Genetic Disruption of Cytosine DNA Methyltransferase Enzymes

Induces Chromosomal Instability in Human Cancer Cells

Adam R. Karpf1and Sei-ichi Matsui

2

Departments of 1Pharmacology and Therapeutics, and 2Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York

Abstract

DNA methyltransferase 1 (DNMT1)–deficient mice are tumor-prone, and this has been proposed to result from theinduction of genomic instability. To address whether loss ofDNMT1, or the related protein DNMT3b, results in genomicinstability in human cancer cells, we used a near-diploidhuman colorectal cancer cell line, HCT116, in which one orboth DNMT genes were disrupted by homologous recombina-tion. Array-based comparative genomic hybridization analysesindicated that double, but not single, DNMT knock-out cellsdisplay two specific alterations in regional DNA copy number,suggesting that DNMT deficiency and genomic DNA hypome-thylation are not associated with widespread genomicamplifications or deletions in human cancer cells. However,spectral karyotype analyses revealed that DNMT-deficientHCT116 cells are highly unstable with respect to large-scalechromosomal alterations; furthermore, this effect is charac-terized by a high degree of individual cell heterogeneity. Theinduction of chromosomal alterations in DNMT-deficient cellswas evidenced both by aneuploidy and by large increases inthe number of novel chromosomal translocations. Studiesof double knock-out cells indicated that the generation ofchromosomal alterations is spontaneous and persistentin vitro , meeting the formal definition of genomic instability.In summary, we show that DNMT deficiency in human cancercells results in constitutive genomic instability manifestedby chromosomal translocations. (Cancer Res 2005; 65(19): 8635-9)

Introduction

The best-established epigenetic alterations in cancer arechanges in the level of cytosine DNA methylation. In manyhuman neoplasms, the methylation of specific genetic loci,methylation of repetitive sequences, or genomic 5-methyl-2V-deoxycytidine levels are reduced (1, 2). To date, the contributionof genomic DNA hypomethylation to human oncogenesis remainsobscure. Originally, it was hypothesized that DNA hypomethyla-tion in cancer is linked to oncogene activation (3). Although thismay be the case in select instances, it does not seem to accountfor the global loss of DNA methylation from genomic DNA foundin many human tumors (1, 2). A second model invoked to explainthe oncogenic role of genomic DNA hypomethylation is genomicinstability (1, 2), and investigations of the effect of geneticdisruption of DNA methylation in murine systems provideevidence for this model (4, 5). Genomic instability is a cellular

state characterized by an increased rate of genetic changes,including DNA sequence changes, aneuploidy, chromosometranslocations, and/or gene amplifications (6). Despite the datafrom murine model systems, it remains plausible that humancancer cells (adult, somatic) respond differently to DNAhypomethylation and/or DNMT loss than do murine embryoniccells. Recently, Vogelstein and coworkers developed human cancercell lines with varying levels of genomic DNA hypomethylationresulting from genetic knock-out of DNMT1 and/or DNMT3b(7, 8). Critically, the parental cell type, HCT116, a humancolorectal carcinoma cell line, has a near-diploid karyotype (9).Thus, the HCT116 DNMT knock-out model is ideally suited forassessing the role of DNA methylation, DNMT1, and DNMT3b, inmaintaining genomic stability in human cancer cells.

Materials and Methods

Cell lines. HCT116 parental and knock-out colon cancer cell lines

[p21�/�; DNMT1�/�; DNMT3b�/�; and DNMT1�/�, DNMT 3b�/� (dual

knock-out cells, DKO)] were a generous gift from Dr. Bert Vogelstein

(Sidney Kimmel Cancer Center, Johns Hopkins University Medical

Institutions, Baltimore, MD). DKO passage number indicates the number

of cellular passages following the dual genetic knock-out. HCT116 Utah

cells were kindly provided by Dr. David Jones (Huntsman Cancer Institute,

University of Utah, Salt Lake City, UT). Cells were cultured as described

previously (10).BAC array comparative genomic hybridization. Genomic DNA from

control and DNMT knock-out cell lines was isolated by standard methods,

and 1 Ag of DNA, which corresponds to f100,000 cells, was used for BAC

array comparative genomic hybridization (aCGH) using the RPCI-11 BAC

array, as described previously (11). DNA labeling, array hybridization, and

digital data acquisition were done as described previously (11). Male and

female DNA pools, each of which contained DNA from 20 cytogenetically

normal individuals, were used as controls.Spectral karyotype analysis. Spectral karyotype analysis (SKY) was

done essentially as described elsewhere (12). Fluorescence color images of

chromosomes developed by Rhodamine, Texas Red, Cy5, FITC, and Cy5.5

were captured under a Nikon microscope, equipped with a Spectral cube

and Interferometer module. SKY View software (version 1.62), was used to

sort numerical changes and structural alterations of chromosomes,

including simple balanced translocations, unbalanced (or nonreciprocal)

translocations, deletions, and duplications.

Quantitative measurement of chromosomal instability. We calcu-

lated two independent measures of chromosomal instability. The first,

Instability index , was defined as the mean number of chromosomal

rearrangements per mitotic cell, after subtraction of the rearrangements

(i.e., stably present translocations) found in the HCT116 parental cell line.

The second measure, Rearrangement diversity, was defined as the total

number of distinct chromosomal rearrangements across a cell popula-

tion, divided by the number of mitotic cells analyzed. Rearrangements

scored included translocations, insertions, deletions, tandem duplications,

and iso-chromosomes. Reciprocal and balanced translocations involving

two chromosomes and/or iso -chromosomes were scored as two

rearrangements.

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

Requests for reprints: Adam R. Karpf, Department of Pharmacology andTherapeutics, Roswell Park Cancer Institute, Buffalo, NY 14263. Phone: 716-845-8305;Fax: 716-845-8857; E-mail: adam.karpf@roswellpark.org.

I2005 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-1961

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Results

DNMT knock-out has subtle effects on genomic DNA locicopy number in HCT116 cells. Genetic knock-out of one or bothkey DNA methyltransferase genes, DNMT1 and DNMT3b , werepreviously generated in the HCT116 colorectal cancer cell line(7, 8). We verified the expected genotype of the DNMT knock-outcell lines by measuring the expression of DNMT1 and DNMT3b byWestern blot or reverse transcription-PCR analyses, respectively(data not shown). In addition, we confirmed the DNA methylationphenotype of the knock-out cells, previously reported by Rhee et al.(8), using a novel mass spectrometry assay (ref. 10; data notshown). Specifically, we found that compared with HCT116parental cells, DNMT1�/� cells had a 20% reduction in genomicDNA methylation, DNMT3b�/� showed no reduction, and DKOhad a reduction of f80%.To assess the inherent stability of the HCT116 genotype, we

compared two independently maintained lines of HCT116 cells,HCT116 Utah and HCT116 parental. The HCT116 parental cells arethe line from which the DNMT knock-out cell lines wereconstructed. aCGH analysis indicated that the two HCT116 celllines are nearly identical, with the exception of two specificgenomic losses in the HCT116 parental cells, an interstitial Ch.13deletion and a telomeric Ch.7p deletion (data not shown). Thisresult suggests that whereas HCT116 cells may occasionallyacquire mutations in vitro , they likely have no underlying genomicinstability, a notion that is also supported by the results of SKYanalyses (see below). Next, we used aCGH to compare the genomeof DNMT knock-out cells to HCT116 parental cells. As a negativecontrol, we analyzed p21�/� HCT116 cells (13). We did not detectany DNA copy number alterations in either of the individualDNMT knock-out lines, or in the p21�/� cells (Fig. 1A ; data notshown). However, DKO cells contained two specific amplifications,one at 11q23 and the other at 18p (Fig. 1A-C , arrows).DNMT knock-out induces aneuploidy in HCT116 cells. We

next did SKY, an individual cell-based analysis, to examine DNMT-deficient HCT116 cell cultures for evidence of chromosomal

instability. Whereas the mean chromosome number of the DNMTknock-out lines was not significantly altered from the HCT116parental cells (data not shown), many of the DNMT-def icient cellsdisplayed a loss or gain of one or a few chromosomes, i.e.,aneuploidy (Fig. 2A). Accordingly, statistical analysis revealed thatchromosome number variance was sharply increased in each ofthe DNMT knock-out lines, as compared with either HCT116parental or HCT116 Utah cells (Fig. 2B ; data not shown). Thiseffect was most pronounced in the DKO cell line, and wasapproximately additive with respect to the contribution of theindividual DNMT knock-outs (Fig. 2B).Karyotype analysis of DNMT knock-out HCT116 cells.

Detailed analysis of mitotic spreads of the HCT116 parental cellline by SKY revealed a few highly conserved chromosomaltranslocations (Fig. 3A , white bars ; Supplementary Table S1). Themajority of these common alterations were also present in each ofthe DNMT knock-out lines (Fig. 3B-D , white bars ; SupplementaryTable S1). Notably, we also observed a number of novelchromosomal rearrangements in the DNMT knock-out cell lines(Fig. 3B-D , yellow bars ; Supplementary Table S1). The novelchromosomal alterations present in DNMT-deficient cells wereprimarily comprised of reciprocal and nonreciprocal transloca-tions. DKO cells in particular displayed a disproportionately highnumber of novel chromosomal rearrangements, involving theformation of bipartite and tripartite chromosomes (Fig. 3D ;Supplementary Fig. S1).DNMT knock-out induces chromosomal instability in

HCT116 cells. To quantify the chromosomal alterations in DNMTknock-out cells, we first calculated an instability index, which is ameasure of the total number of rearrangements observed in eachcell type (see Materials and Methods). The HCT116 Utah linedisplayed the lowest instability index (raw instability value of1.25; Table 1), suggesting again that HCT116 cells have arelatively stable genotype in vitro . p21�/� cells had a 1.2-foldincrease in the instability index relative to HCT116 Utah,indicating that the conditions used to drive homologous

Figure 1. Genome copy numberalterations in DNMT knock-out HCT116 celllines. Genomic DNA was isolated fromthe indicated cell lines and used foraCGH analysis, as described in Materialsand Methods. A, genomic copy numberanalysis of HCT116 parental and DNMTknock-out cell lines. Chromosome numbersare indicated below the graphs. Nodifferences between the HCT116parental cell line and the DNMT1�/�or DNMT3b�/� knock-out cells wereobserved. In contrast, two specificamplifications (arrows ) were present inDKO cells at both passages 8 and 25.B, high-resolution graphs of the DKOcell-specific chromosome 11q23amplification shown in (A). C,high-resolution graphs of the DKOcell-specific chromosome 18pamplification shown in (A).

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recombination in the HCT116 system does not by itself causesizable increases in instability (data not shown). In contrast,DNMT1�/�, and DNMT3b�/� cells showed approximately 2-and 4-fold increases in the instability index, respectively (Table 1).In DKO cells, the effect was far more dramatic, suggestingsynergy (Table 1). The instability index values in both the singleand dual DNMT knock-out cell lines were significantly differentthan that found in HCT116 Utah (Supplementary Table S2). Wealso examined DKO cells harvested at different passage numbers,and these all showed high levels of chromosomal instability(Table 1). In addition to the instability index, we calculatedrearrangement diversity, a measure of the number of distinctrearrangements observed in each cell type (see Materials andMethods). The chief distinction between the instability index andrearrangement diversity is that the instability index value isincreased by conserved rearrangements in the cell population,whereas rearrangement diversity is not. Similar to the instabilityindex, HCT116 Utah cells showed a low value for rearrangementdiversity (Table 1). However, unlike the instability index, therearrangement diversity of DNMT3b�/� cells was only slightlyincreased (1.5-fold) over HCT116 Utah, and was not statisticallysignificant (Supplementary Table S2). In contrast, DNMT1�/�

cells showed a profound enhancement in rearrangement diversity(4.01-fold), and this was highly significant (Supplementary TableS2). The rearrangement diversity of the DKO cell line wassubstantially greater than either of the individual DNMT knock-out lines, again pointing to a synergistic effect (Table 1). Theextraordinary phenotype of DKO cells was also evidenced by thefact that f90% of DKO p25 cells analyzed contained at least oneunique chromosomal rearrangement (data not shown).These striking results encouraged us to directly examine whether

the DKO cell line generates novel chromosomal rearrangementsin vitro or, alternatively, whether the diversity of rearrangementsobserved in this cell type are generated only during the initialestablishment of the line. To this end, we examined the specificchromosomal rearrangements found in the DKO cell line at the threedifferent passage numbers indicated in Table 1. Remarkably, >60% ofthe rearrangements observed in DKO p46 cells were unique to DKOcells of that passage number. In addition, there was a similarpredominance of unique rearrangements in DKO cells at the earlierpassage numbers (data not shown).

Discussion

We have investigated the potential link between DNA hypome-thylation and/or DNMT loss and genomic instability in humancancer cells. Herein, we present a number of novel findings: (a)DNMT knock-out cells display aneuploidy, (b) DNMT knock-outcells show robust increases in multiple types of chromosomalrearrangements, and (c) DNMT deficiency is characterized by thespontaneous and persistent generation of chromosomal rearrange-ments. Taken together, our data reveal that DNMT loss, and theassociated DNA hypomethylation, results in bona fide chromo-somal instability (change/time) in human cancer cells. The extentof chromosomal instability in the DKO cell line, in which f90% ofcells show at least one chromosomal rearrangement, seems to becomparable to, or possibly exceeding, that observed in other well-characterized genetically unstable human cancer cell lines (9, 14,15).We found, using aCGH analyses, that DNMT deficiency and

DNA hypomethylation in human cancer cells does not result inwidespread conserved genomic amplifications or deletions.Because this assay requires thousands of cells for the inputDNA, cellular heterogeneity can mask copy number alterationspresent in subsets of the cell population. aCGH did not revealcopy number alterations in single DNMT knock-out cells,however, in DKO, two specific amplifications were observed, at11q23 and 18p. 11q23 alterations are relatively common inleukemias, and have also been observed in solid tumors; 18palterations, although far less frequent in human cancer, have alsobeen noted (16). These genomic regions contain a number ofestablished and candidate oncogenes, including MLL and Ets1 on11q23, and Yes1 on 18p (16). We hypothesize that the twoamplifications observed in DKO cells do not indicate genomicinstability but instead reflect selectable genetic events thatpromote DKO cell growth or survival.SKY analyses, an individual cell-based assay, revealed that DNMT-

deficient cell lines each display varying degrees of chromosomalinstability. A striking effect was seen in DKO cells, which showedapproximately an order of magnitude increase in chromosomalinstability relative to HCT116 parental cells. Experiments comparingDKO cells of different passage numbers revealed that chromosomalinstability is a constitutively expressed genetic property of these

Figure 2. Aneuploidy in DNMT-deficient HCT116 cell lines. Chromosomenumber was determined by SKY analysis of metaphase spreads fromthe indicated cell lines. A, chromosome number of DNMT-deficient celllines. Each point represents an individually analyzed cell. B, chromosomenumber variance (i.e., the degree of aneuploidy) of each cell line shown in (A)was calculated by ANOVA single factor testing, using Microsoft Excel.

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cells. Moreover, there seemed to be no statistical bias towards theinvolvement of any particular chromosome members in therearrangements (data not shown). The increased structuralrearrangements in DNMT knock-out cells include both balancedand unbalanced translocations, and likely result from increasedrates of DNA recombination. This hypothesis is consistent withearlier work, which found that DNA methylation suppressescrossing-over in Ascobolus , and that DNMT1 knock-out mouse EScells show enhanced rates of gene targeting (17, 18).

One of the key questions arising from our study is whetherchromosomal instability in the DNMT knock-out lines is a directconsequence of genomic DNA hypomethylation, or alternatively,results from the loss of methylation-independent protein functions.In support of the first model, dual DNMT loss has a synergistic,rather than an additive, effect on chromosomal instability, which isanalogous to its effect on genomic DNA hypomethylation. Alsoconsistent with this model, DNMT1�/� cells, which showmoderatelevels of genomic DNA hypomethylation, have significantly higher

Figure 3. SKY analysis of DNMT-deficient HCT116 cell lines. Cells were processed for analysis by SKY as described in Materials and Methods, and a representativemetaphase spread of each cell line is shown. A, HCT116 parental cells. B, DNMT1�/� cells. C, DNMT3b�/� cells. D, DKO p25 cells. White and yellow bars,chromosomal translocations present or not present in the HCT116 parental cell line, respectively.

Table 1. Quantification of genomic instability in DNMT knock-out cell lines

Cell line Cells analyzed Chromosomalrearrangements*

Instability indexc

Fold increaseb

Rearrangement diversity x Fold increaseb

HCT116 Utah 32 40 1.25 1.0 0.343 1.0DNMT1�/� 40 93 2.33 1.86 1.375 4.01

DNMT3b�/� 35 186 5.31 4.25 0.514 1.50

DKO p8 38 456 12.00 9.60 2.89 8.43

DKO p25 54 529 9.80 7.84 2.61 7.61DKO p46 70 842 12.03 9.62 2.46 7.17

*Not observed in HCT116 parental cells.cMean number of chromosomal rearrangements per cell, following subtraction of the conserved rearrangements present in the HCT116 parental cellline.bRelative to the HCT116 Utah cell line.xTotal number of distinct chromosomal rearrangements observed across the cell population, divided by the number of cells analyzed.

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rearrangement diversity than DNMT3b�/� cells, which do not havedetectable genomic DNA hypomethylation. On the other hand,DNMT3b�/� cells, which do not show global DNA hypomethyla-tion, display moderate increases in chromosomal instability,suggesting a role for DNMT3b in maintaining genomic stabilityindependent of its enzymatic activity. This could potentially beexplained by the participation of DNMT3b in the condensincomplex, which is involved in proper segregation of sisterchromatids during mitosis (19). However, even in the case ofDNMT3b�/� cells, we cannot exclude the possibility that loss ofmethylation at specific loci is critical for the instability phenotype.For example, enzymatic region mutations of DNMT3b, which areassociated with the human disease ICF syndrome (immunodefi-ciency, centromere instability, and facial anomalies), lead tohypomethylation of and chromosomal abnormalities involving thepericentromeric regions of chromosomes 1, 9, and 16 (20). In thecurrent study, DNMT3b�/� cells showed two conserved rearrange-ments involving chromosome 1 [t(1;7), der(3)t(1;3)], and oneinvolving chromosome 16 [der(16)t(8;16)] (SupplementaryTable S1). Thus, it will be relevant to determine whether thechromosomal rearrangements observed in DNMT3b�/� cells arerelated to hypomethylation of specific DNA sequences.Cytosine DNA methyltransferases have been proposed as

suitable targets for pharmacologic intervention in cancer basedon the fact that tumor suppressor genes are silenced by DNA

hypermethylation (21). Our data, which indicate that DNAhypomethylation leads to chromosomal instability in humancancer cells, could be interpreted either positively or negativelyfrom the standpoint of epigenetic therapy. The interpretation isdependent on whether instability shows specificity towardstumor cells, and to what extent (and in what direction)instability affects tumor cell fitness. The potentially adverseeffects of DNA hypomethylation have led us to previously suggestthat the most effective clinical use of DNMT inhibitors may be tocombine these drugs, in a temporary and acute fashion, withsecondary agents whose efficacy is enhanced by DNA hypome-thylation (21). Our current findings seem to lend further supportto this idea.

Acknowledgments

Received 6/6/2005; revised 7/8/2005; accepted 7/13/2005.Grant support: Ralph Wilson Medical Research Foundation, Phi Beta Psi, and the

Roswell Park Alliance Foundation (to A.R. Karpf), and CA16056 (to Roswell ParkCancer Institute). The RPCI SKY/FISH Facility was established by a donation from theJ.H. Cummings Foundation.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We are grateful to Dr. Bert Vogelstein for providing the DNMT and p21 knock-outcell lines used in this study, and to Dr. David Jones for providing the HCT116 Utah cellline. We thank Jeff Conroy, Devin McQuaid, and Jeff LaDuca of the RPCI GenomicsCore Facility for excellent technical assistance. We thank Dr. Terry Beerman of RPCIfor critical reading of the manuscript.

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