Chromosomal destabilization during gene amplification.

Abstract

Acentric extrachromosomal elements, such as submicroscopic autonomously replicating circular molecules (episomes) and double minute chromosomes, are common early, and in some cases initial, intermediates of gene amplification in many drug-resistant and tumor cell lines. In order to gain a more complete understanding of the amplification process, we investigated the molecular mechanisms by which such extrachromosomal elements are generated and we traced the fate of these amplification intermediates over time. The model system consists of a Chinese hamster cell line (L46) created by gene transfer in which the initial amplification product was shown previously to be an unstable extrachromosomal element containing an inverted duplication spanning more than 160 kilobases (J. C. Ruiz and G. M. Wahl, Mol. Cell. Biol. 8:4302-4313, 1988). In this study, we show that these molecules were formed by a process involving chromosomal deletion. Fluorescence in situ hybridization was performed at multiple time points on cells with amplified sequences. These studies reveal that the extrachromosomal molecules rapidly integrate into chromosomes, often near or at telomeres, and once integrated, the amplified sequences are themselves unstable. These data provide a molecular and cytogenetic chronology for gene amplification in this model system; an early event involves deletion to generate extrachromosomal elements, and subsequent integration of these elements precipitates a cascade of chromosome instability.

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.…”

“…Breakage resulting in the formation of dicentrics could lead to both delayed and continued instability. Dicentric formation has long been considered a mechanism for another endpoint of genetic instability, gene amplification (24,38,42,45). Dicentrics that are formed during gene amplification can initiate another wave of chromosomal instability (25,38).…”

Chromosomal location of the amplified esterase genes conferring resistance to insecticides in Myzus persicae (Homoptera: Aphididae)

“…The mechanism in M persicae seems to differ from that involved in early gene amplification events in drugresistant Chinese hamster ovary cell lines, where the amplified sequences most often occur on the same chromosome as the original single-copy site, but at some distance from it (Trask & Hamlin, 1989 Possible mechanisms by which identical amplified sequences could occur at multiple loci in M. persicae include: (i) deletion and extrachromosomal amplification as an episome, followed by reintegration into a chromosome (Wahi, 1989); and (ii) association with transposable elements. A common feature of the deletion/episome model is the appearance of the amplified DNA as microscopically visible 'double minute' elements (Ruiz & Wahl, 1990). These have not been detected in M. persicae, but then the probability of detecting such elements would depend on the size!…”