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.

译文

体外建立的大多数红白血病细胞系共表达红细胞和巨核细胞标记,这些标记通常与被称为巨核细胞特异性启动子的反式激活因子的Spi-1和/或Fli-1转录因子的表达相关。在本研究中,我们研究了通过红细胞祖细胞的靶向和条件永生化在体外建立严格保持红细胞特异性特性的新细胞系的可能性。为此,我们建立了几行显示热敏SV40 T抗原的红细胞特异性表达的转基因小鼠。如预期的那样,这些转基因小鼠由于表达T抗原的Ter 119阳性红系有核细胞的大量扩增而出现脾肿大。尽管在体内产生了这种巨大的作用,但促红细胞生成素依赖性红系祖细胞的体外永生化意外地以低频率发生,并且所建立的所有四种细胞系均表达红细胞 (球蛋白) 和巨核细胞标记物 (糖蛋白IIb,血小板因子4) 以及Spi-1和Fli-1转录本在允许的温度下。将细胞切换到非允许温度导致珠蛋白基因表达显着增加,并以促红细胞生成素依赖性方式伴随着Spi-1,Fli-1和巨核细胞基因的表达降低。有趣的是,在体内转基因小鼠脾脏的Ter 119阳性细胞群中已经检测到Spi-1和Fli-1基因的增强表达。然而,像正常的Ter 119红系细胞一样,这些来自转基因小鼠的Ter 119阳性细胞仍表达高水平的 β-珠蛋白和非常低或不可检测的糖蛋白IIb和血小板因子4巨核细胞转录物。综上所述,这些数据表明巨核细胞基因的意外表达是永生化细胞的特定特性,不能仅通过Spi-1和/或Fli-1基因的增强表达来解释。

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