Metallothionein mRNA Induction Research Articles

Induction of metallothionein by superantigenic bacterial exotoxin: probable involvement of the immune system

Metallothionein (MT) can be induced in mouse liver by a bacterial exotoxin, toxic shock syndrome toxin-1 (TSST-1). Hepatic MT was induced by TSST-1 in a dose-dependent manner from 100 μg/kg through 3 mg/kg in CF-1 mice, and by 6 h the induction was almost maximal. The increase of hepatic MT occurred at the mRNA level, also, and both MT-I and II mRNAs increased coordinately. Because TSST-1 is a superantigen, it was investigated whether TSST-1 induces MT through cytokines as a consequences of immunostimulation. In low-cytokine-producing mice (C3H/HeJ), up to a dose of 1 mg/kg of TSST-1, there was only 2- to 3-fold increase of hepatic MT. In contrast, in normal-cytokine-producing mice (C3Heb/FeJ), TSST-1 increased MT in a dose-dependent manner, and at a dose of 1 mg/kg, there was a 25-fold increase in hepatic MT. This suggests that activation of the immune system is probably involved in the induction of MT by TSST-1. Studies on the role of specific hepatic cytokines (IL-1, TNF-α, and IL-6) in TSST-mediated hepatic MT induction showed that TSST-1 did not increase hepatic IL-1 or TNF-α significantly over controls in any of the mouse strains studied. In contrast, TSST-1 induced hepatic IL-6 in all three strains of mice. However, in CF-1 and C3Heb/FeJ mice (normal-cytokine-producing) IL-6 induction preceded MT mRNA induction, but in C3H/HeJ mice (low-cytokine-producing), IL-6 induction did not precede MT and mRNA induction.

Cloning and nucleotide sequence of a complementary DNA encoding Xenopus laevis metallothionein: mRNA accumulation in response to heavy metals.

A cDNA encoding Xenopus laevis metallothionein (MT) was cloned from a cDNA library constructed using liver poly(A+)RNA of X. laevis adult males treated with CdCl2. The probe used to screen the library was a MT-specific DNA fragment obtained by means of the polymerase chain reaction (PCR) and degenerate oligodeoxynucleotide primers. The cDNA clone encodes a putative protein of 62 amino acids, of which 20 are cysteine residues. The position of all the cysteine residues is conserved with respect to mammalian MT sequences. The amino acid sequence of this X. laevis MT, designated XIMT-A, shares between 60% and 67% identity with various vertebrate MTs. Overall, the structure of XIMT-A is no similar in sequence to MT-1 than it is to MT-2 isoforms of various vertebrates. Ten different X. laevis MT cDNA isolates were partially sequenced and turned out to be identical, suggesting a single species of MT mRNA. Southern blot analysis of X. laevis DNA reveals that the XlMT-A gene is present in at least two copies. This result is consistent with the suggestion that a genome duplication occurred in a X. laevis ancestor. The in vivo response to increasing doses of Cd2+, Zn2+, and Cu2+ metal salts was tested. In the liver, all three metals proved to be potent inducers, raising MT mRNA levels between 50- and 100-fold. The maximum response to Cd2+ was at 12 hr after injection and to Zn2+ at 24 hr after injection. High levels of mRNA were maintained for more than 48 hr. Cd2+ and Zn2+ induced XlMT-A mRNA in all tissues examined (kidney, spleen, heart, intestine, testes, and brain). Dexamethasone did not induce MT mRNA synthesis in the liver.

Induction of metallothionein mRNA in the blue crab ( Callinectes sapidus) after treatment with cadmium

1. Dietary treatment of blue crabs with cadmium led to an increase of cadmium metallothionein in the hepatopancreas. 2. Utilizing a unique purification procedure, poly A + RNA was isolated from the hepatopancreas of control and cadmium-treated blue crabs with 260/280 nm ratios of 2.0 or better. 3. Poly A + RNA was hybridized with a series of oligonucleotide probes derived from a portion of the amino acid sequence of lobster metallothionein. A single band of approx. 300 base pairs was present in control and cadmium-treated RNA from the hepatopancreas of blue crab. 4. A four-fold induction of the band occurred following cadmium exposure.

Phorbol ester induction of rat hepatic metallothionein in vivo and in vitro

1. 1. The induction of metallothionein (MT) protein by TPA ( O-tetradecanoyl phorbol acetate), a protein kinase C activator, was demonstrated in vivo in rat liver and in vitro in rat hepatocytes in primary culture. In vivo half maximal induction at 25 hr was seen at 26 nmol TPA/kg body wt. Five- to seven-fold inductions were seen in vivo. De novo protein synthesis was required for this induction as demonstrated by cycloheximide inhibition of [ 35S]cysteine incorporation into MT protein. 2. 2. TPA induction of MT protein in primary cultures of rat hepatocytes reached levels of 2.6–4.1-fold, as assessed by [ 35S]cysteine incorporation, 1.34–2.20-fold, as assessed by 109Cd binding in a metal displacement/HPLC assay, and 2.5–5-fold, as assessed by 109Cd binding in a metal displacement/Sephadex G-75 Superfine assay. 3. 3. The induction of MT mRNA by TPA was demonstrated in vivo in rat liver and in vitro in 2 rat hepatoma cell lines, EC3 and 2M. MT mRNA was quantitated using dot blot and Northern gel assays. In vivo TPA induced hepatic MT mRNA 2.36–5.88-fold (dot blot) and 7.4–22-fold (Northern gels). In vitro TPA induced MT mRNA 1.71–15.26-fold in EC3 cells and 2.23–8.43-fold in 2M cells. MT mRNA was 0.54kb, and α-tubulin mRNA was 1.62kb in size on Northern gels. 4. 4. TPA induction of MT protein and mRNA in vivo and in vitro is rapid and persistent and occurs at low concentrations. The 2 rat hepatoma cell lines provide a useful system in which to study MT induction in vitro without confounding secondary effects which can occur in vivo.

Purinergic agonist induction of metallothionein.

Metallothionein (MT) protein is readily induced in vivo in rat liver by adenosine and adenosine agonists (2-chloroadenosine, 5-(N-ethyl) carboxamido adenosine, and 5-chloro-5-deoxyadenosine). These presumably operate via AMP/adenosine receptors of the P1 (A2) type, which use the cAMP pathway. ATP was ineffective as an inducer for MT. 2-Chloroadenosine was the most effective inducer (7.27-fold at 11 hr). This induction was blockable by the adenosine antagonists, caffeine and theophylline. MT protein induction by 2-chloroadenosine in primary cultured rat hepatocytes was modest (1.55-fold), but this was also blocked by theophylline. MT mRNA induction was assessed using dot blot and Northern gel assays. Large inductions by 2-chloroadenosine (5.1- to 41-fold) were seen, and these were detectable as early as 2 hr in vivo. Two rat hepatoma cell lines (EC3 and 2M) were studied in vitro. Modest inductions of MT mRNA were seen: 2.10-fold for EC3 and 4.12-fold for 2M. Our studies implicate the potential role of the purinergic system in the modulation of transcription of MT genes in rat liver. The sources of adenosine in vivo that might cause induction of MT mRNA and protein are not well defined, but adenosine may be important as a signal in stress response situations involving tissue damage, such as ischemia, hypoxia, and hemorrhagic shock.

Interleukin 1 regulates secretion of zinc-thymulin by human thymic epithelial cells and its action on T-lymphocyte proliferation and nuclear protein kinase C.

Thymic epithelial cells (TEC) are known to secrete thymic hormones that influence maturation of T lymphocytes. One of these peptides, thymulin, requires zinc in an equimolar ratio for biological activity. A previous study [Cousins, R. J. & Leinart, A. S. (1988) FASEB J. 2, 2884-2890] showed that interleukin 1 (IL-1) in vivo stimulates zinc uptake by the thymus. Both the alpha and beta forms of IL-1, which stimulate proliferation of human TEC, also stimulate their uptake of zinc in vitro, and this latter stimulation is both dependent and independent of proliferation. Zinc induces zinc accumulation without proliferation. Two other stimulants of proliferation, bovine pituitary extract and epidermal growth factor, stimulate zinc uptake by TEC, but only in a manner dependent on proliferation. Utilizing in situ hybridization, we show that the IL-1 alpha and beta forms and zinc induce metallothionein mRNA expression TEC. Metallothionein is thought to be involved in the transfer of zinc to thymulin. IL-1 was shown to stimulate the secretion of thymulin as measured both by its ability to stimulate induction of IL-2 receptor-positive lymphocytes from human peripheral blood lymphocytes and by the azathioprine-sensitive rosette assay. In addition, the zinc-thymulin complex in the presence, but not absence, of IL-1 stimulates nuclear protein kinase C in isolated lymphocyte nuclei. IL-1 apparently regulates the synthesis or secretion and delivery of zinc-thymulin complex to the T-lymphocyte system.

Metallothionein induction in cultured fibroblasts and liver of a marine flatfish, the turbot,Scophthalmus maximus

Intraperitoneal injection of turbot with Cd induced the synthesis of a low molecular weight hepatic Cd-binding protein and a 500bp mRNA, which hybridised to a plaice metallothionein (MT) cRNA probe. The Cd-binding protein displayed cross-reactivity in a competitive ELISA with antiserum raised against rainbow trout MT and had the characteristic amino acid composition, metal stoichiometry and spectral characteristics of a Class I MT. Only one isoform was apparent on ion exchange chromatography. Southern blot analysis of DNA cleaved with four restriction enzymes suggested that only a single MT gene is present in turbot.In an established turbot fibroblast cell line, Cd induced MT mRNA and MT levels in a dose and time-dependent manner. MT was also induced by Cu, Hg and Zn but not Pb exposure. Physiological concentrations of glucocorticoids and sex hormones did not induce MT synthesis, although at high concentrations a positive response to corticosterone, dexamethasone, hydrocortisone or progesterone was observedin vitro indicating the possible presence of a functional steroid regulatory element in the fish MT gene.

Induction of zinc metallothionein by calcium ionophore in vivo and in vitro

The calcium ionophore, A23187, can induce rat hepatic metallothionein (MT) when administered in vivo (5.8-fold, 5.0 μM, 11 h) and rat hepatocyte MT when administered in vitro (10.70-fold, 1.0 μM, 24 h). Several rat hepatoma cell lines (2M, 4.55-fold; JM2, 12.29-fold; EC3, 14.12-fold; HTC, 7.99-fold) and a normal rat liver cell line (Clone 9, 39.67-fold) were tested for their inducibility of MT mRNA by Cd 2+ (10 μM, 8 h). Quantitatively, JM2 and 2M made the most MT mRNA, while HTC made the least. A23187 (0.1–7.0 μM) was studied as an inducer of MT mRNA in these cell lines (except for HTC) and in HeLa. A variety of responses and tolerances were seen with inductions ranging up to 32.11-fold. Quantitatively, the best responding cell lines were EC3 and 2M. A combination induction experiment, using TPA, a protein kinase C activator, and A23187 in EC3 cells revealed an additive effect of the two inducers on MT mRNA levels: TPA (10 nM), 11.71-fold; A23187 (3.0μM), 6.71-fold; and TPA + A23187, 20.00-fold. These studies have implicated perturbations in cytosolic calcium ion concentrations, caused by the ionophore A23187, as being involved in the complicated signaling systems which can lead to induction of MT mRNA and protein.

Experimental basis for increasing the therapeutic index ofcis-diamminedicarboxylatocyclobutaneplatinum(II) in brain tumor therapy by a high-zinc diet

Metallothionein (MT), a ubiquitous intracellular protein, confers resistance to the toxic effects of platinum compounds. Since a high-zinc diet has been shown to induce MT synthesis in extracerebral tissues but not in brain, we investigated whether it could provide an experimental basis for decreasing the hematotoxicity of carboplatin without impairing its activity against brain tumors. After 2 weeks on either a high-zinc diet or a control diet (zinc content, 180 vs 10 ppm), mice and rats received various doses of carboplatin or Hanks' balanced salt solution by i.p. injection. The hematotoxicity of carboplatin was evaluated with an assay of colony-forming units of granulocytes and mononuclear cells in mice. The high-zinc diet enabled a 50% increase in the carboplatin dose without increasing hematotoxicity. The antitumor activity was evaluated with an assay of the colony-forming efficiency of gliosarcoma cells from 9L brain tumors in rats. The high-zinc diet did not alter the efficacy of carboplatin against this brain tumor. Northern blot analysis confirmed that the high-zinc diet induced MT mRNA in the kidney but not in the brain of mice and rats; it also showed MT mRNA induction in bone marrow cells of mice but not in rat 9L brain tumors. These results suggest that increasing the dietary intake of zinc might increase the therapeutic index of carboplatin in the treatment of brain tumors.

Triiodothyronine-induced accumulations of malic enzyme, fatty acid synthase, acetyl-coenzyme A carboxylase, and their mRNAs are blocked by protein kinase inhibitors. Transcription is the affected step.

Addition of triiodothyronine (T3) to chick-embryo hepatocytes in culture causes increased accumulations of malic enzyme, fatty acid synthase, acetyl-CoA carboxylase and their mRNAs. H-8 and other protein kinase inhibitors inhibited the T3-induced accumulations of these lipogenic enzymes and their mRNAs but had no effect on the activities of 6-phosphogluconate dehydrogenase and isocitrate dehydrogenase, enzymes not induced by T3 in chick-embryo hepatocytes. H-8 also had no effect on the activities of malic enzyme, fatty acid synthase, and acetyl-CoA carboxylase in hepatocytes not treated with T3. Synthesis of soluble protein, levels of mRNAs for beta-actin and glyceraldehyde-3-phosphate dehydrogenase, and induction of metallothionein mRNA by Zn2+ were unaffected by H-8 at concentrations that inhibited the T3-induced accumulation of lipogenic enzymes and their mRNAs. H-8 inhibited T3-induced transcription of the genes for both malic enzyme and fatty acid synthase but had little effect on transcription of the beta-actin or glyceraldehyde-3-phosphate dehydrogenase genes or on total RNA synthesis in isolated nuclei. H-8 also had no effect on binding of T3 to its nuclear receptor. In isolated nuclei, H-8 inhibited phosphorylation of total protein by 15-20%. Phosphorylation of only one major protein was consistently and substantially inhibited, indicating that the effect of H-8 was selective. These results suggest that on-going protein phosphorylation is required specifically for stimulation of transcription of the lipogenic genes by T3.

Cadmium teratogenicity and its relationship with metallothionein gene expression in midgestation mouse embryos

As an approach toward understanding the mechanisms by which cadmium (Cd) exerts its teratogenic effects, the expression and metal regulation of the metallothionein (MT) genes in midgestation mouse embryos were studied by Northern blot and in situ hybridization. Maternal injection of a teratogenic dosage of Cd (50 μmol Cd/kg body wt) did not induce MT mRNA in day 10 (D10) CD-1 mouse embryos, whereas zinc (Zn) (50 μmol/kg) was an effective inducer. In contrast, Cd was about 10-fold more potent than Zn at rapidly inducing MT mRNA in D10 embryos incubated in vitro in medium containing micromolar concentrations of these metals. This suggests that following maternal injection, Cd but not Zn is prevented from reaching the D10 embryo and establishes that the embryonic MT genes are not refractory to metal induction, which might have explained the sensitivity of the embryo to Cd. MT mRNA was detected at high levels only in the extraembryonic membranes of D9 embryos exposed to Cd in vivo. On days 9 and 10, no embryonic cell types contained detectable levels of MT mRNA. This mRNA was detected first at low levels in hepatocytes on D11, soon after formation of liver and these levels increased dramatically by D12. Therefore, Cd teratogenicity was not associated with high levels of cell type-specific expression of the MT genes in Cd-sensitive regions of the embryo (neural tube, limb bud), that might have served to target Cd to these cells. Taken together, the results of this study suggest that Cd teratogenicity reflects damage to maternal or extraembryonic tissues. However, the results cannot exclude the possibility that certain cells in the embryo are exceptionally sensitive to low levels of Cd.

Endotoxin induction of murine metallothionein gene expression.

Bacterial endotoxin-lipopolysaccharide (LPS) rapidly induced hepatic metallothionein (MT) mRNA levels in the LPS-sensitive CD-1 strain of mice. This LPS effect was severely attenuated in the LPS-resistant C3H/HeJ strain of mice, but could be mimicked by injection of human recombinant interleukin-1 alpha (IL-1 alpha) or human recombinant tumor necrosis factor (TNF-alpha). In the CD-1 strain, LPS induction of MT gene expression occurred in each of 10 organs examined (liver, kidney, pancreas, intestine, lung, heart, brain, ovary, uterus, and spleen). Solution hybridization with probes specific for MT-I or MT-II mRNA established that these genes were co-induced in each of the organs and that the liver and kidney contained the highest absolute levels of these mRNAs, whereas in the intestine and spleen they were 10-20-fold lower. LPS and cytokine induction of hepatic MT gene expression occurred in hypophysectomized mice, which suggests a lack of significant involvement of glucocorticoids. Several recombinant cytokines (TNF-alpha, IL-1 alpha, IL-1 beta, IL-6, interferon-gamma (IFN-gamma), as well as poly(rI.rC) were effective inducers of hepatic MT-I and MT-II genes. As an attempt to determine which of these cytokines may mediate LPS effects on MT gene expression in vivo, CD-1 mice were injected with LPS or various cytokines, and RNA from liver, ovary, and uterus was extracted at various times postinjection and analyzed by Northern blotting using probes specific for IL-1 alpha, IL-1 beta, TNF-alpha, IL-6, and MT mRNA. In each organ examined, LPS, IL-1 alpha, or IL-1 beta injection caused a rapid, coordinate, transient increase in the levels of each of the cytokine mRNAs which peaked by 1 h and declined to low levels by 4 h. In contrast, levels of MT mRNA did not reach a peak until 4-6 h postinjection. TNF-alpha had minimal effects on expression of cytokine and MT genes in organs other than liver. IL-6 had no effect on hepatic cytokine mRNA levels, and induced MT mRNA only in the liver which suggests a direct effect of IL-6 on hepatic MT gene expression. These data suggest that the acute effects of LPS on MT gene expression may include complex paracrine interactions between a variety of cytokines and the cells expressing MT genes in each organ, and tissue-specific cytokine effects on the MT genes.

Differential regulation of metallothionein genes in rainbow trout fibroblasts, RTG-2

Exposure of a trout gonadal fibroblast (RTG-2) cell line to ZnCl 2, CdCl 2 and CuCl 2 resulted in differential levels of accumulation of metallothionein (MT) mRNA. ZnCl 2 being the most effective agent induced MT mRNA in 3 h, with 172-fold induction after 48 h and continued accumulation up to 144 h. Following CdCl 2 treatment, mRNA could be detected after 24 h, reaching peak levels at 72 h. Furthermore, trout MT mRNA could be detected up to 8 days after withdrawal of extraneous ZnCl 2. Using a novel technique of primer extension and DNA sequencing with total RNA as template, specificity of the trout MTa and MTb gene-specific primers was established. Primer extension studies revealed a higher response of MTa to ZnCl 2 and CdCl 2 compared to MTb. Insensitivity of MT mRNA induction to cycloheximide suggested that induction by the metals was independent of de novo protein synthesis. However, simultaneous exposure of cells to actinomycin D and metals completely inhibited MT mRNA synthesis implying control at the transcriptional level.

Metallothionein gene expression and resistance to cisplatin in human ovarian cancer

Intracellular thiols have been proposed as mediators of resistance to alkylating agents and cisplatin. As metallothionein is the predominant protein thiol, we examined its relationship to cisplatin resistance in human ovarian cancer cell lines. A human ovarian carcinoma cell line, A2780, derived from an untreated patient, was treated with cisplatin in several ways and the induced resistance to cisplatin ranged from 13- to 68-fold. The degree of resistance was dependent upon the method of selection. The drug-resistant cell lines also developed low levels of cross-resistance to cadmium. Additional cell lines established from untreated patients or ovarian cancer patients refractory to cisplatin- and/or carboplatin-containing combination chemotherapy were studied. The most cisplatin-resistant cell lines, OVCAR-8 and -10, were from patients previously treated with intensive chemotherapy. OVCAR-8 was relatively cross-resistant to cadmium while OVCAR-10 appeared relatively sensitive. Cell lines were examined for expression of metallothionein mRNA to evaluate the relationship between cisplatin resistance, cadmium cross-resistance and metallothionein expression. Only two of the cell lines with in vitro-induced resistance to cisplatin, 2780E80 and 2780CP70B3, had detectable metallothionein mRNA. The other cell lines selected in vitro for cisplatin resistance, as well as the parental A2780 ovarian cancer cell line, showed no expression at our level of detection. There was variable expression of metallothionein among the OVCAR cell lines. Cell lines from untreated patients, OVCAR-5 and -7, did express metallothionein, while the most cisplatin-resistant cell lines, OVCAR-8 and -10, did not. We also examined cisplatin induction of metallothionein mRNA in the cell lines. Only 2780CP70B3 among the cell lines with in vitro-induced cisplatin resistance showed increased expression after short-term exposure to cisplatin. OVCAR-4 also had a slight increase in expression after exposure to cisplatin. Mouse C127 cells transfected with a bovine papilloma virus-metallothionein gene construct were compared for cisplatin sensitivity to the same cell type transfected with bovine papilloma virus alone. In this model system, metallothionein expression did not influence cisplatin cytotoxicity. On the basis of these studies, we conclude that there is no causal relationship between metallothionein expression and cisplatin resistance.

Molecular cloning of metallothionein cDNA and analysis of metallothionein gene expression in winter flounder tissues

Investigations into the precise role played by metallothionein (MT) in heavy-metal metabolism have been hampered by difficulties in positively identifying and quantifying MT in fish tissues. This study describes the development of an antisense MT RNA (cRNA) probe that will enable MT mRNA levels to be measured with a high degree of specificity and precision. Cadmium chloride administration induces the producton of MT mRNA in the liver and kidney of winter flounder (Pseudopleuronectes americanus). Poly(A)+ RNA purified from liver samples of winter flounder after cadmium chloride injections was used to construct a cDNA library. Several recombinant clones made complementary to MT mRNA were selected from this cDNA library by an oligonucleotide derived from the N-terminal amino acid sequence of winter flounder metallothionein. Sequence analysis of two of the cDNA inserts gave the structure of the entire 3′ untranslated region, a coding region corresponding to winter flounder MT and 49 nucleotides of the 5′ untranslated region. One of the flounder MT cDNAs, pWFMTC4, was subcloned into a RNA probe plasmid and transcribed to produce antisense MT RNA (cRNA). The MT cRNA was then used to detect the induction of MT mRNA production in the liver of winter flounder, following the administration of Cu2+, Zn2+, Cd2+, Pb2+, and Hg2+. The time required for the induction of hepatic MT mRNA by a single injection of Cd2+ was approximately 96 h. Dexamethasone did not induce an increase of MT mRNA in any of the winter flounder tissues examined (liver, kidney, heart, brain, intestinal scrape, and gill filament), whereas Cd2+ induced MT mRNA in all of the tissues except brain, where the constitutive level of expression was high.

Metallothionein mRNA and protein induction by cadmium in peripheral-blood leucocytes.

To help to evaluate the role of metallothionein (MT) in peripheral-blood leucocytes, we examined MT protein and mRNA levels in these cells before and after exposure to CdCl2 in culture. Protein was assayed by 109Cd2+ binding, and RNA by dot-blot hybridization. MT was induced in both lymphocytes and adherent monocytes about 10-fold with a 12 h exposure to 10 microM-CdCl2, but absolute levels were 3-fold higher in monocytes: 57 x 10(5) (+Cd2+) versus 6 x 10(5) (-Cd2+) molecules/cell for monocytes; 18 x 10(5) (+Cd2+) versus 2 x 10(5) (-Cd2+) for lymphocytes. Polymorphonuclear cells expressed relatively little MT (0.6 x 10(5) molecules/cell), and this did not change with phorbol ester stimulation or exposure to Cd2+, arguing against a direct protective role for MT in activated neutrophils. MT mRNA levels corresponded qualitatively to expression of protein in these cells. Our data provide quantitative comparisons of leucocyte MT expression and regulation in the human population. Variation in both basal and induced MT mRNA levels reflects environmental or experimental (intra-individual) and possibly genetic (inter-individual) differences.

Reduction of EGF receptor levels in human tumor cells transfected with an antisense RNA expression vector

An expression vector was constructed from part of pSV2neo with the 3'-ClaI fragment of the epidermal growth factor (EGF) receptor cDNA inserted in an inverted orientation downstream from the human metallothionein (MT) IIa promoter. The human squamous carcinoma cell line NA, which overproduces EGF receptor, was transfected with this vector and selected for resistance to the neomycin derivative G418. One of the stable transfectants had a 90% reduction in cell-surface EGF receptor in response to ZnSO4. The nascent EGF receptor peptide was also decreased with concurrent induction of MT mRNA. These data suggest that the antisense transcript regulated by the MT promoter inhibits the expression of the endogenous EGF receptor genes. Although no transcripts from the antisense gene were detected, the results indicate that transfection with the antisense vector provides a technique by which to modulate the number of EGF receptors on the cell surface of squamous cell carcinomas.

Induction of metallothionein mRNA by tumor promoters in mouse skin and its constitutive expression in papillomas

A single topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) was found to induce mRNA of a metallothionein (MT) gene or genes in the skin of Sencar mice, and papillomas produced by repeated applications of TPA were shown to have elevated levels of MT mRNA. Induction of MT mRNA was maximal 4-8 h after application of TPA and returned to the control level 24 h later. A dose-dependent increase of MT mRNA was observed with doses of TPA of 1-5 micrograms. Of the other promoters tested, phorbol-12, 13-didecanoate, mezerein, and the ionophore A23187 also induced MT mRNA, but 4-O-methyl-TPA and benzoyl peroxide did not. Phorbol and 4 alpha-phorbol-12,13-didecanoate, which are not promoters, also did not induce MT mRNA. Retinoic acid and 1 alpha, 25-dihydroxyvitamin D3, inhibitors of tumor promotion, did not induce MT mRNA themselves or inhibit the induction of MT mRNA by TPA. In C57BL/6 promotion-resistant mice, TPA caused only slight induction of MT mRNA. These data suggest a correlation between induction of MT mRNA and epidermal hyperplasia. The constitutive elevation of MT mRNA levels in papillomas may be due to the loss, during the process of tumor promotion, of some mechanism regulating MT gene expression.

エンドトキシンによる培養肝細胞内メタロチオネインmRNA発現に関する実験的研究

エンドトキシンによる培養肝細胞内メタロチオネインmRNA発現に関する実験的研究

Fish metallothioneins: Molecular cloning studies and induction in cultured cells

A cDNA for plaice metallothionein (MT) was cloned and the sequence for the first 45 amino acid residues was deduced. The sequence of plaice MT was exactly homologous to that of winter flounder MT. Studies of MT induction by Cd and Zn in a rainbow trout (RTH) cell line showed that both metals were strong inducers of the protein in contrast to previous reports which have shown little induction of MT mRNA by Cd. Translational control or differential mRNA and MT stabilities are therefore inferred.