Molecular structure of double-minute chromosomes bearing amplified copies of the epidermal growth factor receptor gene in gliomas - PubMed (original) (raw)

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

Nicolas Vogt et al. Proc Natl Acad Sci U S A. 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.

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Figures

Fig. 1.

Fig. 1.

Characterization of the amplicon in case 22. The amplification levels determined by quantitative PCR are presented relative to the map (scale in megabases) of the region surrounding the EGFR gene (hatched bar). ▪, Fresh tumor; □, xenografted tumor.

Fig. 2.

Fig. 2.

Extent of the amplicons found in the studied gliomas. (A) Map of the EGFR region (scale in megabases; gray bar, EGFR gene). (B) Amplification levels, positions, and extent of the amplified regions in seven gliomas. Dotted lines indicate the localization of the internal deletions found in amplicons of cases 21 and 14.

Fig. 3.

Fig. 3.

Sequence of the junctions resulting from circularization of the amplicons (A) and from formation of internal deletions (B). The sequence of each junction is aligned with respect to the sequences of its two normal counterparts [5′ counterpart above the junction (J) and 3′ counterpart below it]. Microhomologies and inserted sequences are shown in bold.

Fig. 4.

Fig. 4.

Chromosome analysis. (A and B) Metaphases from case 22. (A) Reverse DAPI staining shows the dmins. (B) Cohybridization with probe RP5–1091E12 containing the EGFR gene (red) and a chromosome 7 painting probe (green). Chromosomes are stained in blue with DAPI. Three copies of chromosome 7 are present, and each copy bears the EGFR locus at its expected location (arrowheads). Probe RP5–1091E12 reveals the dmins (extrachromosomal red signals). (C and D) Metaphase from case 21. (C) Reverse DAPI staining. (D) Hybridization with bacterial artificial chromosome CTD-2171F16 (green signal) probing for a sequence deleted from the amplicons of this tumor. FISH signals are visible on each chromosome 7 (arrowheads) but not on the dmins (see reverse DAPI in C).

Fig. 5.

Fig. 5.

Postreplicative models for the formation of extrachromosomal molecules without a chromosome scar. (A) Segregation-based model. The excision of a chromosome fragment gives rise to a deleted chromosome 7 (□) and an extrachromosomal circular element (○). At mitosis, the element may segregate together with three normal copies of chromosome 7 (daughter cell 1) or with two normal copies and one rearranged copy of that chromosome (daughter cell 2′). The model postulates that cell 1, which has four copies of the EGFR gene, is selected. (B) Rereplication-based model. In lane 1, two breaks occur in a replication eye on the same strand (broken arrows). Arrows indicate the directions of fork progression. In lane 2, a chromosome fragment is excised, and two single-strand breaks remain in the chromosome (stars). They may be repaired at this stage or bypassed at stage 3 by fork regression. In lane 3, the linear fragment is circularized and the corresponding chromosomal region rereplicated by forks emanating from flanking regions. In lane 4, an extrachromosomal element has been formed without a chromosome scar.

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