Scholl Sebastian, Bondeva Tzvetanka, Liu Yuantao, Clement Joachim H, Höffken Klaus, Wetzker Reinhard
Department of Internal Medicine II, Medical Faculty at Friedrich Schiller University, Erlanger Allee 101, 07740, Jena, Germany.
J Cancer Res Clin Oncol. 2008 Aug;134(8):861-72. doi: 10.1007/s00432-008-0356-8. Epub 2008 Feb 21.
In acute promyelocytic leukemia (APL) the chromosome translocation t(15;17) resulting in the PML-RAR alpha fusion protein is responsible for a blockage of myeloid differentiation. In this study we investigated the expression of different Phosphatidylinositol 3-kinase (PI3K) isoforms during granulocyte differentiation of NB4 cells induced by all-trans-retinoic acid (ATRA), 9-cis-retinoic acid (9cisRA) or retinoic acid receptor (RAR) agonists.
NB4 cells were analysed for their ability to differentiate into granulocytic lineage by the use of ATRA, 9cisRA or RAR agonists. Expression of signalling proteins was investigated by western blot and real-time PCR. PI3K activity was determined by in vitro kinase assays.
Co-treatment of NB4 cells with either LY294002 to inhibit PI3Ks or PD98059 in order to suppress MEK activity led to significant reduction of CD11b surface expression during ATRA, 9cisRA or the RAR alpha agonist Ro40-6055 dependent NB4 cells granulocyte differentiation. We also show that only the G-protein coupled receptor activated PI3Kgamma isoform demonstrates up-regulated protein and mRNA expression during myeloid differentiation of NB4 cells via RAR alpha and RAR beta-dependent mechanism. Furthermore, activation of MAPK cascade including phosphorylation of MEK increases during retinoid induced differentiation of NB4 cells. Interestingly, protein kinase assays of immunoprecipitated PI3Kgamma revealed a protein of about 50 kDa that is phosphorylated when NB4 cells were treated with the RAR alpha agonist Ro40-6055.
Collectively, our data suggest additive effects of PI3K and MAPK activity on ATRA-dependent NB4 cells granulocyte differentiation.
在急性早幼粒细胞白血病（APL）中，导致PML-RARα融合蛋白的染色体易位t(15;17)是髓系分化受阻的原因。在本研究中，我们调查了全反式维甲酸（ATRA）、9-顺式维甲酸（9cisRA）或维甲酸受体（RAR）激动剂诱导NB4细胞粒细胞分化过程中不同磷脂酰肌醇3激酶（PI3K）亚型的表达。
通过使用ATRA、9cisRA或RAR激动剂分析NB4细胞分化为粒细胞系的能力。通过蛋白质免疫印迹和实时聚合酶链反应研究信号蛋白的表达。通过体外激酶测定确定PI3K活性。
用LY294002抑制PI3K或用PD98059抑制MEK活性共同处理NB4细胞，导致在ATRA、9cisRA或RARα激动剂Ro40-6055依赖的NB4细胞粒细胞分化过程中CD11b表面表达显著降低。我们还表明，只有G蛋白偶联受体激活的PI3Kγ亚型在NB4细胞通过RARα和RARβ依赖性机制进行髓系分化过程中表现出蛋白和mRNA表达上调。此外，在维甲酸诱导的NB4细胞分化过程中，包括MEK磷酸化在内的丝裂原活化蛋白激酶（MAPK）级联反应的激活增加。有趣的是，免疫沉淀的PI3Kγ的蛋白激酶测定显示，当用RARα激动剂Ro40-6055处理NB4细胞时，一种约50 kDa的蛋白会发生磷酸化。
总体而言，我们的数据表明PI3K和MAPK活性对ATRA依赖的NB4细胞粒细胞分化具有累加作用。
