb and 121 ppb, respectively.

Zinc concentrations in serum and follicular fluid during ovarian stimulation and expression of Zn 2+ transporters in human oocytes and cumulus cells

Serum and follicular fluid zinc concentrations were investigated in patients undergoing assisted reproductive treatment. No correlation was found between zinc and oestradiol concentrations in serum. At the time of oocyte retrieval, zinc concentrations in follicular fluid were significantly lower than serum concentrations ( P < 0.0001). The expression of the two families of zinc transporters, ZnT and ZiP, as well as the metal regulatory transcription factors, MTF1 and 2, and metallothioneins, which are both involved in regulatory aspects of zinc transport, was assayed in cumulus cells and in germinal-vesicle oocytes. Most of the zinc transporters, metallothioneins and metal regulatory transcription factor are expressed in oocytes and not in cumulus cells. This may indicate an important role for zinc, in particular with potential linking to genome stability during early embryonic development. In contrast, cumulus cells seem to be at the end of their life’s journey, with weak expression of transcriptional activity linked to cellular housekeeping.

GSK-3β Inhibitors In Overcoming Chemoresistance In Multiple Myeloma

AbstractAbstract 3009Despite recent advances in targeted therapies for myeloma, resistance to therapy, either primary or after initial therapy, is a significant issue. This is especially problematic in myeloma because time to progression is only a modest 6 months and long term maintenance is limited by the toxicity profiles of available drugs. In an effort to identify a non-neurotoxic and steroid-sparing combination of drugs to overcome chemoresistance in myeloma, here we report on the synergy between inhibitors of glycogen synthase kinase-3 beta (GSK-3β) and histone deacetylase inhibitors (HDACi) in reducing the viability of myeloma cells. Our prior studies identified potentiation of apoptosis by HDACi and doxorubicin in a panel of five myeloma cell lines independent of p53 and caspase 3 activation. At clinically relevant concentrations, HDACi and doxorubicin alone had only a marginal effect on the survival of fresh myeloma cells (CD138+) derived from bone marrow aspirates of 18 myeloma patients. Combining HDACi (sodium butyrate and vorinostat) with doxorubicin augmented their antimyeloma activity with a significant reduction in the survival of fresh myeloma samples (p≤0.01, in one-way repeated measures of ANOVA). However, a subset of fresh myeloma samples was resistant to the treatments, and HDACi plus doxorubicin did not attain IC50 in these samples (8/18 with sodium butyrate plus doxorubicin or 6/9 with vorinostat plus doxorubicin).To identify the potential survival, antiapoptotic and drug resistance genes that may confer resistance to vorinostat and doxorubicin combinations, basal gene expression of resistant (n=4) and sensitive (n=4) cohorts were profiled. Non-parametric analysis of gene expression array results identified significantly increased expression (>7.5×, p≤0.05) of several metallothioneins in resistant cohort (MT1M - 67.7×, MT1F - 47.1×, MT1G - 39.8×, MT1H - 28.1×, MT1× -12.4× and MT2A -11.4×) as well as Cyclin D1 (13.2×), Hey1 (9.1×) and JNK3 (7.5×). The ability of metallothioneins to protect cells from metal toxicities and oxidative stress suggested that elevated levels of metallothioneins might have antagonized the cytotoxic activity of both vorinostat and doxorubicin by scavenging the reactive oxygen species (ROS) leading to resistance. Overexpression of multiple metallothioneins with redundant function suggested that inhibition of a specific metallothionein might not be a viable strategy to overcome resistance. Metallothioneins are a family of cysteine-rich proteins that are regulated at transcription by metal regulatory transcription factor 1 (MTF1). The activity of MTF1 is suppressed by HDACs whereas phosphorylated CCAAT/enhancer-binding protein alpha (CEBP-α) is indispensible for its activation. The phosphorylation of CEBP-α is regulated by GSK-3β, a multifunctional serine/threonine kinase which plays a critical role in the survival of myeloma cells. Therefore, we hypothesized that inhibition of GSK-3β would sensitize myeloma cells to therapies including HDACi by silencing the expression of metallothioneins. To test this hypothesis, NCI H929, RPMI 8226 and U266 cells were treated with sub-IC50 concentrations of a cell permeable non-competitive GSK-3β inhibitor TDZD-8 (0.25, 0.5 and 1 μM), vorinostat (75, 150 and 300 nM) or with their combinations for 72 hrs. Results of the viability assays and Chou and Talalay analysis identified strong synergy (combination indices of <1) between the combinations of vorinostat and GSK-3β inhibitor. Further demonstration of the synergy between these two classes of drugs in fresh myeloma cells and in mouse xenograft models will provide a rationale for combining GSK-3β inhibitors that are currently under clinical trials for other indications with existing antimyeloma agents or with HDACi for relapsed or refractory myeloma. Disclosures:No relevant conflicts of interest to declare.

A Novel Loss-of-Function DDAH1 Promoter Polymorphism Is Associated With Increased Susceptibility to Thrombosis Stroke and Coronary Heart Disease

Asymmetrical dimethylarginine (ADMA), an endogenous arginine analogue, inhibits nitric oxide synthases and plays an important role in endothelial dysfunction. In the present study, we tested whether a novel genetic variant in dimethylarginine dimethylaminohydrolase 1 (DDAH1), an important ADMA hydrolyzing gene, was associated with stroke and coronary heart disease (CHD) susceptibility in the Chinese Han population. By resequencing, we identified a novel 4-nucleotide deletion/insertion variant in the DDAH1 promoter. The insertion allele disrupted binding of metal-regulatory transcription factor 1, which resulted in significant reduction of in vitro DDAH1 transcriptional activity and in vivo DDAH1 mRNA level, and in turn, increased plasma ADMA level and the ratio of ADMA to L-arginine. We initially genotyped the polymorphism in 1388 stroke patients and 1027 controls as well as 576 CHD patients and 557 controls and then replicated our study in additional independent case-control cohorts comprising 961 stroke patients and 822 controls and 482 CHD patients and 1072 controls. We identified that the -396 4N ins allele was significantly associated with increased risk of thrombosis stroke and CHD after adjusting for environmental factors in both samples for both diseases (thrombosis stroke discovery set: odds ratio [OR]=1.35, P=0.032; replication set: OR=1.51, P=0.006; CHD discovery set: OR=1.45, P=0.035; replication set: OR=1.47, P=0.003). Our results suggest that the DDAH1 loss-of-function polymorphism is associated with both increased risk of thrombosis stroke and CHD.

Zinc-controlled gene expression by metal-regulatory transcription factor 1 (MTF1) in a model vertebrate, the zebrafish

There is a growing appreciation for the diverse roles of zinc as a signalling substance in biological systems. Zinc signalling is brought about by changes in intracellular concentrations of labile Zn(2+), resulting in both genomic and non-genomic effects. The genomic responses are largely mediated by MTF1 (metal-regulatory transcription factor 1), which binds to MREs (metal-response elements) in the 5' regulatory region of genes in response to zinc. Treatment of cultured zebrafish ZF4 cells with siRNA (small interfering RNA) to MTF1 changed the transcriptional response to zinc for over 1000 genes, as assessed using an oligonucleotide microarray. From this primary list of MTF1-dependent genes, we identified a relatively small cohort that showed a configuration of MREs in their 5' regulatory regions similar to known MTF1 targets. This group showed a remarkable dominance of nucleic acid-binding proteins and other proteins involved in embryological development, implicating MTF1 as a master regulator of gene expression during development.

Cytotoxicities and induction of metallothionein (MT) and metal regulatory element (MRE)-binding transcription factor-1 (MTF-1) messenger RNA levels in the zebrafish ( Danio rerio) ZFL and SJD cell lines after exposure to various metal ions

Using zebrafish liver (ZFL) and caudal fin (SJD) cell-line models, the induction of metallothionein (MT) and metal regulatory element-binding transcription factor-1 (MTF-1) mRNA levels by various metal ions (Zn 2+ , Cd 2+, Cu 2+, Hg +, As 3+, As 5+, Cr 3+ and Cr 6+) were studied using the real-time PCR. The LC 50-24 h values of the metal ions were determined for the two cell lines prior to their exposure to different concentrations (10%, 25%, 50%, 75% and 100% LC 50 values) of the heavy metal ions for RNA assay. The two cell lines were sensitive to As 3+, Cd 2+ and Hg +. Zn 2+and Cu 2+ were moderately toxic, and As 5+ and Cr 3+ were less toxic to both cell lines. Each of the metal ions tested was found to cause significant induction of the mRNA levels in the SJD cells. Only Zn 2+, Cd 2+, Cu 2+, Hg 2+ and As 3+ caused significant induction of MT mRNA levels in ZFL cells. Zn 2+and Cd 2+ were efficient MT inducers in the two cell lines, but As 3+ and As 5+ are strong inducers only in the SJD cell line. In both cell lines, Cu 2+ and Hg 2+ are moderate inducers, and Cr 3+ and Cr 6+ were weak inducers of MT mRNA. MTF-1 induction is believed to be insufficient to cause MT gene induction, but As 3+ and Cd 2+ induced MTF-1 in ZFL cell line. Cd 2+ was the most efficient inducer of MT in the cell lines, and also induced MTF-1 levels with clear dose-responsiveness in SJD cell line. These results indicated that MT induction can occur without MTF-1 induction for many metal ions such as Zn 2+ and Cu 2+.

Copper homoeostasis in Drosophila melanogaster S2 cells.

Copper homoeostasis was investigated in the Drosophila melanogaster S2 cell line to develop an insect model for the study of copper regulation. Real-time PCR studies have demonstrated expression in S2 cells of putative orthologues of human Cu regulatory genes involved in the uptake, transport, sequestration and efflux of Cu. Drosophila orthologues of the mammalian Cu chaperones, ATOX1 (a human orthologue of yeast ATX1), CCS (copper chaperone for superoxide dismutase), COX17 (a human orthologue of yeast COX17), and SCO1 and SCO2, did not significantly respond transcriptionally to increased Cu levels, whereas MtnA, MtnB and MtnD (Drosophila orthologues of human metallothioneins) were up-regulated by Cu in a time- and dose-dependent manner. To examine the effect on Cu homoeostasis, expression of several key copper homoeostasis genes was suppressed using double-stranded RNA interference. Suppression of the MTF-1 (metal-regulatory transcription factor 1), reduced both basal and Cu-induced gene expressions of MtnA, MtnB and MtnD, significantly reducing the tolerance of these cells to increased Cu. Suppression of either Ctr1A (a Drosophila orthologue of yeast CTR1) or Ctr1B significantly reduced Cu uptake from media, demonstrating that both these proteins function to transport Cu into S2 cells. Significantly, Cu induced Ctr1B gene expression, and this could be prevented by suppressing MTF-1, suggesting that Ctr1B might be involved in Cu detoxification. Suppression of DmATP7, the putative homologue of human Cu transporter genes ATP7A and ATP7B, significantly increased Cu accumulation, demonstrating that DmATP7 is essential for efflux of excess Cu. This work is consistent with previous studies in mammalian cells, validating S2 cells as a model system for studying Cu transport and identifying novel Cu regulatory mechanisms.

Polymorphisms in xenobiotic metabolism genes and autism.

Autism is a neurodevelopmental syndrome defined by deficits in social reciprocity and communication and by unusual repetitive behaviors. Although there is an underlying genetic predisposition, the etiology of autism is currently unknown. A recent increase in prevalence suggests that genetically determined vulnerability to environmental exposure might contribute to the causation of autism. We performed family-based association studies of polymorphisms in metal-regulatory transcription factor 1(MTF1), a multispecific organic anion transporter (ABCC1), proton-coupled divalent metal ion transporters (SLC11A3 and SLC11A2), paraoxonase 1 (PON1), and glutathione S-transferase (GSTP1) genes in 196 autistic disorder families. There was deviation from the expected pattern of transmission for polymorphisms in MTF1 (Single nucleotide polymorphism database reference identification number, dbSNP rs3790625, P = .02) and divalent metal ion transporter SLC11A3 (dbSNP rs2304704, P = .07) genes. Although these results might represent chance finding, further investigations of genetic variations of metal metabolism in autism are warranted.

Cadmium‐induced gene expression is regulated by MTF‐1, a key metal‐responsive transcription factor

The transition metal cadmium is a serious occupational and environmental toxin. To inhibit cadmium‐induced damage, cells respond by increasing the expression of genes that encode stress‐responsive proteins. The metal‐regulatory transcription factor 1 (MTF‐1) is a key regulator of heavy‐metal induced transcription of metallothionein‐I and II and other genes in mammals and other metazoans. Transcriptional activation of genes by MTF‐1 is mediated through binding to metal‐responsive elements in the target gene promoters. Phosphorylation of MTF‐1 plays a critical role in the cadmium‐inducible transcriptional activation of metallothionein and other responses. Studies using inhibitors indicate that multiple kinases and signal transduction cascades, including those mediated by protein kinase C, tyrosine kinase and casein kinase II, are essential for cadmium‐mediated transcriptional activation. In addition, calcium signaling is also involved in regulating metal‐activated transcription. In several species, cadmium ...

Putting its fingers on stressful situations: the heavy metal-regulatory transcription factor MTF-1.

It has been suggested that metallothioneins, discovered about 45 years ago, play a central role in heavy metal metabolism and detoxification, and in the management of various forms of stress. The metal-regulatory transcription factor-1 (MTF-1) was shown to be essential for basal and heavy metal-induced transcription of the stress-responsive metallothionein-I and metallothionein-II. Recently it has become obvious that MTF-1 has further roles in the transcriptional regulation of genes induced by various stressors and might even contribute to some aspects of malignant cell growth. Furthermore, MTF-1 is an essential gene, as mice null-mutant for MTF-1 die in utero due to liver degeneration. We describe here the state of knowledge on the complex activation of MTF-1, and propose a model with MTF-1 as an interconnected cellular stress-sensor protein involved in heavy metal metabolism, hepatocyte differentiation and detoxification of toxic agents.

Phosphorylation is involved in the activation of metal-regulatory transcription factor 1 in response to metal ions.

We have studied the role of phosphorylation in the activation of metal-regulatory transcription factor-1 (MTF-1) and metallothionein (MT) gene expression. We showed that MTF-1 is phosphorylated in vivo and that zinc stimulates MTF-1 phosphorylation 2-4-fold. Several kinase inhibitors were used to examine the possible involvement of kinase cascades in the activation of MTF-1. Metal-induced MT gene expression was abrogated by protein kinase C (PKC), c-Jun N-terminal kinase (JNK), phosphoinositide 3-kinase, and tyrosine-specific protein kinases inhibitors, as assayed by Northern analysis and by cotransfection experiments using a metal regulatory element-luciferase reporter plasmid. The extracellular signal-activated protein kinase and the p38 kinase cascades did not appear to be essential for the activation of MT gene transcription by metals. By using dominant-negative mutants of PKC, JNK, mitogen-activated kinase kinase 4 (MKK4), and MKK7, we provide further evidence supporting a role for PKC and JNK in the activation of MTF-1 in response to metals. Notably, increased MTF-1 DNA binding in response to zinc and MTF-1 nuclear localization was not inhibited in cells preincubated with the different kinase inhibitors despite strong inhibition of MTF-1-mediated gene expression. This suggests that phosphorylation is essential for MTF-1 transactivation function. We hypothesize that metal-induced phosphorylation of MTF-1 is one of the primary events leading to increased MTF-1 activity. Thus, metal ions such as cadmium could activate MTF-1 and induce MT gene expression by stimulating one or several kinases in the MTF-1 signal transduction pathway.

Characterization of the mouse metal-regulatory-element-binding proteins, metal element protein-1 and metal regulatory transcription factor-1

Metal activation of metallothionein gene transcription depends mainly on the presence of regulatory DNA sequences termed metal-regulatory elements (MREs) and involves MRE-binding transcription factor-1 (MTF-1) interacting with the MREs in a Zn2+-dependent manner. We previously identified and characterized a nuclear protein, termed metal element protein-1 (MEP-1), specifically binding with high affinity to MRE elements. The precise relationship between MTF-1 and MEP-1 was unclear, and to determine whether MEP-1 and MTF-1 were distinct protein species, we performed DNA binding analyses to characterize the binding properties of both proteins. Electrophoretic mobility-shift assays showed that MTF-1, produced in COS cells, produces a slower-migrating band compared with that obtained with purified MEP-1. Using an anti-MTF-1 antibody, we showed that both the MTF-1–MRE and the MEP-1–MRE complexes are supershifted by an anti-MTF-1 antibody, thus demonstrating that MEP-1 is antigenically related to MTF-1. RNase protection analyses carried out with RNA prepared from different tissues and cell lines failed to reveal the presence of MTF-1 splicing variants. This indicates that MEP-1 may be a proteolytic fragment of MTF-1. MTF-1 DNA-binding activity was rapidly activated in vivo by Zn2+ ions but not by Cd2+, UV irradiation or PMA, and occurred on ice as well as at 21°C. In control and Zn2+-treated cell extracts, DNA-binding activity was not enhanced in vitro following the addition of exogenous Zn2+ or a preincubation at 37°C. However, recombinant MTF-1 produced in vitro required Zn2+ activation for DNA binding. Interestingly, treatment of nuclear extracts with calf intestine phosphatase completely abrogated MTF-1 DNA-binding activity, thus suggesting that phosphorylation is involved in the regulation of MTF-1 activity.

Characterization of the mouse metal-regulatory-element-binding proteins, metal element protein-1 and metal regulatory transcription factor-1.

Metal activation of metallothionein gene transcription depends mainly on the presence of regulatory DNA sequences termed metal-regulatory elements (MREs) and involves MRE-binding transcription factor-1 (MTF-1) interacting with the MREs in a Zn(2+)-dependent manner. We previously identified and characterized a nuclear protein, termed metal element protein-1 (MEP-1), specifically binding with high affinity to MRE elements. The precise relationship between MTF-1 and MEP-1 was unclear, and to determine whether MEP-1 and MTF-1 were distinct protein species, we performed DNA binding analyses to characterize the binding properties of both proteins. Electrophoretic mobility-shift assays showed that MTF-1, produced in COS cells, produces a slower-migrating band compared with that obtained with purified MEP-1. Using an anti-MTF-1 antibody, we showed that both the MTF-1-MRE and the MEP-1-MRE complexes are supershifted by an anti-MTF-1 antibody, thus demonstrating that MEP-1 is antigenically related to MTF-1. RNase protection analyses carried out with RNA prepared from different tissues and cell lines failed to reveal the presence of MTF-1 splicing variants. This indicates that MEP-1 may be a proteolytic fragment of MTF-1. MTF-1 DNA-binding activity was rapidly activated in vivo by Zn(2+) ions but not by Cd(2+), UV irradiation or PMA, and occurred on ice as well as at 21 degrees C. In control and Zn(2+)-treated cell extracts, DNA-binding activity was not enhanced in vitro following the addition of exogenous Zn(2+) or a preincubation at 37 degrees C. However, recombinant MTF-1 produced in vitro required Zn(2+) activation for DNA binding. Interestingly, treatment of nuclear extracts with calf intestine phosphatase completely abrogated MTF-1 DNA-binding activity, thus suggesting that phosphorylation is involved in the regulation of MTF-1 activity.

Nucleo-cytoplasmic Trafficking of Metal-regulatory Transcription Factor 1 Is Regulated by Diverse Stress Signals

The metal-regulatory transcription factor 1 (MTF-1) is a key regulator of heavy metal-induced transcription of metallothionein I and II and other genes in mammals and other metazoans. Transcriptional activation of genes by MTF-1 is mediated through binding to metal-responsive elements of consensus TGCRCNC in the target gene promoters. In an attempt to further clarify the mechanisms by which certain external signals activate MTF-1 and in turn modulate gene transcription, we show here that human MTF-1 has a dual nuclear and cytoplasmic localization in response to diverse stress stimuli. MTF-1 contains a consensus nuclear localization signal located just N-terminal to the first zinc finger that contributes to but is not essential for nuclear import. MTF-1 also harbors a leucine-rich, nuclear export signal. Under resting conditions, the nuclear export signal is required for cytoplasmic localization of MTF-1 as indicated by mutational analysis and transfer to the heterologous green fluorescent protein. Export from the nucleus was inhibited by leptomycin B, suggesting the involvement of the nuclear export protein CRM1. Our results further show that in addition to the heavy metals zinc and cadmium, heat shock, hydrogen peroxide, low extracellular pH (pH 6.0), inhibition of protein synthesis by cycloheximide, and serum induce nuclear accumulation of MTF-1. However, heavy metals alone (and not the other stress conditions) induce a significant transcriptional response via metal-responsive element promoter sequences, implying that nuclear import of MTF-1 is necessary but not sufficient for transcriptional activation. Possible roles for nuclear import under non-metal stress conditions are discussed.

Original MRE-binding Transcriptional Factor Gene in Normal Humans is ZRF, not MTF-1.

Metallothionein gene is transcriptionally regulated by heavy metals through cis-acting metal responsive elements (MREs). Two proteins, metal-regulatory transcription factor-1 (MTF-1) and zinc regulatory factor (ZRF), have been isolated and cloned from human cells as MRE-binding transcriptional factor (MREBT). These proteins are almost identical to each other, except for only one base substitution at codon 185 that causes an amino acid change from histidine to tyrosine. This single amino acid difference has been reported to influence zinc-responsive transcriptional activities. In this study, we determined the nucleotide sequence of the region containing codon 185 in DNA samples obtained from normal Japanese (n = 30) and three human-derived cultured cell lines. The findings indicate that all subjects have the same sequence identical to ZRF, suggesting that ZRF is the original MREBT gene in normal humans, and MTF-1 is its minor variant.

Cell lines from the same cervical carcinomabut with different radiosensitivities exhibit different cDNA microarray patterns of gene expression

Combining chemotherapy with radiotherapy has improved the cure rate among patients with cancers of the cervix. Although one-half to two-thirds of the patients can be cured by radiation alone, such patients cannot be identified at present and must therefore suffer the burden of chemotherapy. Our long-range goal is to identify those cervical cancers that are radiosensitive and could be cured by radiotherapy alone. The advent of methods that permit the simultaneous analysis of expression patterns of thousands of genes, make it feasible to attempt to identify the molecular events related to radiosensitivity and the associated regulatory pathways. We hypothesize that the sensitivity of tumor cells to ionizing radiation (IR) is determined by the level of expression of specific genes that may be identified with the aid of cDNA microarrays. As the first step in testing this hypothesis, we determined the gene expression differences between two cell lines exhibiting different degrees of radiosensitivity. These were derived from the same tumor prior to treatment from a patient with squamous cell carcinoma of the cervix. The mRNA from these cells was subjected to cDNA analysis on a microarray of 5,776 known genes and ESTs. The expression of 52 genes of the total of 5,776 was elevated (maximum 4.1 fold) in the radioresistant cells as compared to the radiosensitive cells. Ten of the 52 sequences are known genes while 42 are ESTs. Conversely, the expression of 18 genes was elevated in the sensitive cells as compared to the resistant cells. Seven of these 18 are known genes while eleven are ESTs. Among the genes expressed differentially between the resistant and sensitive cells were several known to be associated with response to IR and many more genes and ESTs that had not previously been reported to be related to radiosensitivity. The genes that showed the greatest overexpression in the radioresistant cell line were metal-regulatory transcription factor-1, cytochrome P450 CYP1B1, adenomatosis polyposis coli, translation elongation factor-1, cytochrome-c oxidase, whereas in the sensitive cell line, transcription factor NF-kappa-B, metalloproteinase inhibitor-1 precursor, superoxide dismutase-2, insulin-like growth factor binding protein-3, guanine nucleotide-binding protein and transforming growth factor beta-induced protein were overexpressed.

Suppression of Metallothionein Gene Expression in a Rat Hepatoma Because of Promoter-specific DNA Methylation

Metallothionein I can be induced in response to a variety of agents that include heavy metals and oxidative stress. On the contrary, its induction was suppressed in some lymphoid-derived cancer cells. The mechanism of this repression has not been elucidated. Here, we show silencing of MT-I gene in a solid transplanted rat tumor as a result of promoter methylation at all the 21 CpG dinucleotides that span the region from -225 bp to +1 bp. By contrast, none of these CpG dinucleotides were methylated in the livers from the rats bearing the tumor, which was consistent with the efficient induction of the gene in this tissue by zinc sulfate. Genomic footprinting revealed lack of access of the transcriptional activators to the respective cis-acting elements of the methylated MT-I promoter in the hepatoma. The absence of footprinting was not due to inactivation of the metal regulatory transcription factor MTF-1, because it was highly active in the hepatoma. Treatment of the hepatoma bearing rats with 5-azacytidine, a demethylating agent, induced basal as well as heavy metal-activated MT-I gene expression in the hepatoma, implying that methylation was indeed responsible for silencing the gene. Bisulfite genomic sequencing showed significant (>90%) demethylation of CpG dinucleotides spanning MT-I promoter in the hepatoma following treatment with 5-AzaC. The hypermethylation of MT-I promoter was probably caused by significantly higher (as much as 7-fold) level of DNA methyl transferase activity as well as enhanced expression of its gene in the hepatoma relative to the host liver. These data elucidated for the first time the molecular mechanism for the silencing of a highly inducible gene in a solid tumor transplanted in an animal, as compared with the robust induction in the corresponding parental tissue and have discussed the probable reasons for the suppression of this gene in some tumors.