metallothionein and Leukemia

Metallothioneins (MTs), a group of small, cystein-rich proteins, possess various functions, including metal detoxification and homeostasis. We here report new findings on the participation of MT in cellular differentiation processes. MT isogene transcription was significantly increased in phorbol-12-myristate-13-acetate (PMA) incubated leukemic DAMI cells, which supports its role in cellular differentiation. To further address this possibility, we constructed one stable MT-2A overexpressing DAMI cell line. Increase of cell size, intracellular granulation and megakaryocytic specific antigen expression such as CD41 and CD42, and arresting cell proliferation have validated the role of MT in differentiation in this cell line.

Topics: Animals; Biomarkers; Cell Differentiation; Humans; Leukemia; Megakaryocytes; Metallothionein; Phorbol Esters; Tumor Cells, Cultured

PU.1 is a key transcription factor for hematopoiesis and plays important roles in various hematological malignancies. To clarify the molecular function of PU.1, we initially tried to identify bona fide target genes regulated by PU.1. Dual microarrays were employed for this study to compare PU.1-knockdown K562 cells (K562PU.1KD) stably expressing PU.1 short inhibitory RNAs versus control cells and PU.1-overexpressing K562 cells (K562PU.1OE) versus control cells. In these analyses, we found that several genes, including metallothionein (MT)-1 isoforms (MT-1G and MT-1A) and vimentin (VIM), were markedly induced while Jun dimerization protein (JDP) 2 was suppressed in K562PU.1KD cells. Furthermore, the mRNA expressions of the MT-1 and VIM genes were inversely correlated and the mRNA expression of JDP2 was positively correlated with PU.1 mRNA expression in 43 primary acute myeloid leukemia specimens (MT-1G: R = -0.50, p < 0.001; MT-1A: R = -0.58, p < 0.0005; VIM: R = -0.39, p < 0.01; and JDP2: R = 0.30, p < 0.05). Next, we analyzed the regulation of the MT-1 and VIM genes. We observed increased associations of acetylated histones H3 and H4 with the promoters of these genes in K562PU.1KD cells. Sequence analyses of the regions approximately 1 kb upstream from the transcription start sites of these genes revealed numerous CpG sites, which are potential targets for DNA methylation. Chromatin immunoprecipitation assays revealed that methyl CpG-binding protein 2 (MeCP2) and PU.1 bound to the CpG-rich regions in the MT-1 and VIM promoters. Bisulfite sequencing analyses of the PU.1-bound regions of these promoters revealed that the proportions of methylated CpG sites were tightly related to the PU.1 expression levels.

Topics: Biomarkers, Tumor; Chromatin Immunoprecipitation; CpG Islands; DNA Methylation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histones; Humans; K562 Cells; Leukemia; Luciferases; Metallothionein; Methyl-CpG-Binding Protein 2; Oligonucleotide Array Sequence Analysis; Protein Isoforms; Proto-Oncogene Proteins; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Trans-Activators; Vimentin

We have expressed several variants of core binding factor beta (CBFbeta)-smooth muscle myosin heavy chain (SMMHC) from the metallothionein promoter in Ba/F3 cells. Deletion of amino acids 2-11 from the CBFbeta segment, required for interaction with CBFalpha, prevented CBFbeta-SMMHC from inhibiting CBF DNA binding and cell cycle progression. Deletion of 283 carboxyl-terminal residues from the SMMHC domain, required for multimerization, also inactivated CBFbeta-SMMHC. Nuclear expression of CBFbeta(Delta2-11)-SMMHC was decreased relative to CBFbeta-SMMHC. CBFbeta(Delta2-11)-SMMHC linked to a nuclear localization signal still did not slow cell growth. The ability of each CBFbeta-SMMHC variant to inhibit CBF DNA binding and cell proliferation correlated with its ability to inhibit transactivation by an AML1-VP16 fusion protein. Thus, CBFbeta-SMMHC slows cell cycle progression from G1 to S phase by inhibiting CBF DNA binding and transactivation.

Topics: Bone Marrow Cells; Cell Compartmentation; Cell Division; Core Binding Factors; Dimerization; DNA-Binding Proteins; Growth Inhibitors; Hematopoiesis; Leukemia; Lymphoid Tissue; Metallothionein; Muscle, Smooth; Myosin Heavy Chains; Neoplasm Proteins; Oncogene Proteins, Fusion; Peptide Fragments; Promoter Regions, Genetic; Protein Binding; Sequence Deletion; Structure-Activity Relationship; Transcription Factors; Transcriptional Activation; Translocation, Genetic

Topics: Adult; Autoradiography; B-Lymphocytes; Burkitt Lymphoma; Cadmium; Cadmium Chloride; Cadmium Radioisotopes; Cell Line; Cell Line, Transformed; Cell Separation; Chromatography, Gel; Cysteine; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Flow Cytometry; Granulocytes; Herpesvirus 4, Human; Humans; In Vitro Techniques; Leukemia; Lymphocytes; Metallothionein; Monocytes; Sulfur Radioisotopes; T-Lymphocytes

Cd2+-binding proteins of peripheral blood lymphocytes and monocytes have not well been characterized so far, although they are expected to be a clue for understanding Cd2+ toxicity in those immune competent cells. We separated a family of Cd2+-binding proteins from Cd2+-exposed human peripheral blood lymphocytes by gel filtration chromatography, and characterized them by SDS-gel electrophoresis. The proteins showed electrophoretic behaviours closely similar to metallothioneins (MTs) of HeLa cells derived from human cervical carcinoma. The proteins were also found in Cd2+-exposed monocytes, and were inducible by Cd2+ in both lymphocytes and monocytes. Anti-MT serum specifically precipitated these proteins, which were thus identified as MTs. These results suggest that the two classes of the cells involved in the immune system possess a protective mechanism against Cd2+ through MTs. A variety of human lymphoid cell lines derived from both T and B cells were also found to have capacity to synthesize MTs in response to Cd2+.

Topics: Adult; Burkitt Lymphoma; Cadmium; Cell Line; Cell Transformation, Viral; Humans; Leukemia; Lymphocytes; Male; Metallothionein; Monocytes; Multiple Myeloma