Amplification of Mycn, Ddx1, Rrm2, and Odc1 in Rat Uterine Endometrial Carcinomas Åsa Karlsson, 1 Khalil Helou, 1 Anna Walentinsson, 1 Hans J. Hedrich, 2 Claude Szpirer, 3 and Go ¨ ran Levan 1 * 1 Department of Cell and Molecular Biology-Genetics, Go ¨ teborg University, Gothenburg, Sweden 2 Laboratory of Animal Science, Medizinische Hochschule Hannover, Germany 3 IBMM, Universite ´ Libre de Bruxelles, Gosselies, Belgium The BDII rat is genetically predisposed to estrogen-dependent endometrial adenocarcinoma and represents a valuable model for this type of tumor. Tumors arising in strain crosses involving the BDII rats had previously been screened for DNA copy number changes using comparative genome hybridization (CGH). It was found that extra copies of the proximal region of rat chromosome (RNO) 6 commonly could be detected in these tumors. Based on RH-mapping data and comparative mapping with mouse and human, seven cancer-related genes were predicted to be situated in RNO6q14 – q16. Rat PACs were isolated for the N-myc proto-oncogene (Mycn), apolipoprotein B (Apob), the DEAD box gene 1 (Ddx1), ornithine decarboxylase 1 (Odc1), proopiomelanocortin (Pomc1), ribonucleotide reductase, M2 polypeptide (Rrm2), and syndecan 1 (Sdc1). The local- ization of the genes to the region was verified by FISH (fluorescence in situ hybridization) mapping, and the detailed order among them was determined by dual-color FISH. By Southern blot analysis, it was found that the Mycn locus was highly amplified in two out of 10 cell cultures derived from the tumors. In one of them (designated RUT30), the amplification level of Mycn was estimated at 140. Two other genes were coamplified (Ddx1 and Rrm2) at much lower levels. Similarly, in another culture (designated RUT2), Mycn was amplified more than 40, whereas three of the other genes (Ddx1, Rrm2, and Odc1) were coamplified at lower levels. Using FISH on metaphase chromosomes from the cell cultures analyzed, the amplified sequences were shown to be located in typical HSRs. With competitive RT-PCR, distinct overexpression of Mycn and Ddx1 could be demonstrated in both RUT2 and RUT30. In addition, Mycn was overexpressed in two other tumors not exhibiting Mycn amplification. Taken together, our results suggest that overexpression of Mycn plays an important role in the development of endometrial cancer in the BDII rat. In humans, Mycn amplification has been reported mainly from tumors of neuronal origin. To our knowledge, this is the first report of Mycn amplification and overexpression in hormone-dependent tumors. © 2001 Wiley-Liss, Inc. INTRODUCTION Cancer is now known to be a genetic disease that is both polygenic and heterogeneous, in most cases involving changes in several genes in a stepwise fashion (Vogelstein and Kinzler, 1993). The spec- trum of individual genes involved is probably greatly influenced by genetic and environmental factors that are unique for each patient (Mitelman et al., 1997). Consequently, cancer is a very com- plex disease to study in humans, and it is difficult to identify the significant chromosome changes, especially because cancer tumors characteristically display a large number of apparently random chro- mosome changes, so-called “cytogenetic noise.” In a model organism, it is possible reasonably to con- trol both the environment and the genetic back- ground. This would be expected to reduce the complexity of genetic change in a population of tumors, increasing the possibility to identify un- equivocally genetic changes that are significant for tumor development in the particular model. There are inbred rat strains available that are suitable genetic models for different types of cancer. Endometrial carcinoma is one of the most com- mon malignancies among women in the developed countries (Bandera and Boyd, 1997). Yet, not much is known about the molecular mechanisms behind the disease, and our strategy was to use a rodent model to identify molecular changes that might subsequently be extrapolated to human cancer by means of comparative analysis. Female rats belong- ing to the inbred BDII strain are highly predis- posed to cancer development in the endometrial layer of the uterus (Deerberg and Kaspareit, 1987). To study the genetics of endometrial cancer, BDII DNA sequences from the rat Ddx1 gene have been submitted to GenBank and were given accession numbers AF222732, AF222733. Supported by: European Commission; Grant number: ERBBio4CT960562; Swedish Cancer Society; Swedish Strategic Foundation (Genome Research); Royal Swedish Academy of Sci- ences; Royal Physiographical Society in Lund; Royal and Hvit- feldtska Foundation; Inga Britt and Arne Lundberg Research Foun- dation. *Correspondence to: Go ¨ ran Levan, CMB-Genetics, Box 462, SE 405 30 Gothenburg, Sweden. E-mail: Goran.Levan@gen.gu.se Received 30 October 2000; Accepted 16 January 2001 Published online 7 June 2001 GENES, CHROMOSOMES & CANCER 31:345–356 (2001) © 2001 Wiley-Liss, Inc.