GENES, CHROMOSOMES & CANCER 48:943–952 (2009) Selective Elimination of Amplified CDK4 Sequences Correlates with Spontaneous Adipocytic Differentiation in Liposarcoma Zofia He ´ lias-Rodzewicz, 1,2 Florence Pe ´deutour, 3 Jean-Michel Coindre, 4 Philippe Terrier, 5 and Alain Aurias 1,2 * 1 Institut Curie,Genetics and Biology of Cancers, 26 rue d’Ulm, 75248 Paris cedex 05, France 2 INSERM U830, 75248 Paris cedex 05, France 3 Laboratory of Solid Tumors Genetics, Nice University Hospital and CNRS UMR 6543, Faculty of Medicine, 06107 Nice, France 4 Department of Pathology, Institut Bergonie¤ , 229 cours de l’Argonne, 33076 Bordeaux, cedex, France 5 Department of Pathology, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif cedex, France Well-differentiated and undifferentiated liposarcomas are characterized by high-level amplifications of chromosome 12 regions including the CDK4 and MDM2 genes. These amplicons are either localized, in well-differentiated liposarcoma (WDLPS), on extrachromosomal structures (ring or rod chromosomes), or integrated into chromosome arms in undiffer- entiated tumors. Our results reveal that extrachromosomal amplicons are unstable, and frequently lost by micronucleation. This loss correlates with hypermethylation of eliminated sequences and changes of their replication time. Treatment of cells with demethylating agents during early S-phase significantly decreases the rate of micronuclei positive for CDK4. We also demonstrate that, in our model, micronuclei are generated during anaphase as a consequence of anaphase abnormal- ities (chromosome lagging and anaphase bridges). Finally, a dramatic increase of adipocytic differentiation was noted in cells that have eliminated copies of CDK4 gene in micronuclei. These findings provide evidence that, in WDLPS, adipocytic dif- ferentiation could be the consequence of CDK4 loss, an event occurring rarely in undifferentiated tumors in which the amplified sequences are integrated into chromosome arms. V V C 2009 Wiley-Liss, Inc. INTRODUCTION Adult soft tissue sarcomas are rare tumors (around 1% of all cancers). Among them, well-dif- ferentiated liposarcomas (WDLPS) and undiffer- entiated liposarcomas (UDLPS) account for around 15%. WDLPS are tumors of intermediate malignancy, essentially composed of mature non- proliferative adipocytic cells, with a variable amount of nonlipogenic cells (Fletcher et al., 2002). They are associated with a good prognosis, and a very low metastasis rate. On the contrary, UDLPS are composed of uniform areas of nonlipo- genic spindle cells, with rare foci of adipocytic dif- ferentiation. These tumors, which can occur during the progression of a WDLPS, have a worse prognosis, and a metastasis rate around 15%. They were often classified as malignant fibrous histiocy- tomas. Both groups of liposarcomas are character- ized by high-level amplifications of genomic regions of chromosome 12, including the CDK4 and MDM2 genes (Fletcher et al., 2002). These genes play a major role in the oncogenic process: CDK4 protein, in association with D-type cyclins, promotes a robust cell cycle progression, thus pre- cluding a terminal adipocytic differentiation; MDM2 binds to TP53 protein and inhibits its anti- oncogenic functions by targeting its destruction. In liposarcomas, these amplified genes are localized either on supernumerary ring or rod chromosomes, or integrated as homogeneously staining regions in various chromosome arms, often described as marker or derivative chromosomes (these abnor- mal chromosomes will be named ‘‘marker chromo- somes’’ in the text). In most cases, ring and rod chromosomes do not contain a-satellite sequences in centromere regions (Gisselsson et al., 1999; Italiano et al., 2009). WDLPS are tumors that can grow rapidly, in contrast to adipocytes with terminal differentiation Additional Supporting Information may be found in the online version of this article. Supported by: INSERM, Institut Curie; INCa, Grant number: PL007; Conticanet network Fondation de France, Carte d’Identite ´ des Tumeurs (CIT) program of the Ligue Nationale Contre le Cancer, ARC. *Correspondence to: Alain Aurias, Institut Curie, Genetics and Biology of Cancers, 26 rue d’Ulm, 75248 Paris cedex 05, France. E-mail: alain.aurias@curie.fr Received 28 April 2009; Accepted 26 June 2009 DOI 10.1002/gcc.20696 Published online 22 July 2009 in Wiley InterScience (www.interscience.wiley.com). RESEARCH ARTICLES V V C 2009 Wiley-Liss, Inc.