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Gene Knockdown Studies Revealed CCDC50 As a Candidate Gene in Mantle Cell Lymphoma and Chronic Lymphocytic Leukemia

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Gene Knockdown Studies Revealed CCDC50 As a Candidate Gene in Mantle Cell Lymphoma and Chronic Lymphocytic Leukemia Leukemia (2009) 23, 2018–2026 & 2009 Macmillan Publishers Limited All rights reserved 0887-6924/09 $32.00 www.nature.com/leu ORIGINAL ARTICLE Gene knockdown studies revealed CCDC50 as a candidate gene in mantle cell lymphoma and chronic lymphocytic leukemia A Farfsing1, F Engel1, M Seiffert1, E Hartmann2, G Ott2,5, A Rosenwald2, S Stilgenbauer3,HDo¨hner3, M Boutros4, P Lichter1 and A Pscherer1 1Division of Molecular Genetics, German Cancer Research Center (DKFZ), Heidelberg, Germany; 2Institute of Pathology, Junior Research Group ‘Functional Genomics‘, University of Wurzburg, Wurzburg, Germany; 3Department of Internal Medicine III, University hospital Ulm, Ulm, Germany; 4Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and University of Heidelberg, Heidelberg, Germany and 5Institute of Clinical Pathology, Robert-Bosch-Hospital, Stuttgart, Germany The two B-cell non-Hodgkin’s lymphoma entities, chronic 45, 17 and 14% of cases, respectively.14 Candidate genes within lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), these chromosomal regions have been proposed by various groups show recurrent chromosomal gains of 3q25–q29, 12q13–q14 and 18q21–q22. The pathomechanisms affected by these on the basis of profiling data acquired on different microarray aberrations are not understood. The aim of this study was to platforms (single-nucleotide polymorphism array, array compara- 7–13,15–24 identify genes, located within these gained regions, which tive genomic hybridization, expression array). Gains of control cell death and cell survival of MCL and CLL cancer cells. genomic DNA may activate oncogenes through gene dosage Blood samples collected from 18 patients with CLL and 6 effects,20,22 such as CDK4 (cyclin-dependent kinase-4) on 12q13 patients with MCL, as well as 6 cell lines representing both and B-cell lymphoma-2 (BCL2) on 18q21 in MCL.1,8,11,15,18,25 It malignancies were analyzed by gene expression profiling. By a comparison of genomic DNA and gene expression, 72 candi- was recently shown that highly proliferative and clinically date genes were identified. We performed a limited RNA aggressive variants of MCL have a complex karyotype with 11,22,26 interference screening with these candidates to identify genes frequent gains on 3q and 12q. Furthermore, the chromoso- affecting cell survival. CCDC50 (coiled coil domain containing mally gained region, 3q25–q29, shows an association with poor protein 50), SERPINI2 and SMARCC2 mediated a reduction of outcome in MCL patients.22 cell viability in primary CLL cells as well as in cell lines. Gene Chronic lymphocytic leukemia is the most common leukemia knockdown and a nuclear factor kappa B (NFjB) reporter gene assay revealed that CCDC50 is required for survival in MCL and among adults of the western world, with a variable survival time CLL cells and controls NFjB signaling. between ca. 3 and 20 years. CLL is characterized by the Leukemia (2009) 23, 2018–2026; doi:10.1038/leu.2009.144; accumulation of mature, but resting B cells in peripheral blood, published online 30 July 2009 bone marrow and lymphatic or extralymphatic tissues. The Keywords: B-cell chronic lymphocytic leukemia; mantle cell majority of leukemic CLL cells are arrested in the cell cycle, lymphoma; siRNA screen; functional assays in primary CLL cells; mainly in the G0/G1 phase.27,28 Unlike other leukemias, there is CCDC50 only a small proportion of proliferating neoplastic cells that are localized in the so-called ‘pseudofollicular’ proliferation centers Introduction in the lymph nodes or are scattered in the bone marrow of the patients.29,30 The most frequently recurring chromosomal gain, Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma identified in CLL patients, is trisomy 12.27 (MCL) are B-cell non-Hodgkin’s lymphoma subtypes that share The aim of this study was to identify genes with oncogenic patterns of genetic aberrations. The median survival time of potential in recurrently gained chromosomal regions of MCL patients with MCL was reported to be 3–5 years after diagnosis.1 and CLL. To this aim, gene expression profiling was performed, A criterion for diagnosis of MCL is the translocation followed by cell survival and proliferation studies after silencing t(11;14)(q13;q32), resulting in the overexpression of the cyclin of candidate genes. First, we profiled the expression of 18 D1 gene.2–5 In addition to the t(11;14), MCL carries a high primary CLL, 6 primary MCL samples, as well as 6 cell lines, and number of secondary genetic alterations that may contribute to compared all genes identified in the three gained regions (3q, its pathogenesis. Several studies reported the high resolution 12q and 18q) with recently published data. Second, we detection of chromosomal imbalances in MCL using array investigated a set of 72 candidate genes derived from this comparative genomic hybridization,6 accurately defining the analysis by the use of a small interfering RNA (siRNA)-mediated gained regions.7–13 However, these regions still contain too loss of function screen in a multiwell format in MCL cell lines. many genes to enable a reasonable selection of candidates, and Third, we validated the observed changes in cell viability by it is not clear which genes have functional relevance for MCL. gene knockdown in primary CLL cells and analyzed the Recently, the incidence of genomic gains in t(11;14)(q13;q32)- downstream effects of the identified candidate gene, CCDC50 positive MCL cases was assessed by fluorescence in situ (coiled coil domain containing protein 50). hybridization analysis, revealing gains on 3q, 12q and 18q in Correspondence: Professor Dr P Lichter, Division of Molecular Materials and methods Genetics, German Cancer Research Center, Im Neuenheimer Feld 280, Heidelberg 69120, Germany. E-mail: [email protected] Cell lines Received 4 June 2009; accepted 12 June 2009; published online 30 Cell lines were obtained from DSMZ (Braunschweig, Germany) July 2009 and from ATTC (American Type Culture Collection, Manassas, CCDC50 as candidate gene in MCL and CLL A Farfsing et al 2019 VA, USA). Granta (ACC 342), Mec-1 (ACC 497) and HEK-293T RNA isolation, synthesis of cDNA, quantitative (Human embryonic kidney 293-T cells, CRL-1573) cells were real-time PCR and flow cytometry cultured in DMEM (Dulbecco’s modified Eagle’s medium) RNA isolation of primary CLL cells and cell lines was performed (Invitrogen, Karlsruhe, Germany). JVM-2 (Human peripheral using the RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA blood B-cell lymphoma cell line, ACC 12) and LCL-WEI synthesis, quantitative real-time (qRT)-PCR and flow cytometry (Human lympoblastoid B cells, ACC 218) were cultured in of cells were performed as published earlier.32–35 RPMI 1640 medium, including 2 mML-glutamine (Invitrogen, Carlsbad, CA, USA). Both media were supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. siRNA The chemically synthesized siRNAs siCONTROL Non-Targeting Primary cells siRNA Pool 2 (D-001206-14-05) and ON-TARGETplus siCON- TROL Non-targeting Pool (D-001810-10-05) were obtained Peripheral blood samples were collected from patients with CLL from Dharmacon Inc. (Chicago, IL, USA). The siRNAs for and MCL, as well as from healthy donors after informed consent CCDC50 (siCCDC50 1:ID129977, siCCDC50 2: ID129978, was obtained from them (Supplementary Tables S1–S6). All siCCDC50 3: ID129979), Silencer Firefly Luc GL2/3 (AM4629), cases matched standard diagnostic criteria.31 The human bone Negative Control 1 (AM4636) and Silencer Select negative marrow stromal cell line, HS-5, was purchased from the ATCC. control (4390844) were obtained from Ambion Inc. The Cells were cultured and prepared as published earlier.32 negative control Control_AllStars_1 (SI03650318), as well as the 72 siRNA pools for the RNA interference screen, were Nucleofection of cell lines and primary CLL cells obtained from Qiagen. Information about individual siRNA Using the Human B-cell Nucleofector Kit and program U-015, sequences used in the screening can be found in Supplementary 6 5 Â 10 primary B cells were transfected with 500 nM siRNA (in Table S10. 100 ml volume), according to the manufacturer’s instructions (Lonza, Cologne, Germany). After nucleofection, primary cells were added to a sterile filtered conditioned medium, obtained Western blot analysis from HS-5 cells, and cultured as published earlier.32 Transfec- Transfected CLL cells were harvested by centrifugation tions of cell lines were performed by nucleofection (solution T, (800 r.p.m., 10 min, room temperature). Cell pellets were lysed program O-017), according to the manufacturer’s instructions, and protein extracts were purified using the All Prep RNA/ 6 using 2 Â 10 cells and 2 mg DNA or 500 nM siRNA (in 100 ml Protein Kit (Qiagen). Western blot analysis was carried out as volume). Cells were harvested 24, 48 and 72 h after transfection published previously.34 After immunoblotting, polyvinylidene for RNA isolation and functional assays. Using 96-well Shuttle fluoride membranes were probed with primary antibodies system with the 96-well Nucleofector Kit SF and program specific for CCDC50 (HPA001336, Sigma Aldrich, St Louis, 96-DN-100, a total of 4 Â 105 cells of the cell lines JVM-2 or MO, USA) and GAPDH (glyceraldehyde-3-phosphate dehydro- Granta-519 were transfected with 500 nM siRNA (in 20 ml genase) (CB1001, Calbiochem, Darmstadt, Germany). Second- volume), according to the manufacturer’s instructions (Lonza). ary antibodies used were anti-rabbit HRP (horseradish peroxidase) and anti-mouse HRP (Cell Signaling Inc., Danvers, MA, USA). Transfection of HEK-293T cells 5 A total of 4 Â 10 HEK-293T cells were transfected with 50 nM m siRNA or 2.5 g plasmid DNA using the TransIT transfection Cell viability assay reagent, according to the manufacturer’s instructions (Mirus Bio The viability of transfected cells was assayed using the Cell Titer LLC, Madison, WI, USA).
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