Challenging the model for induction of metallothionein gene expression

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich metal-binding proteins found in a wide variety of organisms including bacteria, fungi and all eukaryotic plant and animal species. MTs bind essential and non-essential heavy metals. In mammalian cells MT genes are highly inducible by many heavy metals including Zn, Cd, Hg, and Cu. Aquatic systems are contaminated by different pollutants, including metals, as a result of man's activities. Bivalve molluscs are known to accumulate high concentrations of heavy metals in their tissue and are widely used as bioindicators for pollution in marine and freshwater environments, with MTs frequently used as a valuable marker of metal contamination. We here describe the MT isoform gene expression patterns of marine and freshwater molluscs and fish species after Cd or Zn contamination. Contamination was carried out at a river site polluted by a zinc ore extraction plant or in the laboratory at low, environmentally relevant metal concentrations. A comparison for each species based on the accumulated MT protein levels often shows discrepancies between gene expression and protein level. In addition, several differences observed in the pattern of MT gene expression between mollusc and mammalian species enable us to discuss and challenge a model for the induction of MT gene expression.     

The nucleoside diphosphate kinase activity of DRnm23 is not required for inhibition of differentiation and induction of apoptosis in 32Dcl3 myeloid precursor cells

DRnm23 belongs to a multigene family which includes nm23-H1, the first bona fide metastasis suppressor gene, nm23-H2, nm23-H4, and nm23-H5. Like nm23-H1, nm23-H2, and nm23-H4, DRnm23 possesses nucleoside diphosphate kinase (NDPK) activity. Upon overexpression in myeloid precursor 32Dcl3 cells, DRnm23 inhibits granulocytic differentiation and promotes apoptosis. Two specific mutants of DRnm23 (H134Q and S136P), at residues required for the NDPK activity, inhibit differentiation and promote apoptosis of 32Dcl3 cells. By contrast, substitution of serine 61 with proline (S61P) or deletion of the RGD domain (DeltaRGD) abrogates the effects of wild-type DRnm23. Like wild-type DRnm23, all four mutants show a predominantly mitochondrial subcellular localization. These studies indicate that the enzymatic activity of DRnm23 is not required for the effects observed in 32Dcl3 cells. Moreover, the inability of the S61P and DeltaRGD DRnm23 mutants to inhibit differentiation and promote apoptosis may be due to defective protein-protein interactions at the mitochondria, the predominant site of DRnm23 subcellular localization.     

Differential toll-like receptor 3 (TLR3) expression and apoptotic response to TLR3 agonist in human neuroblastoma cells

Background  

Toll-like receptor-3 (TLR-3) is a critical component of innate immune system against dsRNA viruses and is expressed in the central nervous system. However, it remains unknown whether TLR3 may serve as a therapeutic target in human neuroblastoma (NB).     

Differential gene expression in apoptosis: identification of ribosomal protein S29 as an apoptotic inducer

To identify genes that are specifically involved in apoptosis, poly(A)(+) RNAs were isolated from untreated control rat thymocytes and from adriamycin-induced apoptotic thymocytes. Directionally cloned cDNA libraries were then constructed in UNIZAP-XR vectors followed by biotin-based subtractive hybridization. Three clones were confirmed to be differentially expressed by dot blotting. Sequence analysis revealed homology to two genes previously identified, whereas one clone was novel and did not have homology to any known sequence. One clone was identical to the ribosomal protein S29, and the other was homologous to L8 ribosomal protein. Northern blot analysis revealed a marked increase in the expression of mRNA encoding ribosomal protein S29 in the apoptotic thymocytes compared to the controls. Transfection studies revealed that enhanced S29 expression resulted in increased apoptosis in rat thymocytes and HeLa cells as assessed by various morphological and biochemical characteristics, including cell shrinkage, chromatin condensation, membrane blebbing, formation of apoptotic bodies, TUNEL, FACS, and internucleosomal DNA fragmentation. This was accompanied by upregulation of p53, Caspase 3, and bax, whereas bcl-2 was downregulated as revealed by Western blotting. The current findings provide the first hint of a role for ribosomal protein S29 in the apoptotic process.     

Differential expression of four linked sheep metallothionein genes

Regulation of the closely linked endogenous sheep MT-Ia, MT-Ib, MT-Ic and MT-II genes by heavy metals and dexamethasone was studied in cultured sheep fibroblasts. Only MT-II mRNA was detectable before addition of any inducer. Addition of copper, zinc or cadmium salts to the culture medium increased the level of each mRNA; however, the magnitude of this response varied greatly between the four metallothionein genes. Following induction, levels of MT-Ia mRNA were the highest, followed by MT-II and MT-Ic mRNAs. The MT-Ib mRNA was only present at low levels. Zinc and cadmium were found to be the most effective inducers of each gene. The maximal response of the sMT-Ia, Ib and II genes to copper was only 30% of zinc and cadmium. The sMT-Ic gene responded very weakly to copper, less than 5% of the levels achieved with zinc. Only the MT-II mRNA increased in response to dexamethasone. In the liver of sheep on normal diets, the levels of MT-Ia, Ic and II mRNAs were found to be unexpectedly high and comparable to induced levels in fibroblasts.     

Developmental regulation,induction, and embryonic tissue specificity of sea urchin metallothionein gene expression

Metallothionein (MT) is shown to be present in sea urchin embryos on the basis of its characteristic properties as a small protein (6–7 Da) of extraordinarily high cysteine content, whose biosynthesis is readily induced by heavy metals. Induction by Zn²⁺ results in the accumulation of the cysteine-rich MT protein, a 0.8 kb MT mRNA and a 2.9 kb nuclear RNA. The amount of MT mRNA is regulated intrinsically through the course of embryogenesis to the pluteus stage: A maternal MT mRNA is poly(A)-deficient and is polyadenylated after fertilization. New MT mRNA begins to accumulate between the seventh and eighth cell cleavage, reaches a maximum at the mesenchyme blastula stage, decreases during gastrulation, and rises again in the early pluteus stage. “Animalizing” embryos with Zn²⁺ during early embryogenesis causes a sustained accumulation of MT mRNA to levels greater than 25 times the normal amount. MT mRNA is present in high amount in the ectoderm of the pluteus, but is barely detectable in the mesoderm-endoderm tissue fraction. Treatment of either the pluteus or its isolated tissue fractions with Zn²⁺ results in the induction of MT mRNA accumulation in the mesoderm-endoderm but not in the already MT mRNA-enriched ectoderm. Furthermore, differences in Zn²⁺ induction of the MT gene in the blastula and gastrula are consistent with a developmental pattern in which MT gene expression is maintained constitutively at a high level in the ectoderm and at a low level in the mesoderm-endoderm tissues, which are, however, preferentially inducible by Zn²⁺.     

Coordinate amplification of metallothionein I and II genes in cadmium-resistant Chinese hamster cells: implications for mechanisms regulating metallothionein gene expression.

We describe here the derivation, characterization, and use of clonal cadmium-resistant (Cdr) strains of the Chinese hamster cell line CHO which differ in their metallothionein (MT) induction capacity. By nondenaturing polyacrylamide gel electrophoresis, we showed that the stable Cdr phenotype is correlated with the augmented expression of both isometallothioneins (MTI and MTII). In cells resistant to concentrations of CdCl2 exceeding 20 microM, coordinate amplification of genes encoding both isometallothioneins was demonstrated by using cDNA MT-coding sequence probes and probes specific for 3'-noncoding regions of Chinese hamster MTI and MTII genes. Molecular and in situ hybridization analyses supported close linkage of Chinese hamster MTI and MTII genes, which we have mapped previously to Chinese hamster chromosome 3. This suggests the existence of a functionally related MT gene cluster in this species. Amplified Cdr variants expressing abundant MT and their corresponding Cds parental CHO cells should be useful for future studies directed toward elucidating the mechanisms that regulate expression of the isometallothioneins.     

Quercetin cumulatively enhances copper induction of metallothionein in intestinal cells

Wilson’s disease, a genetic copper-overload condition, is currently treated with zinc because of the ability of zinc to induce metallothionein. We are interested in nonmetal chemicals that may alter intestinal copper metabolism and thus help to alleviate copper toxicity. Previously, we have shown that quercetin, a dietary flavonoid, can chelate copper. This study further examined the interaction of quercetin and copper in intestinal epithelial cells. We found that quercetin enhanced metallothoinein induction by copper and the effect was dose dependent. Quercetin also exerted a cumulative effect after repeated exposure. Repeated low-dose treatment (3–10 μM) of cells with quercetin can lead to the same effect on metallothoinein as one higher concentration treatment (100 μM). This property of quercetin is distinct from its chemical interaction with copper, but both can contribute to a reduction of copper toxicity. Among other flavonoids tested, two other copper chelators, catechin and rutin, did not increase copper induction of metallothionein, whereas genistein, an isoflavone that does not interact with copper chemically, increased copper induction of metallothionein. The effect of quercetin on copper metabolism is unique. Quercetin decreased zinc-stimulated metallothionein expression and had no effect on the cadmium induction of metallothionein. The clinical application of our observation needs to be explored.     

Differential expression of apoptotic markers in jimpy and in Plp overexpressors: evidence for different apoptotic pathways

Point mutations and duplications of proteolipid protein (PLP) gene in mammals cause dysmyelination and oligodendrocyte cell death. The jimpy mouse, which has a lethal Plp point mutation, is the best characterized of the mutants; transgenic mice, which have additional copies of Plp gene, are less characterized. While oligodendrocyte death is a prominent feature in jimpy, the pathways leading to death have not been investigated in jimpy and Plp overexpressors. Using immunohistochemistry and immunobloting, we examined expression of cleaved caspase-3, Poly (ADP-ribose) polymerase (PARP), caspase-12, and mitochondrial apoptotic markers in spinal cord in jimpy males and Plp overexpressors. Compared to controls, cleaved caspase-3 is increased 10× in jimpy white matter spinal cord, and 3× in Plp overexpressor. In jimpy, the number of cleaved caspase-3 cells far exceeds the number of TUNEL⁺ cells. The majority of cleaved caspase-3⁺ cells were not TUNEL⁺ and these cells exhibited staining in perikarya and in processes. Only 30% of the cleaved caspase-3⁺ cells were TUNEL⁺ and exhibited both nuclear and perinuclear staining. This observation suggests that activation of caspase-3 begins earlier and overlaps for a period of time with DNA fragmentation. In both Plp mutants, quantitative immunobloting of PARP showed a 45% increase in total as well as cleaved form, indicating that oligodendrocytes die via apoptosis. Most interestingly, cleavage of caspase-12, a caspase associated with unfolded protein response, is dramatically increased in jimpy but not at all in Plp overexpressors. Mitochondrial markers cytochrome c and Bcl-X_L are upregulated in both Plp mutants but levels of expression are different between mutants, suggesting that apoptosis in these two Plp mutants follows different pathways. In jimpy, mitochondrial apoptotic markers may play a role in amplifying the apoptotic signal. Our data shows for the first time, in vivo, that mutations in Plp gene increase oligodendrocyte death by activating the caspase cascade but the trigger to upregulate this cascade follows different pathways.