[CANCER RESEARCH 61, 2080 –2084, March 1, 2001] Oct-2 and Bob-1 Deficiency in Hodgkin and Reed Sternberg Cells 1 Daniel Re, Markus Mu ¨ schen, Tahamtan Ahmadi, Claudia Wickenhauser, Andrea Staratschek-Jox, Udo Holtick, Volker Diehl, and Ju ¨ rgen Wolf 2 Department of Internal Medicine I [D. R., M. M., T. A., A. S-J., U. H., V. D., J. W.], Institute of Pathology [C. W.], and Institute for Genetics, Department of Immunology [M. M.], University of Cologne, 50931 Cologne, Germany ABSTRACT Hodgkin and Reed Sternberg (H-RS) cells represent the malignant cells in classical Hodgkin’s disease. Although derived from germinal center B cells, they do not express surface immunoglobulin. This has been ex- plained by the presence of crippling mutations within the immunoglobulin genes in numerous cases of Hodgkin’s disease. As immunoglobulin gene expression in B cells requires an interaction between octamer sites and the transactivating factors Oct-2 and Bob-1, this study addresses the expres- sion of the transcription factors Oct-2 and Bob-1 in H-RS cells. In Hodgkin’s disease-derived cell lines, low levels of Oct-2 transcripts but no Oct-2 protein were detected. Transcripts of Bob-1, a B-cell-specific cofac- tor of Oct-2, could not be observed in these cell lines. Absence of Oct-2 and Bob-1 protein expression in primary H-RS cells was demonstrated by performing immunohistochemistry in 20 cases of classical Hodgkin’s dis- ease. H-RS cells stained negative for both proteins in all of the cases analyzed. In conclusion, absence of functional Oct-2 and Bob-1 cells represents a novel mechanism for immunoglobulin gene deregulation in H-RS cells. Lack of Oct-2 and Bob-1 points to a defect in transcription machinery in H-RS cells and is associated with lack of immunoglobulin gene expression in these cells. INTRODUCTION H-RS 3 cells represent the malignant cells in classical HD. In most cases, the H-RS cells are derived from GC B cells because they harbor somatically mutated immunoglobulin-V region genes (1–3), whereas derivation from T cells is rare (4). Notably, in a substantial proportion of classical HD cases, the H-RS cells had lost their capacity to express a functional B-cell receptor because of obviously destructive somatic mutations, rendering potentially functional immunoglobulin gene re- arrangements nonfunctional (2). However, in other cases potentially functional immunoglobulin gene rearrangements were detected in H-RS cells (3, 5), indicating that in these cases, immunoglobulin gene expression might be possible in the lymphoma cells. However, in situ hybridization experiments failed to show detectable amounts of IgL and IgH mRNA in these cases. From these data, it was concluded that in these cases the immunoglobulin gene transcription is deregulated in H-RS cells (3). Expression of rearranged IgH and IgL genes is critical for B-cell differentiation and is regulated by a complex interaction between regulatory DNA elements and transcription factors (6). Among the regulatory DNA elements necessary for B-cell-specific transcription, the octamer motif is an important transcriptional regulatory site that is part of promoters and enhancers of ubiquitously expressed genes. Furthermore, this octamer motif is found in all of the IgH and IgL promoters and in the heavy chain and  light chain enhancer elements (7). It has been shown to be essential for B-cell specificity and activity of the immunoglobulin promoter and enhancer (8, 9). The octamer site interacts with transcription factors belonging to the POU family of homeodomain-proteins binding specifically to this octamer motif via their POU domain (10). Oct-1 was identified as a ubiquitous protein, whereas Oct-2 expres- sion is restricted to B cells and neuronal cells (11). In B cells and neuronal cells, alternative splicing of Oct-2 generates several proteins (12, 13). On the basis of transfection experiments, for Oct-2 a critical role for immunoglobulin promoter transactivation was shown (12). Recent studies (14) demonstrated that in addition to Oct-2, a B-cell- specific cofactor, namely Bob-1, is required. Thus, B-cell specificity of immunoglobulin promoter activity is mediated by the expression of Bob-1 (OCA-B or OBF-1). Bob-1 associates with the POU domain of octamer proteins Oct-1 and Oct-2 and alters their recognition speci- ficity (10). In a Bob-1-deficient mouse model, GC formation was drastically impaired, and class switch recombination was reduced substantially (15). Because H-RS cells derive from GC B cells (16) but lack immu- noglobulin gene expression, we now address the role of Oct-2 and Bob-1 for immunoglobulin gene transcription in classical HD. In this study, we discuss whether a disturbed expression of transcription factors is involved in the deregulation of immunoglobulin gene tran- scription in classical HD. MATERIALS AND METHODS Cell Lines. The characteristics of the seven HD-derived cell lines are summarized by Drexler (17) and Wolf et al. (18). IARC277 is an EBV- immortalized lymphoblastoid cell line (19), and BJA-B is an EBV-negative B-cell lymphoma cell line established from the biopsy of a five-year-old patient suffering from an EBV-negative African Burkitt lymphoma (20). The adherent fibroblastic cell line NIH/3T3 and the human T-lymphoblastic leu- kemia cell line Jurkat were obtained from the American Type Culture Collec- tion (Manassas, VA). All of the cell lines were grown according to standard procedures. Pathological Specimen. Twenty primary cases of classical HD and one nonneoplastic lymph node sample were analyzed by immunohistochemistry. Characteristics are listed in Table 1. Pathological specimen were classified according to the WHO classification (21). All of the diagnoses have been reviewed by the pathologist reference panel of the German Hodgkin’s Lym- phoma Study Group. Cell Separation and Flow Cytometry. B-cell subsets were purified from a reactive tonsil of a child and from peripheral blood of an unrelated donor. CD38 + CD77 + GC B cells were isolated according to Goossens et al. (22). After two cycles of magnetic cell sorting enrichment of CD77 + cells, purity of these GC B cells was more than 90%. After the separation of CD38 + CD77 + cells, tonsillar naı ¨ve (IgD + CD27 - ) B cells were isolated from the flow- through fraction and separated from memory cells on the basis of their CD27 expression according to Klein et al. (23). The naı ¨ve phenotype (IgD + IgM + CD20 + ) was verified by flow cytometry. Purity of these two B-cell subsets was confirmed by amplifying and sequencing of the immunoglobulin V H gene rearrangements from single sorted cells (8 naı ¨ve cells were all unmutated, and 19 GC B cells were all mutated). Received 6/5/00; accepted 1/18/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the Deutsche Forschungsgemeinschaft through SFB502. D. R. is sup- ported by the Friedrich and Maria Sophie Moritz’sche Stiftung (Cologne, Germany). M. M. holds a postdoctoral fellowship from the Cancer Research Institute (Tumor Im- munology Program, New York, NY). 2 To whom requests for reprints should be addressed, at University of Cologne, Department of Internal Medicine I, Joseph-Stelzmann-Str. 9, 50924 Cologne, Germany. Phone: 49-221-478-3410; Fax: 49-221-478-6733; E-mail: ju ¨rgen.wolf@medizin.uni- koeln.de. 3 The abbreviations used are: H-RS, Hodgkin and Reed Sternberg; HD, Hodgkin’s disease; GC, germinal center; RT-PCR, reverse transcriptase PCR. 2080 Research. on November 13, 2017. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from