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 Ãase;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 Â¡ointsassociated with gene amplification in methotrexate- Received October 31,1983; accepted January 24,1984. resistant mouse cell lines, submitted for publication.

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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 ÃŸ- 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 thÃ©fluo 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 a recurring theme, as dis spontaneously in the absence of selection pressure. Johnston ef al. (49) have estimated that the frequency of a 2-fold increase in cussed below. gene copy number is of the order of 1 x 10~3/cell generation in Amplification of a number of genes in somatic cells has now sensitive cells. Thus, the spontaneous frequency of gene ampli been observed, including the CAD gene (96), the metallothionine fication is approximately 102 greater in this CHO cell line than is gene (9), the hypoxanthine-guanine phosphoribosyltransferase gene (16), thymidylate synthetase (76), hydroxymethylglutaryl- estimated from studies based on analysis of emerging MTX- CoA reductase (24, 77), adenosine deaminase (99), glutamine resistant clones. synthetase (99), ornithine decarboxylase (64), and ribonucleotide The frequency with which cells become resistant to MTX can reductase (61). In all such cases, a specific enzyme inhibitor was be increased dramatically by various treatments of cells prior to used to obtain resistant cell variants. Perhaps more interesting MTX selection. Varshafsky (93), as well as our laboratory (90), are a number of reports showing that resistance obtained by has shown that prior treatment of cells with MTX enhances the frequency of MTX resistance approximately 10-fold and that the stepwise selection for Vinca alkaloids (8,10, 56), Colcemid (32), or actinomycin D7 produces a similar cross-resistance phenotype inclusion of the tumor promotor, ^-O-tetradecanoylphorbol-IS- to Vinca alkaloids, actinomycin D, puromycin, and Colcemid that acetate, either at the time of initial MTX treatment or during the results from altered transport. This phenotype is associated with clonal selection process further enhances the frequency of MTX overproduction of specific protein(s), and the karyotypes of such resistance and DHFR gene amplification. Other studies from our cells show the consequences of gene amplification, i.e., HSRs laboratory have shown that inhibition of DMA replication with hydroxyurea, an inhibitor of ribonucleotide reductase (20), or or DMs. In the papers cited, the overproduced protein does not with aphidicolin, an inhibitor of DNA polymerase a,8 enhances appear to have the same size characteristics in all cell lines, the subsequent emergence of MTX-resistant colonies. In addi raising again the question of whether separate amplification tion, Tlsty ef al. (91) have shown that UV radiation and the events can result in the same resistance phenotype. These carcinogen, A/-acetoxy-A/-acetylaminofluorene, can also enhance results, in conjunction with the above-cited resistance to tetra the frequency of MTX resistance and that 12-O-tetradecanoyl- cycline in E. coli, indicate that amplification events can underlie phorbol-13-acetate further increases this frequency. In our stud resistances the physiological phenotype of which is that of ies, there are a number of parameters that are critical in dem altered drug transport. onstrating the enhancement of MTX resistance, including the The general properties of stepwise selection, overproduction growth state of the cells, the subclone of cells used, the dose of of a protein, and either a stable or unstable phenotype also agent utilized, as well as the time following treatment that cells 5 T. D. Tlsty, A. M. Albertini, and J. H. Miller. Gene amplification in the lac region are subjected to MTX selection. Interested readers are referred of E. coli, submitted for publication. to the Brown and Tlsty papers for details. 8S. M. Beverley. J. A. Coderre. D. V. Santi, and R. T. Schimke. Unstable We conclude that any number of agents that affect DNA amplified DNAs are found as extra-chromosomal circular forms in methotrexate- resistant Leishmania tropica, submitted for publication. 7G. Levan, personal communication. â€¢P.C. Brown, unpublished observation.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1984 American Association for Cancer Research. Gene Amplification, Drug Resistance, and Cancer synthesis or introduce damage into DNA may well facilitate sister chromatids and (b) disproportionate replication. With the amplification. This conclusion is in keeping with the proposal of former mechanism, amplification would occur progressively dur Varshafsky (94) that a variety of cell treatments can result in ing each mitosis and is a reciprocal process; i.e., some cells will "misfiring" of replication and can lead to the generation of ampli lose genes, and some will gain genes. The initial events would fied DNA sequences. This concept is further supported by the retain the amplified genes within a chromosome, i.e., HSR, and results of Lavi (58) showing that various treatments of cells, they would subsequently undergo excision to generate DMs. including a wide variety of carcinogens, markedly enhance am Disproportionate replication involves the concept that there can plification of SV40 sequences integrated into the genome of occur more than a single initiation of replication on a portion of CHO cells. a chromosome within a single S phase of the cell cycle. This It is curious in our experiments in 3T6 cells (20, 91 ) that only model is depicted in Chart 1, where the process of DNA repli approximately 50% of emerging MTX-resistant colonies contain cation is shown as the standard linear model (left) or in the loop amplified DHFR genes, irrespective of whether they occur spon configuration. The consequence of multiple initiations is the taneously or are enhanced as much as 100-fold by various generation of free strands of DNA within a replication "bubble," treatments. Thus, such treatments facilitate DHFR gene amplifi the so-called "onion skin" replication model. If one accepts the cation, but they also increase resistance as a result of other concept of Vogelstein et al. (95) that, during DNA synthesis, the physiological mechanisms as well. In at least one cell line we DNA is moving through fixed sites of replication, thereby gener have studied, MTX resistance resulting from alteration in trans ating loop structures, then the loose ends of newly replicated port of MTX is an unstable phenotype.9 The instability of this DNA can readily undergo recombination, generating extrachro- resistance phenotype raises the question of whether gene am mosomal, circular chromosomal structures, I.e., DMs. In addition, plification may also underlie other forms of MTX resistance in a circular DNA structure can undergo rolling circle replication addition to the overproduction of DHFR. with subsequent recombination during a single replication cycle The above results suggest that treatments of cells with agents to generate a tandem head-to-tail repeat of amplified genes at that alter DNA replication or DNA structure may actually facilitate gene amplification and the emergence of resistance. This con cept is supported by studies of Rath ef al.10 in which 3T6 cells LINEAR LOOP were subjected to 3 stepwise protocols in which the time re quired to obtain a specified number of cells resistant to a 10-fold increment in MTX was determined: for a single-step (0 to 200 nM MTX) protocol, the time was 45 days; for a 2-step (0 to 80, 80 to 200 nw MTX) protocol, the time was 21 days; for a multiple- step (0 to 40, 40 to 80, 80 to 120, 120 to 160, 160 to 200 nM MTX) protocol, the time was 6 days. The 2 clones resistant by the single step had no amplified DHFR genes, whereas the cells resistant to 200 nu MTX by both of the step selections had an approximately 6-fold amplification of the DHFR gene. Analysis of the data indicates that the DHFR-amplified cells could not have preexisted in the cell population and therefore were generated in the course of the selection process. Such results suggest that the long-standing principle that resistance of a cell population involves selection of a preexisting "mutant" may not hold for the class of resistances that result from gene amplifications. An obvious and important question arises: does gene amplifi cation occur in "normal" cells? All of the examples of gene (2) amplification in somatic cells, whether cells in culture or tumor cells (see below), involve by definition "abnormal" cells. We do not yet know whether this is specifically a phenomenon of continuous cell lines and tumor cells or whether amplifications can occur in any cell. There are, indeed, differences in the frequency with which gene amplification can be accomplished. In the case of at least one analysis of this question, the difference appears to reside in the properties of the cells.38 In general, cell lines that are more "transformed" appear to have a higher propensity for DHFR gene amplification.

On the Mechanism of Gene Amplification (3)

Two general mechanisms can be envisaged that will result in gene amplification in somatic cells: (a) unequal crossing over of ' R. Cassin. unpublishedobservation. 10H. Rath, T. D. Tlsty, and R. T. Schimke. Rapid emergence of methotrexate resistance in cultured mouse cells, submitted for publication. Chart 1. Saltatory replicationmodel.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1984 American Association for Cancer Research. R. T. Schimke the site of the resident gene, i.e., HSRs (see Ref. 80 for discus Such "extra" DNA is highly unstable, and most is lost rapidly sion of this model). from the cell. However, some rearrangement-recombination The available experimental evidence favors the disproportion events occur, such that some of the additional DNA is retained ate replication mechanism and challenges the assumption that in the genome nucleus. Under appropriate selection conditions, only a single round of DNA replication always occurs in a single those cells retaining the additional, i.e., amplified, DNA corre S phase. For instance, amplification of the ribosomal genes sponding to a necessary gene product will survive. The subset during development in Xenopus leavis is considered to occur by of cells in which amplification occurs in a single cell cycle contains a rolling circle replication of extrachromosomal, circular DNA only twice as many DHFR genes. This minimal increment in gene sequences (46). More recently, the developmental^ regulated copy number, however, will allow such cells to have a greater amplification of the chorion genes in Drosophila, as first demon probability of survival in MTX in the next cell cycle, where they strated by Spradling and Mahowald (86), has been shown to can again undergo overreplication, ultimately generating high result in the generation of amplified DNA sequences of variable gene copy number and resistance. lengths (87), which result from a process of multiple initiations of Aberrant replication has been reported after transient inhibition DNA replication, i.e., multiple replication "bubbles" as observed of DNA synthesis in both prokaryotes (12,72,73) and eukaryotes by electron microscopy (69). (97, 100). We believe that the same general process underlies The structures of the amplified DNA sequences in L tropica amplifications induced by DNA-damaging agents (91). We find resulting from selection for MTX resistance bear on the mecha that the timing of introduction of DNA damage by UV radiation nism of gene amplification.6 Two different DNA sequences are is critical in enhancing MTX resistance and gene amplification; it amplified, resulting in resistance. In both cases, the structure of is only during the third to sixth hr of S phase that UV enhances the amplified DNA is that of a supercoiled, extrachromosomal MTX resistance (91). This suggests that only in cells undergoing circle in newly selected cells. In one class of variant, the amplified active DNA synthesis will DNA-damaging agents promote gene unit is a single circle with a single recombinational joint. In the amplification. other variant, the structure is a circle which is made up of 2 Consistent with the concept that various agents that alter DNA sequence units from the chromosome ligated together as a head- replication patterns can result in extensive overreplication of to-head concatamer. Such a head-to-head circle cannot readily portions of a genome are findings that various ones of the above be the result of a rolling circle replication of a single unit circle, agents result in a variety of chromosomal aberrations in meta- but it could easily result from the ligation of 2 DNA sequences phase spreads of cells obtained while progressing through the rereplicated off opposite DNA strands (see Chart 1). In both first mitosis following treatments. Such aberrations include cells instances, the normal (unamplified) chromosomal sequence re that have undergone endoreduplication, i.e., have gone from 2n mained intact; i.e., it was not excised. These results are far more to An in which the chromosomes appear normal, cells with a consistent with the disproportionate replication-recombination normal 2n complement in which there is a large amount of small model than with any form of unequal crossing-over model. but clearly visible extrachromosomal DNA, and cells with varying That the overreplication of DNA sequences can occur in a degrees of chromosomal fragmentation.11 single-cell cycle has been shown by Mariani and Schimke (62) Although it is not clear that, in all cases of gene amplification using mitotically synchronized CHO cells. Mariani ef a/. (63) as studied in cultured mammalian cells, the mechanism of the showed that DHFR gene expression is regulated in the cell cycle; initial amplification involves overreplication, it should be pointed the rate of DHFR synthesis declines during d and increased out that all of the selections used involve agents or protocols, markedly in the second hr of S. phase. The DHFR gene is the consequence of which is inhibition of DNA replication and replicated in the first 1 to 2 hr of S phase (62). When synchronized cell growth. Thus, the same processes as studied with MTX cells are treated with hydroxyurea for a period of 6 hr beginning resistance and as enhanced by various treatments probably 2 hr after initiation of S phase, following which the cells are occur in these cases as well. Our results raise a number of allowed to resume DNA replication, the following are observed. issues concerning the temporal replication of various genes 1. The frequency of resistance to a level of MTX 100 times during S phase and suggest that genes replicated early in the the normal 50% lethal dose increases as much as 1000-fold cell cycle might be those that are more readily amplified. Our compared to cells not subject to the S-phase block or cells results also raise questions of the possible relationship of DNA treated with hydroxyurea prior to movement into the S phase. replication and transcription, inasmuch as the DHFR gene is 2. Approximately 40% of the cell population subjected to the replicated, and the gene is expressed within the same 1 to 2 hr hydroxyurea block displays a 2- to 4-fold increase in DHFR of the S phase. enzyme content as determined by fluorescence-activated cell sorter analysis. This subset of cells contains approximately 2- Gene Amplification and Clinical Resistance to Chemothera- fold increases in DHFR genes, and it is from this subset of the peutic Agents total cell population that emerge the highly resistant cells with The studies in cultured cells indicate that gene amplification is high DHFR gene copy number. a common mechanism for generating MTX resistance and that 3. There is extensive overreplication of DNA as monitored by gene amplification may be a common process for the emergence incorporation of bromodeoxyuridine. Thus, the rereplication of of resistance to a number of agents under appropriate stepwise DNA involves not only the DHFR gene but essentially all of the selection protocols. Our studies further suggest that various DNA that is normally replicated in the first 2 hr of the S phase agents that inhibit DNA synthesis or introduce damage into DNA (approximately 10% of genomic DNA). We conclude that inhibiting DNA replication in mid-S phase, can markedly facilitate gene amplification and MTX resistance followed by resumption of DNA replication, results in overrepli cation (disproportionate replication) of a portion of the genome. ' A. Hill, and R. T. Schimke, manuscript in preparation.

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as a result of provoking overreplication of a portion of the within cells, proteins that inactivate chemotherapeutic agents, or genome. proteins that facilitate the repair of DNA-damaging agents, our The question arises as to whether gene amplification occurs results concerning the properties of gene amplification as studied in the clinical setting to generate resistance. The answer would in the laboratory have a number of potential implications for appear to be "yes" for the DHFR gene and MTX resistance. clinical therapy. The principles enumerated below address the problem of the emergence of resistant tumor cells, are "idealized" Horns ef a/. (45) and Cardman ef al. (21) have each studied a patient with leukemia in which the cells were analyzed for DHFR from cultured cell systems, and will not be discussed in relation gene copy number prior to and following treatment with MTX. In to experience with various clinical treatment protocols and/or both cases, there was clinical and/or biochemical evidence of individual cases. MTX resistance and an approximately 3- to 6-fold amplification 1. Multiple therapeutic modalities, which are not incompatible, of DHFR genes. Curt ef al. (30) reported that MTX-resistant should be used. These modalities (drugs) should not impart tumor cells from an oat cell lung cancer of a patient treated with resistance by the same mechanism (or the same amplified DNA MTX, as obtained by clonogenic assay techniques, contained sequence). Such combined drug treatments are used commonly approximately 5 times as many DHFR genes as normal cells. and are based on the concept that each resistance phenomenon Finally, Trent ef al. (92) have studied cells from a patient with an is an independent event, such that the probability of 2 resistance ovarian adenocarcinoma which, by clonogenic assay, were re phenomena occurring in the same cell is highly unlikely. However, sistant to very high concentrations of MTX. They observed by if overreplication of DNA is the mechanism of gene amplification, then the "unlikelihood" to 2 independent amplification events is karyological analysis an expanded chromosome in such tumor cells which, by in situ hybridization with a DHFR complementary not as great as is generally envisaged if both genes are replicated DNA clone, contained large (unspecified) numbers of DHFR in the first part of the S phase. This possibility is supported by genes in a HSR configuration. Although this patient had not the finding in L tropica that in some variants both of the DNA received MTX therapy for her cancer prior to diagnosis, she had sequences imparting resistance occur in the same cells.6 received 2 years of treatments of twice-weekly MTX (2.5-mg 2. The dosages of drugs (concentration x time) should be dosages) for chronic psoriasis, and perhaps such treatment had sufficient to ensure that an effective concentration is delivered selected for MTX-resistant cells in the undiagnosed tumor. to the tumor cell to result in cell death. Our results showing that The papers of Horns ef al. (45) and Cardman ef al. (21) studied gene amplification occurs when DNA replication is transiently the entire tumor cell population, and hence, the question of inhibited suggest that, if sufficient concentrations are not used, whether or not DHFR gene amplification occurred during clono or if the drug concentration is allowed to fluctuate above and genic assays is not pertinent. From experience with gene ampli below an effective inhibitory concentration, overreplication of fication protocols and with the time required to obtain a cell DNA, gene amplification, and resistance can be generated. population with high gene copy number, a 6-fold amplification of 3. Agents that damage DNA should not be used in cell pop the DHFR gene by a single-step selection is unlikely to have ulations that are actively cycling. We would propose that alkyl- occurred during the time of the clonogenic assays as used in the ating agents might be most effective when used in noncycling papers of Curt ef al. (30) or Trent ef al. (92). Thus, it can be cells. This conclusion is based on the results indicating that reasonably concluded that DHFR gene amplification is, indeed, introduction of damage into DNA (UV) facilitates overreplication a mechanism for generating clinical resistance. As more resistant only in cells in the S phase. Although our studies have concen tumors are studied, and as more possible DNA sequences that trated on use of UV, we have made preliminary studies indicating impart resistance become available for analysis, the role of gene that A/-acetoxy-A/-acetoaminofluorene enhancement of DHFR amplification in clinical resistance phenomena can be more fully gene amplification is also cell cycle dependent and that other documented. DNA-damaging agents will function in a similar cell cycle-depend It is interesting to note that, in the 3 clinical studies in which ent fashion in facilitating gene amplification. the increase in DHFR gene copy number has been established, 4. The various combined modalities of treatment should not the amplification is only approximately 5-fold. In the studies of be used for prolonged time periods. Rather, they should alter Rath ef a/.10with mouse 3T6 cells cited above, resistance to 200 nate. This conclusion is based on the finding that gene amplifi nM MTX by stepwise protocols was associated with a 5- to 6- cation, when it first occurs, is often unstable, and only when fold amplification of the DHFR genes. In the experimental sys cells are maintained under selective conditions for long periods tems, the MTX concentration is maintained constant, whereas are the amplified genes stable in the emerging resistant popula in most clinical treatments with MTX, the effective concentration tion. Thus, prolonged use of a treatment regimen is more likely at the tumor cell site is subject to various parameters, including to result in stable resistance. A corollary of this conclusion is the diffusion barriers at the tumor site, blood supply, the rates of desirability to determine, subsequent to the development of delivery, metabolism, excretion, etc. Following "conventional" resistance and the cessation of use of the drugs, whether or not treatment dosages, a 200 nM concentration is within bracketed the tumor cells have retained the resistance. If resistance is ranges over a time period of intermittent dosage (22, 47). Thus, unstable, then the same drugs may possibly be used again, the 5-fold DHFR gene amplification may well be sufficient to thereby retaining more flexibility in potential therapeutic modali provide the necessary resistance to conventional clinical dosages ties. of MTX. On the assumption that gene amplification turns out to be an Gene Amplification and Oncogenesis important mechanism for the generation of resistance to various cancer treatment modalities, whether the resistance phenotype The concept that gene amplification may underlie certain as results from overproduction of specific target proteins (such as pects of the generation and/or progression of cancers has been DHFR), proteins that in some fashion prevent drug accumulation proposed by various authors (see Refs. 70, 78, and 94), and

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1984 American Association for Cancer Research. R T. Schimke there are progressively more observations leading credence to Other reports have indicated that various rearrangements are a role of gene amplification in the oncogenic process. Among associated with cancer, including the human 8; 17 translocation in Burkitt's lymphomas, which has been proposed to result in such observations are the following. 1. A number of continuous tumor cell lines, as well as tumor increased expression of the c-myc gene (31, 53, 89; see also cells, generally derived from solid tumors, have the karyological Ref. 78 for speculation on specificity of such translocations). In consequences of gene amplification, i.e., either HSRs or DMs addition, others have reported that a normal cellular "oncogene" (5-7, 29, 59, 74, 75). From such observations, it is not clear can be overexpressed by juxtaposition to a retrovirus promotor whether such chromosomal aberrations (and assumed amplified with high expression efficiency (28, 39, 44, 71). In view of the genes) are causally related to the oncogenic process, or whether fact that a cellular response to DNA-damaging agents in actively they are the result of the establishment of continuous cell lines dividing cells is overreplication of DNA, a consequence of such derived from such tumors. As will be discussed below, more a process may well be the recombination of DNA sequences- recent evidence has indicated that some of these cell lines chromosome fragments, such that a highly active promotor can contain amplified "oncogenes." "activate" a potential oncogene. Thus, the same basic process 2. Levan and Levan (60) have studied a mouse tumor cell line of overreplication of DNA may possibly result in 2 different means (SEWA) which contains multiple DMs (or other chromosomal for overproduction of proteins critical to overcoming normal structures containing amplified genes) when grown as a tumor growth constraints, i.e., gene amplification and recombinational in mice. When these cells are grown under cell culture conditions, activation. the cells progressively lose the DMs. When the cells grown in The results of our studies on overreplication of DNA within a culture are reintroduced into the mouse, the tumor again contains single cell cycle may have further implications for the generation multiple DMs. In addition, the tumorigenicity of the cultured cells and/or progression of cancer, inasmuch as treatment of cells is directly related to the number of DMs that exist in the cells at with inhibitors of DNA replication and DNA-damaging agents the time they are introduced into the mouse.7 This finding has generates extrachromosomal DNA of varying sizes, as well as direct analogy to unstably MTX-resistant cell lines containing occasional cells that undergo endoreduplication or contain frag DMs. In both cases, a selection pressure is imparted. In the case mented chromosomes. Such a process would result in a heter of the SEWA cells, the selection is for growth in the mouse, and ogeneous population of cells with unstable phenotypes, a prop the emerging tumor cells contain multiple DMs. Conversely, erty of a number of tumors. Indeed, there are a number of reports when the tumor cells are placed in nonselective conditions, the showing heterogeneity in DNA content in tumor cells (17, 48, DMs are lost. We would suggest that the selection process in 55). In addition, deVere White ef al. (33) have found an increased the case of SEWA cells is for overcoming normal growth con heterogeneity in DNA content in experimental tumors following straints of the mouse. Nothing is known currently concerning treatment with chemotherapeutic agents. Thus, if various drug how amplification of sequences in the SEWA cells might result treatments can result in a higher frequency of MTX resistance in unconstrained growth, and multiple mechanisms can be envis and gene amplification, is it not also possible that such treat aged. ments can result in a variety of genomic alterations, including 3. In the past several years, a number of investigators have amplifications and rearrangements that facilitate the process of reported on the amplification of oncogenes in tumor cell lines, malignant progression? including c-myc, c-Ki-ras, and c-alb (1, 23, 26, 27, 31, 54, 83, 84). It remains to be determined whether the amplification of Concluding Comments these sequences is causally related to the oncogenic process, In this "Perspectives" article, the phenomenon of gene ampli or whether their amplification is a result of the establishment of continuous cell lines. fication in somatic cells has been reviewed briefly. It would 4. Among the treatments of cells that enhance DHFR gene appear to be a relatively common process in cells in culture and, amplification experimentally are agents that damage DNA, in perhaps, in tumors as well. Currently available studies indicate cluding the well-established carcinogenic agents, UV light, and that amplification occurs by overreplication of portions of the W-acetoxy-N-acetoaminofluorene. Enhancement of gene ampli genome in a single cell cycle, i.e., disproportionate replication, fication occurs at doses lower than generally used in studies of and this process can be enhanced by the very agents used in mutagenesis (doses that produce only 10 to 30% killing), occurs cancer chemotherapy as well as by known carcinogenic agents. only in cells undergoing DNA synthesis and is demonstrated in I have speculated that such overreplication may have a role in terms of enhanced MIX resistance only after DNA synthesis is not only the generation of drug resistances clinically, but also allowed to occur prior to placement of cells in MIX (91). Thus, the progression from a normal to malignant cell. Clearly, it is the demonstration of the role of such agents in facilitating gene more than a platitude to state that far more research is needed amplification can be easily missed if the proper experimental to understand the underlying mechanism(s) of gene amplification conditions are not used. If such DNA-damaging agents facilitate and processes of drug resistance, as well as the possible role of overreplication in a manner similar to that we have found with genome overreplication-recombination in overcoming normal transient inhibition of DNA replication (62), then the question growth constraints. arises as to what types of genes are replicated early in S phase of the cell cycle; specifically, are any oncogenes replicated at ACKNOWLEDGMENTS that time? If so, then they also will be amplified and may lead to the loss of normal growth constraints on cells. It is of interest I would like to thank the many members of my laboratory group whose research, that, in a human neuroblastoma cell line, an HSR-containing both published and in preparation, has been discussed herein, including Steven Bevertey, Peter Brown, Richard Cassin, Nancy Federspeil, Charles Gasser, Anna region replicates early in the cell cycle (57), and Kohl ef al. (54) Hill, Randal Johnston, Brian Mariani, Elise Mosse, Heidi Rath, James Schilling, have just shown that this cell line contains amplified c-myc genes. Allison Schimke, David Smouse, Chris Simonsen, and Thea Tlsty.

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1742 CANCER RESEARCH VOL. 44

Robert T. Schimke

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