[CANCER RESEARCH 57. 5557-5563, December 15. 1997]

Chromosomal Instability and Its Relationship to Other End Points ofGenomic Instability1

Charles L. Limoli,2 Mark I. Kaplan, James Corcoran, Mark Meyers, David A. Boothman, and William F. Morgan

Department i>f Radiation Oncology ¡C.L L, M. l. K., J. C.. W. F. M.] and Graduate Group in Biophysics ¡M.l. K.J. University of California. San Francisco, California94143-0750; Life Sciences Division. Lawrence Berkeley National Laboratory. Berkele\. California 94720 ¡W.F. M.}; ami Department of Hun

Wisconsin Medical School and Comprehensive Cancer Center. Madison, Wisconsin 53792 [M. M.. D. A. B.Iumilii Oncology. Universiry of

ABSTRACT

Chromosomal destabilization is one end point of the more generalphenomenon of genomic instability. We previously established that chromosomal instability can manifest in clones derived from single progenitorcells several generations after X-irradiation. To understand the potential

relationship between chromosomal destabilization and the other endpoints of genomic instability, we generated a series of chromosomallystable and unstable clones by exposure to X-rays. All clones were derivedfrom the human-hamster hybrid line GM10115, which contains a singlecopy of human chromosome 4 in a background of 20-24 hamster chro

mosomes. These clones were then subjected to a series of assays todetermine whether chromosomal instability is associated with a general"imitator phenotype" and whether it modulates other end points of

genomic instability. Thus, we analyzed clones for sister chromatid exchange, delayed reproductive cell death, delayed mutation, mismatchrepair, and delayed gene amplification. Statistical analyses performed oneach group of chromosomally stable and unstable clones indicated that,although individual clones within each group were significantly differentfrom unirradiated clones for many of the end points, there was nosignificant correlation between chromosomal instability and sister chromatid exchange, delayed mutation, and mismatch repair. Delayed geneamplification was found to be marginally correlated to chromosomalinstability (/' < 0.1), and delayed reproductive cell death (the persistent

reduction in plating efficiency after irradiation) was found to be significantly correlated (/' < 0.05). These correlations may be explained by

chromosomal destabilization, which can mediate gene amplification andcan result in cellular lethality. These data implicate multiple molecularand genetic pathways leading to different manifestations of genomic instability in GMI0115 cells surviving exposure to DNA-damaging agents.

INTRODUCTION

Chromosomal instability, an end point of genomic instability, is oneof the many delayed effects associated with acute exposure to ionizingradiation. The multiple phenotypes of genomic instability can includea variety of karyotypic abnormalities, such as chromosomal destabilization. SCE1 and aneuploidy, gene mutation and amplification,

clonal heterogeneity, delayed reproductive cell death, alterations inDNA repair capacity, neoplastic transformation, and metastatic progression (1). These events may contribute to an increased rate ofgenetic change, characteristic of genomic instability, and are hypothesized to be crucial in multistep carcinogenesis (2, 3).

Received 6/30/97; accepted 10/15/97.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by the Office of Health and Environmental Research.United States Department of Energy, Contract 4459-011542. and N1H Grant CM 54189(to W. F. M.). by the NIH National Research Service Award 5-T32-ES07016 from the

National Institute of Environmental Health Sciences (to C. L. L.). and by ContractDE-FG02-93ER61707-05 (to D. A. B.).

2 To whom requests for reprints should be addressed, at Department of RadiationOncology. Box 0750, University of California. San Francisco. CA 94143-0750. Phone:(415)476-2793; Fax: (415)476-1544; E-mail: Limoli@radlab.ucsf.edu.

1The abbreviations used are: SCE, sister chromatid exchange: HPRT. hypoxanthinephosphoribosyl transferase; MMR. mismatch repair: CAD. carfoamoylphosphate synthe-

tase. aspartale transcarbamylase. and dihydrooralase; FBS. fetal bovine serum: MEM.minimal essential media; PALA. AMphosphonacetyD-i.-aspartate; poly(dl-dC).polyldeoxyinosinic-deoxycytidylic acid).

The persistent destabilization of chromosomes has been describedin both clonal and nonclonal populations for a variety of mammaliancell lines surviving exposure to both sparsely and densely ionizingradiations (4-9). Of particular interest is the necessarily dynamic

nature of this phenotype, which can be observed in clonal descendantsof single progenitor cells several generations after irradiation (7).Some fraction of the gross chromosomal change observed in unstableclones might provide the driving force behind a "mutator phenotype"

that could further compromise normal cellular physiology. Support forthis notion is found in the large number of neoplasms that exhibitmany of the same end points, collectively called genomic instability(1-3, 10-12). Other forms of genetic instability have been hypothe

sized to account for a heritable mutator phenotype that can lead toseveral types of delayed mutation ( 13-20), altered tissue responses(21), persistent reduced cloning efficiency (22-24), and neoplastictransformation and carcinogenesis (25-27).

Despite extensive research into each of these cellular processes,very little is known about the potential relationships among themolecular and cellular end points associated with both the mutatorphenotype and genomic instability. In this study, we investigated thepotential relationships between chromosomal instability and other endpoints of genomic instability, specifically, SCE, delayed reproductivecell death, delayed mutation at the HPRT locus, DNA MMR, anddelayed gene amplification at the CAD loci.

MATERIALS AND METHODS

Cell Culture. All chromosomally stable and unstable subclones describedin this report were derived from the parental human/hamster hybrid cell lineCM 10115. which contains one copy of human chromosome 4 in a backgroundof 20-24 hamster chromosomes. Cells were maintained as monolayers inDMEM supplemented with \0% FBS, 2 mM i.-glutamine. I(X) units/ml ofpenicillin. KM)/^g/ml of streptomycin, and 0.2 mM i-proline and grown at34°Cin humidified incubators containing 5% CO, in air. Under these condi

tions, cell doubling times of 21-23 h were obtained.The Chinese hamster ovary cell lines CHO-MT and CHO-B. generous gifts

from Michael Armstrong (Merck Research Laboratories. Philadelphia, PA)and Margerita Bignami (Istituto Superiore di Sanità , Rome. Italy), were usedas the positive and negative controls, respectively, in the MMR assay. Cellswere grown in aMEM. lOVr FBS. I(X) units/ml of penicillin. 100 fig/ml ofstreptomycin, and 2 mM i.-glutaminc.

Isolation and Characterization of Subclones. We generated the following three distinct classes of clones: (a) X-irradiated and chromosomally stable;(b) X-irradiated but chromosomally unstable: and (

CHROMOSOM!: DI-STABILIZATION ANI) (ÃŒHNOMICINSTABILITY

Collection of Metaphase Cells. After 2-3 h exposure to the mitotic spindleinhibitor colcemid (final concentration. 2 X 10~7 M), mitotic cells were

collected from each of the clonally expanded cultures by mitotic shake-off andswollen in hypotonie 0.075 M KCI solution for 15 min at 37°C,dehydrated in

100% methanol. and fixed in methanohacetic acid (3:1 v/v). Mitotic cells weredropped onto precleaned glass microscope slides and allowed to air dry for 1day at ambient temperature before storage at -20°C.

Fluorescence in Situ Hybridization. For analysis of delayed chromosomalinstability, we used fluorescence in siiti hybridization of a labeled probe to thehuman chromosome in the human-hamster hybrid cells. Labeling of thispBluescript vector-based library of human chromosome 4-specific DNA se

quences (pBS4) and subsequent hybridi/.ation protocols were performed asdescribed previously (7). Briefly, slides were hybridized at 37°Cfor 2 days

with 35 /¿Iof hybridi/ation mixture containing 50 ng of biotinylated probe.Indirect detection of the hybridized probe to the human chromosome 4 targetinvolved sequential 15-min incubations with fluorescein- conjugated avidin(Oncor) and an anti-avidin antibody, interspersed with 5-min washes in phosphate-buffered detergent. Chromosomes were counterstained with propidiumiodide in Antifade (Oncor). fitted with glass coverslips. and stored at 4°Cuntil

analysis by fluorescence microscopy.Cytogenetic Analysis. Alter fluorescence in xiiu hybridization, metaphase

chromosomes were analy/.ed by means of a Zeiss Axioskop microscopeequipped with a dual-band pass FITC/Texas Red filter set. In this system, the

hamster chromosomes appear red (propidium iodide emission), and the humanchromosome 4 that hybridi/es with the biotinylated probe appears yellow-

green (fluorescein emission).Chromosomally unstable clones were defined as those having at least three

distinct aberrant metaphase subpopulations involving rearrangements of human chromosome 4 (7). Any clone showing fewer than three such rearrangements was considered chromosomally stable. All clones were derived fromsingle progenitor cells, and 200 individual metaphase spreads were analyzedfor each clone. Chromosome rearrangements were categorized as described byTucker et al. (28). and only those rearrangements involving human chromosome 4 were scored.

Experimental Protocol for Comparison of Clones. In all experiments,unirradiated control clones were compared with subclones surviving 10 Gy ofX-rays. The X-irradiated clones were classified as chromosomally stable (a

total of 14 clones showing 50 cells were scored.

Delayed Gene Amplification Assay. Delayed gene amplification was assayed at the CAD gene locus. CAD is an acronym for the multifunctionalprotein that catalyzes the first three steps of uridine biosynthesis: C, car-bamoylphosphate synthetase; A. aspartate transcarbamylase: D. dihydro-

oratase. Each clone was thawed and grown in the presence of «MEMsupplemented with 10% dialyzed FBS for 3-4 days. CAD amplification was assayed

+/-10Gyof X-rays\

Fig. 1. Schematic of the experimental protocol,outlining Ihc generation and isolation of chromosomally stable and unstable suhclones. and the assays to which each subclone was subjected.

Expand into 6 150-cm2

tissue culture flasks

Delayedreproductive

cell death1Delayed

HPRTgene mutation^Mismatchrepair1

Delayed CAD geneamplification

7_\Fluorescence-plus-Giemsa

differential staining

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CHROMOSOM!-: PESTABII.I/A'IÕON AM) (¡HSOMK INS I ADII 11i

by plating 3 X IO5 cells into each of five 100-mm dishes containing 10 ml ofmedium and 1 X 10~4 M PALA, a transition state inhibitor of aspartate

transcarbamoylase (obtained from the drug synthesis branch of the NationalCancer Institute). To optimize the selection of clones having genuine CAD

amplicons. the concentration of PALA was based on a level determinedpreviously to be nine times the LD5(1(29). Three to 4 weeks of growth weregenerally required before PALA-resistant cells grew into colonies of sufficient

size for counting, isolation, and expansion. Fresh medium containing PALAwas replenished two times each week throughout the selection period.

Sister Chromatic! Exchange Analysis. Each group of clones was culturedin DMEM in the presence of 2 X 10~5 M bromodeoxyuridine (Sigma) for 44 h.

which was optimal for two complete replication cycles and the subsequentdetection of SCE (30. 31 ). Metaphase cells were prepared as described above,and 5-bromodeoxyuridine-substituted chromosomes were stained by a slightlymodified fluorescence-plus-Giemsa procedure (32). Briefly, slides werewashed twice in Sorensen's buffer (15 mM potassium phosphate, pH 7.6) for10 min before incubating them in Sorensen's buffer containing 20 /ig/ml of

Hoechst dye 33258 (Sigma) for 20 min. Slides were rinsed, covered with glasscoverslips, exposed to UVA light (27 watts/irT2 incident output) at 55°Cfor

4 min, and stained for 10 min with a 5% solution of Giemsa in water.Delayed Mutation Assay. Delayed mutation was assayed at the HPRT

locus for each of the three groups of clones. Clones were thawed and grown in«MEMsupplemented with I0

CHROMOSOME DESTABILIZATION AND GENOMIC INSTABILITY

Delayed Reproductive Cell Death. To determine whether theclones exhibited differences in long-term survival, reflecting delayed

reproductive cell death, we diluted and plated cells in triplicate anddetermined plating efficiencies by clonogenic assay (Table 2). Notonly did clones surviving X-irradiation exhibit reduced plating efficiencies compared to unirradiated controls, but irradiated chromo-

somally unstable clones showed reduced plating efficiencies compared to irradiated stable clones (P < 0.05), reflecting a significantlyhigher incidence of delayed reproductive cell death in the unstableclones.

Gene Amplification. The stable and unstable clones were thenanalyzed for their ability to amplify the CAD gene in response toPALA selection. Calculated amplification frequencies for all cloneswere corrected for differences in plating efficiency. CAD gene amplification frequencies for irradiated clones were significantly higherthan those for the unirradiated parental clones (Table 3). A marginallysignificant P was obtained (P < 0.1) in a comparison of stable andunstable clones, indicating a positive correlation between the delayedeffects of gene amplification and chromosomal instability.

SCE. To test whether any of the clones exhibited instability at asecond cytogenetic end point, we measured SCE frequency in met-

aphase chromosomes (Fig. 2). For each clone, the number of chromosomes and the number of SCEs in 30 individual metaphases werescored (Table 4). Statistical analysis indicated no significant difference between stable or unstable clones.

Delayed Mutation. Stable and unstable clones were then analyzedfor mutation frequency at the HPRT locus (Table 5). Mutation frequencies were calculated according to the number of plated cells andwere corrected for differences in plating efficiency as indicated inTable 2. For those clones in which no mutations were observed(clones 7,10, 24, and 129), upper estimates of the mutation frequencyare provided. As with CAD gene amplification frequency, significantincreases in mutation frequency were observed for irradiated compared to unirradiated clones. Unlike CAD, however, no significantincreases in mutation frequency were observed for unstable comparedto stable clones.

Mismatch Repair. Stable and unstable clones were then examinedfor G-T mismatch binding activity. Cell extracts were incubated witha radioactively end-labeled DNA heteroduplex containing a G-T mis

match at a central base pair, and reactions were resolved by PAGE(Fig. 3). A higher molecular weight band indicated the presence of amore slowly migrating heteroduplex bound by protein. As expected,cell extracts isolated from CHO-MT cells were proficient in mismatchbinding, whereas cell extracts prepared from CHO-B cells were not

capable of retarding the mobility of the heteroduplex through the gel

Table 2 Plating efficiency of chromosumally stable and unstable clones

Table 3 Delayed CAD amplification frequency for chnimosiimally stable ana unstableclones

StableclonesGM10ll5fcOM10I15"GM

10115''102103110114118126129130132133141145146152ci

platingefficiency"839285726564746952605769845147592.53.11.26.23.05.58.22.01.75.70.887.55.32.60.672.651

2.9Unstableclones2710ll2324115138147CS9LSILS12%

platingefficiency"31

±3.246±3.950

±2.667±4.745±1.228±1.436±3.428±2.951

±3.546±3.572±3.262±8.1

StableclonesGM10115''GM101I5''102103110114118126129130132133141145146152CAD

amplificationfrequency"17

±3.518±3.178

±1111±3.85.2

±1.63.6±2.215±4.45.1±3.719±2.223±4.9360±1226

±4.190±8.2100±1521±2.5130±13Unstable

clones2710112324115138147CS9LSILS12CADamplification

frequency"220390233445301108204116029018026306.44.23.352224211166.919

'Amplification frequencies corrected for plating efficiency and expressed per IO6

cells; mean ±SE.h One of three independently derived subclones of the unirradiatedparental cell line.

' Values are expressed as mean ±SE.' One of three independently derived subclones of the unirradiated parental cell line.

Fig. 2. Metaphase of an unstable clone (LSI2) showing SCE.

(Fig. 3; Ref. 39). All stable and unstable clones demonstrated proficiency for binding the heteroduplex.

Cell extracts from CHO-MT cells were then used to confirm that

the heteroduplex binding activity was specific for the presence of theG-T mismatch (Fig. 4). At equal concentrations, the heteroduplex wasmore effective at competing G-T mismatch binding than was the

homoduplex. Similar results were obtained when competition experiments were run in the presence of noncompetitive inhibitors ornonspecific DNA templates (data not shown).

To ascertain whether the mismatch binding activity might be due tothe presence of the MSH2 protein, we analyzed cell extracts fromunstable clones and unirradiated clones by immunological blot analysis with an antibody probe specific to the MSH2 protein. Scanningdensitometry indicated no major differences in the levels of MSH2between unstable and unirradiated clones (Fig. 5). Similar results were

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CHROMOSOME DESTABILIZATION AND OENOMIC INSTABILITY

Table 4 SCE in chromosomalk stable and unstable clones

No. of chromosomes/melapha.se"

No. ofSCE/metaphase"

No. of SCE/chromosome

StableclonesOMIOUS*GMIOUS*OMIOUS*102103110114118126129130132133141145146152Unstable

clones2710112324115138147CS9LSILS12232222232222222123232223222323222221222121232222212221230.160.330.280.170.240.250.360.380.210.290.240.210.270.230.130.310.190.330.240.240.230.220.200.190.310.230.280.1420

0.246.15.15.34.74.96.95.06.08.96.45.37.06.34.84.65.75.48.55.59.27.05.46.26.97.23.77.55.30.550.420.550.340.400.430.390.510.560.420.440.540.480.410.370.390.380.520.470.660.440.380.570.650.530.360.510.417.00.470.260.230.240.200.220.310.230.280.390.280.240.300.290.210.210.250.240.400.250.430.330.240.270.320.350.170.350.230.35" Values are expressed as mean ±SE.1One of three independently derived subclones from the unirradiated parental cell line.

Table 5 Delayed HPRT mutation frequency for chromosomalIv stable ana unstableclones

StableclonesGMI0115''GMIOllS*GMIOllS*102103110114118126129130132133141145146152HPRT

mutationfrequency"1.6

±0.981.40.78106.98.32.7166.4

CHROMOSOM!-. DI STABILIZATION AND OENOMIC INSTABILITY

hMSH2

85*.

-220

-100

Fig. 5. Immunological blot of unstable clones and unirradiated GMIOI15 cell extractsusing an antibody probe to MSH2. Arrows on the right, molecular mass (in kDa).

abnormally rearranged chromosomes in the unstable clones contributes to reproductive failure.

A number of studies have emphasized the importance of chromosome déstabilisationand breakage and refusion events in mediatingthe processes responsible for gene amplification (46-49). Thus, we

reasoned that gene amplification, as measured by PALA resistance,might be increased in those clones that exhibited marked chromosomal instability. Irradiated clones did show a significantly higheramplification frequency compared to unirradiated controls, and chro-

mosomally unstable clones also showed a substantially higher amplification frequency than did stable clones. It is uncertain why the levelof gene amplification observed was not higher in those clones (115,CS9, and LS1 ) showing the most chromosome fusions. We shouldpoint out that we are only looking at one chromosome comprising 5%of the genome, and that fusion events involving other chromosomes,including the hamster chromosome with the CAD gene, were notscored. Nevertheless, these observations suggest that chromosomeinstability may be necessary but not sufficient for gene amplification.

Chang and Little (13) have shown that clones derived from CHOcells surviving exposure to X-irradiation demonstrate a persistent

increase in mutation frequency at the HPRT locus. They hypothesizedthat some form of genetic instability results in a heritable mutatorphenotype. To investigate whether a change in mutation frequencycoincides with a change in chromosomal stability, we assayed chro-

mosomally stable and unstable clones for delayed mutation at theHPRT locus. Mutation frequencies were increased significantly insome clones surviving radiation exposure, but there was no overallcorrelation between chromosome stability and delayed mutation.

Because an inverse relationship between plating efficiency andW/?7" mutation frequency was reported by Chang and Little (13), we

plotted plating efficiency against HPRT mutation frequency and CADamplification frequency (Fig. 6). We found no discernible relationshipbetween plating efficiency and mutation frequency (Fig. 6/t) or between plating efficiency and gene amplification (Fig. 6B).

Because it is well established that faithful MMR activity is criticalin maintaining genomic integrity (50-52), we examined stable and

unstable clones for MMR binding capacity and MSH2 and MLH1protein levels. We investigated the functionality and presence ofrepresentative gene products of the MMR pathway in cell extractsderived from our clones by testing their ability to bind to a DNAheteroduplex containing a mismatched base pair. All cell extractswere capable of binding and retarding the mobility of the DNAheteroduplex through the gel (Fig. 3). This binding was specific forthe mismatched heteroduplex. because homoduplex and poly(dl-dC)

templates were unable to compete for binding activity as effectively asthe heteroduplex. Subsequent analysis of cell extracts on immunolog-

ical blots showed no significant differences in levels of hMSH2 orhMLHl protein.

Our results indicate that all GM10115 clones contain functionalMSH2 protein, which can presumably bind specifically to DNAtemplates containing mismatched base pairs. These data indicate thatMMR and chromosomal stability are not interdependent. Nonetheless,

the lack of a correlation between MMR, delayed mutation, and chromosomal stability reported here is consistent when seen in the contextthat functional MMR protects cells against the enhancement of mutation but not necessarily against gross chromosomal change.

Loeb (3) has argued that a mutator phenotype is required formultistep carcinogenesis. This is based on the observation that thespontaneous mutation rate is insufficient to account for the highfrequency of mutations in cancer cells, and that the vast majority ofcancer cells show multiple chromosomal abnormalities. The emergingconcept is that the genomes of cancer cells are unstable, and that thisinstability results in a cascade of mutations, some of which enablecancer cells to bypass host regulatory processes (11). Our data suggestthat chromosomally unstable GM10115 clones do not possess a mutator phenotype per se. Nonetheless, some clones did exhibit increasesin several of the end points associated with genomic instability, suchas gene mutation (i.e., clones 118, 132, 146, and LS12) and geneamplification (i.e., clones 2, 7, 24, 132, 138. and LSI). Collectively,however, chromosomally unstable and stable clones were not significantly different. This is not altogether unexpected, because the multiple end points associated with genomic instability may not be drivenby the same set of upstream events or the same set of downstreamprocesses.

40

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CHKOMOSOMI- Ill -STABILIZATION AND GHNOMIC INSTABILITY

We have presented a comprehensive set of parallel experimentsusing human/hamster hybrid GM10115 cells in which the same subsets of irradiated and chromosomally stable, irradiated and chromo-

somally unstable, and unirradiated subclones were examined andcompared. The data unambiguously demonstrate a close relationshipbetween chromosomal instability and delayed reproductive cell deathand, to a lesser extent, gene amplification. No relationship was foundbetween chromosomal instability and SCE, delayed mutation, orMMR.

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1997;57:5557-5563. Cancer Res Charles L. Limoli, Mark I. Kaplan, James Corcoran, et al. Points of Genomic InstabilityChromosomal Instability and Its Relationship to Other End

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