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Blood, 15 September 2006, Vol. 108, No. 6, pp. 2029-2036. Prepublished online as a Blood First Edition Paper on May 18, 2006; DOI 10.1182/blood-2005-10-014258. This Article Abstract Full Text (PDF) All Versions of this Article: blood-2005-10-014258v1 108/6/2029    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Quintanilla-Martinez, L. Articles by Raffeld, M. Search for Related Content PubMed PubMed Citation Articles by Quintanilla-Martinez, L. Articles by Raffeld, M. Related Collections Oncogenes and Tumor Suppressors Gene Expression Neoplasia Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  NEOPLASIA NPM-ALK–dependent expression of the transcription factor CCAAT/enhancer binding protein in ALK-positive anaplastic large cell lymphoma Leticia Quintanilla-Martinez, Stefania Pittaluga, Cornelius Miething, Margit Klier, Martina Rudelius, Theresa Davies-Hill, Natasa Anastasov, Antonio Martinez, Angelica Vivero, Justus Duyster, Elaine S. Jaffe, Falko Fend, and Mark Raffeld From the Institute of Pathology, GSF–National Research Center for Health and Environment, Neuherberg, Germany; the Hematopathology Section, Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD; and the Department of Internal Medicine III and the Institute of Pathology, Technical University Munich, Germany.    Abstract Top Abstract Introduction Materials and methods Results Discussion References   CCAAT/enhancer binding protein (C/EBP) is one of a 6-member family of C/EBPs. These transcription factors are involved in the regulation of various aspects of cellular growth and differentiation. Although C/EBP has important functions in B- and T-cell differentiation, its expression has not been well studied in lymphoid tissues. We, therefore, analyzed its expression by immunohistochemistry and Western blot in normal lymphoid tissues and in 248 well-characterized lymphomas and lymphoma cell lines. Nonneoplastic lymphoid tissues and most B-cell, T-cell, and Hodgkin lymphomas lacked detectable levels of C/EBP. In contrast, most (40 of 45; 88%) cases of ALK-positive anaplastic large cell lymphoma (ALCL) strongly expressed C/EBP. Western blot analysis confirmed C/EBP expression in the ALK-positive ALCLs and demonstrated elevated levels of the LIP isoform, which has been associated with increased proliferation and aggressiveness in carcinomas. Transfection of Ba/F3 and 32D cells with NPM-ALK and a kinase-inhibitable modified NPM-ALK resulted in the induction of C/EBP and demonstrated dependence on NPM-ALK kinase activity. In conclusion, we report the constitutive expression of C/EBP in ALK-positive ALCL and show its relationship to NPM-ALK. We suggest that C/EBP is likely to play an important role in the pathogenesis and unique phenotype of this lymphoma.    Introduction Top Abstract Introduction Materials and methods Results Discussion References   The CCAAT/enhancer binding proteins (C/EBPs) are a family of leucine zipper transcription factors that are involved in the regulation of various aspects of cellular growth and differentiation in a variety of cell types.1 Six members of the family—C/EBP, C/EBP, C/EBP, C/EBP, C/EBP, C/EBP—have been isolated and characterized to date, and they share a strong homology in the carboxyl-terminal region, which contains a basic DNA-binding and dimerization domain and a leucine-zipper motif.1 In contrast, their N-terminal regions are divergent and contain transcriptional activation and repression domains.2 These factors play major roles in such diverse functions as the acute-phase response and inflammation,3 but they are also important in cellular differentiation programs including adipogenesis,4,5 solid organ development,6-8 and hematopoiesis.1,9-12 Several members of this family have been implicated in tumorigenesis, most notably C/EBP in acute myeloid leukemia (AML),13-15 C/EBP in epithelial tumors,16-19 and C/EBP in myxoid liposarcoma.20,21 CEBPB has a number of interesting characteristics that have led investigators to suggest this gene may have a role in oncogenesis. Like most other members of the C/EBP family, CEBPB is an intronless gene. It is transcribed as a single mRNA that can produce at least 3 isoforms—a 38-kDa liver-enriched activating protein (LAP*), a 35-kDa protein (LAP), and a 21-kDa liver-enriched inhibitory protein (LIP)—with the LAP and LIP forms, constituting the major polypeptides produced in cells.6 LIP is an N-terminal truncated form of C/EBP that lacks most of the transactivation domain, and, although it is able to dimerize with other CEBP family members and bind to DNA, its ability to activate transcription is greatly attenuated; therefore, it appears to act as a repressor of C/EBP-mediated transcription.6 The LIP/LAP ratio appears to be tightly regulated, and changes in this ratio have been shown to affect cell proliferation and differentiation decisions, with increases in the ratio generally favoring proliferation. Consistent with a role in cellular proliferation, aberrant expression of LIP has been reported in breast tumors,18 ovarian carcinoma,16 and colorectal carcinoma,17 where LIP expression levels are associated with the more aggressive tumors. Although C/EBP has been most intensely studied for its role in the acute-phase response3 and in adipogenesis,9 it also plays an important role in the activation and terminal differentiation of macrophages and myeloid cells.10,12 In addition, although C/EBP is not constitutively expressed in mature lymphocytes, it has been implicated in B-cell lymphopoiesis22,23 and is a major regulator of the TH1/TH2 response in T cells.24,25 Not surprisingly, C/EBP-deficient mice display numerous abnormalities in humoral, innate, and cellular immunity26 and develop a lymphoproliferative disorder with plasmacytosis and elevated IL-6 levels similar to what has been observed in Castleman disease in humans. Given a role for C/EBP in lymphoid cells, its complex relationship to proliferation and differentiation, and its link to epithelial cancers, we wanted to explore whether C/EBP might also contribute to lymphoid neoplasias. For this purpose, we assessed its expression in lymphoma cell lines and in a large series of primary B-cell, T-cell, and Hodgkin lymphomas and found that C/EBP is consistently overexpressed in anaplastic lymphoma kinase (ALK)–positive anaplastic large cell lymphoma (ALCL). In addition, we demonstrated that C/EBP expression is transcriptionally induced through the kinase activity of NPM-ALK, opening new insight into the pathogenesis of this lymphoma subgroup.    Materials and methods Top Abstract Introduction Materials and methods Results Discussion References   Cell lines Twenty-one cell lines were selected for this study, 19 lymphoid cell lines (Table 1) and 2 epithelial cell lines. Lymphoid cell lines included 4—KiJK, Karpas 299, SUDHL1, SR786—with ALK expression as a result of the t(2;5) translocation. The rest of the lymphoid cell lines included 2 mantle cell lymphoma cell lines (Granta 519 and NCEB-1), 3 transformed follicular lymphoma cell lines (SUDHL4, SUDHL6, SUDHL10), 3 T-cell lymphoblastic lymphoma cell lines (Molt4, CEM, Jurkat), 5 multiple myeloma cell lines (KMS12, OPM2, JIM3, JD38, KMM1), 1 Burkitt lymphoma cell line (ST 486), and 1 HTLV1+ adult T-cell leukemia/lymphoma (Hut102). The epithelial cell lines HeLa and MCF7 were used as positive controls for C/EBP expression. View this table: [in this window] [in a new window]   Table 1.. Lymphoma cell lines analyzed for C/EBP   Tissue samples Formalin-fixed and paraffin-embedded biopsy specimens from 229 well-characterized lymphomas, including 107 T-cell non-Hodgkin lymphomas (NHLs), 93 B-cell NHLs, 9 nodular lymphocyte–predominant Hodgkin lymphomas (NLPHDs), and 20 classical Hodgkin lymphomas (HLs) (Table 2), were selected from the files of the Hematopathology Section, Laboratory of Pathology, National Cancer Institute, National Institutes of Health (Bethesda, MD) and from the files of the Institute of Pathology, Technical University of Munich (Munich, Germany). All cases were classified according to the guidelines of the World Health Organization (WHO) Classification of Neoplastic Diseases of Hematopoietic and Lymphoid Tissues.27 These lymphomas were chosen to represent all major lymphoma subtypes and included 45 cases of ALK-positive ALCL and 29 cases of ALK-negative ALCL. ALK-negative ALCLs resembled their ALK-positive counterparts, strongly expressing CD30, CD4, or CD3, but were negative for ALK protein. In addition, different samples of reactive lymphoid tissue, including tonsils (5 cases), thymus (5 cases), and lymph nodes (3 cases), were analyzed to determine the distribution of C/EBP in normal lymphoid tissue. All diagnoses were confirmed during the diagnostic workup by immunohistochemistry on paraffin-embedded tissue sections with a panel of antibodies to assess lymphoid phenotype. Anaplastic lymphoma kinase-1 (ALK-1; DakoCytomation, Carpinteria, CA) immunohistochemistry was performed on all cases of ALCL. Frozen tissue from 1 normal reactive lymph node, 4 primary ALK-positive ALCLs, 2 adult T-cell leukemia/lymphomas (ATLLs), 1 precursor T-cell lymphoblastic leukemia/lymphoma (ALL), and 1 unspecified peripheral T-cell lymphoma (PTCL) were selected for Western blot analysis. View this table: [in this window] [in a new window]   Table 2.. Primary lymphoma cases analyzed for C/EBP expression by immunohistochemistry   Immunohistochemistry All cases were previously studied by paraffin section immunohistochemistry (IHC) with a panel of antibodies to assess lymphoid phenotype and ALK in suspected cases of ALCL. The expression of C/EBP (clone H7; Santa Cruz Biotechnology, Santa Cruz, CA) was investigated on paraffin-embedded sections. Cases were scored as C/EBP positive when more than 20% of the tumor cells showed nuclear positivity. All immunohistochemical analyses were reviewed by 3 of the authors (L.Q.-M., S.P., M.R.). Immunohistochemistry was performed on an automated immunostainer (Ventana Medical Systems, Tucson, AZ) or autostainer (DakoCytomation) according to the manufacturer's protocols, with slight modifications. After deparaffinization and rehydration, the slides were placed in a microwave pressure cooker (TenderCooker; NordicWare, Minneapolis, MN) in 0.01 M citrate buffer, pH 6.0, containing 0.1% Tween 20 and were heated in a microwave oven at maximum power for 30 minutes or for 8 minutes (hot start). Thereafter, sections were washed in Tris-buffered saline (pH 7.6) containing 5% fetal calf serum (Life Technologies, Grand Island, NY) for 20 minutes. Antibodies were incubated overnight or for 2 hours at room temperature. The rest of the procedure in the DakoCytomation autostainer was performed using a peroxidase-based detection system (Envision+ System; DakoCytomation), and the slides were counterstained in Gill hematoxylin and mounted in Pertex (Histolab, Göteborg, Germany). Double staining for C/EBP and CD68 (DakoCytomation), S100 (Biogenex, San Ramon, CA), clusterin (Upstate Biotechnology, Lake Placid, NY), or rabbit monoclonal anti-CD3 (Lab Vision, Fremont, CA) was performed with a peroxidase-based system (Envision+ System). The primary antibody C/EBP was incubated overnight, and the secondary antibody was incubated for 30 minutes. Reaction was developed with the Envision+ System and 3,3'-diaminobenzidine as chromogen. Slides were blocked for 10 minutes with 3% peroxide and the second primary antibody. CD3, CD68, or S100 was incubated for 1 hour, and the secondary antibody was incubated for 30 minutes. The reaction was detected again with the Envision+ System and VIP substrate as chromogen (Vector Laboratories, Burlingame, CA) and was counterstained with Vector methyl green (Vector Laboratories). Western blot analysis Frozen tissue samples and cell line pellets were immersed in phosphate-buffered saline (PBS) containing one protease inhibitor cocktail tablet (Complete Mini; Roche Diagnostics, Mannheim, Germany) and were sonicated 3 times for 30 seconds each on ice with 30-second pauses. Protein concentration was determined by the BCA protein assay reagent kit (Pierce, Rockford, IL). Thirty micrograms of protein extracts were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and were transferred to pure nitrocellulose immobilization membranes (BA85; Protran, Dassel, Germany). Membranes were blocked overnight in 5% nonfat dry milk and were incubated with the primary antibody for 2 hours in 3% bovine serum albumin (BSA; Sigma, St Louis, MO). Subsequently, membranes were washed 5 times for 5 minutes each in a wash buffer (10 mM Tris, pH 7.6, 100 mM NaCl, and 0.1% Tween) and were incubated with a biotinylated secondary antibody for 1 hour. Membranes were washed 5 times in the same wash buffer, and detection was performed by SuperSignal West Pico chemiluminescent substrate (Pierce) for 5 minutes. Membranes were exposed to X-OMAT AR film (Eastman Kodak, Rochester, NY). All assays were repeated 3 times and gave similar results. Subcellular fractionation was performed using the NE-PER kit (Pierce). Immunoreagents used for Western blot were a rabbit polyclonal antibody against ALK (Zymed Laboratories, San Francisco, CA) and rabbit polyclonal anti-C/EBP (C-19; Santa Cruz Biotechnology). The C/EBP antibody recognized the 2 major isoforms, 35 kDa LAP and 21 kDa LIP. For loading control, mouse monoclonal anti–-tubulin (Sigma) was used. Additional antibodies used included monoclonal Stat3 (Transduction Laboratories, Lexington, KY) and phospho-Tyr705 Stat3 (pStat3) (clone 3E2; Cell Signaling, Beverly, MA). Cell culture and DNA transfections The murine pro–B-lymphoid cell line Ba/F3 and the murine 32Dc13 myeloid cell line (referred to hereafter as the 32D cell line) were maintained in RPM1 1640 (Gibco BRL, Karlsruhe, Germany) and were supplemented with 10% fetal calf serum (FCS; Biochrom KG, Berlin, Germany) and 1 ng/mL murine recombinant interleukin-3 (mIL-3; R&D Systems, DPC Bierman GmbH, Wiesbaden, Germany). BaF3 is a murine lymphoid cell line that requires IL-3 for proliferation and survival. It expresses only B-220 and contains immunoglobulin genes in germline configuration. Overexpression of NPM-ALK in BaF3 cells converted cells from IL-3–dependent to IL-3–independent growth.28 The 32D cell line is a myeloid progenitor, IL-3–dependent, diploid cell line that undergoes normal granulocytic differentiation in response to G-CSF. DNA transfections in Ba/F3 and 32D cell lines were performed by electroporation, as described previously.28,29 Ba/F3 Mig-NPM-ALK, Ba/F3-Mig-NPM-ALK-ATP-Abl, and Ba/F3-Mig (empty vector used as control) are derived from parental Ba/F3 cells modified by retroviral infection with the respective constructs. Ba/F3 Mig-NPM-ALK, Ba/F3-Mig-NPM-ALK-ATP-Abl, and 32D-Mig-NPM-ALK are mIL-3 independent. In the Mig-NPM-ALK-ATP-Abl (Mig-NAAA) construct, the ATP-binding site of ALK was replaced by the corresponding domain of the Abl kinase. This substitution renders NPM-ALK protein responsive to imatinib while it retains the NPM-ALK kinase substrate phosphorylation pattern (Petra Seipel, C.M., and J.D., manuscript in preparation). The protein band produced by this construct measured 85 kDa, whereas the band produced by Mig-NPM-ALK measured 75 kDa. For Western blot analysis, cells growing exponentially were pelleted and lysed, as described. Ba/F3 Mig-NAAA cells were treated with 5 µM imatinib for 12 hours to inhibit the NPM-ALK kinase activity or were left untreated before lysis and were used as control. Imatinib mesylate was kindly provided by Novartis Pharma (Basel, Switzerland). Real-time quantitative RT-PCR Real-time quantitative RT-PCR analysis was performed with the ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA). For the quantification of C/EBP and TATA box-binding protein (TBP) as the housekeeping gene, we used gene expression assays from Applied Biosystems (C/EBP, Mm00843434_s1; TBP, Mm00446973_m1). Two micrograms RNA was transcribed into cDNA using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) and a mix of Oligo(dT) primer and random hexamers (Roche, Penzberg, Germany) in a final volume of 50 µL according to the manufacturer's instructions. PCR was carried out with the TaqMan Universal Master Mix (Applied Biosystems) using 4 µL diluted cDNA in a 20-µL final reaction mixture (10 minutes at 95°C; 40 cycles of 15 seconds at 95°C and 60 seconds at 60°C). Before the experiments were begun, the linear range of the assays was validated with a dilution curve with transcribed embryonic mouse RNA. Data were analyzed using the CT method. Target gene expression was normalized to TBP by taking the difference between mean threshold PCR cycle values for target and control genes (CT value). This was then calibrated to the control sample in each experiment to give the CT value, where the control had a CT value of 0. The fold target gene expression, compared with the calibrator value, is given by the formula 2–CT. All reactions were performed at least twice in duplicate.    Results Top Abstract Introduction Materials and methods Results Discussion References   C/EBP protein expression in normal lymphoid tissue C/EBP was evaluated by immunohistochemistry performed on paraffin sections of reactive lymph node, tonsil, and thymus. In tonsils and lymph nodes, C/EBP was strongly expressed in the nuclei of scattered cells in the germinal centers, mantle zone, and paracortical region (Figure 1A). The morphology of the C/EBP-positive cells in the germinal centers corresponded to that of follicular dendritic cells (FDCs) (Figure 1A, inset), whereas the tingible body macrophages were negative or weakly positive (Figure 1A). In contrast, histiocytes in the sinuses, interdigitating cells, and monocyte-like cells were strongly positive for C/EBP. Double stainings demonstrated that C/EBP-positive cells were also positive for clusterin, a marker of follicular dendritic cells (Figure 1B), whereas T and B cells were negative for C/EBP (Figure 1C-D). In the tonsils, epithelium surface cells were strongly positive for C/EBP (Figure 1A). In the thymus, neither cortical nor medullary T cells showed expression of C/EBP. CEBP staining was seen only in thymic epithelial cells and in rare macrophage-like cells. C/EBP is highly expressed in ALK-positive ALCL To assess C/EBP expression in malignant lymphoid neoplasms, 229 well-characterized lymphomas—including 93 B-cell lymphomas, 107 T-cell lymphomas (33 PTCL, 74 ALCL), and 29 Hodgkin lymphomas—were immunostained for C/EBP. Intratumoral histiocytes, present in all cases, served as internal positive controls (Figure 1E). Results are summarized in Table 2. Of the 93 B-cell NHLs, only 1 (1%) case of multiple myeloma with anaplastic features stained positively for C/EBP. All 9 cases of nodular lymphocyte-predominant Hodgkin lymphoma were negative for C/EBP, whereas 5 of 20 cases of classical HL showed faint nuclear staining within the Reed-Sternberg cells (Figure 1F-G). Of the 33 cases of PTCL, only 1 showed weak staining for C/EBP in a small number of tumor cells (Figure 1H-J). In some cases, the presence of numerous C/EBP–positive cells made it difficult to evaluate whether these cells corresponded to the neoplastic T-cell population of the reactive myelomonocytic cells. In these cases, double staining with CD3 revealed that the neoplastic T cells were negative for C/EBP (Figure 1K) and that most C/EBP–positive cells were positive for CD68 or S100 (Figure 1J). View larger version (158K): [in this window] [in a new window]   Figure 1.. Immunohistochemical analysis of C/EBP expression in normal lymphoid tissue and in different lymphoid neoplasias. (A) C/EBP protein expression in normal lymphoid tissue (tonsil). In the germinal center, mantle zone, and interfollicular T-cell area, C/EBP is strongly expressed in the nuclei of scattered cells, whereas most surface epithelial cells express high levels of C/EBP. Immunoperoxidase (IP) stain, 100x. C/EBP-positive cells within the germinal center correspond to follicular dendritic cells (inset; IP stain, 400x). (B) Double staining for C/EBP (brown) and clusterin (purple) reveals that the C/EBP-positive germinal center cells express the dendritic cell marker clusterin. Note also that the clusterin stain highlights the dendritic cell processes. IP stain, 650x. (C-D) Double staining for C/EBP (brown) and CD3 (purple) (C) or CD20 (purple) (D) in the germinal center demonstrates that the CD3+ T cells and CD20+ B cells are negative for C/EBP. IP stain, 650x. (E) C/EBP expression in mantle cell lymphoma. The neoplastic cells are negative, whereas the intratumoral histiocytes are positive and serve as an internal positive control. IP stain, 100x. (F-G) C/EBP in 2 cases of classic Hodgkin lymphoma. Faint nuclear reactivity is seen in the Reed-Sternberg cells in 1 case (G), whereas the second case (F) shows no C/EBP expression. In both cases, abundant histiocytes are strongly positive for C/EBP. IP stain, 650x. (H) C/EBP expression in unspecified PTCL. Neoplastic cells are negative, whereas intratumoral histiocytes are positive. IP stain, 100x. (I-J) C/EBP expression in a second case of unspecified PTCL. (I) Double staining for C/EBP (brown) and CD3 (purple) demonstrates that a small population of the neoplastic T cells (arrows) shows weak nuclear staining for C/EBP. In contrast, compare the strong nuclear positivity of the reactive histiocytes. IP stain, 400x. (J) Double staining for C/EBP (brown) and CD68 (purple) confirms the histiocytic origin of the strong C/EBP-positive cells. IP stain, 650x. (K) Unspecified PTCL with abundant histiocytes. Double staining for C/EBP (brown) and CD3 (purple) demonstrates that C/EBP is not expressed in the CD3+ tumor cells. IP stain, 100x. (L) C/EBP expression in ALCL, ALK-negative, with weak nuclear staining in tumor cells. In contrast, reactive histiocytes showed strong nuclear positivity with C/EBP. IP stain, 250x. Images were acquired using a Hitachi camera HW/C20 (Hitachi, Tokyo, Japan) installed in a Zeiss Axioplan microscope (Zeiss, Jena, Germany) using Intellicam software. Plan-Neofluar 10x/0.30 numeric aperture (NA), 20x/0.50 NA, and 40x/0.75 NA objectives as well as a Plan-Apochromat 63x/1.40 oil objective were used. Images were processed using Adobe Photoshop (Adobe Systems, San Jose, CA).   Of the 74 cases of CD30+ ALCL, 45 were ALK positive and 29 were ALK negative (Figure 2). C/EBP was strongly positive in most tumor cells in 40 of 45 (88%) cases of ALK-positive ALCL. C/EBP expression was independent of the cytoplasmic or nuclear localization of ALK (Figure 2). ALK-negative cases were C/EBP negative or were weakly positive in 8 of 29 cases. (Figures 1L, 2). Because of the striking association between C/EBP and ALK-positive ALCL, the remainder of the study focuses on these lymphomas. C/EBP Western blot analysis: LIP is expressed more than LAP Overexpression of the LIP isoform of C/EBP has been associated with high proliferation states in normal tissues and has been observed in several types of epithelial tumors. For these reasons, we were particularly interested in investigating the LIP/LAP ratio in ALK-positive ALCL. To assess the LIP/LAP ratio, 4 ALK-positive ALCL cell lines and 4 primary ALK-positive ALCL cases were studied by Western blot analysis using the C-terminal–specific anti–C/EBP (C-19) antibody that recognizes both LIP and LAP. For comparison, we also assessed expression in a variety of non–ALCL T- and B-cell lines (4 and 11 cell lines, respectively) and in several non–ALCL T-cell neoplasms. The positive control epithelial cell lines HeLa and MCF7 demonstrated 2 bands of the approximate expected sizes for the LIP and LAP isoforms (21 and 35 kDa, respectively) (Figure 3). ALCL lines Ki-JK, Karpas 299, and SUDHL-1 expressed both isoforms of C/EBP, with more LIP than LAP isoforms. SR786 showed only weak expression of the LIP isoform (Figure 3B). Immunocytochemistry of the 4 cell lines showed strong nuclear staining for total C/EBP in Ki-JK, Karpas 299, and SUDHL-1, with weaker nuclear staining observed in SR786 (data not shown). Subcellular fractionation of the SUDHL-1 cell line demonstrated that both isoforms were exclusively expressed in the nucleus (Figure 3A). Other B- and T-cell lymphoma cell lines in this survey expressed LIP weakly (SUDHL-4, SUDHL-6, SUDHL-10, OPM2, KMM1, Jim3, ST 486, MOLT4) or were negative for the 2 isoforms (CEM, Jurkat, Hut102, Granta 519, NCEB1, KMS12) (Figure 3B-C and data not shown). Weak expression of C/EBP in the myeloma and B-cell lymphoma lines may be linked to the endogenous production of IL-6, a known inducer of C/EBP. View larger version (169K): [in this window] [in a new window]   Figure 2.. ALK and C/EBP immunostaining in ALCL. The 2 ALK-positive cases, with nuclear (top) and cytoplasmic (middle) expression, show strong nuclear positivity in most tumor cells for C/EBP. The ALCL, ALK-negative case (bottom) is negative for C/EBP; however, the reactive histiocytes are strongly positive and serve as internal control. IP stain, 400x. Images were otherwise acquired as explained in Figure 1.   View larger version (32K): [in this window] [in a new window]   Figure 3.. Western blot analyses for C/EBP and ALK in lymphoid cell lines and primary lymphoma cases. (A) Subcellular fractionation of the SUDHL-1 cell line. Tubulin is used as control for loading and for the cytoplasmic fraction. (B) KiJk, Karpas 299, SUDHL1, and SR786 represent ALCL cell lines with the t(2;5) translocation. The non-t(2;5) T-cell lines are Molt4, CEM, and Jurkat. Tubulin is used as loading control. (C) Western blot analysis in B-cell lymphoid cell lines including 2 mantle cell lymphoma lines (Granta 519 and NCEB-1), 2 multiple myeloma cell lines (KMS12 and OPM2), and 2 follicular lymphoma cell lines (SUDHL4 and SUDHL10). The ALCL cell line Karpas 299 and the 2 carcinoma cell lines MCF7 and HeLa are used as controls. Tubulin is used as loading control. (D) Western blot analysis in primary T-cell lymphoma cases including 1 normal lymph node (LN), 4 primary ALK-positive (ALCL) lymphomas, 1 acute lymphoblastic leukemia (ALL), 2 adult T-cell leukemia/lymphoma (ATLL), and unspecified PTCL. Tubulin is used as loading control.   The strong expression of C/EBP and high levels of LIP were also confirmed by Western blot in 4 primary ALK-positive ALCL cases (Figure 3D). Reactive lymph node, acute lymphoblastic lymphoma, PTCL, and both cases of adult T-cell lymphoma/leukemia were negative for C/EBP. C/EBP is induced in NPM-ALK–transformed Ba/F3 and 32D cell lines To determine the role of NPM-ALK in the expression of C/EBP in ALK-positive ALCL, Ba/F3 and 32D cells were transfected with NPM-ALK. Both the transfected Ba/F3 cells and the 32D cells showed strong expression of NPM-ALK and activated Stat3 (pStat3), a known downstream target of the NPM-ALK kinase (Figure 4A). The parental Ba/F3 and 32D cell lines were negative for NPM-ALK and pStat3, as expected. All cell lines were positive for total Stat3; however, the transfected cell lines expressed higher amounts of Stat3. Western blot analysis of C/EBP revealed that parental Ba/F3 cells expressed no C/EBP, whereas parental 32D myeloid cell line expressed a modest level of C/EBP. The latter finding was not unexpected because myeloid cells and macrophages expressed C/EBP normally. Significantly, both NPM-ALK–transformed cell lines showed strong induction of C/EBP LIP and LAP isoforms to levels comparable with those of the ALCL cell line Karpas 299. C/EBP expression is induced through the kinase activity of NPM-ALK To demonstrate whether the expression of C/EBP in ALK+ALCL primary cases and cell lines was dependent on NPM-ALK kinase activity, we transformed Ba/F3 cells with an NPM-ALK-ATP-Abl construct. This construct was identical to the NPM-ALK construct except that the ATP-binding site of ALK has been replaced by the corresponding domain of the Abl-kinase, rendering NPM-ALK responsive to imatinib. As a result, the NPM-ALK kinase activity could be completely inhibited. Western blot analysis demonstrated the expression of NPM-ALK in treated and untreated Ba/F3 NPM-ALK-ATP-Abl cells (Figure 4A). In contrast, though pStat3 was expressed in the untreated Ba/F3 NPM-ALK-ATP-Abl cells, it was undetectable in the corresponding imatinib-treated cells, confirming that the NPM-ALK kinase activity was switched off. Significantly, C/EBP was expressed in the untreated Ba/F3 NPM-ALK-ATP-Abl cells but was greatly reduced in the imatinib-treated Ba/F3 NPM-ALK-ATP-Abl cells, indicating the dependence of C/EBP expression on NPM-ALK kinase activity. View larger version (30K): [in this window] [in a new window]   Figure 4.. ALK, Stat3-phosphorylated and C/EBP expression in NPM-ALK and NPM-ALK-ATP-Abl–transformed Ba/F3 and 3D cells. (A) Western blot analysis of NPM-ALK–transformed Ba/F3 and 32D cell lines. Each lane contains 30 µg protein extract. The ALCL cell line Karpas 299 is used as control. The NPM-ALK construct produces a band of 75 kDa. Tubulin is used as loading control. (B) Western blot analysis of Ba/F3 NPM-ALK-ATP-Abl–transformed cell line untreated or treated with 5 µM imatinib for 12 hours. The NPM-ALK-ATP-Abl construct produces a positive protein band of 85 kDa, 5 kDa larger than the protein band detected in the SUDHL-1 cell line used as control. Tubulin is used as loading control.   C/EBP is transcriptionally regulated by NPM-ALK To determine whether the induction of C/EBP by NPM-ALK was transcriptionally or posttranscriptionally regulated, we analyzed C/EBP expression in parallel at the protein and mRNA levels of parental Ba/F3- and NPM-ALK–transformed cells. Western blot analysis showed the induction of C/EBP in NPM-ALK–transformed Ba/F3 cells, whereas the amount of C/EBP mRNA increased approximately 2-fold (Figure 5). To confirm that C/EBP was transcriptionally regulated by NPM-ALK, we also analyzed C/EBP protein and mRNA expression in the NPM-ALK-ATP-Abl–transformed Ba/F3 cells following treatment with imatinib or control buffer after 6 and 20 hours. Western blot analysis confirmed the strong expression of C/EBP in the untreated NPM-ALK-ATP-Abl cell line, weak expression in the imatinib-treated NPM-ALK-ATP-Abl cells after 6 hours, and complete LIP inhibition after 20 hours (Figure 5B). Interestingly, pStat3, used as control for the kinase activity of NPM-ALK, was undetectable in the imatinib-treated NPM-ALK-ATP-Abl cells after 6 hours, signifying that the inhibition of pStat3 occurred shortly after treatment with imatinib and before the inhibition of C/EBP. C/EBP mRNA levels were 5.2-fold higher in the untreated NPM-ALK-ATP-Abl cell line than in the parental BaF3 cell line and steadily decreased with an ongoing blockade of NPM-ALK-ATP-Abl by imatinib 3.5-fold after 6 hours and 1.2-fold after 20 hours of treatment with imatinib. There was a very good correlation between C/EBP protein expression and mRNA level. Together both results indicated that the induction of C/EBP expression by NPM-ALK occurred at the transcriptional level. View larger version (23K): [in this window] [in a new window]   Figure 5.. Qualitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis of C/EBP mRNA levels and protein expression in NPM-ALK and NPM-ALK-ATP-Abl–transformed Ba/F3 cells. (A) Western blot analysis of transformed Ba/F3 cells. Each lane contains 30 µg protein extract. The ALCL cell line Karpas 299 is used as control. Stat3 phosphorylate is used as control for the kinase activity of NPM-ALK. The NPM-ALK-ATP-Abl construct produces a positive protein band of 85 kDa. Tubulin is used as loading control. (B) qRT-PCR analysis of C/EBP was performed relative to the TBP housekeeping gene. Data were analyzed according to the CT method. Results are depicted as mRNA fold induction compared with Ba/F3 parental cells. Error bars indicate SD.      Discussion Top Abstract Introduction Materials and methods Results Discussion References   C/EBP is a multifunctional transcription factor that plays important roles in the proliferation and differentiation of a variety of cell types, including B and T cells, and its aberrant expression has been implicated in the pathogenesis of several epithelial tumors.16-19,30 Limited information is available concerning its expression in normal lymphoid tissues, and virtually no information is available on its expression in lymphoid neoplasms. For this reason, we investigated C/EBP expression in normal lymphoid tissues and in a large number of primary lymphomas and lymphoma cell lines, including examples from most major subgroups. Normal nonneoplastic lymphoid cells from thymus, tonsil, and lymph node and most B-cell and T-cell NHLs did not express immunohistochemically detectable C/EBP. In striking contrast, we found that most (88%) cases of ALK-positive ALCL strongly expressed C/EBP. Significantly, the LIP isoform of C/EBP was preferentially expressed in the ALK-positive ALCL cell lines and in the primary cases examined. Furthermore, we showed in 2 cell line systems that enforced expression of NPM-ALK and kinase-inhibitable modified NPM-ALK resulted in the induction of C/EBP only when the kinase activity was intact, demonstrating not only that NPM-ALK is capable of inducing C/EBP expression but also that its induction is transcriptionally dependent on NPM-ALK kinase activity. ALCL represents a distinct type of NHL of T or null phenotype with unique morphologic features and CD30 antigen expression.31 Recently, it has become evident that there are 2 forms of ALCL based on the presence or absence of the characteristic cytogenetic abnormality, the t(2;5) chromosomal translocation, which juxtaposes the anaplastic lymphoma kinase (ALK) gene at 2p23 to the nucleophosmin (NPM) gene at 5q35, resulting in the expression of a chimeric protein called NPM-ALK.32 Other variant translocations involving fusion of the ALK gene with other partner genes have also been described.33 All translocations generated ALK fusion proteins capable of autodimerization, leading to constitutive activation of the ALK-tyrosine kinase, believed to initiate the process of lymphomagenesis.29,34,35 NPM-ALK interacts with many adaptor proteins and activates several key signaling pathways involved in cell proliferation, transformation, and survival. These include the p85 regulatory subunit of type 1A phosphatidylinositol 3-kinase (PI3K), which activates AKT and other signaling intermediates,28,36 and the JAK/STAT pathway, which plays an important role in the mitogenic signaling initiated by NPM-ALK and protects hematopoietic cells from apoptosis.37,38 In addition, prosurvival PLC- pathways and MAPK pathways have been implicated in NPM/ALK signaling.39,40 In the current study, we have shown that C/EBP expression is dependent on NPM-ALK kinase activity. However, it is unclear how this signal is transduced. The complex signaling networks activated by NPM-ALK suggest several possibilities. Among the possible activators is Stat3. Stat3 has been shown to be directly activated in most ALK-positive ALCLs.37,38,41 We also have found Stat3 activated in a large subset of the current cases (data not shown). Like C/EBP, this transcription factor has a role in the control of inflammatory and native immune responses.3 Although no Stat3-specific binding sites have been observed in the C/EBP promoter region, a novel tethering mechanism is thought to link Stat3 to the C/EBP promoter.42 A second possible pathway of C/EBP activation in ALCL is through MAPK signaling.40 Several adaptor proteins involved in the MAPK pathway, including SHC, GRB-2, and IRS-1, have been coimmunoprecipitated with NPM-ALK. Furthermore, normal ALK has been shown to activate the MAPK pathway in PC12 pheochromocytoma cells.43 Independently, other studies have shown that C/EBP activity can be stimulated by phosphorylation mediated through the RAS/MAPK pathway.44,45 Once activated (phosphorylated), C/EBP can bind to its own promoter and participate in a positive feedback loop, potentially resulting in the high levels of C/EBP seen in ALCL. Finally, several investigators have shown that NPM-ALK activates the P13K/AKT pathway.28,36 Among the many targets of AKT is mammalian target of rapamycin (mTOR), a critical regulator of several translational control proteins, including S6K-1 and translation elongation factors eIF-2 and eIF-4E.46 Activation of these factors through mTOR signaling has recently been shown to affect C/EBP expression and to increase the LIP/LAP ratio through the regulation of translation initiation in ALCL and Hodgkin lymphoma cell lines.47 Given the association of LIP with proliferation in other tumor models, the authors suggested that translationally deregulated expression of C/EBP could play an important role in ALCL and contribute to sustained cellular proliferation47 and that translational control of C/EBP isoforms could be used as a target of proliferation control and therapeutic intervention. Our data not only corroborate their finding that ALCL cell lines express high levels of LIP, they extend this finding to primary ALK-positive ALCL. Although it appears likely that mTOR plays a role in the modulation of C/EBP isoforms in ALCL cell lines, this must be confirmed in primary cases. Whether the PIK3/AKT/mTOR pathway is also involved in the induction of C/EBP or only in its modulation is still unclear. The consequences of C/EBP expression in ALCL are unknown. In addition to its effect on proliferation, C/EBP expression has been shown to promote TH2 responses in T cells.24,48 It has recently been shown that ALCL cells display a chemokine profile similar to that of TH2 cells.49 Thus, it is possible that the expression of C/EBP plays a role in the cytokine and chemokine profiles of ALCL cells. C/EBP has also been associated with activation or terminal differentiation in the monocyte/macrophage lineages.1,3 ALCL shows a loss of important T-cell antigens such as CD3 and ZAP-70,50 and it expresses some proteins shared with monocytic and dendritic cells such as clusterin,51 CD4, and CD68. A recent report indicates that enforced expression of either C/EBP or C/EBP in differentiated B cells leads to their rapid and efficient reprogramming into macrophages, with a loss of B-cell markers and an up-regulation of macrophage markers.52 Similar transdifferentiation effects have been preliminarily reported in thymocytes, in which stage-specific enforced expression of C/EBP and C/EBP induced the down-regulation of T-cell markers and the up-regulation of myelo-monocytic lineage markers.53 These experiments suggest that the aberrant expression of C/EBP could be responsible for some of the phenotypic characteristics seen in ALCL T cells. In conclusion, we report for the first time the constitutive activation of the transcription factor C/EBP in ALK-positive ALCL. In addition, we demonstrate that NPM-ALK is capable of inducing C/EBP and that the induction is dependent on NPM/ALK kinase activity. The link between CEBP LIP overexpression and proliferation and the role of C/EBP in TH2 T-cell commitment and in myelomonocytic differentiation suggest that C/EBP likely plays an important role in the pathogenesis and unique phenotype of this lymphoma.    Acknowledgements   We thank Daniela Angermeier for assistance with Western blot analysis, and we thank Jacqueline Müller, Elenore Samson, and Thomas Fountaine III and the staff at the Immunohistochemistry Unit of the Laboratory of Pathology, National Cancer Institute, for assistance with the immunohistochemical stains.    Footnotes   Submitted October 7, 2005; accepted May 11, 2006. Prepublished online as Blood First Edition Paper, May 18, 2006; DOI 10.1182/blood-2005-10-014258. Supported in part by grant FE 597/1-1 from the Deutsche Forschungsgemeinschaft (L.Q.-M., F.F.). L.Q.-M. designed and performed research, analyzed data, and wrote the manuscript; S.P. performed research and analyzed data; C.M. performed research and contributed vital new reagents; M.K., M.R., T.D.-H., N.A., A.M., and A.V. performed research; J.D. analyzed data and contributed vital new reagents; E.S.J. contributed cases and analyzed data; F.F. analyzed data and contributed to the design and writing of the manuscript; and M.R. designed research, analyzed data, and wrote the manuscript. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. 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