Unexpected and coordinated expression of Spi-1, Fli-1, and megakaryocytic genes in four Epo-dependent cell lines established from transgenic mice displaying erythroid-specific expression of a thermosensitive SV40 T antigen. 
Most erythroleukemic cell lines established in vitro coexpress erythrocytic and megakaryocytic markers that often are associated with expression of Spi-1 and/or Fli-1 transcription factors known as transactivators of megakaryocyte-specific promoters. In the present study, we examined the possibility of establishing new cell lines keeping strictly erythroid-specific properties in vitro through the targeted and conditional immortalization of erythrocytic progenitors. For that purpose, we established several lines of transgenic mice displaying erythroid-specific expression of a thermosensitive SV40 T antigen. As expected, these transgenic mice developed splenomegaly due to the massive amplification of Ter 119 positive erythroid nucleated cells expressing T antigen. Despite this drastic effect in vivo, the in vitro immortalization of erythropoietin-dependent erythroid progenitors unexpectedly occurred at low frequency, and all four cell lines established expressed both erythrocytic (globins) and megakaryocytic markers (glycoprotein IIb, platelet factor 4) as well as Spi-1 and Fli-1 transcripts at permissive temperature. Switching the cells to the nonpermissive temperature led to a marked increase in globin gene expression and concomitant decrease in expression of Spi-1, Fli-1, and megakaryocytic genes in an erythropoietin-dependent manner. Interestingly, enhanced expression of Spi-1 and Fli-1 genes already was detected in the Ter 119 positive cell population of transgenic mice spleen in vivo. However, like normal Ter 119 erythroid cells, these Ter 119 positive cells from transgenic mice still expressed high levels of beta-globin and very low or undetectable glycoprotein IIb and platelet factor 4 megakaryocytic transcripts. Taken together, these data indicate that the unexpected expression of megakaryocytic genes is a specific property of immortalized cells that cannot be explained only by enhanced expression of Spi-1 and/or Fli-1 genes.