Metallothionein Isoforms Research Articles

Metallothionein 2A genetic polymorphisms and risk of prostate cancer in a Polish population

Metallothionein 2A (MT2A) is the most expressed metallothionein (MT) isoform in prostate cells. A number of studies have demonstrated altered MT2A expression in various human tumors, including prostate cancer. We conducted an association study to examine whether MT2A gene polymorphisms are associated with a risk of prostate cancer. Genotyping was conducted using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Three single nucleotide polymorphisms (SNPs), rs28366003, rs1610216, and rs10636, were genotyped in 358 prostate cancer cases and 406 population controls. One SNP in MT2A (rs28366003) showed a positive association with prostate cancer. Compared to homozygous common allele carriers, heterozygosity for the G variant (odds ratio (OR)=2.30, 95% confidence interval (CI): 1.50-3.47, P-trend<0.0001; the OR assuming a dominant model 2.43 (95% CI: 1.62-3.61, P(dominant)=0.001) after adjustment for age) had a significantly increased risk of prostate cancer in a Polish population. Our data suggest that the rs28366003 SNP in MT2A is associated with the risk of prostate cancer in a Polish population.

Monitoring of the prostate tumour cells redox state and real-time proliferation by novel biophysical techniques and fluorescent staining

The present paper is focused on zinc(ii) treatment effects on prostatic cell lines PC-3 (tumour) and PNT1A (non-tumour). Oxidative status of cells was monitored by evaluation of expression of metallothionein (MT) isoforms 1A and 2A at the mRNA and protein level, glutathione (oxidised and reduced), and intracellular zinc(ii) after exposition to zinc(ii) treatment at concentrations of 0-150 μM using electrochemical methods, western blotting and fluorescent microscopy. A novel real-time impedance-based growth monitoring system was compared with widely used end-point MTT assay. Impedance-based IC(50) for zinc(ii) is 55.5 and 150.8 μM for PC-3 and PNT1A, respectively. MTT-determined IC(50) are >1.3-fold higher. Impedance-based viability correlates with viable count (r > 0.92; p < 0.03), not with MTT. Two-fold lower intracellular zinc(ii) in the tumour PC-3 cell line was found. After zinc(ii) treatment >2.6-fold increase of intracellular zinc(ii) was observed in non-tumour PNT1A and in tumour PC-3 cells. In PC-3 cells, free and bound zinc(ii) levels were enhanced more markedly as compared to PNT1A. PNT1A produced 4.2-fold less MT compared to PC3. PNT1A cells showed a 4.8-fold increase trend (r = 0.94; p = 0.005); PC-3 did show a significant trend at MT1 and MT2 protein levels (r = 0.93; p = 0.02) with nearly ten-fold increase after 100 μM zinc(ii) treatment. In terms of redox state, PNT1A had a predominance of reduced GSH forms (GSH : GSSG ratio > 1), when exposed to zinc(ii) compared to PC3, where predominance of oxidised forms remains at all concentrations. IC(50) differs significantly when determined by MTT and real-time impedance-based assays due to dependence of impedance on cell morphology and adhesion. When real-time growth monitoring, precise electrochemical methods and fluorescent microscopy are performed together, accurate information for metal fluxes, their buffering by thiol compounds and monitoring of the redox state become a powerful tool for understanding the role of oxidative stress in carcinogenesis.

Effects of sublethal, environmentally relevant concentrations of hexavalent chromium in the gills of Mytilus galloprovincialis

Hexavalent chromium Cr(VI) is an important contaminant released from both domestic and industrial effluents, and represents the predominant chemical form of the metal in aquatic ecosystems. In the marine bivalve Mytilus galloprovincialis exposure to non-toxic, environmentally relevant concentrations of Cr(VI) was shown to modulate functional parameters and gene expression in both the digestive gland and hemocytes. In this work, the effects of exposure to Cr(VI) (0.1–1–10μgL−1animal−1 for 96h) in mussel gills were investigated. Gill morphology and immunolocalization of GSH-transferase (GST), of components involved in cholinergic (AChE and ChAT), adrenergic (TH) and serotoninergic (5-HT3 receptor) systems, regulating gill motility, were evaluated. Total glutathione content, activities of GSH-related enzymes (glutathione reductase – GSR, GST), of catalase, and of key glycolytic enzymes (phosphofructokinase – PFK and pyruvate kinase – PK) were determined. Moreover, mRNA expression of selected Mytilus genes (GST-π, metallothionein isoforms MT10 and MT20, HSP70 and 5-HT receptor) was assessed by RT-q-PCR. Cr(VI) exposure induced progressive changes in gill morphology and in immunoreactivity to components involved in neurotransmission that were particularly evident at the highest concentration tested, and associated with large metal accumulation. Cr(VI) increased the activities of GST and GSR, and total glutathione content to a different extent at different metal concentrations, this suggesting Cr(VI) detoxication/reduction at the site of metal entry. Cr(VI) exposure also increased the activity of glycolytic enzymes, indicating modulation of carbohydrate metabolism. Significant changes in transcription of different genes were observed. In particular, the mRNA level for the 5-HTR was increased, whereas both decreases and increases were observed for GST-π, MT10, MT20 and HSP70 mRNAs, showing sex- and concentration-related differences. The results demonstrate that Cr(VI) significantly affected functional and molecular parameters in mussel gills, and indicate that this tissue represents the major target of exposure to environmentally relevant concentrations of the metal.

Friedreich's Ataxia Causes Redistribution of Iron, Copper, and Zinc in the Dentate Nucleus

Friedreich's ataxia (FRDA) causes selective atrophy of the large neurons of the dentate nucleus (DN). High iron (Fe) concentration and failure to clear the metal from the affected brain tissue are potential risk factors in the progression of the lesion. The DN also contains relatively high amounts of copper (Cu) and zinc (Zn), but the importance of these metals in FRDA has not been established. This report describes nondestructive quantitative X-ray fluorescence (XRF) and "mapping" of Fe, Cu, and Zn in polyethylene glycol–dimethylsulfoxide (PEG/DMSO)-embedded DN of 10 FRDA patients and 13 controls. Fe fluorescence arose predominantly from the hilar white matter, whereas Cu and Zn were present at peak levels in DN gray matter. Despite collapse of the DN in FRDA, the location of the peak Fe signal did not change. In contrast, the Cu and Zn regions broadened and overlapped extensively with the Fe-rich region. Maximal metal concentrations did not differ from normal (in micrograms per milliliter of solid PEG/DMSO as means ± S.D.): Fe normal, 364 ± 117, FRDA, 344 ± 159; Cu normal, 33 ± 13, FRDA, 33 ± 18; and Zn normal, 32 ± 16, FRDA, 33 ± 19. Tissues were recovered from PEG/DMSO and transferred into paraffin for matching with immunohistochemistry of neuron-specific enolase (NSE), glutamic acid decarboxylase (GAD), and ferritin. NSE and GAD reaction products confirmed neuronal atrophy and grumose degeneration that coincided with abnormally diffuse Cu and Zn zones. Ferritin immunohistochemistry matched Fe XRF maps, revealing the most abundant reaction product in oligodendroglia of the DN hilus. In FRDA, these cells were smaller and more numerous than normal. In the atrophic DN gray matter of FRDA, anti-ferritin labeled mostly hypertrophic microglia. Immunohistochemistry and immunofluorescence of the Cu-responsive proteins Cu,Zn-superoxide dismutase and Cu++-transporting ATPase α-peptide did not detect specific responses to Cu redistribution in FRDA. In contrast, metallothionein (MT)-positive processes were more abundant than normal and contributed to the gliosis of the DN. The isoforms of MT, MT-1/2, and brain-specific MT-3 displayed only limited co-localization with glial fibrillary acidic protein. The results suggest that MT can provide effective protection against endogenous Cu and Zn toxicity in FRDA, similar to the neuroprotective sequestration of Fe in holoferritin.

Arsenic Trioxide (ATO) Influences the Gene Expression of Metallothioneins in Human Glioblastoma Cells

Arsenic trioxide (As(2)O(3); ATO, TRISENOX®) is used to treat patients with refractory or relapsed acute promyelocytic leukaemia while its application for treatment of solid cancers like glioblastoma is still under evaluation. In the present study, we investigated the interaction of arsenic trioxide with metallothionein (MT) isoforms as a possible (protective response) resistance of glioblastoma cells to arsenic-induced cytotoxicity. Special attention was focused on MT3, the isoform expressed mainly in the brain. MT3 has low metal inducibility, fast metal binding/releasing properties and outstanding neuronal inhibitory activity. The human astrocytoma (glioblastoma) cell line U87 MG was treated with 0.6, 2 and 6-7 μM arsenic (equivalent to 0.3, 1 and 3-3.5 μM As(2)O(3)) for 12, 24 or 48 h and gene expression for different MT isoforms, namely MT2A, MT1A, MT1F, MT1X, MT1E and MT3, was measured by real time qPCR using SYBR Green I and Taqman® gene expression assays. TfR, 18S rRNA, GAPDH and AB were tested as reference genes, and the last two evaluated to be appropriate in conditions of low (GAPDH) and high (AB) arsenic exposure. The gene expression of MT3 gene was additionally tested and confirmed by restriction enzyme analysis with PvuII. In the given conditions the mRNAs of six MT isoforms were identified in human glioblastoma cell line U87 MG. Depending on arsenic exposure conditions, an increase or decrease of MT gene expression was observed for each isoform, with the highest increase for isoforms MT1X, MT1F and MT2A mRNA (up to 13-fold) and more persistent decreases for MT1A, MT1E and MT3 mRNA. Despite the common assumption of the noninducibility of MT3, the evident MT3 mRNA increase was observed during high As exposure (up to 4-fold). In conclusion, our results clearly demonstrate the influence of As on MT isoform gene expression. The MT1X, MT1F and MT2A increase could represent brain tumour acquired resistance to As cytotoxicity while the MT3 increase is more enigmatic, with its possible involvement in arsenic-related induction of type II cell death.

A critical role for increased labile zinc in reducing sensitivity of cultured sheep pulmonary artery endothelial cells to LPS-induced apoptosis

We previously noted an important signaling role for decreased labile intracellular zinc ([ Zn ] (i)) in LPS-induced apoptosis in cultured sheep pulmonary artery endothelial cells (SPAEC) (Tang ZL, Wasserloos KJ, Liu X, Stitt MS, Reynolds IJ, Pitt BR, St Croix CM. Mol Cell Biochem 234-235: 211-217, 2002; Thambiayya K, Wasserloos KJ, Huang Z, Kagan VE, St Croix CM, Pitt BR. Am J Physiol Lung Cell Mol Physiol 300: L624-632, 2011). In the present study, we used small interfering RNA (siRNA) to important contributors of zinc homeostasis [ SLC39A14 or Zrt/Irt-like protein 14 (ZIP14), a zinc importer; metallothionein (MT), a zinc binding protein ] to define molecular pathways by which extracellular zinc or nitric oxide (NO) increase labile [ Zn ] (i) [ e.g., zinc-sensitive fluorophore (FluoZin-3) detectable and/or chelatable by N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine ] and reduce the sensitivity of SPAEC to LPS. Addition of 10 μM zinc to serum-free medium of SPAEC increased [ Zn ] (i) and abolished LPS-induced apoptosis (e.g., increased annexin V binding). The increase in [ Zn ] (i) and the protective effect of extracellular zinc were sensitive to reduction in ZIP14 expression (by siRNA), but not affected by collectively knocking down major isoforms of sheep MT (sMT-Ia, -Ib, -Ic, and -II). Pretreatment of wild-type SPAEC with 250 μM of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) increased labile zinc in a relatively similar fashion to addition of extracellular zinc and reduced sensitivity of SPAEC to LPS-induced apoptosis (e.g., caspase-3/7 activation) in a N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine-sensitive fashion. The antiapoptotic effects of SNAP were insensitive to siRNA knockdown of ZIP14, but were abolished (along with SNAP-induced increase in [ Zn ] (i)) when SPAEC were pretreated with siRNA to sheep MT. Zinc was able to directly inhibit recombinant caspase-3 activity in an in vitro assay. Collectively, these data show that increases in labile [ Zn ] (i) are an important component of ZIP14- or NO-mediated resistance to LPS-induced apoptosis. Cytoprotection via ZIP14 appeared to be secondary to transcellular movement of extracellular zinc, whereas NO-mediated protection was secondary to S-nitrosation of MT and redistribution of [ Zn ] (i).

Zinc‐metallothionein ratios reflect cellular zinc status

Animal and human studies show that intracellular zinc levels are sensitive to changes in dietary zinc. The protein metallothionein (MT) is maintained in proportion to intracellular zinc content, and therefore may be a useful marker of zinc status. Measuring MT levels is difficult due to the unique properties of the protein, so most studies focus on MT gene expression. Our objective was to develop new methods to quantify MT protein and MT‐bound zinc content. Zinc deficiency or excess was modeled in the Jurkat leukocytic cell line using the zinc‐selective chelator TPEN or exogenous zinc sulfate, respectively. Cellular protein was separated using size‐exclusion filtration to isolate an MT‐rich fraction. Then, endogenous zinc content was released, apo‐MT was loaded with cadmium, and MT‐bound cadmium levels were quantified by ICP; this method permitted the determination of cellular MT‐bound zinc content (MBZC) and MT zinc saturation (%MTsat). Gene expression of the major MT and zinc transporter isoforms were measured in parallel by RT‐PCR, which reflected the change in zinc balance in these cells. MBZC and %MTsat levels correlated with intracellular zinc content and changes in MT gene expression, showing bimodal responses to zinc deficiency and zinc excess. These measures of the cellular zinc‐metallothionein ratio may have utility as markers of zinc homeostasis. This work was supported by the HarvestPlus.

Downregulation of Metallothionein 1F, a putative oncosuppressor, by loss of heterozygosity in colon cancer tissue

PurposeDownregulation of metallothionein (MT) genes has been reported in several tumors with discrepant results. This study is to investigate molecular mechanism of MT gene regulation in colon cancer which is characterized by tumor suppressor gene alterations. Experimental designIntegral analysis of microarray data with loss of heterozygosity (LOH) information was employed. Quantitative real-time PCR and immunohistochemistry were used to validate MT isoform expression in colon cancer tissues and cell lines. The effects of MT1F expression on RKO cell survival and tumorigenesis was analyzed. Bisulphite sequencing PCR (BSP) and methylation-specific PCR were employed to detect the methylation status of the MT1F gene in colon cancer tissues and cell lines. DNA sequencing was used to examine the LOH at the MT1F locus. ResultsMT1F, MT1G, MT1X, and MT2A gene expression was significantly downregulated in colon cancer tissue (p<0.05). Exogenous MT1F expression increased RKO cell apoptosis and inhibited RKO cell migration, invasion and adhesion as well as in vivo tumorigenicity. Downregulation of MT1F gene in majority of human colon tumor tissues is mainly through mechanism by loss of heterozygosity (p=0.001) while CpG island methylation of MT1F gene promoter region was only observed in poorly differentiated, MSI-positive RKO and LoVo colon cancer cell lines. ConclusionsMT1F is a putative tumor suppressor gene in colon carcinogenesis that is downregulated mainly by LOH in colon cancer tissue. Further studies are required to elucidate a possible role for MT1F downregulation in colon cancer initiation and/or progression.

Expression of antioxidant molecules after peripheral nerve injury and regeneration

Oxidative stress is considered to be one of the main causes of neural damage after injury. However, little is known about the changes in mRNA expression of antioxidant molecules that occur after injury and regeneration of the peripheral nerve. In the present study, the rat median nerve was transected, and transcriptional changes were studied at day 6 and day 12 after injury in both the proximal and the distal stumps, in the absence or presence of microsurgical repair. The expression profiles of the following genes were investigated: three metallothionein isoforms (MT-1, MT-2, and MT-3), the main antioxidant enzymes (catalase, superoxide dismutase, and glutathione-S-transferase), and the marker of cellular damage poly(ADP-ribose) polymerase-1 (PARP-1). The results showed that, in the proximal nerve stump, MT-3 mRNA expression was significantly and markedly up-regulated in the absence of surgical repair, whereas MT-1 and MT-2 showed significant down-regulation. In the distal nerve portion, mRNA expression of all MT isoforms decreased significantly in the absence of microsurgical reconstruction, whereas, after repair, MT-3 mRNA expression alone was up-regulated. Expression of all the antioxidant enzymes decreased significantly after repair in the proximal nerve portion, but a significant general increase in their mRNA expression was revealed in the distal nerve stump. PARP-1 expression was significantly up-regulated in the proximal nerve portion without repair but dramatically reduced after reconstruction. In contrast, PARP-1 expression increased markedly in the distal stump after surgical repair. Taken together, these findings indicate that antioxidant molecules are differentially modulated and might, therefore, play an important role in peripheral nerve injury and regeneration.

Is MtnE, the fifth Drosophila metallothionein, functionally distinct from the other members of this polymorphic protein family?

Metallothioneins (MTs) are a super-family of small, Cys-rich, non-homologous proteins that bind metal ions through the formation of metal-thiolate bonds. Although universally ubiquitous, they exhibit distinct metal-binding preferences, either for divalent (Zn-thioneins) or monovalent (Cu-thioneins) metal ions. Drosophila constitutes a bizarre exception, since it is currently the only case of metazoans synthesizing only Cu-thioneins, which are similar to the paradigmatic yeast Cup1 protein. Until recently, the Drosophila MT system was assumed to be composed of 4 isoforms (MtnA, MtnB, MtnC and MtnD), all of them responsive to heavy metal load through the dMTF1 transcription factor. The significance of this polymorphism has been analyzed in depth both at genomic and proteomic levels. Singularly, a fifth MT isoform was recently annotated and named MtnE. The analysis of the MtnE expression pattern revealed some differential traits with regard to the other MTs. We analyze here the peculiarities of the metal binding abilities of the MtnE polypeptide and compare them with those of the other Drosophila MTs determined through the same rationale. Characterization by ESI-MS spectrometry and CD and UV-visible spectrophotometry of the Zn(II)-, Cd(II)- and Cu(I)-MtnE complexes obtained by recombinant synthesis demonstrates that MtnE is the least metal-specific isoform of the Drosophila MTs, and therefore it could play a role when/where a broad spectrum of metal coordination abilities are advantageous in terms of physiological needs.

Distribution of mercury in metallothionein-null mice after exposure to mercury vapor: amount of metallothionein isoform does not affect accumulation of mercury in the brain

To examine the contribution of metallothionein (MT) to mercury accumulation in mouse tissues, 129 strain female mice and MT null mice were exposed to metallic mercury vapor at a sub-toxic level, and Hg levels in the brain, kidney and liver were determined on 1, 3 and 7 days after the exposure. After exposure to mercury vapor, significant Hg accumulation was observed in the brains of wild-type and MT-I/II null and MT-III null mice, as well as in the liver and kidneys. No strain difference was observed in the tissue Hg accumulations 24 hr after the exposure except for the kidneys, where the highest accumulation was found in MT-III null mice. Although the brains of MT-III null mice showed slightly higher Hg accumulation than the other two strains, no significant difference was observed except in the cerebrum on Day 7. Gel chromatograms of cerebrum soluble fractions revealed that a significant amount of Hg existed as an MT-bound form in all the mouse strains. On the other hand, MT-bound Hg was found as a minor fraction in soluble fractions of the kidneys and livers in wild-type and MT-III null mice. Despite a significant strain difference in total MT levels in the cerebrum, there was no difference among the three strains in the amount of Hg accumulated in the cerebrum and its distribution rates in MT fractions. The present study demonstrated that brain uptake of Hg(0) and its accumulation as Hg(2+) did not depend on the amount of MT isoform in the tissue, at least in the early phase.

Structural changes in metallothionein isoforms revealed by capillary electrophoresis and Brdicka reaction

Metallothionein (MT) as a potential cancer marker is at the center of interest and its properties, functions and behavior under various conditions is intensively studied. In the present study, two major mammalian MT isoforms (MT-1 and MT-2) were separated using capillary electrophoresis (CE) coupled with UV detector in order to describe their basic behavior. Under the optimized conditions, the separation of both isoforms was enabled as well as estimation of detection limits as subunits and units of ng per μL for MT-2 and MT-1, respectively. Further, the effects of thermal treatment and the presence of denaturing agent such as urea on MT-1 and MT-2 isoforms were studied by CE-UV. Thermal treatment caused an increase in the signals of both isoforms. A new parameter called precipitation rate has been defined based on this finding. This parameter can be expressed as a slope of the linear regression of the time dependency curve recalculated on the MT concentration. The thermal precipitation rate for MT-1 and MT-2 was determined as 1.1 and 0.9 ng of MT/min, respectively. The chemical precipitation rate calculated from the linear regression for both isoforms provided the same value of 0.25 ng of MT/min. The results were confirmed by manual spectrometric measurements and by differential pulse voltammetry Brdicka reaction. Based on these results, a model of MT behavior under the conditions studied was suggested.

The metal binding abilities of Megathura crenulata metallothionein (McMT) in the frame of Gastropoda MTs

Metallothioneins (MTs) are proteins that play a major role in metal homeostasis and/or detoxification in all kind of organisms. The MT gene/protein system of gastropod molluscs provides an invaluable model to study the diversification mechanisms that have enabled MTs to achieve metal-binding specificity through evolution. Most pulmonate gastropods, particularly terrestrial snails, harbor three paralogous isogenes encoding three MT isoforms with different metal binding preferences: the highly specific CdMT and CuMT isoforms, for cadmium and copper respectively, and the unspecific Cd/CuMT isoform. Megathura crenulata is a non-pulmonate gastropod in which only one MT isogene has so far been reported. In order to elucidate the metal binding character of the corresponding peptide (McMT), it has been recombinantly synthesized in the presence of Cd2+, Zn2+ or Cu2+, and the corresponding metal complexes have been analyzed using electrospray mass spectrometry, and CD and UV–visible spectroscopy. The metal-binding traits exhibited by McMT revealed that it is an unspecific MT, similarly to the pulmonate Cd/CuMT isoforms. This is in full concordance with the protein sequence distance analysis in relation to other gastropod MTs.

Comparison of capillary electrophoretic techniques for analysis and characterization of metallothioneins

To explore and understand the significance of individual metallothionein isoforms, the methods of their identification are needed. Separation of these isoforms requires a high resolution technique which can exploit very small differences in mass, charge, and hydrophobicity. In this report, three different techniques of CE were analyzed and used for metallothionein separation: detection using capillary gel electrophoresis, capillary zone electrophoresis, and capillary isoelectric focusing. Also, three different metallothionein samples were used from horse kidney, rabbit liver, and human liver. We identified metallothionein isoforms based on the determination of their relative molecular masses, on the charge differences in different pH buffers, and based on the pI value. Methods used in this report allow metallothionein identification, permit to quantify the purity and content of its isoforms, and allow studying its polymerization. This report supports and endorses the increased application of CE methodology in proteomics.

Nematode and snail metallothioneins

Metallobiologists have, at large, neglected soil dwelling invertebrates; exceptions are the nematode (Caenorhabditis elegans) and snails (Helix pomatia and Cantareus aspersus). This review aims to compare and contrast the molecular, protein and cellular mechanisms of the multifunctional nematode and snail metallothioneins (MTs). The C. elegans genome contains two MT genes, mtl-1, which is constitutively expressed in the pharynx and likely to act as an essential and/or toxic metal sensor, and mtl-2, which plays a negligible role under normal physiological conditions but is strongly induced (as mtl-1) in intestinal cells upon metal exposure. It has been possible to follow the intricate phenotypic responses upon the knockdown/knockout of single and multiple MT isoforms and we have started to decipher the multifunctional role of C. elegans MTs. The snails have contributed to our understanding regarding MT evolution and diversity, structure and metal-specific functionality. The H. pomatia and C. aspersus genomes contain at least three MT isoform genes. CdMT is responsible for cadmium detoxification, CuMT is involved in copper homeostasis and Cd/CuMT is a putative ancestral MT possibly only of minor importance in metal metabolism. Further investigations of nematode, snail and other invertebrate MTs will allow the development of alternative biomarker approaches and lead to an improved understanding of metallobiology, protein evolution and toxicogenomics.

Metallothionein expression in colorectal cancer: relevance of different isoforms for tumor progression and patient survival

Metallothioneins are a family of small, cysteine-rich proteins with many functions. Immunohistochemical evaluation of all metallothionein 1 + 2 isoforms in colorectal tumors has demonstrated an important down-regulation compared with normal tissue, although its prognostic significance is unclear. Moreover, the contribution of individual isoforms to overall metallothionein down-regulation is not known. To address these important issues, we analyzed the messenger RNA expression levels of all functional metallothionein 1 + 2 isoforms by quantitative reverse transcription polymerase chain reaction in 22 pairs of normal and tumor-microdissected epithelia and correlated these to the overall immunohistochemical protein expression. Our results showed that 5 isoforms (MT1G, 1E, 1F, 1H, and 1M) were lost during the transition from normal mucosa to tumor, whereas MT1X and MT2A were less down-regulated, and their expression was correlated with overall protein positivity. Second, we showed that MT1G hypermethylation occurred in cell lines and in 29% of tumor samples, whereas histone deacetylase inhibitors are able to induce most isoforms. Furthermore, we analyzed by immunohistochemistry 107 normal mucosae, 25 adenomas, 81 carcinomas, and 19 lymph node metastases to evaluate metallothionein expression during different stages of cancer development and to assess its relationship to patient survival. A lower immunohistochemical expression was associated with poorer survival, although it was not an independent predictor. Overall, this study identifies for the first time the relevant metallothionein isoforms for colorectal cancer progression, supports the concept that their loss is associated with worse prognosis, and suggests 2 mechanisms for epigenetic repression of metallothionein expression in colorectal tumors.

Chemistry and biology of mammalian metallothioneins

Metallothioneins (MTs) are a class of ubiquitously occurring low molecular mass, cysteine- and metal-rich proteins containing sulfur-based metal clusters formed with Zn(II), Cd(II), and Cu(I) ions. In mammals, four distinct MT isoforms designated MT-1 through MT-4 exist. The first discovered MT-1/MT-2 are widely expressed isoforms, whose biosynthesis is inducible by a wide range of stimuli, including metals, drugs, and inflammatory mediators. In contrast, MT-3 and MT-4 are noninducible proteins, with their expression primarily confined to the central nervous system and certain squamous epithelia, respectively. MT-1 through MT-3 have been reported to be secreted, suggesting that they may play different biological roles in the intracellular and extracellular space. Recent reports established that these isoforms play an important protective role in brain injury and metal-linked neurodegenerative diseases. In the postgenomic era, it is becoming increasingly clear that MTs fulfill multiple functions, including the involvement in zinc and copper homeostasis, protection against heavy metal toxicity, and oxidative damage. All mammalian MTs are monomeric proteins, containing two metal-thiolate clusters. In this review, after a brief summary of the historical milestones of the MT-1/MT-2 research, the recent advances in the structure, chemistry, and biological function of MT-3 and MT-4 are discussed.

Physiological relevance and contribution to metal balance of specific and non-specific Metallothionein isoforms in the garden snail, Cantareus aspersus

Variable environmental availability of metal ions represents a constant challenge for most organisms, so that during evolution, they have optimised physiological and molecular mechanisms to cope with this particular requirement. Metallothioneins (MTs) are proteins that play a major role in metal homeostasis and as a reservoir. The MT gene/protein systems of terrestrial helicid snails are an invaluable model for the study of metal-binding features and MT isoform-specific functionality of these proteins. In the present study, we characterised three paralogous MT isogenes and their expressed products in the escargot (Cantareus aspersus). The metal-dependent transcriptional activation of the three isogenes was assessed using quantitative Real Time PCR. The metal-binding capacities of the three isoforms were studied by characterising the purified native complexes. All the data were analysed in relation to the trace element status of the animals after metal feeding. Two of the three C. aspersus MT (CaMT) isoforms appeared to be metal-specific, (CaCdMT and CaCuMT, for cadmium and copper respectively). A third isoform (CaCd/CuMT) was non-specific, since it was natively recovered as a mixed Cd/Cu complex. A specific role in Cd detoxification for CaCdMT was revealed, with a 80-90% contribution to the Cd balance in snails exposed to this metal. Conclusive data were also obtained for the CaCuMT isoform, which is involved in Cu homeostasis, sharing about 30-50% of the Cu balance of C. aspersus. No apparent metal-related physiological function was found for the third isoform (CaCd/CuMT), so its contribution to the metal balance of the escargot may be, if at all, of only marginal significance, but may enclose a major interest in evolutionary studies.

A Review of Metallothionein Isoforms and their Role in Pathophysiology

The Metallothionein (MT) is a protein which has several interesting biological effects and has been demonstrated increase focus on the role of MT in various biological systems in the past three decades. The studies on the role of MT were limited with few areas like apoptosis and antioxidants in selected organs even fifty years after its discovery. Now acknowledge the exploration of various isoforms of MT such as MT-I, MT-II, MT-III and MT-IV and other isoforms in various biological systems.Strong evidence exists that MT modulates complex diseases and the immune system in the body but the primary function of MT still remains unknown. This review's main objective is to explore the capability to specifically manipulate MT levels in cells and in animals to provide answers regarding how MT could impact those complex disease scenarios.The experimental result mentioned in this review related among MT, zinc, cadmium, diabetic, heart disease, bone retardation, neuro toxicity, kidney dysfunction, cancer, and brain suggest novel method for exploration and contribute significantly to the growing scientist to research further in this field.

Comparative genomics analysis of metallothioneins in twelve Drosophila species

Metallothioneins (MTs) are a superfamily of Cys-rich polypeptides that bind heavy metal ions, both for physiological and detoxification purposes. They are present in all organisms, but their origin is probably polyphyletic, so that MT evolutionary studies are rather scarce. We present a thorough search and analysis of the MT coding sequences in the 12 Drosophila genomes completely sequenced, taking as reference the features reported for D. melanogaster, where four isogenes ( MtnA to MtnD) are known and deeply characterized. We include a fifth isoform in this study, named MtnE, and recently annotated. The MTs polymorphism pattern is essentially the same for the 12 Drosophila species. Invariably, a MtnA form and an MtnB-cluster, comprising the MtnB-to-MtnE forms in tandem array, are observed. The whole set of genes are kept in the same synteny element (Muller E), but implicated in rearrangement events (mainly inversions), encompassing all or some of the isogenes. Gene exon/intron architecture, and cDNA and protein sequences appear highly conserved through Drosophila speciation, concordantly with an essential function for MT isoforms in flies, even for those previously considered as minor products. Data presented here will be comprehensively analyzed to provide a valuable guide for future MT evolutionary, structure and function studies.