Hodgkin-Reed-Sternberg Cells in Classical Hodgkin Lymphoma Show Alterations of Genes Encoding the NADPH Oxidase Complex and Impaired Reactive Oxygen Species Synthesis Capacity
Hodgkin-Reed-Sternberg Cells in Classical Hodgkin Lymphoma Show Alterations of Genes Encoding the NADPH Oxidase Complex and Impaired Reactive Oxygen Species Synthesis Capacity
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DOI：
10.1371/journal.pone.0084928
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发表时间：
2013-12-23
期刊：
影响因子：
3.7
通讯作者：
Schuetze, Stefan
中科院分区：
文献类型：
--
作者：
Giefing, Maciej;Winoto-Morbach, Supandi;Schuetze, Stefan
参与活性氧(ROS)合成的膜结合型NADPH氧化酶是一种由CybA、CyBB、NCF1、NCF2和NCF4基因编码的多蛋白质酶。越来越多的证据表明，ROS在非吞噬细胞信号通路的调节中发挥作用，包括B细胞前体细胞的分化和增殖。在B细胞来源的经典型霍奇金淋巴瘤(CHL)的细胞系中，已有报道CYBB基因转录下调。因此，我们探讨了CYBB下调对NADPH复合体的功能影响。用流式细胞术检测和定量CHL细胞系中超氧阴离子的合成，我们发现在所有刺激的CHL细胞系中，与刺激的非霍奇金淋巴瘤细胞系相比，超氧阴离子产生的经常性损失。由于CYBB缺失对CHL细胞中NADPH氧化酶复合体产生有害影响，我们用原位杂交法分析了原代CHL活检组织中Hodgkin和Reed-Sternberg(HRS)细胞中的CYBB基因，发现8/18例出现了该基因的反复缺失。对其中14例的免疫组织化学分析显示，在所有研究的病例中，所有HRS细胞中都完全缺乏可检测到的CYBB蛋白表达。此外，通过对CHL细胞系的基因芯片分析，我们发现了NADPH氧化酶基因的额外变化，包括3/7细胞系的CybA拷贝数丢失，以及与正常B细胞亚群相比，NCF1转录显著下调(p=0.006)。此外，NCF1蛋白表达显著下调(p
The membrane bound NADPH oxidase involved in the synthesis of reactive oxygen species (ROS) is a multi-protein enzyme encoded by CYBA, CYBB, NCF1, NCF2 and NCF4 genes. Growing evidence suggests a role of ROS in the modulation of signaling pathways of non-phagocytic cells, including differentiation and proliferation of B-cell progenitors. Transcriptional downregulation of the CYBB gene has been previously reported in cell lines of the B-cell derived classical Hodgkin lymphoma (cHL). Thus, we explored functional consequences of CYBB downregulation on the NADPH complex. Using flow cytometry to detect and quantify superoxide anion synthesis in cHL cell lines we identified recurrent loss of superoxide anion production in all stimulated cHL cell lines in contrast to stimulated non-Hodgkin lymphoma cell lines. As CYBB loss proved to exert a deleterious effect on the NADPH oxidase complex in cHL cell lines, we analyzed the CYBB locus in Hodgkin and Reed-Sternberg (HRS) cells of primary cHL biopsies by in situ hybridisation and identified recurrent deletions of the gene in 8/18 cases. Immunohistochemical analysis to 14 of these cases revealed a complete lack of detectable CYBB protein expression in all HRS cells in all cases studied. Moreover, by microarray profiling of cHL cell lines we identified additional alterations of NADPH oxidase genes including CYBA copy number loss in 3/7 cell lines and a significant downregulation of the NCF1 transcription (p=0.006) compared to normal B-cell subsets. Besides, NCF1 protein was significantly downregulated (p