Inactivation of a c-Myb/estrogen receptor fusion protein in transformed primary cells leads to granulocyte/macrophage differentiation and down regulation of c-kit but not c-myc or cdc2

Abstract

Primary murine fetal hemopoietic cells were transformed with a fusion protein consisting of the ligand-binding domain of the estrogen receptor and a carboxylterminally truncated c-Myb protein (ERMYB). The ERMYB-transformed hemopoietic cells exhibit an immature myeloid phenotype when grown in the presence of b-estradiol. Upon removal of b-estradiol, the ERMYB cells display increased adherence, decreased clonogenicity and di erentiate to cells exhibiting granulocyte or macrophage morphology. The expression of the c-myc, ckit, cdc2 and bcl-2 genes, which are putatively regulated by Myb, was investigated in ERMYB cells grown in the presence or absence of b-estradiol. Neither c-myc nor cdc2 expression was down-regulated after removal of bestradiol demonstrating that di erentiation is not a consequence of decreased transactivation of these genes by ERMYB. While bcl-2 expression was reduced by 50% in ERMYB cells grown in the absence of bestradiol, there was no increase in DNA laddering, suggesting that Myb was not protecting ERMYB cells from apoptosis. In contrast, a substantial (200-fold) decrease in c-kit mRNA level was observed following di erentiation of ERMYB cells, and c-kit mRNA could be partially re-induced by the re-addition of b-estradiol. Furthermore, a reporter construct containing the c-kit promoter was activated when cotransfected with a Myb expression vector, providing further evidence of a role for Myb in the regulation of c-kit.

Transcription factors and translocations in lymphoid and myeloid leukemia

“…Following terminal differentiation to monocytes, neutrophils, or erythrocytes, c-Myb is downregulated. 81,92 Genes implicated as targets of c-Myb which are involved in its role in proliferation include the cell cycle regulator gene Cdc2, 90 the DNA polymerase ␣ gene, 93 c-kit [94][95][96] and c-myc, [97][98][99] however recent studies question the regulation of c-myc by c-Myb. 89 In addition, antisense oligonucleotides to c-myb inhibit myeloid cell proliferation 100 and overexpression of c-Myb is known to inhibit cellular differentiation in erythroid and myeloid cell lines.…”

Regulation of the gene encoding glutathione S-transferase M1 (GSTM1) by the Myb oncoprotein

“…A number of studies have demonstrated that c-Myb plays an important role in cellular proliferation and in the maintenance of cells in an immature, undifferentiated state (reviewed in Weston, 1998). Myb has also been shown to be involved in regulating apoptosis in some systems (Frampton et al, 1996;Taylor et al, 1996;Hogg et al, 1997). Overexpression of activated or normal forms of c-Myb has been shown to induce cellular transformation (Ferrao et al, 1995;Fu and Lipsick, 1997).…”

“…In order to do this, we have utilized both cDNA array screening and PCR-based subtractive hybridization in conjunction with a conditionally Myb-transformed cell line, ERMYB (Hogg et al, 1997). This cell line was derived by transforming murine fetal liver cells with an activated form of Myb fused to the ligand-binding domain of the human estrogen receptor.…”

“…A portion of the duplicate filters is shown with cDNA spots corresponding to GSTM1 on the array circled. (c) Northern blot analysis of polyA þ RNA (2 mg) prepared (as described; Hogg et al, 1997) from ERMYB cells grown in the absence of b-estradiol for varying periods of time (as indicated), or grown in the absence of b-estradiol for 24 h and then reinduced with b-estradiol for a further 24 h (24 þ 24). The blot was hybridized with a-32 Plabelled probes for GSTM1 or GAPDH as indicated.…”

MYB IS Required for B-Selection During Thymopoises

“…(Methionine adenyl-transferase II, alpha) [236,243,244], Gata-3 [245], Rag-2 [168,169,246], c-kit [247][248][249], CD4 [245,250,251], and cyclin B1 [252]. However, many of these genes were identified by testing the ability of exogenously expressed c-Myb to activate promoter-reporter gene constructs in cells that do not normally express c-Myb [138,253] and it has recently been reported that the ability of Myb proteins to activate test constructs varied greatly from their ability to activate endogenous genes [154].…”