Inthal Andrea, Krapf Gerd, Beck Dominik, Joas Ruth, Kauer Max O, Orel Lukas, Fuka Gerhard, Mann Georg, Panzer-Grümayer E Renate
Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria.
Clin Cancer Res. 2008 Nov 15;14(22):7196-204. doi: 10.1158/1078-0432.CCR-07-5051.
We explored the mechanisms leading to the distinct overexpression of EPOR as well as the effects of EPO signaling on ETV6/RUNX1-positive acute lymphoblastic leukemias.
ETV6/RUNX1-expressing model cell lines and leukemic cells were used for real-time PCR of EPOR expression. Proliferation, viability, and apoptosis were analyzed on cells exposed to EPO, prednisone, or inhibitors of EPOR pathways by [3H]thymidine incorporation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and Annexin V/propidium iodide staining. Western blot analysis was done to detect activation of signaling proteins. Serum EPO levels and sequences of the EPOR (n = 53) as well as hemoglobin levels were taken from children with acute lymphoblastic leukemia enrolled in Austrian protocols.
We show here that ectopic expression of ETV6/RUNX1 induced EPOR up-regulation. Anemia, however, did not appear to influence EPOR expression on leukemic cells, although children with ETV6/RUNX1-positive leukemias had a lower median hemoglobin than controls. Exposure to EPO increased proliferation and survival of ETV6/RUNX1-positive leukemias in vitro, whereas blocking its binding site did not alter cell survival. The latter was not caused by activating mutations in the EPOR but might be triggered by constitutive activation of phosphatidylinositol 3-kinase/Akt, the major signaling pathway of EPOR in these cells. Moreover, prednisone-induced apoptosis was attenuated in the presence of EPO in this genetic subgroup.
Our data suggest that ETV6/RUNX1 leads to EPOR up-regulation and that activation by EPO might be of relevance to the biology of this leukemia subtype. Further studies are, however, needed to assess the clinical implications of its apoptosis-modulating properties.
我们探究了导致促红细胞生成素受体（EPOR）明显过表达的机制以及促红细胞生成素（EPO）信号传导对ETV6/RUNX1阳性急性淋巴细胞白血病的影响。
使用表达ETV6/RUNX1的模型细胞系和白血病细胞进行EPOR表达的实时聚合酶链反应。通过[³H]胸腺嘧啶核苷掺入、3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐法和膜联蛋白V/碘化丙啶染色，对暴露于EPO、泼尼松或EPOR途径抑制剂的细胞进行增殖、活力和凋亡分析。进行蛋白质免疫印迹分析以检测信号蛋白的激活情况。血清EPO水平、EPOR的序列（n = 53）以及血红蛋白水平取自参与奥地利方案的急性淋巴细胞白血病患儿。
我们在此表明，ETV6/RUNX1的异位表达诱导了EPOR上调。然而，贫血似乎并未影响白血病细胞上的EPOR表达，尽管ETV6/RUNX1阳性白血病患儿的血红蛋白中位数低于对照组。体外暴露于EPO可增加ETV6/RUNX1阳性白血病的增殖和存活，而阻断其结合位点并未改变细胞存活。后者并非由EPOR中的激活突变引起，而是可能由磷脂酰肌醇3-激酶/蛋白激酶B（PI3K/Akt）的组成性激活触发，PI3K/Akt是这些细胞中EPOR的主要信号传导途径。此外，在该基因亚组中，EPO存在时泼尼松诱导的凋亡减弱。
我们的数据表明ETV6/RUNX1导致EPOR上调，并且EPO的激活可能与这种白血病亚型的生物学特性相关。然而，需要进一步研究以评估其凋亡调节特性的临床意义。
