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Gene Amplification, Drug Resistance, and Cancer1

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Gene Amplification, Drug Resistance, and Cancer1 [CANCER RESEARCH 44,1735-1742, May 1984] Perspectives in Cancer Research Gene Amplification, Drug Resistance, and Cancer1 Robert T. Schimke2 Department of Biological Sciences, Stanford University, Stanford, California 94305 Introduction absence of selection (MTX), in some cell lines, the resistance and amplified DHFR genes are stable (over a period of 6 months), The finding that genetic alterations, i.e., mutations, can involve whereas in other cell lines, the amplified DHFR genes are unst quantitative changes, as contrasted to the qualitative changes, able such that 50% of the genes and MTX resistance can be in a genome was first made in Drosophila (88) and more recently lost in 20 cell doublings. When the genes are stable, they are has been examined extensively as a developmental process with present on chromosomes, often on a single of the 2 homologous ribosomal genes in amphibians (18; see Ref. 13 for review). The chromosomes at the site of the resident nonamplified gene (68, phenomenon of selective gene amplification in cultured mam 80). Such chromosomes are often expanded in length and were malian cells was first documented in 1978 as a mechanism for originally denoted as HSRs by Siedler and Spengler (11), who the acquisition of resistance to MTX3 (2, 79). Since that time, first reported such a chromosomal abnormality in MTX-resistant increasing numbers of examples of gene amplification in mam Chinese hamster lung cell lines. Such HSR-containing chromo malian cells have been reported, including amplification of so- called "oncogenes" in tumor cell lines as well as gene amplifica somes have been reported in a number of cases in which genes are stably amplified (see Ref. 78). The size of the DNA sequence tion resulting in the emergence of clinical chemotherapy resis amplified may vary extensively, with estimates of the amplified tance. This "Perspective" will attempt to provide a brief overview unit varying from 135 kilobases (66) to over 500 kilobases of of this newly emerging area of research, using in large part DNA (68) in separate, MTX-resistant isolates of CHO cells. research from the author's own laboratory as examples. The Studies from our laboratory with a number of mouse cell lines reader is referred to Ref. 78 for more extensive details of this with amplified DHFR genes4 indicate that the amplified DNA field. sequence is the same as the normal, unamplified DNA within DHFRGene AmplificationandMTX Resistancein Cultured and surrounding the DHFR gene, and that the actual unit of MammalianCells amplified DNA can be of variable length. In the unstably amplified state, the DHFR genes reside on There are 3 well-documented mechanisms whereby cultured small, often paired extrachromosomal elements, called DMs. mammalian cells become resistant to MTX: an alteration in affinity Such elements replicate in the cell cycle, but they contain no of DHFR for MTX (36, 41); altered MTX transport (85); and centromers. Because they lack centromers, they can be propor overproduction of DHFR (3, 43). In all cases studied, the over tioned unequally into daughter cells at mitosis and/or undergo production of DHFR is the consequence of a proportional ampli micronucleation and hence can be lost rapidly (see Ref. 80 for fication of the DHFR gene (2, 14, 34, 37, 65-67), i.e., a gene discussion of this process). It is not at all clear why some cell dosage relationship. The general properties of MTX resistance lines tend to generate amplified DHFR genes on DMs, whereas resulting from DHFR gene amplification include the following. others tend to generate cell lines with DHFR genes in a HSR 1. Cells with amplified DHFR genes are selected characteris configuration. In general, cell lines that maintain a stable kary- tically by stepwise selection. Cell variants can be obtained readily otype tend to have HSRs, whereas cell lines that are highly with as many as 100 to 1000 DHFR genes by gradual step aneuploid tend to have DMs (19). increments of MTX in the medium. We interpret this to indicate When cells are initially selected for MTX resistance, the emerg that the amplifications occur in small steps and, hence, that high ing population is vastly heterogeneous for gene copy number in MTX resistance resulting from gene amplification cannot be the cells (51) as studied with a fluorescein conjugate of MTX and obtained by large, single-step selection protocols. the fluorescence-activated cell sorter. The majority of such cells 2. Resistance is the result of overproduction of a normal contain unstably amplified DHFR genes. However, when this cell protein. In the case of MTX, resistance results from the failure population is maintained under selection pressure for as short a of variant cells containing sufficient free enzyme to generate time as 60 cell doublings, the population of cells that emerges enough tetrahydrofolate for cellular synthetic processes. contains amplified genes (see Ref. 80 for more extensive discus 3. The resistance phenotype and amplified genes can be sions). This is an important point: although the instability of a either stable or unstable; i.e., when cells are grown in the phenotype is an excellent clue to the possibility that an amplifi cation phenomenon has occurred, the lack of an unstable phe notype does not rule out amplification as a mechanism, in partic 1Studies described herein from the author's laboratory have been supported by ular when such cells have been maintained under selective research grants from the American Cancer Society (NP148), the National Cancer Institute (CA 16318), and the National Institute of General Medical Sciences (GM conditions for 2 to 4 months of growth. 14931). 2American Cancer Society Research Professor of Biology. 3 The abbreviations used are: MTX, methotrexate; DHFR, dihydrofolate reduc íase;HSR, homogeneously staining region; DM, double minute chromosome; CHO, 4 N. Federspeil, S. M. Beverley, C. Simonsen, J. Schilling, and R. T. Schimke. Chinese hamster ovary. Novel recombination ¡ointsassociated with gene amplification in methotrexate- Received October 31,1983; accepted January 24,1984. resistant mouse cell lines, submitted for publication. MAY 1984 1735 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1984 American Association for Cancer Research. R. T. Schimke How Common Is Gene Amplification? describe certain of the resistances to insecticides (81). This brief enumeration of examples of gene amplification is by no means Gene amplification is a more common phenomenon than gen inclusive. However, we conclude that gene amplification occurs erally considered, especially under selective conditions where under many different circumstances and is not an uncommon cell growth inhibition can be overcome by overproduction of a phenomenon throughout biology. protein. Gene duplications are common in bacteria, and such duplications are highly unstable. In Salmonella, Anderson and Frequency of Gene Amplification Roth have shown that duplications of the histidine operan occur in approximately 0.1% of all cells (see Ref. 4 for review). In more What is the frequency of spontaneous gene amplification in recent studies, resistance to penicillin in Escherichia coli has cultured cells? In experiments in which cells are selected for single-step resistance to a MTX concentration 10 times the been shown to result from duplications of the chromosomal ß- normal 50% lethal dose, the frequency is approximately 2 x 10~5 lactamase gene (35), and resistance to tetracycline in E. coli can result from duplication of a chromosomal sequence resulting in in CHO cells (90). Such experiments require growth of the cells rapid efflux of the antibiotic (40). Tlsty ef al.5 have reported for some 2 to 3 weeks in MTX and, hence, may not reflect the recently that extensive amplification of the /3-galactosidase gene frequency of gene amplification if such genes are highly unstable coding for a partially defective enzyme occurs readily in E. coli and are lost within the period of growth required for colony under growth in limiting lactose. Thus, it appears that, under detection. Johnston ef al. (49) have addressed this question in appropriate selective conditions, a common mechanism whereby CHO cells, using a fluorescein conjugate of MTX and théfluo rescence-activated cell sorter, a technique we have utilized to E. coli overproduces a required enzyme is by gene duplication amplifications. Gene amplification and the generation of drug study the heterogeneity and changes in cell populations with resistance also occur in Protozoa. Our laboratory has reported respect to DHFR enzyme and gene copy number (42, 52, 80). that Leishmania tropica becomes resistant to MIX by virtue of By sorting from various populations of CHO cells those cells gene amplification (25). Of interest is the finding that 2 entirely which are on the upper end of a fluorescence intensity distribu different DMA sequences can be amplified to impart MTX resis tion (those cells with more DHFR enzyme content), growing such tance; one sequence codes for a fused thymidylate synthetase- cells in the absence of MTX, and subsequently resorting the DHFR protein, whereas the physiological mechanism of the other upper end of the fluorescence distribution repeatedly, we can amplification event is unknown.6 Thus, 2 different amplification obtain cells under nonselective conditions with a 50-fold increase events can occur in the same organism to impart resistance to in DHFR gene copy number. Thus, gene amplification can occur the same drug. This may become
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