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ORIGINAL ARTICLE Amplification at 7Q22 Targets Cyclin-Dependent Kinase 6 in T-Cell Lymphoma

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ORIGINAL ARTICLE Amplification at 7Q22 Targets Cyclin-Dependent Kinase 6 in T-Cell Lymphoma Leukemia (2008) 22, 387–392 & 2008 Nature Publishing Group All rights reserved 0887-6924/08 $30.00 www.nature.com/leu ORIGINAL ARTICLE Amplification at 7q22 targets cyclin-dependent kinase 6 in T-cell lymphoma S Nagel1, E Leich2, H Quentmeier1, C Meyer1, M Kaufmann1, HG Drexler1, A Zettl2, A Rosenwald2 and RAF MacLeod1 1Department of Human and Animal Cell Cultures, DSMZ, Braunschweig, Germany and 2Institute of Pathology, University of Wu¨rzburg, Wu¨rzburg, Germany Recurrent chromosomal aberrations in hematopoietic tumors Organization, T-cell lymphomas include anaplastic large-cell target genes involved in pathogenesis. Their identification and lymphoma (ALCL) and peripheral T-cell lymphoma, not other- functional characterization are therefore important for the wise specified (PTCL NOS).4 ALCL is rareFrepresenting roughly establishment of rational therapies. Here, we investigated F genomic amplification at 7q22 in the T-cell lymphoma cell line 3% of all lymphomas and the clinical course differs signifi- SU-DHL-1 belonging to the subtype of anaplastic large-cell cantly between ALK-positive (ALK þ ) and ALK-negative (ALKÀ) lymphoma (ALCL). Cytogenetic analysis mapped this amplicon ALCL patients.3 The main chromosomal rearrangement in ALCL, to 86–95 Mb. Copy-number determination quantified the ampli- t(2;5)(p23;q35), fuses NPM1 to ALK, which generates a fication level at 5- to 6-fold. Expression analysis of genes constitutively active kinase-enhancing proliferation.5,6 Genes located within this region identified cyclin-dependent kinase 6 dysregulated by non-chromosomal mechanisms in ALCL cells (CDK6) as a potential amplification target. In comparison with control cell lines, SU-DHL-1 expressed considerably higher include CCND3 and CDKN1B coding for cyclin D3 and p27, levels of CDK6. Functionally, SU-DHL-1 cells exhibited reduced respectively, both key cell cycle regulators, highlighting the sensitivity to rapamycin treatment, as indicated by cell growth proliferative aspect of this disease.7,8 PTCL NOS is predomi- and cell cycle analysis. Rapamycin reportedly inhibits degrada- nantly a nodal lymphoma and comprises up to 70% of T-cell tion of the CDK inhibitor p27 with concomitant downregulation lymphomas in western countries.3 Cytogenetic abnormalities of cyclin D3, implying a proliferative advantage for CDK6 are common, although few recurrent aberrations have been overexpression. Amplification of the CDK6 locus was analyzed 9 in primary T-cell lymphoma samples and, while detected described. infrequently in those classified as ALCL (1%), was detected in Cyclin-dependent kinases (CDKs) are key regulators of the cell 23% of peripheral T-cell lymphomas not otherwise specified. cycle. Diverse CDKs are known, namely CDK1–10 in mammals. Taken together, analysis of the 7q22 amplicon identified CDK6 The G1–S phase transition of the cell cycle depends on CDK2, as an important cell cycle regulator in T-cell lymphomas, CDK4 and CDK6, which interact with D-type cyclins and representing a novel potential target for rational therapy. phosphorylate retinoblastoma protein family members.10 CDK Leukemia (2008) 22, 387–392; doi:10.1038/sj.leu.2405028; inhibitors repress the activity of CDKs and encompass INK4 and published online 8 November 2007 11 Keywords: ALCL; PTCL NOS; amplification; CDK6; BRCA1 KIP protein families, which include p27. Here, we describe the analysis of an amplicon in the ALCL- derived cell line SU-DHL-1 located at 7q22. This region is part of a larger amplicon described previously in ALCL cells. The reduced amplicon in SU-DHL-1 allows more focused analysis of Introduction genes located within this region. Accordingly, the aim of this study was the identification and characterization of candidate Recurrent chromosomal translocations and amplifications spe- genes targeted by 7q22 amplification. cifically target oncogenes involved in tumor development and maintenance. This underlines the importance of their character- ization for understanding pathomechanisms of cancer develop- Materials and methods ment and uncovering rational targets for therapeutic intervention. Unlike tumor-suppressor genes and oncogenes Cell culture and patient samples targeted for genomic amplification, leukemic oncogenes acti- ALCL-derived cell lines DEL, KARPAS-299, L-82, SR-786, SU- vated by translocation are often developmental and, thus, rarely DHL-1 and SUP-M2, as well as the cell lines HSB-2 and MOLT- involved in solid tumors.1 Gene amplification within chromo- 3 are held at the DSMZ, Braunschweig, Germany somal amplicons, manifesting as ‘double minute’ chromosomes (www.dsmz.de); MAC-2A was provided by M Kadin, Boston, or as ‘homogeneously staining regions’, occurs widely in solid MA, USA. Cell lines were cultured as recommended by tumors where it is believed to facilitate upregulated transcription originators. Rapamycin was obtained from Sigma, Taufkirchen, of selected genes located therein. This phenomenon is less Germany. Patient material was represented by 156 paraffin- widespread in lymphoid and myeloid neoplasia where chromo- embedded tumor samples (43 PTCL NOS, 45 ALK þ ALCL and some translocations affecting gene juxtapositions and fusions, 68 ALKÀ ALCL) from the Institute of Pathology, University of respectively, predominate.2 Wuerzburg, Germany. All tumors were classified according to T-cell lymphomas represent about 12% of the total.3 the World Health Organization criteria. This study was According to the cancer classification of the World Health authorized by the Ethics Committee of the University of Wuerzburg, Germany. Correspondence: Dr S Nagel, Department of Human and Animal Cell Cultures, DSMZ, Inhoffenstrasse 7B, Braunschweig 38124, Germany. E-mail: [email protected] Fluorescence in situ hybridization Received 9 October 2007; accepted 12 October 2007; published Fluorescence in situ hybridization (FISH) analysis was per- online 8 November 2007 formed on metaphases prepared from cell lines as described CDK6 in T-cell lymphoma S Nagel et al 388 previously12 using the following RP11 BAC clones obtained Using a BAC contig for FISH analysis, we determined both the from the Sanger Institute, Cambridge, UK (www.genome.ucs- size (9 Mb) and location (86–95 Mb) of the amplicon (Figure 1b). c.edu): 315P14, 30D21, 371H19, 90H9, 232H24 and 124G15. Our results are consistent with genomic data provided by the Primary patient samples were analyzed by interphase FISH as Sanger Institute (www.sanger.ac.uk/cgi-bin/genetics/CGP/ described previously.13 Briefly, FISH was performed in paraffin- 10kCGHviewer.cgi?dna ¼ SU-DHL-1), which show a sharp loss embedded material in a tissue microarray format. Cases showing of heterozygosity peak and concomitant copy-number gains at a gain/amplification of the CDK6 locus were subsequently B90 Mb. For precise copy-number determination at the 7q22 analyzed on whole paraffin-embedded tissue sections as well. amplicon, we used a PCR-based MLPA assay, which simulta- The following RP11 BAC clones, used as spanning probes to neously analyzed 40 genes, including CDK6 located within the detect the CDK6 locus, were purchased from the German amplicon at 92.1–92.3 Mb. Data indicated a 5- to 6-fold Resource Centre for Genome Research, Berlin, Germany amplification of this gene in SU-DHL-1 (Figure 1c). Additional (www.rzpd.de): 467N23, 332M5 and 514K1. The centromeric data obtained by this assay revealed a threefold amplification of probe cep7 (Abbott, Wiesbaden, Germany) as well as probes BRCA1 which is located at 17q21 (38.5 Mb), in keeping with detecting the region slightly telomeric of the T-cell receptor Sanger Institute SNP-array data, which also show copy number genes TRG (7p14–15) and TRB (7q35) loci were used as increase at 37–50 Mb, i.e. covering 17q21, albeit unaccompa- reference probes.14 For negative controls, CDK6 and cep7 nied by loss of heterozygosity. Furthermore, according to our probes were tested simultaneously in seven paraffin-embedded MPLA analysis, the tumor-suppressor gene CHFR (at 12q24.3), reactive lymph nodes as well as on cells from the cell lines which is reportedly hypermethylated in T-cell lymphoma,17 MOLT-3 and HSB-2. The cell line SU-DHL-1 served as a seems to be deleted in SU-DHL-1 (Figure 1c). positive control. Reverse transcription-PCR Reverse transcription (RT)-PCR analysis was performed as described previously.15 Oligonucleotides are listed in Table 1 and were purchased from MWG, Martinsried, Germany. Quantitative real-time RT-PCR analysis was performed on an ABI 7500 SDS cycler, using commercial expression assays for CDK6 and TBP (Applied Biosystems, Darmstadt, Germany). Immunocytology Cytospin preparation and immunodetection were performed as described previously.16 Antibody anti-CDK6 was obtained from Santa Cruz (Heidelberg, Germany). Flow cytometry Flow cytometry and cell cycle analysis were performed as described previously.16 Multiplex ligation-dependent probe amplification Genomic copy-number determination was performed using a PCR-based multiplex ligation-dependent probe amplification (MLPA) kit, amplifying 40 cancer-related genes, including CDK6 (SALSA MS-MLPA kit ME001 tumor suppressor-1, MRC- Figure 1 (a) Karyotype analysis showing the homogeneously staining Holland, Amsterdam, The Netherlands). The mean amplification region on 7q in SU-DHL-1 cells. (b) Fluorescence in situ hybridization (FISH) analysis was used to map the amplicon at 7q22. A painting signal was set to 1. probe for chromosome 7 (red) was co-hybridized with BAC probe RP11-90H9 (green), demonstrating CDK6 amplification on der(7) in SU-DHL-1. (c) Copy
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