A central role for chromosome breakage in gene amplification, deletion formation, and amplicon integration.

Abstract

A CHO cell line with a single copy of the DHFR locus on chromosome Z2 was used to analyze the structure of the amplification target and products subsequent to the initial amplification event. Dramatic diversity in the number and cytogenetic characteristics of DHFR amplicons was observed as soon as eight to nine cell doublings following the initial event. Two amplicon classes were noted at this early time: Small extrachromosomal elements and closely spaced chromosomal amplicons were detected in 30-40% of metaphases in six of nine clones, whereas three of nine clones contained huge amplicons spanning >50 megabases. In contrast, the incidence of metaphases containing extrachromosomal amplicons fell to 1-2% in cells analyzed at 30-35 cell doublings, and most amplicons localized to rearranged or broken derivatives of chromosome Z2 at this time. Breakage of the Z2 chromosome near the DHFR gene, and deletion of the DHFR gene and flanking DNA was also observed in cells that had undergone the amplification process. To account for these diverse cytogenetic and molecular consequences of gene amplification, we propose that chromosome breakage plays a central role in the amplification process by (1) generating intermediates that are initially acentric and lead to copy number increase primarily by unequal segregation, (2) creating atelomeric ends that are either incompletely replicated or resected by exonucleases to generate deletions, and (3) producing recombinogenic ends that provide preferred sites for amplicon relocalization.

Polyomavirus large T antigen-dependent DNA amplification

“…We, therefore, asked whether PyLT might still lead to genome destabilization and gene ampli®cation and found that this is indeed the case as long as the amino acid sequence decisive for the interaction of PyLT with pRB and the other pocket proteins is intact. Cells selected for the presence of an ampli®ed CAD gene by addition of PALA have higher chromosome number and in addition show the same structures of ampli®ed genes as previously observed in several other cases Windle et al, 1991;Ma et al, 1993;Toledo et al, 1993). The PALA resistant cells appear normal with respect to p53.…”

“…However, upon closer inspection of the karyotype of PALA resistant clones ( Figure 2) it became clear that there was not only a higher number of chromosomes but there were chromosomal aberrations visible, in particular dicentric chromosomes, which are thought to be responsible for the generation of ampli®ed genes through bridge breakage fusion (BBF) cycles (reviewed by Stark, 1993). We have, therefore, carried out¯uorescence in situ hybridization (FISH) experiments to analyse ampli®ed CAD gene structures in more detail and indeed found many examples of structures ( Figure 3) as they were repeatedly shown to be characteristic for ampli®ed genes originating through interference with the p53-mediated checkpoint control Windle et al, 1991;Ma et al, 1993;Toledo et al, 1993).…”

Double minute chromosomes carrying the human multidrug resistance 1 and 2 genes are generated from the dimerization of submicroscopic circular DNAs in colchicine-selected KB carcinoma cells.

“…In addition, in vitro studies imply that many of the structural chromosomal abnormalities occurring in tumors and tumorigenic cell lines (Bishop, 1987;Tlsty et al, 1989) may result from molecular mechanisms common to those mediating extrachromosomal gene amplification. For example, recent studies have provided firm evidence that some extrachromosomal elements have the potential to integrate into chromosomes, resulting in either HSRs, ECRs (Carroll et al, 1988;Ruiz and Wahl, 1990;Von Hoff et al, 1990;Windle et al, 1991), or other chromosome abnormalities such as ring chromosomes (Windle et al, 1991). These studies support earlier observations of gene amplification in hamster cell lines in which Biedler (1982) observed that the extrachromosomal circular DMs rapidly integrated into chromosomes, resulting in HSR structures (Biedler, 1982).…”

“…Several studies have used in situ hybridization/fluorescent techniques to identify very early amplification structures. These studies indicate that gene amplification can be mediated by either intra-or interchromosomal recombination events, such as sister chromatid exchange (Trask and Hamlin, 1989;Smith et al, 1990), or by extrachromosomal circular DNA intermediates (Wahl, 1989;Ruiz and Wahl, 1990;Windle et al, 1991).…”

Delayed chromosomal instability induced by DNA damage.

“…Although the observed induction of gross chromosome rearrangements in these clones did not influence the reciprocal exchange of homologous DNA sequences between sister chromatids, it is reasonable to assume that delayed chromosomal instability plays a significant role in gene mutation (7,16,32,40), gene amplification (17,18,33,38,50), cellular transformation (26,28), teratogenesis (15,35), and even carcinogenesis (13,30) after exposure of cells to DNA-damaging agents.…”