metallothionein and hexamethylene-bisacetamide

Friend murine erythroleukemia (MEL) cells are transformed erythroid precursors that are held in an immature and proliferating state but can be induced to differentiate in vivo by treatment with a variety of chemical agents such as N, N-hexamethylene bisacetamide (HMBA). To investigate the role of Myb proteins in maintaining MEL cells in an immature and proliferating state we have produced stable transfectants in the C19 MEL cell line that contain a dominant interfering Myb allele (MEnT) under the control of an inducible mouse metallothionein I promoter. When expression of MEnT protein was induced with ZnCl2, the stable transfectants differentiated with kinetics that were similar to wild type C19 MEL cells treated with HMBA, including induction of alpha-globin mRNA expression, assembly of hemoglobin and growth arrest. Expression of endogenous c-myb and c-myc was also decreased in response to MEnT. Expression of mad-1 mRNA was rapidly increased in response to expression of MEnT resulting in a shift from predominantly c-Myc/Max complexes to predominantly Mad/Max containing complexes. These results strongly suggest that C19 MEL cells are held in an immature and proliferating state by a pathway that is dependent on Myb activity.

Topics: Acetamides; Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Basic-Leucine Zipper Transcription Factors; Cell Cycle Proteins; Cell Division; DNA-Binding Proteins; Friend murine leukemia virus; Genes, myc; Globins; Hemoglobins; Leukemia, Erythroblastic, Acute; Metallothionein; Mice; Nuclear Proteins; Phosphoproteins; Plasmids; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myb; Repressor Proteins; RNA, Messenger; Trans-Activators; Transcription Factors; Tumor Cells, Cultured; Zinc

Heme-hemopexin coordinately regulates genes encoding protective proteins including metallothionein-I (MT-I) and heme oxygenase 1 (HO-1). Hexamethylene-bisacetamide (HMBA), which induces differentiation and activates protein kinase C (PKC), synergistically augments the induction of both MT-I and MT-II mRNAs in response to heme-hemopexin, but attenuates the induction of HO-1. HMBA also augments the increase in MT mRNA in response to cobalt protoporphyrin-hemopexin, a hemopexin (HPX) receptor ligand that activates signaling cascades without tetrapyrrole uptake. Unlike the PKC-activating phorbol esters that induce MT-I and HO-1, HMBA has minimal effects on MT-I or HO-1. HMBA is an amphipathic molecule, and is shown here to interact physically with lipids in model membranes using differential scanning calorimetry (DSC). The data are consistent with a stabilization of the lipid bilayer and an HMBA-induced segregation of lipids into separate domains each relatively enriched in one of the lipids. HMBA also perturbs membrane-protein interactions, and causes a loss of PKC and G-protein subunits from plasma membranes in vitro. Taken together, these observations reveal an additional level of complexity in the regulation of protective proteins induced by HPX, and which may take place in vivo in response to natural compounds that reorganize membrane phospholipids. A model is proposed whereby a reorganization of lipids by HMBA alters signaling pathways and fusion events considered to be the etiology of the differential response of the MT-1 (and MT-II) and the HO-1 genes to HMBA and heme-HPX.

Topics: Acetamides; Animals; Cell Membrane; Hematinics; Heme; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Hemopexin; Membrane Proteins; Metallothionein; Mice; Phospholipids; RNA, Messenger; Tumor Cells, Cultured

During chemically induced differentiation of murine erythroleukemia (MEL) cells, cAMP-dependent protein kinase activity increases, and the enzyme's isozyme pattern changes. To examine the enzyme's role during MEL cell differentiation, we stably transfected MEL cells with recombinant plasmids in which the mouse metallothionein I promoter controlled expression of either a mutant form of the type I regulatory subunit of cAMP-dependent protein kinase (RI) or the enzyme's specific peptide inhibitor (PKI); expressing either sequence rendered cells cAMP-dependent protein kinase-deficient. Chemically induced differentiation of MEL cells as assessed by beta-globin mRNA and hemoglobin accumulation was inhibited in RI mutant and PKI transfectants; adding zinc further inhibited differentiation in the transfectants but had no effect on parental MEL cells. The inhibition of differentiation correlated with the amount of RI mutant mRNA and protein in the RI mutant transfectants and with the cells' degree of cAMP-dependent protein kinase deficiency in both the RI mutant and PKI transfectants. Overexpression of wild type RI did not interfere with differentiation or enzyme activity. We conclude that cAMP-dependent protein kinase activity is important for chemically induced differentiation of MEL cells and that the down-regulation of RI protein which occurs during MEL cell differentiation is not essential for differentiation to proceed.

Topics: Acetamides; Animals; Blotting, Northern; Blotting, Western; Cell Differentiation; Enzyme Repression; Globins; Isoenzymes; Leukemia, Erythroblastic, Acute; Macromolecular Substances; Metallothionein; Mice; Plasmids; Promoter Regions, Genetic; Protein Kinases; RNA, Messenger; Sulfuric Acid Esters; Transfection; Tumor Cells, Cultured; Zinc