Gordiienko I, Shlapatska L, Kholodniuk V M, Kovalevska L, Ivanivskaya T S, Sidorenko S P
Department of Molecular and Cellular Pathobiology, R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv 03022, Ukraine.
Department of Oncohematology, R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv 03022, Ukraine.
Exp Oncol. 2017 Dec;39(4):291-298.
Sequential stages of B-cell development is stringently coordinated by transcription factors (TFs) network that include B-lineage commitment TFs (Ikaros, Runx1/Cbfb, E2A, and FOXO1), B-lineage maintenance TFs (EBF1 and PAX5) and stage specific set of TFs (IRF4, IRF8, BCL6, BLIMP1). Deregulation of TFs expression and activity is often occurs in malignant B cells. The aim of this study was to evaluate TFs expression in chronic lymphocytic leukemia cells taking into consideration CD150 cell surface expression. From other side we attempted to regulate TFs expression via CD150 and CD180 cell surface receptors.
Studies were performed on normal peripheral blood B-cell subpopulations and chronic lymphocytic leukemia (CLL) cells isolated from peripheral blood of 67 primary untreated patients with CLL. Evaluation of TFs expression was performed on mRNA level using qRT-PCR and on protein level by western blot analysis.
Median of PAX5 and EBF1 mRNA expression was higher in cell surface CD150 positive (csCD150) compared to csCD150 CLL cases or normal CD19 and CD19CD5 B-cell subsets. Differences in mRNA expression of IRF8, IRF4 and BLIMP1 between studied groups of CLL and normal B cells were not revealed. All CLL cases were characterized by downregulated expression of PU.1 and BCL6 mRNAs in comparison to normal B cells. At the same time elevated SPIB mRNA expression level was restricted to CLL cells. Protein expression of IRF4, IRF8 and BCL6 was uniformly distributed between csCD150 and csCD150 CLL cases. PU.1 protein and CD20 that is direct PU.1 target gene positively correlated with CD150 cell surface expression on CLL cells. Ligation of CD150 and CD180 alone or in combination upregulated IRF8 and PU.1 while downregulated the IRF4 mRNA expression. Signaling via CD150 or CD180 alone elevated the level of BCL6 mRNA. Strong downregulation of IRF4 mRNA was observed after CD150, CD180 or CD150 andCD180 coligation on CLL cells. We found that in CLL cells CD150 is a negative regulator of SPIB while CD180 is involved in upregulation of EBF1 expression level. Moreover, CD180 ligation on CLL cells caused increase of CD150 mRNA level that is a one of the EBF1 target genes.
Analysis of TFs expression profile revealed upregulated SPIB mRNA level and downregulated PU.1 in CLL cells. CD150 and CD180 receptors may modulate transcriptional program in CLL cells by regulating the TFs expression levels.
B细胞发育的连续阶段由转录因子（TFs）网络严格协调，该网络包括B谱系定向转录因子（Ikaros、Runx1/Cbfb、E2A和FOXO1）、B谱系维持转录因子（EBF1和PAX5）以及特定阶段的转录因子组（IRF4、IRF8、BCL6、BLIMP1）。TFs表达和活性的失调在恶性B细胞中经常发生。本研究的目的是考虑CD150细胞表面表达情况，评估慢性淋巴细胞白血病细胞中TFs的表达。另一方面，我们试图通过CD150和CD180细胞表面受体调节TFs的表达。
对67例未经治疗的原发性慢性淋巴细胞白血病患者外周血中分离出的正常外周血B细胞亚群和慢性淋巴细胞白血病（CLL）细胞进行研究。使用qRT-PCR在mRNA水平评估TFs的表达，并通过蛋白质印迹分析在蛋白质水平进行评估。
与CD150阴性的CLL病例或正常CD19和CD19CD5 B细胞亚群相比，细胞表面CD150阳性（csCD150）的PAX5和EBF1 mRNA表达中位数更高。未发现所研究的CLL组与正常B细胞组之间IRF8、IRF4和BLIMP1 mRNA表达存在差异。与正常B细胞相比，所有CLL病例的特征均为PU.1和BCL6 mRNA表达下调。同时，SPIB mRNA表达水平升高仅限于CLL细胞。IRF4、IRF8和BCL6的蛋白质表达在csCD150和CD150阴性的CLL病例中均匀分布。PU.1蛋白和直接受PU.1调控的靶基因CD20与CLL细胞上的CD150细胞表面表达呈正相关。单独或联合连接CD150和CD180可上调IRF8和PU.1，同时下调IRF4 mRNA表达。单独通过CD150或CD180发出的信号可提高BCL6 mRNA水平。在CLL细胞上连接CD150、CD180或CD150与CD180联合后，观察到IRF4 mRNA的强烈下调。我们发现，在CLL细胞中，CD150是SPIB的负调节因子，而CD180参与EBF1表达水平的上调。此外，在CLL细胞上连接CD180会导致EBF1靶基因之一的CD150 mRNA水平升高。
TFs表达谱分析显示，CLL细胞中SPIB mRNA水平上调，PU.1下调。CD150和CD180受体可能通过调节TFs表达水平来调节CLL细胞中的转录程序。
