MT Protein Research Articles

N-Methyltransferase involved in gramine biosynthesis in barley: Cloning and characterization

The indole alkaloid gramine occurs in leaves of certain barley ( Hordeum vulgare L.) cultivars but not in others. A gene sequence in barley that earlier was characterized as a jasmonate-induced O-methyltransferase ( MT) (EC 2.1.1.6, GenBank accession U54767) was here found to be absent in some barley cultivars and breeding lines that all lacked gramine. The cDNA was cloned and expressed in Escherichia coli and the recombinant protein purified. The purified recombinant protein methylated two substrates in the pathway to gramine: 3-aminomethylindole (AMI) and N-methyl-3-aminomethylindole (MAMI) at a high rate, with Km-values of 77 μM and 184 μM, respectively. In contrast, the protein did not exhibit any detectable methylation with the earlier suggested substrate for O-methylation, caffeic acid. A number of cultivars and breeding lines of barley were analyzed for presence of the U54767 gene sequence and MT protein and the enzyme activity in vitro with MAMI or caffeic acid as substrates. The results showed a clear relationship between the presence of the MT gene, the MT protein and N-methyltransferase activity, and confirmed the identification of the gene as coding for an N-methyltransferase (NMT, EC 2.1.1) and being involved in gramine biosynthesis.

Arsenite and monomethylarsonous acid generate oxidative stress response in human bladder cell culture

Arsenicals have commonly been seen to induce reactive oxygen species (ROS) which can lead to DNA damage and oxidative stress. At low levels, arsenicals still induce the formation of ROS, leading to DNA damage and protein alterations. UROtsa cells, an immortalized human urothelial cell line, were used to study the effects of arsenicals on the human bladder, a site of arsenical bioconcentration and carcinogenesis. Biotransformation of As(III) by UROtsa cells has been shown to produce methylated species, namely monomethylarsonous acid [MMA(III)], which has been shown to be 20 times more cytotoxic. Confocal fluorescence images of UROtsa cells treated with arsenicals and the ROS sensing probe, DCFDA, showed an increase of intracellular ROS within five min after 1 microM and 10 microM As(III) treatments. In contrast, 50 and 500 nM MMA(III) required pretreatment for 30 min before inducing ROS. The increase in ROS was ameliorated by preincubation with either SOD or catalase. An interesting aspect of these ROS detection studies is the noticeable difference between concentrations of As(III) and MMA(III) used, further supporting the increased cytotoxicity of MMA(III), as well as the increased amount of time required for MMA(III) to cause oxidative stress. These arsenical-induced ROS produced oxidative DNA damage as evidenced by an increase in 8-hydroxyl-2'-deoxyguanosine (8-oxo-dG) with either 50 nM or 5 microM MMA(III) exposure. These findings provide support that MMA(III) cause a genotoxic response upon generation of ROS. Both As(III) and MMA(III) were also able to induce Hsp70 and MT protein levels above control, showing that the cells recognize the ROS and respond. As(III) rapidly induces the formation of ROS, possibly through it oxidation to As(V) and further metabolism to MMA(III)/(V). These studies provide evidence for a different mechanism of MMA(III) toxicity, one that MMA(III) first interacts with cellular components before an ROS response is generated, taking longer to produce the effect, but with more substantial harm to the cell.

Induction and activity of oxidative stress-related proteins during waterborne Cu-exposure in brown trout ( Salmo trutta)

Induction of gene transcription for proteins and enzymes involved in metal-mediated oxidative stress were studied in brown trout transferred to a Cu-contaminated river in the Røros region in Central Norway. In addition to metallothionein (MT-A), Cu/Zn-superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) gene transcription, protein levels of MT and enzyme activities of SOD and CAT were analyzed in gill, liver and kidney. MT-A, SOD and GR transcription increased significantly along with uptake of Cu in gills, while only transcription of MT-A was found to respond in liver and kidney during the exposure. Already present MT proteins in gills seemed to be oxidized during the exposure, probably caused by Cu-mediated oxidative stress, and no increase in MT protein levels were observed in gills. SOD and CAT enzyme levels were affected in all tissues during the exposure. A negative correlation between SOD and CAT activities was observed in gills, and we suggest that the activities of these enzymes were influenced not only through transcription. GPx and GR transcription levels correlated positively with each other in gills and liver, indicating their shared function in GSH-turnover. Levels of MT and activity of SOD and CAT dealing with metal-induced oxidative stress appear to be regulated not only through gene transcription, but also through post-translational mechanisms.

Identification and measurement of multimeric forms of metallothionein in mouse tissues utilizing the Ames dwarf model of delayed aging

Metallothionein 1 and 2 (MT) are the most widely distributed and abundant of the four known vertebrate MT isoforms. These small proteins (61–63 amino-acids) are metal- binding thiols (20 cysteine residues) and likely serve as metal detoxifying agents that assist in redox pathways and free radical scavenging. In solutions similar to the internal millieu, monomeric MT readily forms homo- and/or heterodimers and multimers via oxidative cystine bridges and metal-bridged clusters. Therefore, monomeric MT probably does not accumulate intracellularly. Evidence suggests that the long-lived Ames dwarf mouse has an elevated antioxidant defense capability that may lower oxidative stress. We speculate that MT levels might also be elevated in these tissues and here report a two-fold increase of multimeric MT protein in dwarf heart, liver and kidney, compared to controls, using a competitive ELISA assay. The ELISA was modified in order to estimate the percent of the total MT that is oxidized and our data indicate that in control animals it is 10–16% and 0% in dwarfs. In addition, our comparative Western blot data for dwarf and control mice show that in heart, liver and kidney, multimeric forms of MT within the same tissue are similar, but are disparate between different tissues. The apparent absence of oxidized MT in the dwarf tissues may indicate a reduction in the level of oxidative stress in these mice, and the biological importance of these several multimeric forms of MT awaits further study.

Identification and characterization of metallothionein-1 and -2 gene expression in the context of (+/-)3,4-methylenedioxymethamphetamine-induced toxicity to brain dopaminergic neurons.

In mice, the recreational drug (+/-)3,4-methylenedioxymethamphetamine [MDMA ("ecstasy")] produces a selective toxic effect on brain dopamine (DA) neurons. Using cDNA microarray technology in combination with an approach designed to facilitate recognition of relevant changes in gene expression, the present studies sought to identify genes potentially involved in murine MDMA-induced toxicity to DA neurons. Of 15,000 mouse cDNA fragments studied, metallothionein (Mt)-1 and Mt2 emerged as candidate genes possibly involved in MDMA-induced toxicity to DA neurons. Northern blot analysis confirmed the microarray findings and revealed a dynamic upregulation of Mt1 and Mt2 mRNA in the ventral midbrain within 4-12 hr after MDMA treatment. Western blot analysis showed a similar increase in MT protein levels, with peak times occurring subsequent to increases in mRNA levels. Mt1-2 double knock-out mice were more vulnerable to MDMA-induced toxicity to DA neurons than corresponding wild-type mice. Stimulation of endogenous expression of MT protein with zinc acetate conferred complete protection against MDMA-induced toxicity to DA neurons, and administration of exogenous MT protein afforded partial protection. Collectively, these results indicate that MDMA-induced toxicity to DA neurons is associated with increased Mt1 and Mt2 gene transcription and translation, possibly as part of a neuroprotective mechanism. The present findings may have therapeutic implications for neuropathological conditions involving DA neurons.

Induction of metallothionein I by phenolic antioxidants requires metal-activated transcription factor 1 (MTF-1) and zinc.

Phenolic antioxidants, such as tBHQ [2,5-di-(t-butyl)-1,4-hydroquinone], induce Mt1 (metallothionein 1) gene expression and accumulation of MT protein. Induction of Mt1 mRNA does not depend on protein synthesis, and correlates with oxidation-reduction functions of the antioxidants. In the present study, we analysed the biochemical pathway of the induction. Induction depends on the presence of MTF-1 (metal-activated transcription factor 1), a transcription factor that is required for metal-induced transcription of Mt1, but does not require nuclear factor erythroid 2-related factor 2, a tBHQ-activated CNC bZip (cap 'n' collar basic leucine zipper) protein, that is responsible for regulating genes encoding phase II drug-metabolizing enzymes. Moreover, tBHQ induces the expression of MRE-beta Geo, a reporter gene driven by five metal response elements that constitute an optimal MTF-1 binding site. Reconstitution of Mtf1 -null cells with MTF-1 restores induction by both zinc and tBHQ. Unlike activation of phase II genes by tBHQ, induction of Mt1 expression does not occur in the presence of EDTA, when cells are cultured in zinc-depleted medium, or in cells with reduced intracellular 'free' zinc due to overexpression of ZnT1, a zinc-efflux transporter, indicating that induction requires zinc. In addition, fluorescence imaging reveals that tBHQ increases cytoplasmic free zinc concentration by mobilizing intracellular zinc pools. These findings establish that phenolic antioxidants activate Mt1 transcription by a zinc-dependent mechanism, which involves MTF-1 binding to metal regulator elements in the Mt1 gene promoter.

Stable transfection and overexpression of metallothionein isoform 3 inhibits the growth of MCF-7 and Hs578T cells but not that of T-47D or MDA-MB-231 cells.

The third isoform of metallothionein (MT-3) is overexpressed in some breast cancers and its expression is associated with a poor disease outcome. In the PC-3 prostate cancer cell line, MT-3 expression has been shown to inhibit cell growth and increase drug resistance. The goal of the present study was to determine if MT-3 overexpression would influence the growth of human breast cancer cell lines. To determine this, the coding sequence of the MT-3 gene was stably transfected into two estrogen receptor positive (MCF-7 and T-47D) and two estrogen receptor negative cell lines (Hs578T and MDA-MB-231) having no basal expression of MT-3. Cell growth was determined by counting DAPI-stained nuclei, cadmium resistance by the colony formation assay, MT mRNA expression by RT-PCR, and MT protein by immuno-blot. It was demonstrated that MCF-7 and Hs578T cells that overexpress the MT-3 gene were growth inhibited compared to untransfected cells. In contrast, T-47D and MDA-MB-231 cells that overexpress MT-3 were not growth inhibited. Stable transfection of the MT-1E gene had no effect on the growth of any of the four cell lines. It was also demonstrated that the overexpression of both MT-3 and MT-1E only increased the resistance of MCF-7 cells to Cd(+2). In all instances, stable transfection of the MT-3 or MT-1E gene had no effect on the expression of the other MT isoforms. The study shows that MT-3 can influence the growth of some breast cancer cell lines.

Induction of metallothionein by zinc protects from daunorubicin toxicity in rats

Daunorubicin (DNR) is an anthracyline antibiotic used in the treatment of a variety of human cancers. Reactive oxygen species (ROS) produced in the metabolism of DNR have severe cardiotoxicity which consequently compromises its clinical use as anticancer drug. This study aimed to investigate the effect of DNR administration on both cardiac and hepatic tissues, and the possible protective role of zinc on the cardiotoxicity and hepatotoxicity produced by DNR. Administration of 10 or 20 mg/kg DNR to Sprague–Dawley rats, increases serum creatine kinase activity, and blood troponin T levels (as cardiotoxicity indices), alanine aminotransferase activity (as hepatotoxicity index), as well as cardiac and hepatic 2-thiobarbituric acid reactive substances (as an index of lipid peroxidation). Treatment with 20 mg/kg Zn prior to DNR, dramatically induced metallothionein-1 (MT-1) mRNA and MT protein in both heart and liver while DNR alone induced MT, but to a much lower degree than Zn. The analysis of MT protein isoforms revealed that MT-1 was the form induced, while metallothionein-2 (MT-2) levels remained practically unchanged. The increases in both MT protein and MT-1 mRNA ran parallel with the reduction of cardiac and hepatic toxicities. Our results indicate that MT induction by Zn is a highly effective approach in preventing cardiotoxicity and hepatotoxicity caused by DNR. These animal data suggest the use of Zn to reduce DNR-induced cardiotoxicity and hepatotoxicity in the chemotherapy of cancer patients.

Metallothionein isoform 3 expression inhibits cell growth and increases drug resistance of PC-3 prostate cancer cells.

The third isoform of metallothionein (MT-3) is overexpressed in prostate cancers and PIN lesions. The expression of MT-3 is highly variable but appears to correlate to Gleason score. The goal of the present study was to determine the effect of MT-3 overexpression on the growth of the PC-3 prostate cancer cell line. PC-3 cells were stably transfected with either the MT-3 or MT-1E gene. Cell growth was determined by counting DAPI-stained nuclei, drug resistance by the colony formation assay, MT mRNA expression by reverse transcriptase-polymerase chain reaction, and MT protein by immunoblot. PC-3 cells that overexpress the MT-3 gene are growth inhibited compared with either untransfected cells, cells with blank vector, or cells with similar overexpression of the MT-1E gene. Furthermore, increased chemotherapeutic drug resistance occurred in PC-3 clones derived from MT-3- and MT-1E-transfected cells. The overexpression of MT-3 can influence the growth and chemotherapeutic drug resistance of the PC-3 prostate cancer cell line.

The plant S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase is located in both envelope and thylakoid chloroplast membranes.

Chlorophyll biosynthesis requires a metabolic dialog between the chloroplast envelope and thylakoids where biosynthetic activities are localized. Here, we report the first plant S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase (MgP(IX)MT) sequence identified in the Arabidopsis genome owing to its similarity with the Synechocystis sp. MgP(IX)MT gene. After expression in Escherichia coli, the recombinant Arabidopsis thaliana cDNA was shown to encode a protein having MgP(IX)MT activity. The full-length polypeptide exhibits a chloroplast transit peptide that is processed during import into the chloroplast. The mature protein contains two functional regions. The C-terminal part aligns with the Synechocystis full-length protein. The corresponding truncated region binds to Ado-met, as assayed by UV crosslinking, and is shown to harbor the MgP(IX)MT activity. Downstream of the cleaved transit peptide, the 40 N-terminal amino acids of the mature protein are very hydrophobic and enhance the association of the protein with the membrane. In A. thaliana and spinach, the MgP(IX)MT protein has a dual localization in chloroplast envelope membranes as well as in thylakoids. The protein is active in each membrane and has the same apparent size corresponding to the processed mature protein. The protein is very likely a monotopic membrane protein embedded within one leaflet of the membrane as indicated by ionic and alkaline extraction of each membrane. The rationale for a dual localization of the protein in the chloroplast is discussed.

Suppressed oxidant-induced apoptosis in cadmium adapted alveolar epithelial cells and its potential involvement in cadmium carcinogenesis

Apoptosis involves a series of genetically programmed events associated with endonucleolytic cleavage of DNA. This process is triggered by a variety of agents, including oxidants such as hydrogen peroxide (H 2O 2) and it plays a key role in eliminating pre-neoplastic cells from the lung. Failure to do so could favor tumor promotion. The current study demonstrated that alveolar epithelial cells, adapted to cadmium (CdCl 2) by repeated in vitro exposure, exhibit lower levels of H 2O 2-induced apoptosis than similarly challenged non-adapted cells. An immunologic assay, measuring cytoplasmic histone-associated DNA fragments, indicated maximal apoptosis 24 h after exposure to 400 μM H 2O 2. Non-adapted cells showed a 13-fold increase in oxidant-induced apoptosis while Cd-adapted cells had only a 4-fold elevation. A terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method was used to assess the percentage of cells with DNA breaks consistent with apoptosis. Cd-adapted and non-adapted cells that were not exposed to H 2O 2 did not differ in TUNEL positivity. However, after H 2O 2 treatment, the percentage of TUNEL positive cells was 4-fold higher in non-adapted cultures than in adapted ones. Suppression of oxidant-induced apoptosis is due, in part, to up-regulation in the gene expression of several resistance factors including metallothioneins (MT-1 and MT-2), glutathione S-transferases (GST-α and GST-π), and γ-glutamylcysteine synthetase catalytic subunit (γ-GCS). These steady-state mRNA changes, determined by Northern blotting, were accompanied by increased levels of MT and γ-GCS protein, GST activity, and glutathione (GSH). Suppressed oxidant-induced apoptosis, resulting at least in part from these response modifications, could leave pre-neoplastic or neoplastic cells alive, favor clonal expansion, and ultimately lead to cancer development.

Effect of non-toxic mercury, zinc or cadmium pretreatment on the capacity of human monocytes to undergo lipopolysaccharide-induced activation.

1. Metal salts can inhibit cell activity through direct toxicity to critical cellular molecules and structures. On the other hand, they can also change cell behaviour by inducing specific genes (including genes encoding members of the metallothionein [MT] gene family). Therefore, transition metals may affect cell functions either by acting as a toxin, or by transmitting or influencing signals controlling gene expression. 2. To explore the latter possibility, we measured the ability of low, non-toxic metal pretreatment to alter immune cell behaviour. We previously found that pretreatment of human monocytes with zinc induces metallothionein gene expression and alters their capacity to undergo a bacterial lipopolysaccharide-induced respiratory burst. We showed here that cadmium and mercury salts, at concentrations that exert no discernible toxicity, inhibit activation of human monocytic leukemia (THP-1) cells. CdCl2 1 microM, ZnCl2 20-40 microM or HgCl2 2 microM pretreatment for 20 h induced MT-2 mRNA and total MT protein accumulation and had no effect on proliferation potential or metabolic activity, but significantly inhibited the ability of subsequent lipopolysaccharide treatment to induce the oxidative burst, increased adhesion to plastic, and MT-2 and interleukin-1 beta (IL-1 beta) mRNA accumulation. 3. The phenomenon of metal-induced suppression of monocyte activation, at metal concentrations that have no effect on cell viability, has important implications for assessment of acceptable levels of human exposure to cadmium, zinc and mercury.

Arsenic induces and enhances rat hepatic metallothionein production in vivo

Metallothionein genes (MT) are inducible by a variety of agents, including heavy metals. We report the induction of MT expression by arsenite (As 3+) in rat liver in vivo. As 3+ (but not arsenate [As 5+]) injection increased MT protein and MT-1 and MT-2 mRNA accumulation in liver only, but not in kidney or pancreas. In addition, As 3+ enhanced zinc-induced MT protein accumulation in liver without any increase in MT mRNA levels. These data indicate that arsenic may increase MT expression either directly (by inducing MT mRNA accumulation), or indirectly by altering post-transcriptional events. This constitutes an unusual mechanism of enhancement of MT gene expression and appears to be mediated by processes not specifically associated with binding of arsenite to MT in vivo.

Metallothionein gene expression in the intestinal cell: modulation of mRNA and protein synthesis by copper and zinc

Metallothionein is a small protein that binds heavy metals, is transcriptionally regulated by several factors (heavy metals, glucocorticoids) is involved in regulation of heavy metals homeostasis and is also supposed to play a role in cell differentiation (1). The of MT mRNA and protein was studied after induction with zinc and copper in the CaCo2 cells, a human colon carcinoma cell line able to differentiate with time into enterocytes (2). The toxicity of zinc and copper in the CaCo2 cells was assessed and viable concentrations of ZnSOq (400pM ) and CuSO4 (400pM) used. MTmRNA accumulation is shown in fig.1. MTmRNA is well detectable after 2 h of induction with zinc, reaches the maximum of accumulation already at 3 h and is steady until 8 h (lanes 2-8 ) . Differently copper treatment. elicits a response only after 5 h but the accumulation of MTmRNA increases until 9 h (lanes 3-9). MT is detectable in CaCo2 cells after 6 h of induction with zinc and the increases until 18 h (fig.2 lanes 6-18,left). The of MT after copper addition is very litt-le and increases sligtly from 6 until 18 h ( fig.2, lanes 6-18,right). As shown in fig. 3 zinc accumulates earlier and faster than copper into the cells reaching a plateau between 10-14h; copper is transported into the cell more slowly but increases its int r-acellular concent-ration until 14 h (Fig. 3) . Thi' :ifferent time course of MTmRNA aftel zinc and copper induction could then be explained in part by the different accumulation of copperand zinc into the cells. When total protein was measured (CaCo2 cells were treated with copper and zinc, labelled with 10kCi/ml of 35s methionine for lh and the incorporated label counted) we found that 400p.M CuSO4 inhibit to 80% after 10 hours and to 10% after 18 hours the total protein and that 400p.M ZnSOq decreases it to 70% after 10 hours and to 6% after 18 hours . So total protein is affected similarly by zinc and copper whereas the 'I de novo synthesis of MT is very limited only when copper is added to the cells. From these results we could conclude that either MTmRNA is less efficiently translated or MT is more rapidly degraded after addition of copper to the CaCo2 cell.

Heavy metal tolerant transgenic Brassica napus L. and Nicotiana tabacum L. plants

A chimeric gene containing a cloned human metallothionein-II (MT-II) processed gene was introduced into Brassica napus and Nicotiana tabacum cells on a disarmed Ti-plasmid of Agrobacterium tumefaciens. Transformants expressed MT protein as a Mendelian trait and in a constitutive manner. Seeds from self-fertilized transgenic plants were germinated on media containing toxic levels of cadmium and scored for tolerance/ susceptibility to this heavy metal. The growth of root and shoot of transformed seedlings was unaffected by up to 100 μM CdCl2, whereas control seedlings showed severe inhibition of root and shoot growth and chlorosis of leaves. The results of these experiments indicate that agriculturally important plants such as B. napus can be genetically engineered for heavy metal tolerance/sequestration and eventually for partitioning of heavy metals in non-consumed plant tissues.

Regulation of the isoforms of metallothionein

The objective of this research was to study the regulation of hepatic MT-I and MT-II in rats. Induction of MTs following Cd, Zn, and glucocorticoid treatments was studied in adult rats and the ontogeny of MTs was evaluated in developing rats. The isoforms were quantitated by anion-exchange HPLC and their respective mRNAs were evaluated by Northern blot hybridization with mouse cRNA probes. In untreated adult rat liver, MT-II was more abundant than MT-I. Cd treatment increased MT-I and MT-II to similar concentrations. Low dosages of Zn increased MT-II levels three times higher than MT-I levels, whereas higher dosages increased the isoforms to similar levels. Differences in the degradation of Zn-induced MTs were observed: apparent half-lives for MT-I and MT-II were 12.2 and 21.9 h, respectively. MT-II was the predominant glucocorticoid-inducible isoform. MT-I and MT-II mRNAs increased coordinately after glucocorticoid treatment, but MT-II mRNA was more abundant than MT-I mRNA. In immature rats, MT-I and MT-II were first detected in fetal liver on gestation day 18 and increased to maximum hepatic concentrations during the first week postpartum. Thereafter, MT levels decreased, reaching adult levels by 35 d. In contrast, MT mRNAs reached maximal levels during late gestation that were maintained throughout the first three weeks postpartum. Discoordination between MT mRNA and protein levels is suggestive of translational control of MTs in developing rats. In summary, MT-II is more abundant than MT-I in untreated rats and in rats treated with glucocorticoids or low dosages of zinc. In addition to transcriptional regulations of MTs, translational control of MTs may be important in regulating hepatic MT concentrations, particularly in developing rats, and differences in the degradation of MT-I and MT-II may influence tissue concentrations of the isoforms.

Overexpression of metallothionein in CHO cells and its effect on cell killing by ionizing radiation and alkylating agents

Metallothionein protein protects cells from the toxic effects of heavy metal ions. To establish its protective function against ionizing radiation and alkylating agents, a model system was created by transfecting two CHO cell lines (wild-type, K1-2 and X-ray sensitive, xrs-2 subclone Bc11) with the human metallothionein II-A (hMTII-A) gene integrated in a bovine papilloma derived autonomously replicating vector. The isolated transfectants are cadmium-resistant (Cdr), due to the overexpression of the hMTII-A gene. Their steady-state level of hMTII-A mRNA can be increased up to 40-fold after Cd treatment and 20-fold after induction with ionizing radiation. The transfected cell lines proved to be as sensitive as the recipient cell lines to ionizing radiation and bleomycin but the transfectants were significantly more resistant to N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG) and mitomycin C (MMC). These results lead to the conclusion that the MT protein does provide a defence mechanism to protect cells from monofunctional alkylating and cross-linking agents but not from free radicals.

Butyrate selectively activates the metallothionein gene in teratocarcinoma cells and induces hypersensitivity to metal induction.

The expression of metallothionein genes (MT-I and MT-II) was shown to be enhanced within 2 h of addition of 2.5-5 mM sodium butyrate to cultures of teratocarcinoma cells. Both undifferentiated stem cells (F9 and OC15) and differentiated cells (PSA5E and OC15 END) reacted similarly to butyrate by increased accumulation of MT mRNAs. As expected, all of the teratocarcinoma cells that were tested also responded to Zn2+ and Cd2+ by 5- to 10-fold increases in MT mRNA accumulation within 2-24 h of metal addition to the culture media. Surprisingly, MT genes in cells pretreated with butyrate were hypersensitive to metal induction, and this was demonstrated by accumulated transcript levels and by synthesis of MT protein. The maximal metal response was obtained by exposure of cells to butyrate for around 5-8 h together with 10 microM heavy metals. Metal additions to culture media over a range of concentrations and times only induced half the levels of MT mRNA that were achieved by butyrate plus metals. Butyrate enhanced the rate of accumulation of MT mRNA in response to metals, increased the sensitivity of the MT gene to metals, and protected cells from toxic effects of high concentrations of metals. The butyrate and metal ion responses were selective in that no accumulation of c-myc, c-fms, HSP-70, or AFP mRNA was detected. However, c-fos mRNA accumulated in cells exposed to toxic concentrations of metals (50 microM and higher) and this was also potentiated by butyrate treatment. These results suggest that butyrate alters the chromatin conformation of both the MT-I and MT-II genes leading to an accentuated transcriptional response to metals.

Mapping regions of the matrix protein of vesicular stomatitis virus which bind to ribonucleocapsids, liposomes, and monoclonal antibodies

The matrix (M) protein of vesicular stomatitis virus (VSV) appears to function as a bridge between the ribonucleocapsid (RNP) core and the envelope in assembly of the virion. Two such properties would necessitate at least one site for interaction with the nucleocapsid and one with the envelope. In this study M protein was found to mediate the in vitro binding to RNP cores of phospholipid vesicles, representing membrane structures. The M protein could bind initially to either the vesicles or the RNP cores to promote RNP-vesicle association. A trypsin-resistant fragment (MT) of M protein, missing the initial 43 amino acids from its amino terminus, reconstituted with acidic phospholipid vesicles with the same binding efficiency as did whole M protein, suggesting that the carboxy-terminal 81% retained those regions of the M protein which interact with a lipid bilayer. The MT protein, however, was considerably less efficient than intact M protein as an inhibitor of in vitro virus transcription; almost 2.5-fold more MT protein than intact M protein was required for 50% inhibition of VSV transcription, indicating that a site for interaction with the RNP core may have been lost. A monoclonal antibody which is able to reverse the in vitro inhibition of transcription by M protein did not react by immunoblotting with MT protein. Partial tryptic digests of the M protein probed with this monoclonal antibody indicated that epitope 1 lies between amino acid residues 18 and 43. This region appears to be a site that promotes interaction of the M protein with the RNP core of VSV. Monoclonal antibodies to epitopes 2 and 3, which exhibit some overlap in binding to M protein but do not reverse transcription inhibition, were mapped by cleavage with N-chlorosuccinimide at regions in a carboxy direction from epitope 1.

Depletion of O6-methylguanine-DNA-methyltransferase in human fibroblasts increases the mutagenic response to N-methyl-N'-nitro-N-nitrosoguanidine.

We showed previously that resistance of a series of human fibroblast cell lines to the cytotoxic and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is highly correlated with their level of O6-methylguanine-DNA-methyltransferase (MT) activity. In the present study, MT activity in normal fibroblasts was decreased to between 40 and 20% of the constitutive level by 15 or 24 h exposure of the cells to exogenous O6-methylguanine (O6-MeG). MT-depleted and non-depleted populations were then challenged with various doses of MNNG and assayed for cytotoxicity and mutagenicity. At every dose the frequency of 6-thioguanine resistant cells induced by MNNG was higher in the MT-depleted populations than in the controls. Since the MT activity in these cells does not remove methyl from the O4 position of thymine, these results strongly support the hypothesis that O6-methylguanine is the principal mutagenic lesion induced by MNNG. Cells with decreased levels of MT were not significantly more sensitive to the cytotoxic effect of MNNG. If O6-methylguanine is a potentially cytotoxic lesion, this lack of increased sensitivity may reflect the fact that regeneration of MT protein occurred rapidly enough to remove these lesions before they resulted in cell death (i.e., inability to form a clone). Consistent with this explanation is the fact that 7 h after removal of the exogenous O6-MeG, the level of MT activity had regenerated to 51% of normal; by 18 h, it was 65% of normal.