A Myb dependent pathway maintains Friend murine erythroleukemia cells in an immature and proliferating state

Abstract

Friend murine erythroleukemia (MEL) cells are transformed erythroid precursors that are held in an immature and proliferating state but can be induced to dierentiate 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 ZnCl 2 , the stable transfectants dierentiated with kinetics that were similar to wild type C19 MEL cells treated with HMBA, including induction of aglobin mRNA expression, assembly of hemoglobin and growth arrest. Expression of endogenous c-myb and cmyc 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.

The carbonic anhydrase I locus contains a c-Myb target promoter and modulates differentiation of murine erythroleukemia cells

“…entiation in C19 MEL cells and these cells provide a useful system to identify physiologically relevant c-Myb target promoters (Chen et al, 2002). Our experiments using this model have identified the CAIe promoter, which is downregulated during MEL cell differentiation, as a potential target of c-Myb activity.…”

“…MEnT consists of the mouse c-Myb DBD fused to the transcription suppression domain of the Drosophila Engrailed protein (Badiani et al, 1994). Expression of MEnT in these stable transfectants is regulated by the ZnCl 2 -inducible mouse metallothionein I (MTH-I) promoter and induced expression of MEnT in C19 MEL cells provides a sufficient signal to induce differentiation (Chen et al, 2002). One of these transfectants, clone 17, was grown in culture for 4, 24 and 72 h with or without ZnCl 2 and total cellular RNA from each sample was analysed by differential display.…”

“…Clone 17 cells are stable transfectants of the C19 MEL cell line that contain a dominant interfering Myb cDNA (MEnT) under the inducible transcriptional control of a mouse metallothionein I promoter and were cultured in the same medium containing G418 at a concentration of 400 mg/ml (Chen et al, 2002). CV1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% newborn calf serum and 5% fetal bovine serum.…”

“…Expression of c-Myb is downregulated during MEL cell differentiation, and forced expression of c-Myb blocks chemically induced MEL cell differentiation (McClinton et al, 1990). To gain insight into the mechanism of cMyb-regulated differentiation in MEL cells, we developed C19 MEL cell lines that carry an inducible dominant interfering Myb protein (MEnT) and induction of MEnT expression provides a sufficient signal for MEL cells to differentiate (Chen et al, 2002). We now identify the carbonic anhydrase I erythroid-specific promoter (CAIe) as a target of c-Myb activity.…”

v-Myb represses the transcription of Ets-2

“…Studies from a number of different laboratories have shown that the v-Myb protein is nuclear, binds to specific DNA sequences as a monomer, and can activate the expression of reporter genes (Boyle et al, 1984;Klempnauer et al, 1984;Biedenkapp et al, 1988;Garcia et al, 1991). In addition, both v-Myb and its normal cellular homologue c-Myb have been shown to be capable of activating gene expression in a variety of cell types Nishina et al, 1989;Weston and Bishop, 1989;Ibanez and Lipsick, 1990;Nakagoshi et al, 1992;Siu et al, 1992;Cogswell et al, 1993;Ku et al, 1993;Melotti and Calabretta, 1994;Burk et al, 1997;Worpenberg et al, 1997;Schlichter et al, 2001;Chen et al, 2002;Bartley et al, 2003;Rushton et al, 2003;Braas et al, 2004;Lang et al, 2005;Liu et al, 2006).…”