Giefing Maciej, Winoto-Morbach Supandi, Sosna Justyna, Döring Claudia, Klapper Wolfram, Küppers Ralf, Böttcher Sebastian, Adam Dieter, Siebert Reiner, Schütze Stefan
Institute of Human Genetics, Christian-Albrechts University Kiel & University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany ; Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.
Institute of Immunology, Christian-Albrechts University Kiel & University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.
PLoS One. 2013 Dec 23;8(12):e84928. doi: 10.1371/journal.pone.0084928. eCollection 2013.
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<0.005) in cHL compared to other lymphoma cell lines. Together this findings show recurrent alterations of the NADPH oxidase encoding genes that result in functional inactivation of the enzyme and reduced production of superoxide anion in cHL.
参与活性氧（ROS）合成的膜结合NADPH氧化酶是一种由CYBA、CYBB、NCF1、NCF2和NCF4基因编码的多蛋白酶。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细胞祖细胞的分化和增殖。先前已报道在B细胞来源的经典霍奇金淋巴瘤（cHL）细胞系中CYBB基因的转录下调。因此，我们探讨了CYBB下调对NADPH复合物的功能影响。使用流式细胞术检测和定量cHL细胞系中超氧阴离子的合成，我们发现与受刺激的非霍奇金淋巴瘤细胞系相比，所有受刺激的cHL细胞系中超氧阴离子产生反复缺失。由于CYBB缺失被证明对cHL细胞系中的NADPH氧化酶复合物产生有害影响，我们通过原位杂交分析了原发性cHL活检组织中霍奇金和里德-斯腾伯格（HRS）细胞中的CYBB基因座，发现在8/18例病例中该基因反复缺失。对其中14例病例的免疫组织化学分析显示，在所有研究病例的所有HRS细胞中完全缺乏可检测到的CYBB蛋白表达。此外，通过对cHL细胞系的微阵列分析，我们发现NADPH氧化酶基因的其他改变，包括3/7细胞系中CYBA拷贝数缺失以及与正常B细胞亚群相比NCF1转录的显著下调（p = 0.006）。此外，与其他淋巴瘤细胞系相比，cHL中NCF1蛋白显著下调（p < 0.005）。这些发现共同表明，NADPH氧化酶编码基因的反复改变导致该酶功能失活，并减少了cHL中超氧阴离子的产生。
