Co-amplified markers alternate in megabase long chromosomal inverted repeats and cluster independently in interphase nuclei at early steps of mammalian gene amplification.

Abstract

Two‐colour in situ hybridization with probes for two co‐amplified markers located several megabases apart on chromosome 1 has been used to analyse early stages of adenylate deaminase 2 (AMPD2) gene amplification in Chinese hamster cells. In the amplified chromosomal structures, the distribution of hybridization spots identifies megabase‐long inverted repeats. Their organization is remarkably well accounted for if breakage‐fusion‐bridge cycles involving sister chromatids drive the amplification process at these early stages. During interphase the markers often segregate into distinct nuclear domains. Many nuclei have bulges or release micronuclei, carrying several copies of one or both markers. These observations indicate that the amplified units destabilize the nuclear organization and eventually lead to DNA breakage during interphase. We propose a model in which interphase breakage has a role in the progression of gene amplification.

Sustained mitotic block elicits DNA breaks: one-step alteration of ploidy and chromosome integrity in mammalian cells

“…At later time points, cells slipped out of mitosis and underwent re-replication, as indicated (Figure 1Ba-c) revealed the presence of chromosome fragments in almost all of them. Two-color fluorescence in situ hybridization (FISH) with markers that, respectively, label the p and q arms of Chinese hamster chromosome 1 (Toledo et al, 1992) showed that around 30% of these tetraploid mitotic cells display major rearrangements of this chromosome. Extensive cell death did not occur at that stage as cells often cycled up to three or four times upon prolonged growth in the presence of the drug (Figure 1Bd).…”

“…In situ hybridization and FACS analysis Cosmids probes and FISH procedure were described previously (Toledo et al, 1992;Coquelle et al, 1997). FACS analyses were performed with a FACScalibur flow cytometer (Becton Dickinson France S.A.S., Le Pont de Claix, France).…”

New pattern of EGFR amplification in glioblastoma and the relationship of gene copy number with gene expression profile

“…1,3,4 According to the breakage-fusion-bridge model of amplification, the initiating event in HSR formation is double chromatid breakage at a fragile site or telomere erosion, 2,26,27 fused sister chromatids and breaking of the anaphase bridges. 28 Breakage-fusion-bridge cycles could then result in inverted amplified structures, 29 and the mutated sister chromatids are distributed to the daughter cells giving rise to intra-tumor heterogeneity. 30 Gene amplifications are also acquired by selection and unequal segregation of circular extrachromosomal chromatin (dmin and episomes).…”

Cytokinesis-block micronucleus cytome assay

“…1-5) [3][4][5][6][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] . This method is now also used to measure NPBs, a biomarker of dicentric chromosomes resulting from telomere end-fusions or DNA misrepair, and to measure NBUDs, a biomarker of gene amplification [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39] . The significance of these developments and the concept of the CBMN assay as a ''cytome'' assay of chromosomal instability are explained in the following sections.…”

“…This process has been observed in cultures grown under strong selective conditions that induce gene amplification as well as under moderate folic acid deficiency [27][28][29][30][31][32][33][34][35][36][37] . Shimizu et al 31,32 showed, using in vitro experiments with mammalian cells, that amplified DNA is selectively localized to specific sites at the periphery of the nucleus and eliminated via nuclear budding to form MNi during S phase of mitosis.…”