Molecular structure of double-minute chromosomes bearing amplified copies of the epidermal growth factor receptor gene in gliomas

doi:10.1073/pnas.0402979101

Molecular structure of double-minute chromosomes bearing amplified copies of the epidermal growth factor receptor gene in gliomas

^*Instabilité du Génome et Cancer, FRE 2584, Centre National de la Recherche Scientifique, Institut Curie, 26 Rue d'Ulm, 75248 Paris, Cedex 5, France; ^†Institut for Histology and Embryology, Medical Faculty, 1000 Ljubljana, Slovenia; and ^‡Laboratoire de Biologie des Interactions Neurones-Glie, Institut National Français de Recherche Médicale U495, Pitié-Salpêtrière, 75013 Paris, France

Edited by Frederick W. Alt, Harvard Medical School, Boston, MA, and approved June 18, 2004 (received for review April 28, 2004)

Abstract

Amplification of the epidermal growth factor receptor gene on double minutes is recurrently observed in cells of advanced gliomas, but the structure of these extrachromosomal circular DNA molecules and the mechanisms responsible for their formation are still poorly understood. By using quantitative PCR and chromosome walking, we investigated the genetic content and the organization of the repeats in the double minutes of seven gliomas. It was established that all of the amplicons of a given tumor derive from a single founding extrachromosomal DNA molecule. In each of these gliomas, the founding molecule was generated by a simple event that circularizes a chromosome fragment overlapping the epidermal growth factor receptor gene. In all cases, the fusion of the two ends of this initial amplicon resulted from microhomology-based nonhomologous end-joining. Furthermore, the corresponding chromosomal loci were not rearranged, which strongly suggests that a postreplicative event was responsible for the formation of each of these initial amplicons.

Footnotes

↵ § To whom correspondence should be addressed. E-mail: bernard.malfoy{at}curie.fr.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: dmin, double minute; EGFR, epidermal growth factor receptor; NHEJ, nonhomologous end-joining; FISH, fluorescence in situ hybridization; DAPI, 4′,6-diamidino-2-phenylindole.