[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

The finding that genetic alterations, i.e., mutations, can involvequantitative changes, as contrasted to the qualitative changes,in a genome was first made in Drosophila (88) and more recentlyhas been examined extensively as a developmental process withribosomal genes in amphibians (18; see Ref. 13 for review). Thephenomenon of selective gene amplification in cultured mammalian cells was first documented in 1978 as a mechanism forthe acquisition of resistance to MTX3 (2, 79). Since that time,

increasing numbers of examples of gene amplification in mammalian cells have been reported, including amplification of so-called "oncogenes" in tumor cell lines as well as gene amplifica

tion resulting in the emergence of clinical chemotherapy resistance. This "Perspective" will attempt to provide a brief overview

of this newly emerging area of research, using in large partresearch from the author's own laboratory as examples. The

reader is referred to Ref. 78 for more extensive details of thisfield.

DHFRGene Amplificationand MTX Resistancein CulturedMammalianCells

There are 3 well-documented mechanisms whereby cultured

mammalian cells become resistant to MTX: an alteration in affinityof DHFR for MTX (36, 41); altered MTX transport (85); andoverproduction of DHFR (3, 43). In all cases studied, the overproduction of DHFR is the consequence of a proportional amplification of the DHFR gene (2, 14, 34, 37, 65-67), i.e., a gene

dosage relationship. The general properties of MTX resistanceresulting from DHFR gene amplification include the following.

1. Cells with amplified DHFR genes are selected characteristically by stepwise selection. Cell variants can be obtained readilywith as many as 100 to 1000 DHFR genes by gradual stepincrements of MTX in the medium. We interpret this to indicatethat the amplifications occur in small steps and, hence, that highMTX resistance resulting from gene amplification cannot beobtained by large, single-step selection protocols.

2. Resistance is the result of overproduction of a normalprotein. In the case of MTX, resistance results from the failureof variant cells containing sufficient free enzyme to generateenough tetrahydrofolate for cellular synthetic processes.

3. The resistance phenotype and amplified genes can beeither stable or unstable; i.e., when cells are grown in the

1Studies described herein from the author's laboratory have been supported by

research grants from the American Cancer Society (NP148), the National CancerInstitute (CA 16318), and the National Institute of General Medical Sciences (GM14931).

2American Cancer Society Research Professor of Biology.3The abbreviations used are: MTX, methotrexate; DHFR, dihydrofolate reduc

íase;HSR, homogeneously staining region; DM, double minute chromosome; CHO,Chinese hamster ovary.

Received October 31,1983; accepted January 24,1984.

absence of selection (MTX), in some cell lines, the resistanceand amplified DHFR genes are stable (over a period of 6 months),whereas in other cell lines, the amplified DHFR genes are unstable such that 50% of the genes and MTX resistance can belost in 20 cell doublings. When the genes are stable, they arepresent on chromosomes, often on a single of the 2 homologouschromosomes at the site of the resident nonamplified gene (68,80). Such chromosomes are often expanded in length and wereoriginally denoted as HSRs by Siedler and Spengler (11), whofirst reported such a chromosomal abnormality in MTX-resistantChinese hamster lung cell lines. Such HSR-containing chromo

somes have been reported in a number of cases in which genesare stably amplified (see Ref. 78). The size of the DNA sequenceamplified may vary extensively, with estimates of the amplifiedunit varying from 135 kilobases (66) to over 500 kilobases ofDNA (68) in separate, MTX-resistant isolates of CHO cells.Studies from our laboratory with a number of mouse cell lineswith amplified DHFR genes4 indicate that the amplified DNA

sequence is the same as the normal, unamplified DNA withinand surrounding the DHFR gene, and that the actual unit ofamplified DNA can be of variable length.

In the unstably amplified state, the DHFR genes reside onsmall, often paired extrachromosomal elements, called DMs.Such elements replicate in the cell cycle, but they contain nocentromers. Because they lack centromers, they can be proportioned unequally into daughter cells at mitosis and/or undergomicronucleation and hence can be lost rapidly (see Ref. 80 fordiscussion of this process). It is not at all clear why some celllines tend to generate amplified DHFR genes on DMs, whereasothers tend to generate cell lines with DHFR genes in a HSRconfiguration. In general, cell lines that maintain a stable kary-

otype tend to have HSRs, whereas cell lines that are highlyaneuploid tend to have DMs (19).

When cells are initially selected for MTX resistance, the emerging population is vastly heterogeneous for gene copy number inthe cells (51) as studied with a fluorescein conjugate of MTX andthe fluorescence-activated cell sorter. The majority of such cells

contain unstably amplified DHFR genes. However, when this cellpopulation is maintained under selection pressure for as short atime as 60 cell doublings, the population of cells that emergescontains amplified genes (see Ref. 80 for more extensive discussions). This is an important point: although the instability of aphenotype is an excellent clue to the possibility that an amplification phenomenon has occurred, the lack of an unstable phenotype does not rule out amplification as a mechanism, in particular when such cells have been maintained under selectiveconditions for 2 to 4 months of growth.

4 N. Federspeil, S. M. Beverley, C. Simonsen, J. Schilling, and R. T. Schimke.

Novel recombination ¡ointsassociated with gene amplification in methotrexate-resistant mouse cell lines, submitted for publication.

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R. T. Schimke

How Common Is Gene Amplification?

Gene amplification is a more common phenomenon than generally considered, especially under selective conditions wherecell growth inhibition can be overcome by overproduction of aprotein. Gene duplications are common in bacteria, and suchduplications are highly unstable. In Salmonella, Anderson andRoth have shown that duplications of the histidine operan occurin approximately 0.1% of all cells (see Ref. 4 for review). In morerecent studies, resistance to penicillin in Escherichia coli hasbeen shown to result from duplications of the chromosomal ß-lactamase gene (35), and resistance to tetracycline in E. coli canresult from duplication of a chromosomal sequence resulting inrapid efflux of the antibiotic (40). Tlsty ef al.5 have reported

recently that extensive amplification of the /3-galactosidase gene

coding for a partially defective enzyme occurs readily in E. coliunder growth in limiting lactose. Thus, it appears that, underappropriate selective conditions, a common mechanism wherebyE. coli overproduces a required enzyme is by gene duplicationamplifications. Gene amplification and the generation of drugresistance also occur in Protozoa. Our laboratory has reportedthat Leishmania tropica becomes resistant to MIX by virtue ofgene amplification (25). Of interest is the finding that 2 entirelydifferent DMA sequences can be amplified to impart MTX resistance; one sequence codes for a fused thymidylate synthetase-

DHFR protein, whereas the physiological mechanism of the otheramplification event is unknown.6 Thus, 2 different amplification

events can occur in the same organism to impart resistance tothe same drug. This may become a recurring theme, as discussed below.

Amplification of a number of genes in somatic cells has nowbeen observed, including the CAD gene (96), the metallothioninegene (9), the hypoxanthine-guanine phosphoribosyltransferasegene (16), thymidylate synthetase (76), hydroxymethylglutaryl-

CoA reductase (24, 77), adenosine deaminase (99), glutaminesynthetase (99), ornithine decarboxylase (64), and ribonucleotidereductase (61). In all such cases, a specific enzyme inhibitor wasused to obtain resistant cell variants. Perhaps more interestingare a number of reports showing that resistance obtained bystepwise selection for Vinca alkaloids (8,10, 56), Colcemid (32),or actinomycin D7 produces a similar cross-resistance phenotype

to Vinca alkaloids, actinomycin D, puromycin, and Colcemid thatresults from altered transport. This phenotype is associated withoverproduction of specific protein(s), and the karyotypes of suchcells show the consequences of gene amplification, i.e., HSRsor DMs. In the papers cited, the overproduced protein does notappear to have the same size characteristics in all cell lines,raising again the question of whether separate amplificationevents can result in the same resistance phenotype. Theseresults, in conjunction with the above-cited resistance to tetra

cycline in E. coli, indicate that amplification events can underlieresistances the physiological phenotype of which is that ofaltered drug transport.

The general properties of stepwise selection, overproductionof a protein, and either a stable or unstable phenotype also

5T. D. Tlsty, A. M. Albertini, and J. H. Miller. Gene amplification in the lac region

of E. coli, submitted for publication.8S. M. Beverley. J. A. Coderre. D. V. Santi, and R. T. Schimke. Unstable

amplified DNAs are found as extra-chromosomal circular forms in methotrexate-resistant Leishmania tropica, submitted for publication.

7G. Levan, personal communication.

describe certain of the resistances to insecticides (81). This briefenumeration of examples of gene amplification is by no meansinclusive. However, we conclude that gene amplification occursunder many different circumstances and is not an uncommonphenomenon throughout biology.

Frequency of Gene Amplification

What is the frequency of spontaneous gene amplification incultured cells? In experiments in which cells are selected forsingle-step resistance to a MTX concentration 10 times thenormal 50% lethal dose, the frequency is approximately 2 x 10~5

in CHO cells (90). Such experiments require growth of the cellsfor some 2 to 3 weeks in MTX and, hence, may not reflect thefrequency of gene amplification if such genes are highly unstableand are lost within the period of growth required for colonydetection. Johnston ef al. (49) have addressed this question inCHO cells, using a fluorescein conjugate of MTX and théfluorescence-activated cell sorter, a technique we have utilized to

study the heterogeneity and changes in cell populations withrespect to DHFR enzyme and gene copy number (42, 52, 80).By sorting from various populations of CHO cells those cellswhich are on the upper end of a fluorescence intensity distribution (those cells with more DHFR enzyme content), growing suchcells in the absence of MTX, and subsequently resorting theupper end of the fluorescence distribution repeatedly, we canobtain cells under nonselective conditions with a 50-fold increase

in DHFR gene copy number. Thus, gene amplification can occurspontaneously in the absence of selection pressure. Johnston efal. (49) have estimated that the frequency of a 2-fold increase ingene copy number is of the order of 1 x 10~3/cell generation in

sensitive cells. Thus, the spontaneous frequency of gene amplification is approximately 102 greater in this CHO cell line than is

estimated from studies based on analysis of emerging MTX-

resistant clones.The frequency with which cells become resistant to MTX can

be increased dramatically by various treatments of cells prior toMTX selection. Varshafsky (93), as well as our laboratory (90),has shown that prior treatment of cells with MTX enhances thefrequency of MTX resistance approximately 10-fold and that theinclusion of the tumor promotor, ^-O-tetradecanoylphorbol-IS-

acetate, either at the time of initial MTX treatment or during theclonal selection process further enhances the frequency of MTXresistance and DHFR gene amplification. Other studies from ourlaboratory have shown that inhibition of DMA replication withhydroxyurea, an inhibitor of ribonucleotide reductase (20), orwith aphidicolin, an inhibitor of DNA polymerase a,8 enhances

the subsequent emergence of MTX-resistant colonies. In addi

tion, Tlsty ef al. (91) have shown that UV radiation and thecarcinogen, A/-acetoxy-A/-acetylaminofluorene, can also enhancethe frequency of MTX resistance and that 12-O-tetradecanoyl-phorbol-13-acetate further increases this frequency. In our stud

ies, there are a number of parameters that are critical in demonstrating the enhancement of MTX resistance, including thegrowth state of the cells, the subclone of cells used, the dose ofagent utilized, as well as the time following treatment that cellsare subjected to MTX selection. Interested readers are referredto the Brown and Tlsty papers for details.

We conclude that any number of agents that affect DNA

•P. C. Brown, unpublished observation.

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Gene Amplification, Drug Resistance, and Cancer

synthesis or introduce damage into DNA may well facilitateamplification. This conclusion is in keeping with the proposal ofVarshafsky (94) that a variety of cell treatments can result in"misfiring" of replication and can lead to the generation of ampli

fied DNA sequences. This concept is further supported by theresults of Lavi (58) showing that various treatments of cells,including a wide variety of carcinogens, markedly enhance amplification of SV40 sequences integrated into the genome ofCHO cells.

It is curious in our experiments in 3T6 cells (20, 91 ) that onlyapproximately 50% of emerging MTX-resistant colonies contain

amplified DHFR genes, irrespective of whether they occur spontaneously or are enhanced as much as 100-fold by various

treatments. Thus, such treatments facilitate DHFR gene amplification, but they also increase resistance as a result of otherphysiological mechanisms as well. In at least one cell line wehave studied, MTX resistance resulting from alteration in transport of MTX is an unstable phenotype.9 The instability of this

resistance phenotype raises the question of whether gene amplification may also underlie other forms of MTX resistance inaddition to the overproduction of DHFR.

The above results suggest that treatments of cells with agentsthat alter DNA replication or DNA structure may actually facilitategene amplification and the emergence of resistance. This concept is supported by studies of Rath ef al.10 in which 3T6 cells

were subjected to 3 stepwise protocols in which the time required to obtain a specified number of cells resistant to a 10-foldincrement in MTX was determined: for a single-step (0 to 200nM 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 nMMTX) protocol, the time was 6 days. The 2 clones resistant bythe single step had no amplified DHFR genes, whereas the cellsresistant to 200 nu MTX by both of the step selections had anapproximately 6-fold amplification of the DHFR gene. Analysis ofthe data indicates that the DHFR-amplified cells could not have

preexisted in the cell population and therefore were generatedin the course of the selection process. Such results suggest thatthe long-standing principle that resistance of a cell populationinvolves 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

amplification in somatic cells, whether cells in culture or tumorcells (see below), involve by definition "abnormal" cells. We do

not yet know whether this is specifically a phenomenon ofcontinuous cell lines and tumor cells or whether amplificationscan occur in any cell. There are, indeed, differences in thefrequency with which gene amplification can be accomplished.In the case of at least one analysis of this question, the differenceappears to reside in the properties of the cells.38 In general, celllines that are more "transformed" appear to have a higher

propensity for DHFR gene amplification.

On the Mechanism of Gene Amplification

Two general mechanisms can be envisaged that will result ingene 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.

sister chromatids and (b) disproportionate replication. With theformer mechanism, amplification would occur progressively during each mitosis and is a reciprocal process; i.e., some cells willlose genes, and some will gain genes. The initial events wouldretain the amplified genes within a chromosome, i.e., HSR, andthey would subsequently undergo excision to generate DMs.Disproportionate replication involves the concept that there canoccur more than a single initiation of replication on a portion ofa chromosome within a single S phase of the cell cycle. Thismodel is depicted in Chart 1, where the process of DNA replication is shown as the standard linear model (left) or in the loopconfiguration. The consequence of multiple initiations is thegeneration of free strands of DNA within a replication "bubble,"the so-called "onion skin" replication model. If one accepts the

concept of Vogelstein et al. (95) that, during DNA synthesis, theDNA is moving through fixed sites of replication, thereby generating loop structures, then the loose ends of newly replicatedDNA can readily undergo recombination, generating extrachro-

mosomal, circular chromosomal structures, I.e., DMs. In addition,a circular DNA structure can undergo rolling circle replicationwith subsequent recombination during a single replication cycleto generate a tandem head-to-tail repeat of amplified genes at

LINEAR LOOP

(2)

(3)

Chart 1. Saltatory replicationmodel.

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R. T. Schimke

the site of the resident gene, i.e., HSRs (see Ref. 80 for discussion of this model).

The available experimental evidence favors the disproportionate replication mechanism and challenges the assumption thatonly a single round of DNA replication always occurs in a singleS phase. For instance, amplification of the ribosomal genesduring development in Xenopus leavis is considered to occur bya rolling circle replication of extrachromosomal, circular DNAsequences (46). More recently, the developmental^ regulatedamplification of the chorion genes in Drosophila, as first demonstrated by Spradling and Mahowald (86), has been shown toresult in the generation of amplified DNA sequences of variablelengths (87), which result from a process of multiple initiations ofDNA replication, i.e., multiple replication "bubbles" as observed

by electron microscopy (69).The structures of the amplified DNA sequences in L tropica

resulting from selection for MTX resistance bear on the mechanism of gene amplification.6 Two different DNA sequences are

amplified, resulting in resistance. In both cases, the structure ofthe amplified DNA is that of a supercoiled, extrachromosomalcircle in newly selected cells. In one class of variant, the amplifiedunit is a single circle with a single recombinational joint. In theother variant, the structure is a circle which is made up of 2sequence units from the chromosome ligated together as a head-to-head concatamer. Such a head-to-head circle cannot readily

be the result of a rolling circle replication of a single unit circle,but it could easily result from the ligation of 2 DNA sequencesrereplicated off opposite DNA strands (see Chart 1). In bothinstances, the normal (unamplified) chromosomal sequence remained intact; i.e., it was not excised. These results are far moreconsistent with the disproportionate replication-recombinationmodel than with any form of unequal crossing-over model.

That the overreplication of DNA sequences can occur in asingle-cell cycle has been shown by Mariani and Schimke (62)

using mitotically synchronized CHO cells. Mariani ef a/. (63)showed that DHFR gene expression is regulated in the cell cycle;the rate of DHFR synthesis declines during d and increasedmarkedly in the second hr of S. phase. The DHFR gene isreplicated in the first 1 to 2 hr of S phase (62). When synchronizedcells are treated with hydroxyurea for a period of 6 hr beginning2 hr after initiation of S phase, following which the cells areallowed to resume DNA replication, the following are observed.

1. The frequency of resistance to a level of MTX 100 timesthe normal 50% lethal dose increases as much as 1000-foldcompared to cells not subject to the S-phase block or cells

treated with hydroxyurea prior to movement into the S phase.2. Approximately 40% of the cell population subjected to the

hydroxyurea block displays a 2- to 4-fold increase in DHFRenzyme content as determined by fluorescence-activated cellsorter analysis. This subset of cells contains approximately 2-

fold increases in DHFR genes, and it is from this subset of thetotal cell population that emerge the highly resistant cells withhigh DHFR gene copy number.

3. There is extensive overreplication of DNA as monitored byincorporation of bromodeoxyuridine. Thus, the rereplication ofDNA involves not only the DHFR gene but essentially all of theDNA that is normally replicated in the first 2 hr of the S phase(approximately 10% of genomic DNA).

We conclude that inhibiting DNA replication in mid-S phase,

followed by resumption of DNA replication, results in overreplication (disproportionate replication) of a portion of the genome.

Such "extra" DNA is highly unstable, and most is lost rapidly

from the cell. However, some rearrangement-recombination

events occur, such that some of the additional DNA is retainedin the genome nucleus. Under appropriate selection conditions,those cells retaining the additional, i.e., amplified, DNA corresponding to a necessary gene product will survive. The subsetof cells in which amplification occurs in a single cell cycle containsonly twice as many DHFR genes. This minimal increment in genecopy number, however, will allow such cells to have a greaterprobability of survival in MTX in the next cell cycle, where theycan again undergo overreplication, ultimately generating highgene copy number and resistance.

Aberrant replication has been reported after transient inhibitionof DNA synthesis in both prokaryotes (12,72,73) and eukaryotes(97, 100). We believe that the same general process underliesamplifications induced by DNA-damaging agents (91). We find

that the timing of introduction of DNA damage by UV radiationis critical in enhancing MTX resistance and gene amplification; itis only during the third to sixth hr of S phase that UV enhancesMTX resistance (91). This suggests that only in cells undergoingactive DNA synthesis will DNA-damaging agents promote gene

amplification.Consistent with the concept that various agents that alter DNA

replication patterns can result in extensive overreplication ofportions of a genome are findings that various ones of the aboveagents result in a variety of chromosomal aberrations in meta-

phase spreads of cells obtained while progressing through thefirst mitosis following treatments. Such aberrations include cellsthat have undergone endoreduplication, i.e., have gone from 2nto An in which the chromosomes appear normal, cells with anormal 2n complement in which there is a large amount of smallbut clearly visible extrachromosomal DNA, and cells with varyingdegrees of chromosomal fragmentation.11

Although it is not clear that, in all cases of gene amplificationas studied in cultured mammalian cells, the mechanism of theinitial amplification involves overreplication, it should be pointedout that all of the selections used involve agents or protocols,the consequence of which is inhibition of DNA replication andcell growth. Thus, the same processes as studied with MTXresistance and as enhanced by various treatments probablyoccur in these cases as well. Our results raise a number ofissues concerning the temporal replication of various genesduring S phase and suggest that genes replicated early in thecell cycle might be those that are more readily amplified. Ourresults also raise questions of the possible relationship of DNAreplication and transcription, inasmuch as the DHFR gene isreplicated, and the gene is expressed within the same 1 to 2 hr

of the S phase.

Gene Amplification and Clinical Resistance to Chemothera-

peutic Agents

The studies in cultured cells indicate that gene amplification isa common mechanism for generating MTX resistance and thatgene amplification may be a common process for the emergenceof resistance to a number of agents under appropriate stepwiseselection protocols. Our studies further suggest that variousagents that inhibit DNA synthesis or introduce damage into DNAcan markedly facilitate gene amplification and MTX resistance

' A. Hill, and R. T. Schimke, manuscript in preparation.

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as a result of provoking overreplication of a portion of thegenome.

The question arises as to whether gene amplification occursin the clinical setting to generate resistance. The answer wouldappear to be "yes" for the DHFR gene and MTX resistance.

Horns ef a/. (45) and Cardman ef al. (21) have each studied apatient with leukemia in which the cells were analyzed for DHFRgene copy number prior to and following treatment with MTX. Inboth cases, there was clinical and/or biochemical evidence ofMTX resistance and an approximately 3- to 6-fold amplificationof DHFR genes. Curt ef al. (30) reported that MTX-resistant

tumor cells from an oat cell lung cancer of a patient treated withMTX, as obtained by clonogenic assay techniques, containedapproximately 5 times as many DHFR genes as normal cells.Finally, Trent ef al. (92) have studied cells from a patient with anovarian adenocarcinoma which, by clonogenic assay, were resistant to very high concentrations of MTX. They observed bykaryological analysis an expanded chromosome in such tumorcells which, by in situ hybridization with a DHFR complementaryDNA clone, contained large (unspecified) numbers of DHFRgenes in a HSR configuration. Although this patient had notreceived MTX therapy for her cancer prior to diagnosis, she hadreceived 2 years of treatments of twice-weekly MTX (2.5-mg

dosages) for chronic psoriasis, and perhaps such treatment hadselected for MTX-resistant cells in the undiagnosed tumor.

The papers of Horns ef al. (45) and Cardman ef al. (21) studiedthe entire tumor cell population, and hence, the question ofwhether or not DHFR gene amplification occurred during clonogenic assays is not pertinent. From experience with gene amplification protocols and with the time required to obtain a cellpopulation with high gene copy number, a 6-fold amplification ofthe DHFR gene by a single-step selection is unlikely to have

occurred during the time of the clonogenic assays as used in thepapers of Curt ef al. (30) or Trent ef al. (92). Thus, it can bereasonably concluded that DHFR gene amplification is, indeed,a mechanism for generating clinical resistance. As more resistanttumors are studied, and as more possible DNA sequences thatimpart resistance become available for analysis, the role of geneamplification in clinical resistance phenomena can be more fullydocumented.

It is interesting to note that, in the 3 clinical studies in whichthe increase in DHFR gene copy number has been established,the amplification is only approximately 5-fold. In the studies ofRath ef a/.10with mouse 3T6 cells cited above, resistance to 200

nM MTX by stepwise protocols was associated with a 5- to 6-

fold amplification of the DHFR genes. In the experimental systems, the MTX concentration is maintained constant, whereasin most clinical treatments with MTX, the effective concentrationat the tumor cell site is subject to various parameters, includingdiffusion barriers at the tumor site, blood supply, the rates ofdelivery, metabolism, excretion, etc. Following "conventional"

treatment dosages, a 200 nM concentration is within bracketedranges over a time period of intermittent dosage (22, 47). Thus,the 5-fold DHFR gene amplification may well be sufficient to

provide the necessary resistance to conventional clinical dosagesof MTX.

On the assumption that gene amplification turns out to be animportant mechanism for the generation of resistance to variouscancer treatment modalities, whether the resistance phenotyperesults from overproduction of specific target proteins (such asDHFR), proteins that in some fashion prevent drug accumulation

within cells, proteins that inactivate chemotherapeutic agents, orproteins that facilitate the repair of DNA-damaging agents, our

results concerning the properties of gene amplification as studiedin the laboratory have a number of potential implications forclinical therapy. The principles enumerated below address theproblem of the emergence of resistant tumor cells, are "idealized"

from cultured cell systems, and will not be discussed in relationto experience with various clinical treatment protocols and/orindividual cases.

1. Multiple therapeutic modalities, which are not incompatible,should be used. These modalities (drugs) should not impartresistance by the same mechanism (or the same amplified DNAsequence). Such combined drug treatments are used commonlyand are based on the concept that each resistance phenomenonis an independent event, such that the probability of 2 resistancephenomena occurring in the same cell is highly unlikely. However,if overreplication of DNA is the mechanism of gene amplification,then the "unlikelihood" to 2 independent amplification events is

not as great as is generally envisaged if both genes are replicatedin the first part of the S phase. This possibility is supported bythe finding in L tropica that in some variants both of the DNAsequences imparting resistance occur in the same cells.6

2. The dosages of drugs (concentration x time) should besufficient to ensure that an effective concentration is deliveredto the tumor cell to result in cell death. Our results showing thatgene amplification occurs when DNA replication is transientlyinhibited suggest that, if sufficient concentrations are not used,or if the drug concentration is allowed to fluctuate above andbelow an effective inhibitory concentration, overreplication ofDNA, gene amplification, and resistance can be generated.

3. Agents that damage DNA should not be used in cell populations that are actively cycling. We would propose that alkyl-

ating agents might be most effective when used in noncyclingcells. This conclusion is based on the results indicating thatintroduction of damage into DNA (UV) facilitates overreplicationonly in cells in the S phase. Although our studies have concentrated on use of UV, we have made preliminary studies indicatingthat A/-acetoxy-A/-acetoaminofluorene enhancement of DHFR

gene amplification is also cell cycle dependent and that otherDNA-damaging agents will function in a similar cell cycle-depend

ent fashion in facilitating gene amplification.4. The various combined modalities of treatment should not

be used for prolonged time periods. Rather, they should alternate. This conclusion is based on the finding that gene amplification, when it first occurs, is often unstable, and only whencells are maintained under selective conditions for long periodsare the amplified genes stable in the emerging resistant population. Thus, prolonged use of a treatment regimen is more likelyto result in stable resistance. A corollary of this conclusion is thedesirability to determine, subsequent to the development ofresistance and the cessation of use of the drugs, whether or notthe tumor cells have retained the resistance. If resistance isunstable, then the same drugs may possibly be used again,thereby retaining more flexibility in potential therapeutic modalities.

Gene Amplification and Oncogenesis

The concept that gene amplification may underlie certain aspects of the generation and/or progression of cancers has beenproposed by various authors (see Refs. 70, 78, and 94), and

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R T. Schimke

there are progressively more observations leading credence toa role of gene amplification in the oncogenic process. Amongsuch observations are the following.

1. A number of continuous tumor cell lines, as well as tumorcells, generally derived from solid tumors, have the karyologicalconsequences of gene amplification, i.e., either HSRs or DMs(5-7, 29, 59, 74, 75). From such observations, it is not clear

whether such chromosomal aberrations (and assumed amplifiedgenes) are causally related to the oncogenic process, or whetherthey are the result of the establishment of continuous cell linesderived from such tumors. As will be discussed below, morerecent evidence has indicated that some of these cell linescontain amplified "oncogenes."

2. Levan and Levan (60) have studied a mouse tumor cell line(SEWA) which contains multiple DMs (or other chromosomalstructures containing amplified genes) when grown as a tumorin mice. When these cells are grown under cell culture conditions,the cells progressively lose the DMs. When the cells grown inculture are reintroduced into the mouse, the tumor again containsmultiple DMs. In addition, the tumorigenicity of the cultured cellsis directly related to the number of DMs that exist in the cells atthe time they are introduced into the mouse.7 This finding has

direct analogy to unstably MTX-resistant cell lines containing

DMs. In both cases, a selection pressure is imparted. In the caseof the SEWA cells, the selection is for growth in the mouse, andthe emerging tumor cells contain multiple DMs. Conversely,when the tumor cells are placed in nonselective conditions, theDMs are lost. We would suggest that the selection process inthe case of SEWA cells is for overcoming normal growth constraints of the mouse. Nothing is known currently concerninghow amplification of sequences in the SEWA cells might resultin unconstrained growth, and multiple mechanisms can be envisaged.

3. In the past several years, a number of investigators havereported on the amplification of oncogenes in tumor cell lines,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 ofthese sequences is causally related to the oncogenic process,or whether their amplification is a result of the establishment ofcontinuous cell lines.

4. Among the treatments of cells that enhance DHFR geneamplification experimentally are agents that damage DNA, including the well-established carcinogenic agents, UV light, andW-acetoxy-N-acetoaminofluorene. Enhancement of gene amplification occurs at doses lower than generally used in studies ofmutagenesis (doses that produce only 10 to 30% killing), occursonly in cells undergoing DNA synthesis and is demonstrated interms of enhanced MIX resistance only after DNA synthesis isallowed to occur prior to placement of cells in MIX (91). Thus,the demonstration of the role of such agents in facilitating geneamplification can be easily missed if the proper experimentalconditions are not used. If such DNA-damaging agents facilitateoverreplication in a manner similar to that we have found withtransient inhibition of DNA replication (62), then the questionarises as to what types of genes are replicated early in S phaseof the cell cycle; specifically, are any oncogenes replicated atthat time? If so, then they also will be amplified and may lead tothe loss of normal growth constraints on cells. It is of interestthat, in a human neuroblastoma cell line, an HSR-containing

region replicates early in the cell cycle (57), and Kohl ef al. (54)have just shown that this cell line contains amplified c-myc genes.

Other reports have indicated that various rearrangements areassociated with cancer, including the human 8; 17 translocationin Burkitt's lymphomas, which has been proposed to result in

increased expression of the c-myc gene (31, 53, 89; see also

Ref. 78 for speculation on specificity of such translocations). Inaddition, others have reported that a normal cellular "oncogene"

can be overexpressed by juxtaposition to a retrovirus promotorwith high expression efficiency (28, 39, 44, 71). In view of thefact that a cellular response to DNA-damaging agents in actively

dividing cells is overreplication of DNA, a consequence of sucha process may well be the recombination of DNA sequences-

chromosome fragments, such that a highly active promotor can"activate" a potential oncogene. Thus, the same basic process

of overreplication of DNA may possibly result in 2 different meansfor overproduction of proteins critical to overcoming normalgrowth constraints, i.e., gene amplification and recombinationalactivation.

The results of our studies on overreplication of DNA within asingle cell cycle may have further implications for the generationand/or progression of cancer, inasmuch as treatment of cellswith inhibitors of DNA replication and DNA-damaging agents

generates extrachromosomal DNA of varying sizes, as well asoccasional cells that undergo endoreduplication or contain fragmented chromosomes. Such a process would result in a heterogeneous population of cells with unstable phenotypes, a property of a number of tumors. Indeed, there are a number of reportsshowing heterogeneity in DNA content in tumor cells (17, 48,55). In addition, deVere White ef al. (33) have found an increasedheterogeneity in DNA content in experimental tumors followingtreatment with chemotherapeutic agents. Thus, if various drugtreatments can result in a higher frequency of MTX resistanceand gene amplification, is it not also possible that such treatments can result in a variety of genomic alterations, includingamplifications and rearrangements that facilitate the process ofmalignant progression?

Concluding Comments

In this "Perspectives" article, the phenomenon of gene ampli

fication in somatic cells has been reviewed briefly. It wouldappear to be a relatively common process in cells in culture and,perhaps, in tumors as well. Currently available studies indicatethat amplification occurs by overreplication of portions of thegenome in a single cell cycle, i.e., disproportionate replication,and this process can be enhanced by the very agents used incancer chemotherapy as well as by known carcinogenic agents.I have speculated that such overreplication may have a role innot only the generation of drug resistances clinically, but alsothe progression from a normal to malignant cell. Clearly, it ismore than a platitude to state that far more research is neededto understand the underlying mechanism(s) of gene amplificationand processes of drug resistance, as well as the possible role ofgenome overreplication-recombination in overcoming normal

growth constraints.

ACKNOWLEDGMENTS

I would like to thank the many members of my laboratory group whose research,both published and in preparation, has been discussed herein, including StevenBevertey, Peter Brown, Richard Cassin, Nancy Federspeil, Charles Gasser, AnnaHill, Randal Johnston, Brian Mariani, Elise Mosse, Heidi Rath, James Schilling,Allison Schimke, David Smouse, Chris Simonsen, and Thea Tlsty.

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Gene Amplification, Drug Resistance, and Cancer

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