Gene Amplification in Cultured Animal Cells Review

Robert T. Schimke Department of Biological Sciences Stanford University, Stanford, California 94305 Quantitative changes in gene copy number are found frequently in biology. They range from polytenization of chromosomes to generation of polyploid cells to selective gene amplification, including amplification of deleted rDNA in Drosophila melanogaster (Ritossa, 1968) and develop- mental amplification of rDNA in amphibian oocytes (Brown and Dawid, 1968) and chorion genes in Drosophila mela- nogaster ovaries (Spradling and Mahowald, 1980). Gene duplications are detected readily when bacteria are placed under selection conditions (see Anderson and Roth, 1977). During the past six years the study of gene amplification has received new impetus from the findings that a number of specific genes can undergo amplification when somatic mammalian cells are placed under selective conditions (see Schimke, 1982; Stark and Wahl, 1984; Hamlin et al., 1984). The rate at which such cells, whether bacterial or cultured mammalian cells, can be selected for gene am- plification is greater than standard mutation rates, sug- gesting that gene amplifications are relatively frequent. This review describes current studies of gene amplification in mammalian cells, with emphasis on the acquisition of resistance to methotrexate as a result of amplification of the dihydrofolate reductase gene. Gene Amplification and Acquisition of Methotrexate Resistance Methotiexate resistance in cultured cells can result from at least three mechanisms: altered transport of MTX (Sirotnak et al., 1981); reduced affinity of DHFR for MTX (Flintoff et al., 1976; Haber et al., 1981); and overproduction of DHFR (Alt et al., 1976). The overproduction of DHFR is the result of a dose-related amplification of the DHFR gene (Alt et al., 1978; Bostock and Tyler-Smith, 1981; Melera et al., 1980). The general properties of MTX resistance resulting from DHFR gene amplification include the following: -Cells with amplified DHFR genes are selected charac- teristically by stepwise selection, and cell variants can be obtained eventually with as many as 100 to 1000 DHFR genes. This is interpreted to indicate that amplifications occur in small increments; hence high MTX resistance resulting from gene amplification cannot be obtained by large, single-step selection protocols. The stringency of selection is critical in obtaining amplified cell variants. This property is seen in the studies of Tlsty et al. (1984a) where P-galactosidase gene amplification in Escherichia coli was obtained only with a mutant enzyme that retained approx- imately 8% of normal enzyme activity. -Resistance is the result of overproduction of the target protein. In the case of MTX, resistance results from the variant cells’ containing sufficient free enzyme to generate tetrahydrofolate for cell-synthetic processes. Haber and Schimke (1981) have reported an example in which a mutation in the DHFR gene occurred secondary to initial gene amplification that imparted resistance as a result of a 300-fold decrease in affinity of DHFR for MTX. Con- versely, Flintoff et al. (1976) reported that low MTX resist- ance resulted from mutation of DHFR to low affinity for MTX, and subsequent higher MTX resistance resulted from overproduction of the altered enzyme. Thus various mech- anisms of resistance can be superimposed on other mech- anisms -The resistance phenotype and amplified genes can be stable or unstable. When cells are grown in the absence of MTX in some cell lines the amplified genes decline in the population slowly over a period of 6 to 12 months (stable), whereas in others half of amplified genes the population can be lost in 20 cell doublings, a period of 2 weeks (unstable) (Kaufman et al., 1979). When the amplified genes are stable, they are present on chromo- somes (Nunberg et al., 1978; Dolnick et al., 1979). Elon- gated chromosomal structures were first observed by Biedler and Spengler (1976) in MTX-resistant Chinese hamster lung cell lines and were named ‘homogeneously staining regions’ (HSRs). The absence of an HSR does not rule out gene amplification. Fougere-Deschatrette et al. (1984) have reported DHFR amplification in a number of rat hepatoma cell lines with comparable degrees of gene amplification; in one of these there is no karyological evidence of an HSR whereas by in situ localization the genes are present on a single chromosome that cannot be distinguished from the same chromosome in sensitive cells. Inasmuch as the size of the DNA sequence that is amplified can vary markedly (see below), the detection of an HSR is a function of both the unit length and the number of amplified genes. In the unstably amplified state, the DHFR genes reside on small, often paired, extrachromosomal elements, called double minute chromosomes (DMs). Such elements vary in size between different cell lines or within the same cells, replicate in the cell cycle, but contain no centromeres (Levan et al., 1977; Barker et al., 1980). Because they lack centromeres, they can be proportioned unequally into daughter cells at mitosis and/or undergo micronucleation and hence be lost rapidly under nonselective conditions (Schimke et al., 1981). It is unclear why some cell lines tend to generate amplified DHFR genes on DMs, whereas others tend to generate cell lines with DHFR genes in an HSR configuration. In general, cell lines that maintain a stable karyotype (e.g. hamster) tend to have HSRs whereas highly aneuploid lines (e.g. mouse) tend to have DMs. -The majority of cells, when first selected for MTX resist- ance, contain unstably amplified DHFR genes, and when selection is maintained for some time, the emerging pop- ulation often contains stably amplified genes. When Chinese hamster ovary cells (in which amplified DHFR genes are classically stable) are initially selected for MTX