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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Radboud Repository PDF hosted at the Radboud Repository of the Radboud University Nijmegen The following full text is a publisher's version. For additional information about this publication click this link. http://hdl.handle.net/2066/25861 Please be advised that this information was generated on 2017-12-05 and may be subject to change. ELSEVIER Fine Mapping of the Human Renal Oncocytoma- Associated Translocation (5;ll)(q35;ql3) Breakpoint Richard J. Sinke, Trynie Dijkhuizen, Bert Janssen, Daniël Olde Weghuis, Gérard Merkx, Eva van den Berg, Ed Schuuring, Aurelia M. Meloni, Bauke de Jong, and Ad Geurts van Kessel ABSTRACT: Recent cytogenetic analysis of a series of human renal oncocytomas revealed the presence of a recurring chromosomal translocation (5;ll)(q35;ql3) as sole anomaly in a subset of the tumors. The molecular characterization of this translocation was initiated using two primary t(5;ll)-positive renal oncocytomas and a panel of soma tic cell hybrids derived from one of these tumors, in conjunction with fluorescence in situ hybridization (FISH) and Southern blot analysis. The breakpoint in chromosome band llql3 could be located within a genomic interval of at maximum 400 Kb immediately centromeric to the BCLl locus. © Elsevier Science Inc., 1997 INTRODUCTION cytogenetic anomaly in at least 3 independent cases and, as such, should be considered as a primary change. Deletion of Oncocytomas are benign tumors that occur predominantly 3p material that occurs in the majority of renal cell carci in the kidney, accounting for approximately 5% of all pri nomas [16,17] has not been observed in renal oncocytomas. mary tumors at that site. Histologically, oncocytomas are The presence of mitochondrial DNA alterations has been characterized by the presence of epithelial cells with a strong reported by some investigators [18,19]. These latter obser granular eosinophilic cytoplasm. Ultras true tural examina vations, however, could not be substantiated by others [20]. tion revealed that these so-called oncocytic cells are In view of the steadily accumulating evidence that chro densely packed with mitochondria, many of which show mosomal changes play causal roles in cancer development abnormal morphologic characteristics [l]. This phenotype, [21-23], we assume that in this case, too, the chromosomal however, also can be found in some of the malignant neo translocation points at a gene(s) that plays a crucial role in plasms of the kidney such as the granular and chromopho the pathogenesis of renal oncocytomas. Therefore, we ini bic variants of renal cell carcinomas [2], Although long tiated the fine mapping of the t(5;ll)(q35;ql3) breakpoint. term follow-up studies have shown that oncocytomas do Because band llql3 is involved in a variety of malignant not metastasize, several reports indicate the invasive disorders, including mantle cell lymphomas, squamous potential of these tumors [3-5]. Preliminary cytogenetic cell carcinomas, breast cancer, multiple endocrine neopla studies performed on a limited series of renal oncocyto sia type 1, and extragonadal germ cell tumors [24-30], and mas have revealed a heterogeneous chromosomal constitu because llql3 is relatively well mapped [24, 31, 32], we tion [3, 5-7] Basically, two subgroups can be distinguished: selected this region for detailed analysis. To this end, fluo [1) those with numerical anomalies, including -Y and -1 rescence in situ hybridization (FISH) with breakpoint re [8-10]; and (2) those with recurring structural anomalies, gion-specific probes was employed. In addition, a panel of in particular t(5;ll)(q35;ql3) [11, 12] or variants thereof oncocytoma-derived interspecies somatic cell hybrids was [13-15]. The t(5;ll)(q35;ql3) has been observed as sole generated to facilitate our studies. The resulting mapping data are useful (1) to construct a detailed physical map of From the Department of Human Genetics [R. J. S., B.J., D. O, W., the breakpoint region; and (2) to provide a basis to isolate G. M., A, G.v, K.], University Hospital Nijmegen, The Netherlands; the breakpoint itself and the gene[s) involved in the devel the Department of Medical Genetics [T. D., E, v. d. B., B. de f.L opment of renal oncocytomas. University of Groningen, The Netherlands; the Department of Pathology [E. S.], University Hospital, Leiden, The Netherlands; and the Southwest Biomedical Research Institute and Genzyme MATERIALS AND METHODS Genetics /A, M. M.], Scottsdale, Arizona, USA. Address reprint requests to: Dr. A, Geurts van Kessel, Depart Tumors and Somatic Cell Hybrids ment of Human Genetics, University Hospital, P. O. Box 9101, 6500 HB Nijmegenf The Netherlands. Samples of the t(5;ll)(q35;ql3)-positive renal oncocytomas Received May 29, 1996; accepted Avgust 9, 1996. T88-1665 and T92-12439 were obtained from. 2 different Cancer Genet Cytogenet 96:95-101 (1997) © Elsevier Scionce Inc., 1997 0165-4608/9 7/$l 7.00 655 Avenue of the Americas, New York, NY 10010 P1I SOI 65*4600(96)00250-6 96 R, J, Sinke et al. patients after radical nephrectomy at surgery. Following dis- hybrid-derived metaphase spreads. Visualization of the aggregation of tumor cells with collagenase and subsequent hybridization signals was accomplished using Texas Red- short-term culturing, metaphase spreads were prepared conjugated avidin and biotinylated goat antiavidin anti using standard procedures. In addition, a panel of somatic bodies (Vector Laboratories, Burlingame, CA} USA) for cell hybrids was isolated after fusion of the thymidine kinase biotin-labeled probes and fluorescein-conjugated sheep deficient (tk~) Chinese hamster cell line A3 with T92-12439 antidigoxigenin (Boehringer, Mannheim, Germany) and cells. Fusion and hybrid selection were performed essen fluorescein-conjugated donkey antisheep antibodies (Jack tially as described before [33], using Sendai virus as a fuso- son Immunoresearch, West Grove, PA, USA) for digoxigenin- gen and a combined HAT/Ouabain procedure for selection of labeled probes. Slides were immersed in antifade-solution, hybrid cells in which the reciprocally translocated chromo supplemented with 4,6-diamino-2-phenylindole (DAPI, 0.5 somes segregate, The chromosomal constitution of hybrid |ig/mL; Sigma, St, Louis, MO, USA) for counterstaining of cells was evaluated by cytogenetic analysis using R-banding the chromosomes. Evaluation of the FISH results was per after heat dénaturation and by FISH using a combination of formed using a Zeiss Axiophot epifluorescence microscope, centromere-specific probes and whole-chromosome paints. equipped for the visualization of fluorescein isothiocyan- ate (FITC), Texas Red, and DAPI fluorescence. Digital images Fluorescence in Situ Hybridization were captured using a high-performance CH250/A cooled Multicolor FISH experiments were carried out as reported CCD-camera (Photometries, Tucson, AZ, USA) interfaced before [28, 34], In short, probe DNAs were labeled with onto a Macintosh Quadra 950 computer. Acquisition, pro biotin-14-dATP (Life Technologies, Gaithersburg, MD, USA) cessing, and display were accomplished using the BDS- or digoxigenin-ll-dUTP (Boehringer, Mannheim, Germany) image™ FISH software package (Oncor Imaging, Gaithers following nick translation, All labeled probes, except the burg, MD, USA). Photographs were made using a Polaroid alphoid probe, were subjected to preannealing to compete Quick-Print with Kodak EPP 100 Plus color slide film. out repetitive sequences using 25- to 50-fold excess of Cot.l DNA (Life Technologies, Gaithersburg, MD, USA), Southern Blot Analysis For efficient hybridizations, 10 ng labeled alphoid and 200 High-molecular-weight DNAs were isolated as described ng labeled cosmid probe DNAs were applied under a 20 X before [35], digested to completion, electrophoresed dirough 20 mm coverslip onto primary tumor or somatic cell 0.8% agarose gels, and blotted onto Genescreen Plus nylon Figure 1 Renal oncocytoma-derived karyotype exhibiting t(5;ll)(q35;ql3) as the sole cytogenetic abnormality (arrows). 13 14 15 16 17 18 19 20 Y 21 22 Fine Mapping of the Renal (5;ll)(q35;ql3) Breakpoint 97 membranes (Dupont) using standard protocols. Blots were RESULTS AND DISCUSSION hybridized with probes (labeled with a-32P-dCTP by ran- Characterization of t(5;ll)(q35;ql3) in Primary Tumors dom priming) in 0.5 M phosphate, 1 mM Na2-EDTA, and 7% SDS (w/v) at 65°C overnight and subsequently washed Cytogenetic analysis of two primary renal oncocytomas (T88- in 40 mM or 10 mM phosphate 0.1% SDS at 65°C. 1665 and T92-12439) revealed the presence of t(5;ll) (q35;ql3) as sole anomaly (Fig. 1). These primary tumors were used for a more detailed characterization of the translo Probes cation. Starting from the llq l3 region, FISH analyses were For the FISH and Southern blot analyses we used 27 cosxnid performed on metaphase spreads obtained from short-term and/or plasmid probes specific for chromosomes 5 and 11. cultured primary tumor cells. Double hybridizations with As references in our FISH experiments we used a plasmid combinations of llql3-specific cosmids (Table 1) and the probe specific for the centromeric (alphoid) region of chro alphoid probe (pCLllA) specific for the centromeric region mosome 11 (pCLllA), and whole-chromosome painting 5- of chromosome 11 were carried out to verify the location of and 11-specific plasmid libraries pBS5 and pBSll, respec the translocation breakpoint within this cytogenetic band. In tively [36]. A summary of all the probes used is listed in both primary tumors, the cosmid probes for PGA (cgHGPl8) Table 1 [37-39]. and BCLl (cCLBCLl) mapped proximal and distal to the Figure 2 FISH analysis of primary renal oncocytoma T88-1665 (c) and oncocytoma (T92-12439)-derived somatic cell hybrid A30N17B (a,b,d) metaphase spreads, (a) Cosm id clone cCI5-20 (green; arrows) in combination with the chromosome 11 centromere-specific probe pCLllA (red; arrowheads); cCI5~20 remains on der(5).