Division of Molecular Medicine, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97239, USA
* To whom correspondence should be addressed. Tel: +1 5034942447; Fax: +1 5034947368; Email: [email protected]
Received June 13, 2005; Accepted August 10, 2005
An open article from Human Molecular Genetics 2005 14(19):2813-2827; doi:10.1093/hmg/ddi314.
Certain chromosome rearrangements, found in cancer cells orin cells exposed to ionizing radiation, exhibit a chromosome-widedelay in replication timing (DRT) that is associated with adelay in mitotic chromosome condensation (DMC). We have developeda chromosome engineering strategy that allows the generationof chromosomes with this DRT/DMC phenotype. We found that 10%of inter-chromosomal translocations induced by two distinctmechanisms, site-specific recombination mediated by Cre or non-homologousend joining of DNA double-strand breaks induced by I-Sce1, resultin DRT/DMC. Furthermore, on certain balanced translocationsonly one of the derivative chromosomes displays the phenotype.Finally, we show that the engineered DRT/DMC chromosomes acquiregross chromosomal rearrangements at an increased rate when comparedwith non-DRT/DMC chromosomes. These results indicate that theDRT/DMC phenotype is not the result of a stochastic processthat could occur at any translocation breakpoint or as an epigeneticresponse to chromosome damage. Instead, our data indicate thatthe replication timing of certain derivative chromosomes isregulated by a cis-acting mechanism that delays both initiationand completion of DNA synthesis along the entire length of thechromosome. Because chromosomes with DRT/DMC are common in tumorcells and in cells exposed to ionizing radiation, we proposethat DRT/DMC represents a common mechanism responsible for thegenomic instability found in cancer cells and for the persistentchromosomal instability associated with cells exposed to ionizingradiation.
In addition to structural alterations of chromosomes, morphologicaldifferences between chromosomes within the same cell have alsobeen observed in tumor-derived cells. Some of these morphologicalalterations have been referred to as ‘incomplete condensation’or ‘pulverization’ of chromosomes or regions ofchromosomes during mitosis (reviewed in 5). Furthermore, theseabnormally condensed chromosomes synthesize DNA after the normallycondensed chromosomes have ceased replication (6,7). However,the nature of the chromosome abnormalities associated with thesemorphological changes and the molecular basis for the apparentreplication asynchrony between chromosomes were not determinedin these earlier studies. More recently, we characterized anabnormal chromosomal phenotype that was associated with certaintumor-derived chromosome alterations (8). We found that fourdifferent chromosome rearrangements displayed a significantdelay in replication timing (DRT) of the entire chromosome.This DRT phenotype is characterized by a 2–3-h delay inboth the initiation and the completion of DNA synthesis alongthe entire length of the chromosome, whereas the other chromosomeswithin the same cell show normal patterns of DNA synthesis.Chromosomes with the DRT phenotype also display a significantdelay in mitotic chromosome condensation (DMC) that is characterizedby an under-condensed appearance during mitosis. This under-condensedappearance is accompanied by a delay in the mitosis-specificphosphorylation of histone H3 on serine 10. These observationssuggested that certain tumor-derived chromosome rearrangementsdisplay this abnormal chromosomal phenotype. Importantly, chromosomeswith DRT/DMC were present in five of seven tumor-derived celllines and five of 13 primary tumor samples, indicating thatchromosomes with this phenotype are common in tumor cells invitro and in vivo (8).
More recently, we found that exposing cell lines, primary bloodlymphocytes or mice to ionizing radiation (IR) resulted in thegeneration of chromosomes with DRT/DMC in as many as 25% ofsurviving cells (9). Furthermore, we found that DRT/DMC occurredpredominantly on inter-chromosomal translocations (ICTs) andthat it occurred on a surprisingly large fraction of the ICTs,estimated to be 5% of all translocations induced by IR. Importantly,DRT/DMC was not detected on the majority of ICTs or on non-rearrangedchromosomes, suggesting that the DRT/DMC phenotype only occursfollowing specific chromosomal exchanges (9). Unfortunately,the exact nature of the IR-induced chromosome alterations thatresulted in the DRT/DMC phenotype could not be determined, especiallygiven the propensity of DRT/DMC chromosomes to undergo secondaryrearrangements (8,9). Furthermore, the possibility that theDRT/DMC phenotype was the result of a stochastic process thatcould potentially occur at any translocation breakpoint couldnot be ruled out (9). Therefore, to generate a methodology thatwould allow for the systematic analysis of the DRT/DMC phenotype,we have developed a ‘chromosome engineering’ systemthat allows us to: (1) target the genome in a random fashion,(2) create reciprocal chromosome translocations, (3) generatethe same translocations in multiple independent events usingtwo distinct mechanisms and (4) characterize the chromosomesboth before and after the translocation events. A preliminaryanalysis of 10 chromosome rearrangements generated using thisapproach identified a single balanced translocation with theDMC phenotype (9).In this article, we show the analysis of 83 independent celllines that contain chromosome rearrangements generated usingthis Cre/loxP system. We found that 10% of chromosome translocationsgenerated using this system display DRT/DMC. We also found thattwo distinct mechanisms for generating the same translocations(site-specific homologous recombination mediated by Cre or non-homologousend joining of DNA double-strand breaks induced by I-Sce1) giverise to DRT/DMC, suggesting that DRT/DMC is not the consequenceof a specific DNA repair process. Furthermore, on certain balancedtranslocations that display DRT/DMC, only one of the derivativechromosomes displays the phenotype. These observations indicatethat the replication timing of certain chromosome translocationsis regulated in cis by a mechanism that results in delayed replicationalong the entire length of the chromosome. Finally, we showthat cells containing engineered chromosomes with DRT/DMC acquirenew chromosomal rearrangements at an increased rate and thereforedisplay genomic instability.
Chromosome engineering strategy
The chromosome engineering strategy that we employed uses theCre/loxP site-specific recombinase system to generate ICTs (fora recent review, see 10). Because the Cre/loxP system is relativelyinefficient at generating ICTs (<1x10–3), we used reconstitutionof a selectable marker to isolate the cells that had undergoneCre-mediated recombination. A schematic representation of thisstrategy is shown in Figure 1 (9). Briefly, this strategy involvesreconstitution of a cloned murine Aprt gene in a human cellline, HTD114, which is deficient for APRT (11). This strategyinvolves the generation of a collection of parental cell clones(P-clones), each containing two independently inserted plasmidcassettes. One cassette contains the 5' portion of the Aprtgene linked to a neomycin resistance gene (5'-AP-Neo) and theother cassette contains the 3' portion of the Aprt gene linkedto a hygromycin resistance gene (Hyg-3'RT). Each cassette containsloxP sites in the second intron of the Aprt gene. Thus, followingCre-mediated homologous recombination the Aprt gene is reconstitutedat the loxP sites and a reciprocal translocation is generated.Thus, following selection in media containing azaserine andadenine (AA), Aprt+ cells can be isolated. In addition, workfrom Dr M. Jasin's laboratory has indicated that chromosometranslocations can be generated from two DNA double-strand breaks(DSBs) induced by the rare cutting restriction enzyme I-Sce1(12). Therefore, as an alternative approach to generate thetranslocations, we also introduced I-Sce1 recognition sequencesinto the second intron of the Aprt gene in the 5' and 3' cassettes(Fig. 1). Thus, this system allows the generation of chromosometranslocations at the same translocation breakpoints in multipleindependent events using two distinct mechanisms: (1) homologousrecombination mediated by Cre and (2) non-homologous end joiningof DSBs generated by I-Sce1.
Screen for DMC
We isolated 97 P-clones that contained independent random insertionsof the two Aprt cassettes. Subsequently, each P-clone was transientlytransfected with a Cre-expression plasmid and cultured in mediathat selects for Aprt-expressing cells (azaserine plus adenine).We were able to generate Aprt+ colonies from 83 different P-clones.During development of this system, we measured the efficiencyof Cre-mediated recombination for 33 different P-clones. Inthese clones, Aprt+ colonies were generated at frequencies between1x10–2 and 1x10–6 per transfected cell (SupplementaryMaterial, Table S1). The Aprt+ colonies were pooled from eachP-clone to generate 83 independent recombinant pools (R-pools).We pooled the colonies for three reasons. First, every cellfrom a given P-clone is expected to generate the same translocation(or limited number of translocations depending on the numberof plasmid cassette insertions) following any Cre-mediated event,and assaying multiple clones from 83 different P-clones wouldbe prohibitive. Second, if DRT/DMC occurs in a stochastic mannerand could occur at any translocation breakpoint, pooling thecolonies would allow us to detect chromosomes with DRT/DMC inevery pool at similar frequencies. Third, chromosomes that displayDRT/DMC are unstable (8) and pooling the Aprt+ colonies wouldallow for fewer generations prior to karyotypic analysis. One critical aspect in the analysis of chromosomes with DMCis that a 2–3-h pre-treatment of the cultures with colcemidprior to mitotic harvest interferes with our ability to detectthe under-condensed phenotype (8). Therefore, to determine whetherany of the R-pools contained chromosomes with DMC, each R-poolwas harvested for mitotic cells in the absence of colcemid.Mitotic spreads from each R-pool were analyzed for the presenceof chromosomes with DMC. We scored a cell as DMC-positive ifit contained one or more chromosomes with at least two of thefollowing characteristics: (1) at least twice as long as anyother chromosome within the same spread, (2) less than halfas wide as any other chromosome within the same spread and/or(3) contained a bend of greater than 180° as previouslydescribed (8,9). Note that the parental HTD114 cells containthree pre-existing ICTs (Supplementary Material, Fig. S1),but display a relatively low frequency of spontaneous DRT/DMC,0.5% [Table 1 and (9)]. We found that the majority of the R-poolsdisplayed a relatively low frequency of DMC, ranging from 0to 4% (Table 1). In contrast, 13 pools contained chromosomeswith DMC in greater than 5% of mitotic cells, ranging from 6to 50%. This observation indicated that DMC did not occur inall R-pools with similar frequencies, suggesting that only certainCre-mediated chromosome rearrangements resulted in DMC. Examplesof mitotic spreads containing chromosomes with DMC from thisinitial screen are shown in Figure 2.
Because there is a low but detectable frequency of DMC in theparental HTD114 cells, each engineered translocation had tobe analyzed separately using chromosome-specific probes. Therefore,to determine which chromosomes were affected in each clone,we performed a series of karyotypic studies on nine P-clonesand their respective R-pools that contained DMC in >6% ofthe mitotic spreads, as well as on four P-clones and their respectiveR-pools that showed a low frequency of DMC. This initial karyotypicanalysis was carried out in the presence of colcemid to facilitatethe characterization of the chromosomes involved. Initially,we used fluorescence in situ hybridization (FISH), with theAprt plasmid cassettes as probes, followed by R-banding to identifythe sites of insertion. We next used whole chromosome paints(WCPs) to confirm the insertion sites in the P-clones and tovisualize the Cre-dependent translocations in the R-pools. Finally,the chromosome translocations present in the R-pools were confirmedusing G-banding. An example of this analysis is shown for P175and its Aprt+ pool R175 in Figure 3. P175 contains plasmid cassetteinsertions in 6q14–15 and 10q11.2 (Fig. 3A and B).As expected, R175 contains a new translocation involving chromosomes6 and 10 (Fig. 3C–E). The breakpoints for this newtranslocation are at the plasmid cassette insertion sites andgenerated a balanced translocation, t(6;10)(q14–15;q11.2).A schematic view of the plasmid cassette insertions and thet(6;10) is shown in Figure 3F. A summary of this karyotypicanalysis for all 13 clones is shown in Table 1. In addition,we have characterized the plasmid cassette insertions in thisset of P-lines and in their respective R-pools for copy numberand for reconstitution of the Aprt cassettes using southernblot hybridizations (Supplementary Material, Fig. S2).
One of the advantages of this approach is that the same translocationscan be generated in multiple, independent events. Therefore,to determine if a particular translocation displayed DMC followingindependent Cre events, additional R-pools were generated andanalyzed for DMC in mitotic spreads prepared in the absenceof colcemid. We analyzed two independently derived pools ofR175 (R175A and R175B) using FISH with chromosome 6 and 10 WCPs.Figure 4A–D shows examples of this analysis and indicatedthat chromosomes with DMC hybridized to both the chromosome6 and 10 probes in both isolates of R175. Analysis of P268 andtwo Aprt-expressing pools, R268A and R268B, indicated that abalanced translocation involving the long arm of chromosome15 and the long arm of chromosome 16, t(15q;16q)(q24;q12.1)also displays DMC (Table 1 and Supplementary Material, Fig. S3).These results indicate that DMC was induced in two independentlyderived pools of R175 and R268, and it occurred on the Cre-dependentt(6;10) and t(15;16), respectively. A similar analysis of R27,R186 and R276 showed that DMC also occurred on Cre-dependentbalanced translocations (Table 1). In contrast, DMC was notdetected on balanced translocations generated by Cre in twoindependent pools each of R38, R161, R244, R248 or R263, withover 200 metaphase spreads analyzed from each pool (Table 1,Supplementary Material, Fig. S4, data not shown). Theseresults indicate that DMC was detected in independent isolatesof certain Cre-dependent chromosome translocations and not inothers, indicating that this abnormal chromosomal phenotypeis not the consequence of a stochastic process that could occurat any translocation breakpoint.
Only one or both derivative chromosomes display DMC
Because the DMC phenotype occurred on balanced translocationsand every balanced translocation generates two derivative chromosomes,we next determined whether one or both derivative chromosomesdisplayed the DMC phenotype. To address this question, we analyzedmitotic spreads for chromosomes with DMC using FISH with chromosome-specificcentromeric probes. Figure 4E and F shows two examples of thisanalysis for R175 and indicated that the chromosomes with DMChybridized to the chromosome 10 centromeric probe and not tothe chromosome 6 centromeric probe. In total, we detected 10DMC chromosomes that hybridized to the chromosome 10 probe andno DMC chromosome that hybridized to the chromosome 6 probe.A similar analysis on R268 indicated that the chromosome 15derivative, not the chromosome 16 derivative, displayed DMC(Supplementary Material, Fig. S3). Analysis of a thirdtranslocation with DMC, a t(3;16)(p13;p13.3) present in R27,also indicated that only one product, the 16 derivative, displayedDMC (data not shown). Although it would be impossible to provethat any given chromosome ‘never’ displays DMC,these observations suggest that only one of the products fromthese three translocations displays the phenotype. In contrast,analysis of a fourth translocation, a t(3;13) present in R186,indicated that both derivative chromosomes displayed DMC (Fig. 5).Note that P186 contains three independent sites of plasmid cassetteinsertion, 3(q29), 11(p15) and 13(q14). However, the only newtranslocation detected in two independent R186 pools was a t(3;13)(q29;q14),indicating that the 11(p15) insertion site did not participatein the Cre events that generated the Aprt+ cells in the R186pools. The reason that the 11p cassette has not been detectedin a translocation in R186 is unknown, but it may contain adeleted or rearranged cassette that prevents reconstitutionof a functional Aprt gene, or perhaps the frequency at whichthe 11p15 cassette can participate in a Cre event with the othercassette may simply be much lower than the frequency for theother insertion sites (Supplementary Material, Table S1). Regardless,using FISH with chromosome 3 and 13 centromeric probes, we detectedDMC on both derivative chromosomes from t(3;13) (Fig. 5Fand G). These observations indicate that either one or bothof the products from certain balanced translocations displaythe DMC phenotype.
Engineered chromosomes with DMC also display DRT
The DMC phenotype is preceded by a DRT of the entire chromosome(8). Therefore, to confirm that the engineered chromosomes withDMC also display DRT, we analyzed replication timing of thechromosomes with DMC using a BrdU incorporation assay combinedwith FISH using chromosome-specific centromeric probes. Figure6A shows a schematic illustration of this replication timingassay. Analysis of mitotic spreads from R175B harvested forlate replication indicated that the chromosomes with DMC hybridizedto the chromosome 10 centromeric probe and incorporated BrdUalong their length at a time when the fully condensed chromosomesdid not show any detectable BrdU incorporation. An example ofthis analysis is shown in Figure 6B–D. This analysis indicatesthat the derivative chromosome 10 present in R175 displays bothDRT and DMC.
Plasmid insertions and small intra-chromosomal deletions are not sufficient to induce DRT/DMC
The results described earlier indicate that DRT/DMC occurs onbalanced translocations induced by Cre-mediated recombination,and that one or both of the derivative chromosomes display thephenotype. These observations suggest that DRT/DMC occurs followinginter-chromosomal exchanges at specific chromosome locations.However, it was also possible that simple plasmid cassette insertionor Cre-mediated recombination at these specific chromosome locationswas responsible for inducing DRT/DMC. Therefore, to determinewhether DRT/DMC was simply the result of plasmid cassette insertion,we analyzed mitotic spreads for DMC in P-clones that generatedDMC at a high frequency in their R-pools. None of the P-clonesassayed displayed DMC in greater than 4% of the spreads (Table1). In addition, P175 and P27 were assayed for DMC combinedwith FISH with centromeric probes. This analysis indicated thatP175 and P27 did not retain DMC chromosomes that hybridizedto the chromosome 6 and 10, or 3 and 16 centromeric probes,respectively (Table 2). Therefore, simple plasmid cassette insertionat these chromosomal locations is not sufficient to induce DRT/DMC.
Next, to determine whether DRT/DMC was simply the consequenceof Cre-mediated recombination at these specific chromosomallocations, we took advantage of the fact that our 5' and 3'cassettes contain Neo and Hyg genes flanked by loxP sites (i.e.floxed; Fig. 1). Previous studies indicate that intra-chromosomalCre events are markedly more efficient than inter-chromosomalCre events (13). Thus, the design of this set of experimentswas to transiently transfect P-clones with a Cre-expressionvector and isolate clones that experienced Cre-mediated intra-chromosomaldeletion of the Neo and Hyg genes but did not generate ICTs.To aid in the isolation of Cre-transfected cells, we co-transfecteda GFP-expression vector and isolated the GFP-positive cellsby fluorescence-activated cell sorting (FACS). The GFP-positivecells, and presumably Cre-positive cells, were plated at clonaldensities, allowed to form colonies in the absence of Aprt selectionand isolated as individual clones. For this analysis, we chosetwo clones, P175 and P27, that generated DRT/DMC on Cre-dependentICTs. Deletion of the Neo and Hyg genes occurred in >90%of these clones as assayed by southern blot hybridization (SupplementaryMaterial, Fig. S2C), and the absence of the translocationswas confirmed by southern blot hybridization for reconstitutionof the Aprt gene and FISH with WCPs (data not shown). This setof experiments resulted in the generation of cell lines thatexperienced Cre-mediated intra-chromosomal deletion of the Neoand Hyg genes at the same chromosomal locations involved inthe translocation events produced in R175 and R27. The cloneswith the Neo and Hyg genes removed were designated P175NH andP27NH, respectively. Three independent P175NH clones were assayedfor DMC after hybridization with chromosome 6 and 10 centromericprobes and two independent P27NH lines were assayed for DMCafter hybridization with chromosome 3 and 16 centromeric probes.Chromosomes with DMC were not detected in any of the P175NHor P27NH clones with any of the centromeric probes (Table 2).Taken together, these observations indicate that insertion ofthe plasmid cassettes and Cre-mediated intra-chromosomal deletionsat these specific chromosome locations is not sufficient toinduce DRT/DMC and suggest that DRT/DMC is the result of inter-chromosomalexchanges at specific chromosomal locations. However, it isimportant to point out that this analysis does not rule outthe possibility that other intra-chromosomal deletions or rearrangementscould result in DRT/DMC.
DRT/DMC occurs on translocations
The results described earlier indicate that DRT/DMC can occurfollowing Cre-mediated site-specific recombination, but onlyon specific translocation derivatives. To determine if a differentmechanism for generating the translocations could produce DMCon one of these derivatives, we took advantage of the fact thatour 5' and 3' cassettes have I-Sce1 sites in the second intronof the Aprt gene (Fig. 1). We transiently transfected P175cells with an I-Sce1-expression vector and selected for Aprt-expressingcells. Because DSBs induced by I-Sce1 are subject to exonucleaseactivity prior to NHEJ and could potentially result in translocationswith different breakpoints (12), we isolated Aprt+ clones, namedthe RS175 series. Karyotypic analysis of RS175-1 indicated thatthe expected balanced translocation, a t(6;10)(q12–13;q11.2),was generated (Fig. 7A–C). Although we have not sequencedthe breakpoints in RS175-1, t(6;10) is cytogenetically identicalto the translocation produced by Cre in the R175 pools. In addition,southern blot analysis indicated that the Aprt gene was reconstitutedin the second intron, and the Neo and Hyg genes were still intact(Fig. 1; data not shown). Therefore, the translocationbreakpoints in RS175-1 are located within the plasmid cassettes,indicating that the DSBs induced by I-Sce1 did not experiencesignificant exonuclease digestion prior to the generation ofthe t(6;10) in this clone. To determine if DMC was occurringon the chromosome 10 derivative, as it is in R175, we analyzedmitotic spreads from RS175-1 using FISH with the chromosome6 and 10 centromeric probes. An example of this analysis shownin Figure 7D indicated that the chromosome 10 derivative, notthe chromosome 6 derivative, displayed DMC. A similar analysison I-Sce1-induced translocations from P244 and P38, which didnot generate DMC following Cre, showed undetectable levels ofDMC on the expected translocations (data not shown). Therefore,this analysis indicates that ICTs produced by two distinct mechanisms,Cre-mediated homologous recombination or NHEJ of DSBs, can generatechromosomes with DRT/DMC.
Chromosomes with DRT/DMC are unstable
We previously found that chromosomes with DRT/DMC participatein frequent secondary rearrangements and translocations (8).During characterization of the R-pools described here, we alsoobserved unexpected rearrangements involving the chromosomeswith DRT/DMC (Fig. 5 and Supplementary Material, Fig. S3).To further characterize these secondary rearrangements and tocompare the stability of DRT/DMC chromosomes to non-DRT/DMCchromosomes, clones containing Cre-induced translocations wereisolated and expanded through a defined number of generations(20–22) and examined for unexpected chromosome rearrangements.For this analysis, we isolated Aprt+ clones from four differentP-clones: three that generated DRT/DMC following Cre-mediatedrecombination (P175, P186 and P268) and one that did not (P244);these clones were named the R175F, R186F, R268F and R244F series,respectively. In addition, to characterize the stability ofthe chromosomes prior to the generation of the Cre-induced translocations,the P-clones were transfected with an intact Aprt-expressionvector and stable Aprt-expressing clones isolated; these cloneswere named the P175F, P186F, P268F and P244F series. Analysisof these Aprt-transfected clones would allow us to determinethe stability of the chromosomes prior to creation of the translocationsand to control for any variability in chromosome stability betweendifferent P-clones subjected to transfection, Aprt selectionand clonal expansion in AA media. Because the cells constitutingeach clone were cultured through a similar number of generations(20–22) and were grown under identical conditions, thestability of the chromosomes could be directly compared bothbefore and after the translocation events and between DRT/DMCand non-DRT/DMC containing clones. In addition, we analyzeda series of P268F clones that had experienced Cre transienttransfection, but did not generate the translocation. Theseclones, designated P268NH, were generated by co-transfectionwith Cre and GFP-expression vectors, FACS for GFP-expressingcells, expansion in the absence of selection and screening fordeletion of the Hyg gene by southern blot hybridization (datanot shown). These clones were cultured through a similar numberof generations as the other clones (20–22) and servedas an additional control for non-specific affects of Cre transienttransfection.
Mitotic spreads from each clone were analyzed using FISH withWCPs that hybridized to the Cre-dependent translocations, and100 mitotic spreads were scored for each clone with each WCP.This set of experiments allowed us to determine the frequencyat which unexpected gross chromosomal rearrangements (GCRs:translocations, deletions, insertions and rearrangements) occurredon Cre-dependent translocations, with or without DRT/DMC.
These cell lines could clearly be segregated into two groupsbased on the frequency of new GCRs. All the control Aprt-transfectedclones (P175F1–4, P186F1–4, P268F1–4 and P244F1–4),the non-DRT/DMC translocation containing clones (R244F1–5)and the P268NH clones displayed low or undetectable frequenciesof new GCRs involving the appropriate chromosomes. For example,we did not detect any GCRs involving chromosomes 15 and 16 inthe P268F or P268NH clones, and we did not detect any GCRs involvingchromosomes 13 and 16 in the P244F clones (Table 3). In addition,we detected only one GCR involving chromosome 13 in one cellfrom one R244F clone (Table 3 and Supplementary Material, Fig. S5).In contrast, the DRT/DMC containing clones showed dramaticallyhigher frequencies of GCRs involving the DRT/DMC chromosomes.For example, every R268F clone contained GCRs that hybridizedto the chromosome 15 and/or 16 WCPs, and cells with GCRs werealso quite frequent within each clone, occurring in 19–100%(Table 3 and Supplementary Material, Fig. S6). Furthermore,each R268F clone retained a different array of GCRs and manyof these GCRs represented translocations to other, and presumablynon-DRT/DMC chromosomes (Fig. 8). Interestingly, and consistentwith our observation that only the chromosome 15 derivativedisplayed DRT/DMC in the R268 pools (Supplementary Material,Fig. S3), the chromosome 16 derivative was still presentand un-rearranged in all of the R268F clones (Fig. 8).This observation indicates that the chromosome with DRT/DMC,the 15 derivative, generated the GCRs in the R268F clones. Inaddition, to determine whether any of the Cre-treated cloneshad integrated the Cre-expression vector, which could potentiallycontribute to new GCRs via Cre-mediated recombination betweenendogenous or cryptic loxP sites (14,15), we carried out southernblot hybridizations on genomic DNA extracted from all of theCre-treated clones shown in Table 3. This analysis indicatedthat none of these clones retained the Cre-expression vector(data not shown).
Finally, using a fluctuation analysis (16), as modified by Linet al. (17) to allow for estimating high mutation rates,we estimate that the rate at which cells acquired new GCRs involvingthe DRT/DMC chromosomes was 7x10–1 (or one new GCR every14 cell divisions) for the t(6;10) in R175, 1.6x10–1 (orone new GCR every six cell divisions) for the t(3;13) in R186and 1.7x10–1 (or one new GCR every five cell divisions)for the t(15;16) in R268. We also point out that this is likelyto be an underestimate of the rate for GCR formation involvingthese translocations, as many of the R-clones retained GCRsin a large fraction of the cells, ranging from 2 to 100% ofthe cells (Table 3). In contrast, we estimate that the rateat which cells acquired new GCRs involving the appropriate non-rearrangedchromosomes (i.e. in P175F, P186F, P244F and P268F clones),non-DRT/DMC translocations (i.e. the t(13;16) in R244F) andin Cre-transfected but non-translocation carrying clones (i.e.P268NH) was 2.5x10–3 (or one new GCR every 400 cell divisions).Therefore, we estimate that chromosomes with DRT/DMC have an30–80-fold increase in the rate at which new GCRs occuron the affected chromosomes.
Genetic instability can occur at distinct levels. In most cancers,the instability occurs at the chromosome level, resulting ingains or losses of whole chromosomes (4). This type of instabilityis a dominant trait and is independent of p53 mutations (18).Another common form of genetic instability found in cancer cellsis characterized by the generation of frequent chromosome rearrangements,including the formation of marker chromosomes and gene amplifications.We believe that these two forms of genetic instability are distinct,and refer to the process that results in gains or losses ofintact chromosomes as chromosome instability (CIN) (4) and tothe process that results in the generation of frequent translocationsand rearrangements as translocation instability (TIN). Unfortunately,the molecular mechanisms responsible for either CIN or TIN intumor cells are still poorly understood (4,19).
Another example of genetic instability found in mammalian cellsoccurs at a delayed time following exposure to IR (20,21). Thisdelayed or persistent chromosomal instability occurs in vitroand in vivo and is characterized by the appearance of new chromosometranslocations and rearrangements in subsequent generationsafter the initial exposure to IR (22). Interestingly, many aspectsof this delayed instability cannot be explained by simple mutationalinactivation of trans-acting factors. For example, sub-cloningexperiments of irradiated cells indicate that this type of genomicinstability is unevenly transmitted to sibling sub-clones andthat the chromosomal rearrangements that occur within the unstableclones are non-randomly distributed throughout the karyotype(23,24). So, alternative epigenetic and/or cis-acting mechanismshave been proposed to explain this poorly understood process(reviewed in 25,26).
Given our limited understanding of genomic instability in mammaliancell systems, it is currently not known whether the delayedchromosomal instability observed in irradiated cells and theTIN associated with cancer cells are caused by similar or distinctmechanisms. However, we previously showed that chromosomes withDRT/DMC are common in tumor cells in vitro and in vivo (8).In addition, we found that chromosomes with DRT/DMC were presentin as many as 25% of cells exposed to IR (9). In this study,we found that chromosomes with DRT/DMC have a 30–80-foldincrease in the rate at which new GCRs occur on the affectedchromosomes. Therefore, because chromosomes with DRT/DMC arecommon in tumor cells and in cells exposed to IR, we proposethat chromosomes with DRT/DMC represent a common source of thegenomic instability observed in cancer cells (i.e. TIN) andin cells exposed to IR (i.e. delayed chromosomal instability).In addition, because the DRT/DMC phenotype occurs only on certainderivative chromosomes, our data provide support for previousmodels that genomic instability is driven by a cis-acting mechanism(26).
DRT/DMC is regulated in cis
How do ICT events at specific chromosome locations result inthe DRT/DMC phenotype? This is an intriguing question giventhat the translocation breakpoints that induced DRT/DMC in thisstudy occurred within the plasmid cassette sequences and notwithin the chromosomal DNA. In addition, it appears that inter-chromosomalexchanges are required for the phenotype, as plasmid cassetteinsertions and small Cre-mediated intra-chromosomal deletionsat the same chromosome locations did not result in DRT/DMC.Furthermore, on certain balanced translocations, only one ofthe derivative chromosomes displayed the phenotype. Taken together,these observations indicate that the DRT/DMC phenotype is regulatedby a cis-acting mechanism that occurs following specific chromosomalexchange. However, it is important to point out that intra-chromosomalrearrangements, involving chromosomal DNA instead of plasmidDNA, may be capable of generating DRT/DMC. Regardless, we areconsidering two possibilities to explain how specific chromosomalexchanges can give rise to the DRT/DMC phenotype. First, itis possible that these chromosomal exchanges result in deletionor mutation of a cis element that normally establishes earlyreplication timing for the entire chromosome. Loss of this elementwould then result in delayed replication of the entire chromosome.Second, it is possible that these specific chromosomal exchangesgenerate dominant interfering elements that act in cis to delaynormal chromosome replication timing by some unknown mechanism.Although we cannot distinguish between these possibilities atthe present time, the chromosome engineering strategy describedhere, combined with ‘mixing and matching’ of loxP-taggedchromosomes and directing loxP sites to specific chromosomelocations, should provide for a system in which the molecularmechanisms responsible for the DRT/DMC phenotype can be elucidated.
Cells were exposed to 10 ng/ml of colcemid (Sigma) for1 h. Trypsinized cells were centrifuged at 1000 rpm(300 g) for 10 min in a swinging bucket rotor. Thecell pellet was resuspended in 75 mM potassium chloridefor 15 min at 37°C, recentrifuged at 1000 rpmfor 10 min and fixed in 3 : 1 methanol : aceticacid. Fixed cells were added drop-wise to microscope slidesto make mitotic chromosome spreads using standard methods (27).
Fluorescence in situ hybridization
Slides with mitotic spreads were baked at 85°C for 20 minand then treated with 0.1 mg/ml RNase for 1 h at 37°C.After RNase treatment, the slides were washed in 2x SSC (1xSSC is 150 mM NaCl and 15 mM sodium citrate) withthree changes for 3 min each and dehydrated in 70, 90 and100% ethanol for 3 min each. The chromosomes were denaturedin 70% formamide in 2x SSC at 70°C for 3 min and WCPswere used according to the manufacturer's recommendations (AmericanLaboratory Technologies and Vysis). Detection of digoxigenin-dUTPprobes used a three-step incubation of slides with sheep FITC-conjugatedanti-digoxigenin antibodies (Roche) followed by rabbit FITC-conjugatedanti-sheep antibodies (Roche) followed by goat FITC-conjugatedanti-rabbit antibodies (Jackson Laboratories). Slides were stainedwith DAPI (12.5 µg/ml) or propidium iodide (0.3 µg/ml),cover slipped and viewed under UV fluorescence with FITC filters(Zeiss).
Mitotic chromosome spreads were prepared as described in theprevious section. Slides were treated with RNase at 100 µg/mlfor 1 h at 37°C and washed in 2x SSC and dehydratedin 70, 90 and 100% ethanol. Chromosomes were denatured at 75°Cfor 3 min in 70% formamaide/2x SSC, followed by dehydrationin ice cold 70, 90 and 100% ethanol. Probe cocktails (Vysis)were denatured at 75°C for 10 min and pre-hybridizedat 37°C for 30 min. Probes were applied to slides andincubated overnight at 37°C. Post-hybridization washes consistedof three 3-min rinses in 50% formamide/2x SSC, three 3-min rinsesin 2x SSC and finally three 3-min rinses in PN buffer (0.1 MNa2HPO4+0.0 M NaH2PO4, pH 8.0, +2.5% Nonidet NP-40), allat 45°C. Slides were then counterstained with either propidiumiodide (2.5 µg/ml) or DAPI (15 µg/ml)and viewed under UV fluorescence (Zeiss).
Replication timing and immunofluorescence
The BrdU replication timing assay was performed as previouslydescribed (8). Asynchronously growing R175B cells were exposedto a pulse of 20 µg/ml of BrdU (Sigma) for 15 min,washed with PBS and chased in media containing 0.2 mM thymidine.Mitotic cells were harvested in the absence of colcemid. Thecells were treated with 75 mM KCl for 15 min at 37°C,fixed in 3 : 1 methanol : acetic acid anddropped on wet slides. The chromosomes were denatured in 70%formamide in 2x SSC (1x SSC is 150 mM NaCl and 15 mMsodium citrate) at 70°C for 3 min. Incorporated BrdUwas detected using a FITC-labeled anti-BrdU antibody (BectonDickinson). Slides were stained with propidium iodide (0.3 µg/ml),cover slipped and viewed under UV fluorescence (Zeiss).
Three micrograms of the 5'-AP-Neo plasmid was linearized ata unique NotI site, electroporated (300 V, 950 µFin PBS; Bio-Rad) into HTD114 cells and grown under 500 µg/mlG418 (Geneticin, Gibco) selection for 10–14 days. G418-resistant(NeoR) colonies were then pooled and expanded. Three microgramsof the Hyg-3'RT plasmid was linearized at a unique NotI site,electroporated (300 V, 950 µF in PBS; Bio-Rad)into the pooled NeoR cells and grown under 500 µg/mlG418 and 200 µg/ml Hygromycin B selection for 10–14days. One hundred and ten individual NeoR, HygR colonies (‘P-clones’)were picked, expanded and subsequently transiently co-transfected(Lipofectamine, Gibco) with 1 µg of a green fluorescentprotein (GFP) expression plasmid (pCSGFP) and either 3 µgof a Cre recombinase expression plasmid (pBS185, Gibco) or anempty vector control (pBluescript SK, Stratagene). Average transfectionefficiencies were between 5 and 10%, as determined by GFP expression.Cre-transfected cells were grown for 14–21 days in 10 µg/mlazaserine and 10 µg/ml adenine (AA selection). Theresulting Aprt+ colonies were pooled to generate 83 independentpools and expanded. A preliminary description of this systemwas described previously (9). Alternatively, individual Aprt+clones were picked and expanded to approximately 4x106 cellsand analyzed for the presence of ‘new’ rearrangementsusing FISH with WCPs.
Deletion of neomycin and hygromycin genes
P27 and P175 were co-transfected (Lipofectamine, Gibco) with3 µg of a Cre-expression plasmid (pBS185) and 1 µgof a GFP-expression plasmid (pcsGFP). After 24 h, the cellswere trypsinized, washed once in Hank's balanced salt solution(Gibco) and 1x106 cells were subjected to FACS with gating setto collect GFP-expressing cells. Typical transfection efficienciesbefore FACS, based on the number of GFP-positive cells, werebetween 15 and 25%. After FACS, the collected cell fractionswere suspended in 1 ml of fresh Hank's balanced salt solutionand consisted of approximately 3x105 cells with 85–95%being GFP+. The GFP+ cells were plated in serial dilutions,to allow single colony growth, onto 15-cm tissue culture dishescontaining DMEM supplemented with 10% fetal bovine serum. Individualcolonies were picked, expanded and challenged to grow in mediacontaining 500 µg G418 or 200 µg HygromycinB. Clones that failed to grow in both G418 and Hygromycin Bwere expanded and subjected to southern analysis to confirmloss of the Neo and Hyg markers. The absence of translocationswas confirmed using FISH with appropriate WCPs.
Summary of karyotypic analysis, and frequency of DMC
The plasmid cassette insertion sites in each P-clone were determined by FISH, using the Aprt plasmids as probes, followed by R-banding. The plasmid insertion sites are denoted by brackets and translocation breakpoints by parentheses. The identity of each translocation was determined by FISH, using WCPs as probes, and subsequently confirmed by G-banding. P27/R27 and P38/R38 were described previously [From Breger et al., (9)]. The frequency of DMC was determined for each R-pool and selected P-clones and listed as percent (%). ND, not determined; N/A, not applicable.
Cre-mediated deletion of floxed Neo or Hyg genes in P-lines does not cause DMC
Metaphase spreads were analyzed for DMC after FISH with centromere-specific probes relevant to the chosen cell lines (Chr.16-cen and Chr.3-cen probes for P27 derivative lines or Chr.6-cen and Chr.10-cen probes for P175 derivative lines). The frequency of DMC for the R-lines is from the initial screen for DMC and is shown for comparison. The actual numbers of metaphase spreads scored are shown in parentheses.
*P<0.05 compared to R-line.
Frequencies of GCRs involving Cre-dependent translocations
Mitotic spreads from each clone were analyzed by FISH using WCPs which hybridized to the Cre-dependent translocations: 6 and 10 for P175/R175, 3 and 13 for P186/R186, 13 and 16 for P244/R244, and 15 and 16 for P268/R268/P268NH. One hundred mitotic spreads were scored for each clone with each WCP. The frequencies of GCRs involving each chromosome are indicated. The frequencies of GCRs involving the Cre-dependent translocations (T) represent the product of GCRs involving both WCPs. N/A, not applicable.
Schematic diagram of the Cre/loxP chromosome engineering strategy. A diagram of the mouse genomic Aprt gene, with a unique HindIII site in intron 2, is shown. The 5' portion of the Aprt gene was separated from the 3' portion at this unique HindIII site. A floxed Neo or Hyg resistance gene was inserted at the HindIII site in both the 5' and 3' portions of the Aprt
gene, respectively, resulting in the 5'-AP-Neo and Hyg-3'RT cassettes,
as shown. The 5'-AP-Neo and Hyg-3'RT cassettes integrate randomly
throughout the genome following linearization and electroporation.
After Cre transient transfection, reciprocal translocations are
generated in a two-step process. First, due to the close proximity of
the loxP sites flanking the Neo and Hyg genes, and the fact
that they are aligned in the same orientation, the Neo and Hyg genes
are excised as circles via highly efficient (determined to be nearly
90%; data not shown) intra-chromosomal events. Next, Cre directs the
remaining loxP sites to proceed through a low efficiency (<1x10–3) inter-chromosomal reciprocal exchange. This results in reconstitution of the Aprt gene on one derivative chromosome, and a single loxP site on the other derivative, converting cells from APRT– (P-clones) to APRT+ (R-pools). A preliminary description of this system was described elsewhere (9).
(Click image to enlarge)
Representative examples of DMC from the initial screen. Following Cre
transient transfection and pooling of Aprt+ colonies, 30–50 metaphase
spreads from each R-pool were scored for the presence of chromosomes
with DMC. Examples of DMC from R27A (A–C), R186A (D–F), R230A (G, H) and R268A (I) are shown. Arrows mark chromosomes with DMC. Chromosomes were stained with propidium iodide (PI).
(Click image to enlarge)
Cytogenetic characterization of P175 and R175. (A) FISH on P175
using the plasmids containing the 5'-AP-Neo and Hyg-3'RT cassettes as
probes. Arrows mark the chromosome 10 and 6 insertion sites on a
representative mitotic spread. Chromosomes were stained with propidium
iodide (PI). (B) R-banding of the mitotic spread shown in (A).
Arrows mark the chromosome 10 and 6 insertion sites and are: 6q12–13
and 10q11.2. (C) FISH on a representative mitotic spread from
R175A using the plasmids containing the 5'-AP-Neo and Hyg-3'RT
constructs as probes. R-banding indicated that a balanced
t(6;10)(q12–13;q11.2) was generated at the plasmid cassette insertion
sites (not shown). Arrows mark the chromosome 10 (der10) and 6 (der6)
derivatives. Chromosomes were stained with propidium iodide (PI). (D)
FISH using chromosome 6 (red) and 10 (green) WCPs on a representative
mitotic spread from R175A. Arrows mark the balanced t(6;10).
Chromosomes were stained with DAPI. (E) Representative G-banded
chromosomes from P175 and R175A (R175) showing the normal chromosomes 6
and 10 in P175 and the t(6;10) in R175A. (F) A schematic
representation of the plasmid insertion sites in P175 and the
Cre-dependent t(6;10) generated in R175. A similar analysis of an
independent R175 pool (R175B) indicated that the same balanced t(6;10)
was generated (not shown).
(Click image to enlarge)
DMC on the Cre-dependent t(6;10) in R175. (A) FISH using a
chromosome 6 WCP as probe on a mitotic spread from R175A. The arrow
marks a chromosome with DMC that partially hybridizes to the WCP. (B)
FISH using a chromosome 10 WCP as probe on a mitotic spread from R175A.
The arrow marks a chromosome with DMC that partially hybridized to the
WCP. The inset shows the DAPI image of the delayed chromosome. (C, D)
FISH, using both chromosome 6 (red) and 10 (green) WCPs, on a mitotic
spread from R175B. The arrows mark a chromosome with DMC that
hybridizes to both WCPs. (E, F) FISH, using chromosome 6-
(red) and 10- (green) specific centromeric probes, on mitotic spreads
from R175B. Arrows mark chromosomes with DMC. A similar analysis on
R175A indicated that the chromosome 10, not the chromosome 6
derivative, displayed DMC (not shown). The chromosomes were stained
(Click image to enlarge)
Cytogenetic analysis of P186 and R186. (A) FISH, using the Aprt
plasmid (P) cassettes, and R-banding (R) on chromosomes from P186.
Hybridization was detected at 3q29, 13q14 and 11p15. (B)
G-banding of the chromosomes involved in the Cre-dependent t(3;13)
present in R186A. A similar analysis of R186B indicated that the same
t(3;13) was generated (not shown). (C) A schematic representation of the Cre-dependent t(3;13) generated in R186. (D, E)
DMC of the Cre-dependent t(3;13) in R186A. (D) A representative mitotic
spread from R186A. (E) FISH using a chromosome 3 WCP as probe on the
mitotic spread from panel D. The arrows mark chromosomes with DMC that
hybridize to the chromosome 3 WCP, the asterisks (*) mark
non-rearranged chromosome 3s, the arrowhead marks the pre-existing
t(3;11) (Supplementary Material, Fig. S1), the pluses (+) mark
unexpected translocations involving chromosome 3 (see below) and the
double arrows mark the chromosome 13 derivative from the t(3;13). (F, G)
FISH, using chromosome 3- (green) and chromosome 13- (red) specific
centromeric probes, on two mitotic spreads from R186A. Arrows mark
chromosomes with DMC that hybridize to either the chromosome 3 (F) or
chromosome 13 (G) centromeric probes. Chromosomes were stained with
(Click image to enlarge)
Engineered chromosomes with DMC also have DRT. (A) A schematic
representation of the replication timing assay. The average duration of
the G2 phase for R175B was 4–5 h (not shown). R175B cells were pulsed
with BrdU for 15 min and mitotic harvests were prepared at 3-, 4- and
5-h time points. (B–D) FISH, using a chromosome
10-specific centromeric probe (red) combined with immunostaining with
an anti-BrdU antibody (green), on a mitotic spread from R175B pulsed
4 h earlier with BrdU. Note that all of the normally condensed
chromosomes showed no BrdU incorporation and that the chromosome 10
derivative (arrow) showed BrdU incorporation along the entire length of
(Click image to enlarge)
Cytogenetic analysis of RS175-1. (A, B) The t(6;10)
generated from P175 following expression of I-Sce1 is cytogenetically
identical to t(6;10) generated following expression of Cre. FISH, using
chromosome 6- (red) and 10- (green) specific WCPs, on a mitotic spread
harvested with a colcemid pre-treatment step from RS175-1 (B).
R-banding of mitotic spreads following the WCPs indicated the t(6;10)
was indistinguishable from the t(6;10)(q12–13;q11.2) generated in R175A
and R175B. Chromosomes were stained with DAPI, and arrows mark the
t(6;10). (C) A schematic representation of the t(6;10) generated following expression of I-Sce1 in P175. (D)
DMC on the chromosome 10 derivative in RS175-1. FISH, using chromosome
6- (red) and 10- (green) specific centromeric probes, on a mitotic
spread prepared in the absence of a colcemid pre-treatment step from
RS175-1. The arrow marks a chromosome with DMC that hybridized to the
chromosome 10 centromeric probe.
(Click image to enlarge)
Chromosomes with DRT/DMC are unstable. Examples of FISH using WCPs as
probes on cells from independent clones either before (P244F and P268F)
or after Cre (R244F and R268F). All mitotic spreads were prepared in
the presence of a colcemid pre-treatment step. (A) P244 was transfected with an Aprt-expression
vector, selected in AA media, and individual clones (P244F1–5) were
isolated and expanded through 20–22 cell doublings. Mitotic spreads
were hybridized simultaneously to chromosome 13 (red) and 16 (green)
WCPs. (B) P244 was transfected with a Cre-expression vector,
selected in AA containing media, and individual clones (R244F1–5) were
isolated and expanded through 20–22 cell doublings. Mitotic spreads
were hybridized simultaneously with chromosome 13 (red) and 16 (green)
WCPs. (C) P268 was transfected with an Aprt-expression
vector, selected in AA media, and individual clones (P268F1–4) were
isolated and expanded through 20–22 cell doublings. Mitotic spreads
were hybridized simultaneously to chromosome 15 (green) and 16 (red)
WCPs. (D) P268 was transfected with a Cre-expression vector,
selected in AA media, and individual clones (R268F1–5) were isolated
and expanded through 20–22 cell doublings. Mitotic spreads were
hybridized simultaneously to chromosome 15 (red) and 16 (green) WCPs.
One hundred mitotic spreads were analyzed for each clone. Each panel
shows representative FISH+ chromosomes from each clone.
(Click image to enlarge)