metallothionein and Lymphoma--Non-Hodgkin

Cadmium, a ubiquitous heavy metal, is a toxic industrial and environmental pollutant. The initial biological response to cadmium exposure is induction of metallothioneins (MTs), a family of cysteine-rich, low-molecular-weight proteins that bind primarily zinc, cadmium, or both. This MT induction protects against cadmium toxicity by quenching cadmium. However, the effects of long-term cadmium exposure on MT1 gene expression are largely unknown. To investigate these effects, we used P1798 mouse lymphosarcoma cells, in which the MT1 gene is suppressed. As previously reported, MT1 expression remained unchanged after cadmium treatment. However, MT1 induction was observed in cells treated with 0.1 µM cadmium for 7 days, then exposed to 10 µM cadmium for 3 hr. In cells treated with 0.1 µM cadmium for 7 days, the transfected MT1 promoter reporter gene transcription and the cadmium incorporation in response to 10 µM cadmium induction were similar to those in untreated P1798 cells. Bisulfite genomic sequencing revealed that 7 day treatment with 0.1 µM cadmium slightly decreased CpG methylation in the 5´ flanking region of the MT1 gene. Our results together show that cadmium treatment results in MT1 induction and epigenetic modification of the MT1 gene.

Topics: 5' Flanking Region; Animals; Cadmium; Dose-Response Relationship, Drug; Epigenesis, Genetic; Gene Expression; Lymphoma, Non-Hodgkin; Metallothionein; Methylation; Mice; Promoter Regions, Genetic; Protein Binding; Time Factors; Transcription, Genetic; Tumor Cells, Cultured

Dnmt3a and Dnmt3b are de novo DNA methyltransferases that also act as transcriptional repressors independent of methyltransferase activity. To elucidate the underlying mechanism of transcriptional repression, Dnmt3a was purified from mouse lymphosarcoma cells (P1798) by extensive fractionation on five different chromatographic matrices followed by glycerol density gradient centrifugation. Liquid chromatography electrospray tandem mass spectrometry analysis of Dnmt3a-associated polypeptides identified the methyl CpG binding protein Mbd3, histone deacetylase 1(Hdac1), and components of Brg1 complex (Brg1, Baf155, and Baf57) in the purified preparation. Association of Dnmt3a with Mbd3 and Brg1 was confirmed by coimmunoprecipitation and coimmunolocalization studies. Glutathione S-transferase pulldown assay showed that the NH2-terminal ATRX homology domain of Dnmt3a interacts with the methyl CpG binding domain of Mbd3 and with both bromo and ATPase domains of Brg1. Chromatin immunoprecipitation assay revealed that all three proteins are associated with transcriptionally silent methylated metallothionein (MT-I) promoter in the mouse lymphosarcoma cells. To understand the functional significance of their association with the promoter, their role on the MT-I promoter activity was analyzed by transient transfection assay. The results showed that Mbd3 and Dnmt3a specifically inhibited the methylated promoter, and the catalytic activity of Dnmt3a was dispensable for the suppression. In contrast, the wild-type but not the ATPase-inactive mutant of Brg1 suppressed MT-I promoter irrespective of its methylation status, implicating involvement of ATP-dependent chromatin remodeling in the process. Coexpression of two of the three interacting proteins at a time augmented their repressor function. This study shows physical and functional interaction of Dnmt3a with components of nucleosome remodeling machinery.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Nucleus; DNA (Cytosine-5-)-Methyltransferases; DNA Helicases; DNA Methyltransferase 3A; DNA-Binding Proteins; Humans; Immunoprecipitation; Liver Neoplasms; Lymphoma, Non-Hodgkin; Metallothionein; Mice; Nuclear Proteins; Promoter Regions, Genetic; Protein Structure, Tertiary; Transcription Factors; Transfection; X-linked Nuclear Protein

Metallothionein (MT) promoter was methylated in rat hepatoma and in mouse lymphosarcoma cells by methylation of cytosine within the CpG dinucleotide region. After demethylation of MT-I promoter in mouse lymphosarcoma cells or in the transplanted rat hepatoma with 5-azacytidine, a potent inhibitor of DNA methyltransferase, the promoter was activated in response to heavy metal treatment. MT-I promoter was also suppressed in human prostate cancer lines PC3 and DU145, probably by promoter methylation, whereas cadmium induced MT-I in the human prostate cancer line LNCaP. In the prostate cancer lines where MT-I was suppressed, glutathione-S-transferase-pi (GST-pi) was expressed. On the contrary, GST-pi gene was repressed in the cell line where MT-I was induced, which suggests an inverse relationship between MT-I induction and GST-pi expression in some prostate cancer lines. The expressions of GST-pi and gamma-glutamyl cysteine synthase were also significantly higher (5- to 12-fold) in the lymphosarcoma cells and the hepatoma relative to the parental tissues. The higher expressions of these two genes suggest a compensatory mechanism in the cells where the gene for the antioxidant MT-I/II is not induced. MT-I/II may function as a growth suppressor either alone or in concert with other factor(s), and consequently their lack of expression could facilitate the tumor growth. In addition to suppression of MT-I/II expression by promoter methylation, the lack of MT induction could also be brought about by nuclear factor I (NFI), probably by interaction with the metal transcription factor MTF-1. An inverse relationship was observed between the level of NFI and MT-I expression in some cells, which suggests a role for NFI in the relatively low constitutive levels of MT-I expression in these cells.

Topics: Animals; Carcinoma, Hepatocellular; CCAAT-Enhancer-Binding Proteins; Cytosine; DNA-Binding Proteins; Down-Regulation; Humans; Lymphoma, Non-Hodgkin; Male; Metallothionein; Methylation; Mice; NFI Transcription Factors; Nuclear Proteins; Promoter Regions, Genetic; Prostatic Neoplasms; Rats; Transcription Factors; Tumor Cells, Cultured; Y-Box-Binding Protein 1

Metallothionein-I (MT-I) gene is silenced by methylation of CpG islands in mouse lymphosarcoma P1798 cells but not in the thymus, the cell type from which the tumor was derived. Bisulfite genomic sequencing revealed that all 21 CpG dinucleotides present within -216 bp to +1 bp with respect to transcription start site are methylated in the tumor cell line, but none is methylated in the thymus. The lymphosarcoma cells induced MT-I in response to heavy metals only after demethylation with 5-azacytidine (5-AsaC). The electrophoretic mobility shift assay using specific oligonucleotide probes showed that the key transcription factors regulating MT-I gene (e.g., MTF-1, Sp 1 and MLTF/USF) are active in P1798 cells. In vivo footprinting of the proximal promoter region showed that none of the metal regulatory elements (MREs) or MLTF/USF are occupied in response to heavy metals. Demethylation of the lymphosarcoma cells with 5-AzaC resulted in constitutive footprinting at MLTF/ARE, and zinc-inducible footprinting at MRE-c, MRE-d and MRE-e sites. Demethylation of just 10-20% of the CpG islands was sufficient to render the gene inducible by cadmium or zinc. The MT-I induction persisted in the cancer cells for several generations even after withdrawal of 5-AzaC from the culture medium.

Topics: Animals; Antimetabolites, Antineoplastic; Azacitidine; Base Sequence; CpG Islands; DNA Footprinting; DNA Methylation; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; Gene Silencing; Lymphoma, Non-Hodgkin; Metallothionein; Metals, Heavy; Mice; Molecular Sequence Data; Neoplasm Proteins; Promoter Regions, Genetic; Regulatory Sequences, Nucleic Acid; Thymus Gland; Transcription Factors; Tumor Cells, Cultured