Molecular Mechanisms of Inhibition of Erythroid Differentiation by Overexpression of Ets Family Oncogenes in MEL cells

我们之前报道过，ets家族癌基因成员PU.1的过表达可诱导二甲亚砜（DMSO）处理小鼠红细胞白血病（MEL）细胞的分化抑制、生长停滞和凋亡（Blood 89: 1383- 1393,1997）。在本研究中，我们研究了PU- 0.1在MEL细胞中诱导作用的分子机制。在检测的几种凋亡相关蛋白中，Bcl-2和c-Myc蛋白的表达在pu .1过表达的MEL细胞中被显著抑制。在MEL细胞中引入bcl-2或c-myc基因均可阻止细胞凋亡，提示bcl-2和c-myc基因的下调参与了pu .1诱导的MEL细胞凋亡。此外，在DMSO处理过表达pu .1的MEL细胞中，对红细胞存活至关重要的红细胞特异性转录因子GATA-1的DNA结合活性显著降低。通过双杂交系统，我们发现PU.1与共激活子CBP（CREB结合蛋白）之间存在物理和功能上的相互作用，表明通过限制CBP的量，转录因子之间存在正、负互扰。事实上，c-myb的过表达抑制了pu .1介导的转激活。在MEL细胞中，过表达CBP可阻止pu .1诱导的生长停滞和细胞凋亡，但不抑制分化。过表达PU.1不仅能抑制MEL细胞的红细胞分化，还能促进MEL细胞的髓单核细胞分化。通过差异显示的方法，我们发现了几个与MEL细胞中PU.1过表达相关的新基因。我们现在正在克隆这些基因来分析它们的生物活性。

We previously reported that overexpression of PU.1, a member of ets family oncogenes, induces differentiation inhibition, growth arrest and apoptosis in murine erythroleukemia (MEL) cells treated with dimethylsulfoxide (DMSO) (Blood 89 : 1383-1393, 1997).In the present study, we have investigated molecular mechanisms of PU-.1-induced effects in MEL cells. Among several apoptosis-related proteins examined, expression of the Bcl-2 and c-Myc protein was significantly suppressed in PU.1-overexpressing MEL cells. Introduction of either bcl-2 or c-myc gene in the cells prevented the apoptosis, suggesting that down-regulation of the bcl-2 and c-myc genes is involved in PU.1-induced apoptosis in MEL cells. Furthermore, the DNA binding activity of GATA-1, an erythroid-specific transcription factor critical for survival of erythroid cells, was markedly reduced in PU.1-overexpressing MEL cells treated with DMSO. By using two-hybrid system, we found that physical and functional interactions between PU.1 and the coactivator CBP(CREB binding protein), suggesting that positive and negative cross-talk between transcription factors through limiting amount of CBP. Indeed, overexpression of c-myb inhibited PU.1-mediated transactivation. PU.1-induced growth arrest and apoptosis but not differentiation inhibition was prevented by overexpression of CBP in MEL cells. Overexpression of PU.1 not only inhibited erythroid differentiation but also promoted myelomonocytic differentiation of MEL cells. We have identified several novel genes associated with overexpression of PU.1 in MEL cells by the method of differential display. We are now cloning the genes to analyze their biological activities.