The spleen focus-forming virus envelope glycoprotein, gp55, and the block in the erythropoietin-induced differentiation of Friend murine erythroleukemia cells

Thomas Samuel Lin, Yale University.

This is an Open Access Thesis


Unlike normal committed erythroid progenitor cells, which require erythropoietin (EPO) for both proliferation and terminal differentiation, Friend murine erythroleukemia (MEL) cells are EPO-independent and do not differentiate in response to the cytokine. Dimethylsulfoxide (DMSO) induces differentiation and restores responsiveness to EPO. DMSO-treated Friend cells responded to EPO with both increased proliferation and further hemoglobin synthesis. The effects of DMSO upon the expression of EPO receptor (EPOR) mRNA and cell surface receptor were examined. Clone DMSO/R1, an MEL subline resistant to the differentiation-inducing and EPO-sensitizing effects of DMSO, expressed a higher level of EPOR mRNA than parental Friend cells, and DMSO treatment increased mRNA levels in both lines. Friend cells expressed 180 receptors per cell of a single class with an affinity constant (k$\rm \sb{d})$ of 1.0 nM. DMSO treatment decreased expression to 130 receptors per cell with a k$\rm \sb{d}$ of 0.9 nM after 2 days, and to 110 receptors per cell with a k$\rm \sb{d}$ of 0.6 nM after 4 days. Despite a higher level of EPOR mRNA than Friend cells, DMSO/R1 cells expressed only 60 receptors per cell with a k$\rm \sb{d}$ of 0.41-0.46 nM.Friend virus is a complex of two retroviruses: the Friend murine leukemia virus (F-MuLV), a helper virus, and the polycythemia-inducing strain of spleen focus-forming virus (SFFVp), a replication-defective virus responsible for the acute erythroleukemia induced in mice. Unlike other acute transforming retroviruses, the tumorigenic oncogene of the Friend virus is the SFFVp envelope glycoprotein, gp55. Gp55 binds to and constitutively activates the EPOR, resulting in growth independence. The possible role or gp55 in the block in the EPO-induced differentiation of Friend cells was examined. DMSO/R1 cells expressed a similar level of gp55 synthesis but a greatly increased level of total cellular gp55 protein, compared to parental Friend cells. Friend cells were transfected with vectors expressing SFFVp gp55 cDNA in the sense or antisense direction to increase or decrease the amount of total cellular gp55. An increased level of gp55 resulted in resistance to the ability of DMSO to restore sensitivity to the maturation-inducing property of EPO, but not in resistance to the differentiating action of DMSO. A decreased level of gp55 resulted in increased sensitivity to both the maturation-inducing and EPO-sensitizing effects of DMSO.Chemical crosslinking and coimmunoprecipitation studies examined whether DMSO treatment resulted in dissociation of gp55 from the the EPOR. Radioiodinated EPO was crosslinked to a 60-65 kDa protein consistent with the cell surface form of SFFVp gp55, and DMSO did not affect this crosslinking. Gp55 and the EPOR were coimmunoprecipitated from cellular lysates of Friend and DMSO/R1 cells, and this association did not change upon induction of differentiation by DMSO. These studies indicated that, although gp55 plays a role in the block in the EPO-induced differentiation of Friend cells, DMSO does not restore EPO responsiveness through dissociation of gp55 from the EPOR.