tetracycline and Leukemia--Erythroblastic--Acute

The functions of AML1 in hematopoietic differentiation are repressed by AML1-mutants including the AML1/ETO chimeric protein, which is seen in t(8;21) acute myeloid leukemia. Erythroid progenitors of the patients with t(8;21) AML expressed AML1/ETO. To investigate the effect of AML1/ETO in erythroid cells, we made a tetracycline-regulated AML1/ETO overexpression system in mouse erythroleukemic (MEL) cells. Enforced AML1/ETO repressed the terminal erythroid differentiation. Furthermore, we performed representational difference analysis using this MEL cell system to clone the downstream targets of AML1 in erythroid cell differentiation. We cloned a novel transmembrane protein, Art-1 (AML1-regulated transmembrane protein 1), which is a member of tetramembrane spanning superfamily. Art-1 expression was restricted in hematopoietic cells. It was upregulated by AML1 and downregulated by AML1/ETO in both erythroid and myeloid cells, and increased during erythroid cell differentiation. Art-1 may play an important role in the differentiation of erythroid cells, possibly as a direct downstream target of AML1.

Topics: Amino Acid Sequence; Animals; Cell Differentiation; Cloning, Molecular; Core Binding Factor Alpha 2 Subunit; DNA-Binding Proteins; Enzyme Inhibitors; Erythrocytes; Hydroxamic Acids; Leukemia, Erythroblastic, Acute; Membrane Proteins; Mice; Molecular Sequence Data; Myeloid Cells; Oncogene Proteins, Fusion; Proto-Oncogene Proteins; RNA, Messenger; RUNX1 Translocation Partner 1 Protein; Tetracycline; Tissue Distribution; Transcription Factors; Tumor Cells, Cultured

The widespread occurrence of AU-rich elements (AREs) in mRNAs encoding proteins with diversified functions and synthesized under a vast variety of physiological conditions suggests that AREs are involved in finely tuned and stringent control of gene expression. Thus it is important to investigate the regulation of ARE-mediated mRNA decay in a variety of mammalian cells in different physiological states. The tetracycline (Tet)-regulatory promoter system appears appropriate for these investigations. However, we found that efficient degradation of mRNAs bearing different AREs cannot be observed simply by blocking constitutive transcription from the Tet-regulated promoter with Tet, possibly due to saturation of the cellular decay machinery. In addition, deadenylation kinetics and their relationship to mRNA decay cannot be adequately measured under these conditions. To overcome these obstacles we have developed a new strategy that employs the Tet-regulated promoter system to achieve a transient burst of transcription that results in synthesis of a population of cytoplasmic mRNAs fairly homogeneous in size. Using this new system we show that ARE-destabilizing function, necessary for down-regulating mRNAs for cytokines, growth factors and transcription factors, is maintained in quiescent or growth-arrested cells as well as in saturation density-arrested NIH 3T3 cells. We also demonstrate that the ARE-mediated decay pathway is conserved between NIH 3T3 fibroblasts and K562 erythroblasts. These in vivo observations support a broader role for AREs in the control of cell growth and differentiation. In addition, we observed that there is a significant difference in deadenylation and decay rates for beta-globin mRNA expressed in these two cell lines. Deadenylation and decay of beta-globin mRNA in K562 cells is extraordinarily slow compared with NIH 3T3 cells, suggesting that the increased stability gained by beta-globin mRNA in K562 cells is mainly controlled at the deadenylation step. Our strategy for studying mammalian mRNA turnover now permits a more general application to different cell lines harboring the Tet-regulated system under various physiological conditions.

Topics: 3T3 Cells; Adenosine; Animals; Base Sequence; Globins; Humans; Leukemia, Erythroblastic, Acute; Mice; Mutagenesis, Site-Directed; Plasmids; Promoter Regions, Genetic; Regulatory Sequences, Nucleic Acid; Repressor Proteins; RNA, Messenger; Tetracycline; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Uridine

When mouse erythroleukemia (MEL) cells were incubated in the presence of chloramphenicol (a specific inhibitor for mitochondrial protein synthesis) during the early stage of in vitro erythroid differentiation, the number of induced erythroid cells was greatly reduced. By use of cell fusion between two genetically marked MEL cells, this finding was further investigated. We found that the drug, along with other agents which inhibit mitochondrial protein synthesis, blocked the induction and turnover of the DMSO-inducible intracellular-erythroid-inducing activity (differentiation-inducing factor II) in a manner similar to that of cycloheximide, an inhibitor for nuclear protein synthesis. The inhibitory effect was confirmed by directly assaying differentiation-inducing factor II in the cell extracts. These results strongly suggest that mitochondrial protein synthesis is closely associated with in vitro erythroid differentiation of MEL cells.

Topics: Animals; Cell Differentiation; Cell Line; Chloramphenicol; Cycloheximide; Dimethyl Sulfoxide; Friend murine leukemia virus; Hemoglobins; Kinetics; Leukemia, Erythroblastic, Acute; Mice; Mitochondria; Protein Biosynthesis; Protein Synthesis Inhibitors; Tetracycline; Ultraviolet Rays

Topics: Aged; Ampicillin; Bone Marrow; Bone Marrow Cells; Carbamates; Cephaloridine; Cyclophosphamide; Cytarabine; Daunorubicin; Environmental Exposure; Humans; Imines; Insecticides; Leukemia, Erythroblastic, Acute; Lysosomes; Macrophages; Male; Microscopy, Electron; Mitochondria; Pneumonia; Prednisone; Sulfides; Tetracycline; Thioguanine; Vincristine