Topology of Double Minutes (dmins) and Homogeneously Staining Regions (HSRs) in Nuclei of Human Neuroblastoma Cell Lines Irina Solovei, 1 Dirk Kienle, 2 Graham Little, 3 Roland Eils, 3 Larissa Savelyeva, 4 Manfred Schwab, 4 Willi Ja ¨ger, 3 Christoph Cremer, 5 and Thomas Cremer 1 * 1 Institute for Anthropology and Humangenetics, University of Munich (LMU), Munich, Germany 2 Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany 3 Interdisciplinary Center of Scientific Computing (IWR), University of Heidelberg, Heidelberg, Germany 4 Deutsches Krebsforschungszentrum, Abteilung Zytogenetik, Heidelberg, Germany 5 Institute for Applied Physics, University of Heidelberg, Heidelberg, Germany Amplification of the MYCN gene is a characteristic feature of many neuroblastomas and is correlated with aggressive tumor growth. Amplicons containing this gene form either double minutes (dmins) or homogeneously staining regions (HSRs). To study the nuclear topology of these tumor-specific and transcriptionally active chromatin structures in comparison to chromosome territories, we performed fluorescence in situ hybridization with a MYCN probe and various chromosome paint probes, confocal laser scanning microscopy, and quantitative three-dimensional image analysis. The dmins formed dot-like structures in interphase nuclei and were typically located at the periphery of complexly folded chromosome territories; dmins noted in the chromosome territory interior were often detected within an invagination of the territory surface. Interphase HSRs typically formed extremely expanded structures, which we have never observed for chromosome territories of normal and tumor cell nuclei. Stretches of HSR-chromatin often extended throughout a large part of the cell nucleus, but appeared well separated from neighboring chromosome territories. We hypothesize that dmins are located within the interchromo- somal domain (ICD) space and that stretches of HSR-chromatin align along this space. Such a topology could facilitate access of amplified genes to transcription and splicing complexes that are assumed to localize in the ICD space. © 2000 Wiley-Liss, Inc. INTRODUCTION Each interphase chromosome occupies a distinct territory in the cell nucleus (Manuelidis, 1985; Cre- mer et al., 1988; Lichter et al., 1988; Pinkel et al., 1988). The organization of chromosome territories is considered an important factor affecting nuclear functions (Cremer et al., 1993, 1996; Lamond and Earnshaw, 1998). The compartmentalized struc- ture of the nucleus ensures that molecules neces- sary for certain biological processes, such as tran- scription and splicing, are present in the right place and at the right time (Spector, 1996; Misteli et al., 1997; Misteli and Spector, 1998). Although the to- pology of chromatin in tumor and normal cell nu- clei may differ profoundly, a detailed analysis of these differences is still lacking. In this study we investigated the nuclear topology of aberrant chro- matin structures in neuroblastomas, the most com- mon solid tumor type in young children. Neuroblastoma cells are often characterized by amplification of the MYCN gene, which correlates with both enhanced expression of the gene (Schwab et al., 1984b) and aggressive tumor growth (Schwab et al., 1983). The MYCN gene maps to 2p23–24 (Schwab et al., 1984a) where it is retained in single copy during amplification (Corvi et al., 1995; Schwab et al., 1995). Amplicons containing the MYCN gene range in size from 100 – 800 kbp and form multiple tandem arrays present in two types of aberrant chromatin structures, double min- utes (dmin; Kohl et al., 1983) and “homogeneously stained regions” (HSRs; Amler and Schwab, 1989; Amler et al., 1992). Dmins are often seen as small extra chromosomes in direct metaphase prepara- tions from neuroblastomas, whereas HSRs are in- tegrated as extra segments in abnormal chromo- somes and are typically found in established neuroblastoma cell lines (Schwab et al., 1984a). HSRs can reach a size of up to 80 Mbp (Amler and Schwab, 1989; Amler et al., 1992), and their inte- gration sites vary in different neuroblastoma cell Supported by Faculty of Medicine, University of Heidelberg and, in part, by the Dr. Mildred Scheel Stiftung. *Correspondence to: Prof. Dr. Thomas Cremer, Institut fu ¨r An- thropologie und Humangenetik, Universita ¨t Mu ¨ nchen, LMU, Rich- ard-Wagner-Strasse, 10/I, 80333 Munich, Germany. E-mail: Thomas.Cremer@lrz.uni-muenchen.de Received 25 May 1999; Accepted 2 June 2000 GENES, CHROMOSOMES & CANCER 29:297–308 (2000) © 2000 Wiley-Liss, Inc.