Abstract
Alterations in the expression of two proto-oncogenes, c-myb and c-myc, have been implicated in the differentiation of transformed erythroid cells induced by chemical inducers, such as dimethyl sulfoxide (Me2SO). In the present study, we compared the expression of c-myb and c-myc during erythropoietin (Epo) and Me2SO induction of Rauscher erythroleukemia cells, which differentiate in response to both inducers, and Friend erythroleukemia cells, in which Epo-induced differentiation is blocked. Our results demonstrate that Epo induces specific changes in expression of c-myb and c-myc in both Rauscher and Friend cells. Epo increases c-myc transcript, in contrast to a decreased caused Me2SO, indicating that the biphasic mode of c-myc regulation seen with Me2SO is not required for erythropoiesis. The Epo-induced changes in c-myb and c-myc do not require new protein synthesis, thus identifying these proto-oncogenes as early response genes for Epo. Both cell types also exhibit rapid changes in membrane protein phosphorylation in response to Epo. Since the signal pathway from Epo receptor activation to the nucleus appears equally functional in both Rauscher and Friend cells, the data suggest that the inability of Friend cells to differentiate in response to Epo is due to a block at a later step in the induction process.
Highlights
Friend cell line [2]: they can bienduced by a variety of chemical reagents, including dimethyl sulfoxide (Me,SO), to undergo adifferentiation process resemblingthat of normal erythroid cells [7, 9, 10]
Alterations in the expression of two proto-oncogenes, lished from circulating leukemia cells of mice infected with c-myb and c-myc, have been implicated in the differ- the Rauscher virus, differentiate in response to either Epoor entiation of transformed erythroid cells induced by MezSO [8, 11]
Our results demonstrate that Epo induces specific changes in expression of c-myb and cmyc in both Rauscher and Friend cells
Summary
Total Cytoplasmic R N A Extraction and Northern Blot AnalysisRauscher cells [8, 11, 12] (clone R28) and Friend cells [7, 9] (clone 745A) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Gibco). Cytoplasmic RNA was preparedusingguanidiniumisothiocyanate [13]. Twenty-five micrograms of total RNA were fractionated electrophoreticallyon 1.2%agarose containing 5.5% formaldehyde andtransferredto GeneScreen Plus (Du Pont-New England Nuclear). The filterswere hybridized sequentially with 32P-labeledplasmids containing exon 3 subcloned from the mouse c-myc gene [14],a 0.7-kilobase SstI-EcoRI fragment of the mouse c-myb gene [15], or a cDNA of a “houseErythropoietin (Epo),’ a 34-kDa glycoprotein, is the major keeping” gene, glyceraldehyde-3-phosphate dehydrogenase gene [16]. The action of Epo has been studied very actively in the past few years [1,2,3,4,5,6], andchangesin calciumionflux [1, 3, 4]and membrane protein phosphorylation [5, 6] have been implicated
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