title: Hodgkin-Reed-Sternberg cells in classical Hodgkin lymphoma show alterations of genes encoding the NADPH oxidase complex and impaired reactive oxygen species synthesis capacity.
abstract: 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.
authors.affiliation: 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.
authors.forename: Maciej
authors.initials: M
authors.lastname: Giefing
authors.affiliation: Institute of Immunology, Christian-Albrechts University Kiel & University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.
authors.forename: Supandi
authors.initials: S
authors.lastname: Winoto-Morbach
authors.affiliation: Institute of Immunology, Christian-Albrechts University Kiel & University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.
authors.forename: Justyna
authors.initials: J
authors.lastname: Sosna
authors.affiliation: Senckenberg Institute of Pathology, University of Frankfurt, Medical School, Frankfurt, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Döring
authors.affiliation: Department of Pathology, Hematopathology Section and Lymph Node Registry, Kiel, Germany.
authors.forename: Wolfram
authors.initials: W
authors.lastname: Klapper
authors.affiliation: Institute of Cell Biology (Cancer Research), University of Duisburg-Essen, Faculty of Medicine, Essen, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Küppers
authors.affiliation: Second Department of Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Böttcher
authors.affiliation: Institute of Immunology, Christian-Albrechts University Kiel & University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.
authors.forename: Dieter
authors.initials: D
authors.lastname: Adam
authors.affiliation: Institute of Human Genetics, Christian-Albrechts University Kiel & University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.
authors.forename: Reiner
authors.initials: R
authors.lastname: Siebert
authors.affiliation: Institute of Immunology, Christian-Albrechts University Kiel & University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Schütze
mesh_terms: D045744:Cell Line, Tumor; D056915:DNA Copy Number Variations; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology*; D006689:Hodgkin Disease / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008562:Membrane Glycoproteins / Q000378:metabolism; D046228:Microarray Analysis; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D016539:Reed-Sternberg Cells / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism
chemical_list: D008562:Membrane Glycoproteins; D017382:Reactive Oxygen Species; D013481:Superoxides; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1