Human Molecular Genetics Advance Access originally published online on August 22, 2005
Human Molecular Genetics 2005 14(19):2813-2827; doi:10.1093/hmg/ddi314
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Engineering translocations with delayed replication: evidence for cis control of chromosome replication timing
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: thayerm{at}ohsu.edu
Received June 13, 2005; Accepted August 10, 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Certain chromosome rearrangements, found in cancer cells or in cells exposed to ionizing radiation, exhibit a chromosome-wide delay in replication timing (DRT) that is associated with a delay in mitotic chromosome condensation (DMC). We have developed a chromosome engineering strategy that allows the generation of chromosomes with this DRT/DMC phenotype. We found that
10% of inter-chromosomal translocations induced by two distinct mechanisms, site-specific recombination mediated by Cre or non-homologous end joining of DNA double-strand breaks induced by I-Sce1, result in DRT/DMC. Furthermore, on certain balanced translocations only one of the derivative chromosomes displays the phenotype. Finally, we show that the engineered DRT/DMC chromosomes acquire gross chromosomal rearrangements at an increased rate when compared with non-DRT/DMC chromosomes. These results indicate that the DRT/DMC phenotype is not the result of a stochastic process that could occur at any translocation breakpoint or as an epigenetic response to chromosome damage. Instead, our data indicate that the replication timing of certain derivative chromosomes is regulated by a cis-acting mechanism that delays both initiation and completion of DNA synthesis along the entire length of the chromosome. Because chromosomes with DRT/DMC are common in tumor cells and in cells exposed to ionizing radiation, we propose that DRT/DMC represents a common mechanism responsible for the genomic instability found in cancer cells and for the persistent chromosomal instability associated with cells exposed to ionizing radiation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Genetic changes occur in virtually all types of cancers. The extent of these changes can range from single nucleotide alterations to loss, duplication or rearrangement of multiple chromosomes. Chromosome rearrangements represent one of the more common types of genetic alteration and are found in nearly every type of cancer. Recent surveys describe more than 2000 recurrent chromosomal aberrations present in many different types of cancer cells (1
,2). In addition to these recurrent chromosomal aberrations, many cancers also contain numerous other non-recurrent chromosome rearrangements; more than 100 000 independent aberrations have been described (3). These non-recurrent changes are seen as simple unbalanced rearrangements, including deletions, insertions, inversions and translocations, or as complex rearrangements involving multiple chromosomes, i.e. marker chromosomes. In addition, gene amplifications, in the form of double minutes or homogeneously staining regions, are also common in tumor cell karyotypes. Unfortunately, the relationship between oncogenesis and the majority of these chromosomal aberrations is currently unknown. Furthermore, the continuously evolving karyotypes of cancer cells in vitro and in vivo suggest that an underlying genetic instability is present and is responsible for these ongoing chromosomal changes (4).
In addition to structural alterations of chromosomes, morphological differences between chromosomes within the same cell have also been observed in tumor-derived cells. Some of these morphological alterations have been referred to as ‘incomplete condensation’ or ‘pulverization’ of chromosomes or regions of chromosomes during mitosis (reviewed in 5). Furthermore, these abnormally condensed chromosomes synthesize DNA after the normally condensed chromosomes have ceased replication (6,7). However, the nature of the chromosome abnormalities associated with these morphological changes and the molecular basis for the apparent replication asynchrony between chromosomes were not determined in these earlier studies. More recently, we characterized an abnormal chromosomal phenotype that was associated with certain tumor-derived chromosome alterations (8). We found that four different chromosome rearrangements displayed a significant delay in replication timing (DRT) of the entire chromosome. This DRT phenotype is characterized by a 2–3-h delay in both the initiation and the completion of DNA synthesis along the entire length of the chromosome, whereas the other chromosomes within the same cell show normal patterns of DNA synthesis. Chromosomes with the DRT phenotype also display a significant delay in mitotic chromosome condensation (DMC) that is characterized by an under-condensed appearance during mitosis. This under-condensed appearance is accompanied by a delay in the mitosis-specific phosphorylation of histone H3 on serine 10. These observations suggested that certain tumor-derived chromosome rearrangements display this abnormal chromosomal phenotype. Importantly, chromosomes with DRT/DMC were present in five of seven tumor-derived cell lines and five of 13 primary tumor samples, indicating that chromosomes with this phenotype are common in tumor cells in vitro and in vivo (8).
More recently, we found that exposing cell lines, primary blood lymphocytes or mice to ionizing radiation (IR) resulted in the generation of chromosomes with DRT/DMC in as many as 25% of surviving cells (9). Furthermore, we found that DRT/DMC occurred predominantly on inter-chromosomal translocations (ICTs) and that 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-rearranged chromosomes, suggesting that the DRT/DMC phenotype only occurs following specific chromosomal exchanges (9). Unfortunately, the exact nature of the IR-induced chromosome alterations that resulted in the DRT/DMC phenotype could not be determined, especially given the propensity of DRT/DMC chromosomes to undergo secondary rearrangements (8,9). Furthermore, the possibility that the DRT/DMC phenotype was the result of a stochastic process that could potentially occur at any translocation breakpoint could not be ruled out (9). Therefore, to generate a methodology that would allow for the systematic analysis of the DRT/DMC phenotype, we have developed a ‘chromosome engineering’ system that allows us to: (1) target the genome in a random fashion, (2) create reciprocal chromosome translocations, (3) generate the same translocations in multiple independent events using two distinct mechanisms and (4) characterize the chromosomes both before and after the translocation events. A preliminary analysis of 10 chromosome rearrangements generated using this approach identified a single balanced translocation with the DMC phenotype (9).
In this article, we show the analysis of 83 independent cell lines that contain chromosome rearrangements generated using this Cre/loxP system. We found that 10% of chromosome translocations generated using this system display DRT/DMC. We also found that two distinct mechanisms for generating the same translocations (site-specific homologous recombination mediated by Cre or non-homologous end joining of DNA double-strand breaks induced by I-Sce1) give rise to DRT/DMC, suggesting that DRT/DMC is not the consequence of a specific DNA repair process. Furthermore, on certain balanced translocations that display DRT/DMC, only one of the derivative chromosomes displays the phenotype. These observations indicate that the replication timing of certain chromosome translocations is regulated in cis by a mechanism that results in delayed replication along the entire length of the chromosome. Finally, we show that cells containing engineered chromosomes with DRT/DMC acquire new chromosomal rearrangements at an increased rate and therefore display genomic instability.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Chromosome engineering strategy
The chromosome engineering strategy that we employed uses the Cre/loxP site-specific recombinase system to generate ICTs (for a recent review, see 10
). Because the Cre/loxP system is relatively inefficient at generating ICTs (<1x10–3), we used reconstitution of a selectable marker to isolate the cells that had undergone Cre-mediated recombination. A schematic representation of this strategy is shown in Figure 1 (9). Briefly, this strategy involves reconstitution of a cloned murine Aprt gene in a human cell line, HTD114, which is deficient for APRT (11). This strategy involves the generation of a collection of parental cell clones (P-clones), each containing two independently inserted plasmid cassettes. One cassette contains the 5' portion of the Aprt gene linked to a neomycin resistance gene (5'-AP-Neo) and the other cassette contains the 3' portion of the Aprt gene linked to a hygromycin resistance gene (Hyg-3'RT). Each cassette contains loxP sites in the second intron of the Aprt gene. Thus, following Cre-mediated homologous recombination the Aprt gene is reconstituted at the loxP sites and a reciprocal translocation is generated. Thus, following selection in media containing azaserine and adenine (AA), Aprt+ cells can be isolated. In addition, work from Dr M. Jasin's laboratory has indicated that chromosome translocations 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 the translocations, we also introduced I-Sce1 recognition sequences into the second intron of the Aprt gene in the 5' and 3' cassettes (Fig. 1). Thus, this system allows the generation of chromosome translocations at the same translocation breakpoints in multiple independent events using two distinct mechanisms: (1) homologous recombination mediated by Cre and (2) non-homologous end joining of DSBs generated by I-Sce1.
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Screen for DMC
We isolated 97 P-clones that contained independent random insertions of the two Aprt cassettes. Subsequently, each P-clone was transiently transfected with a Cre-expression plasmid and cultured in media that 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 efficiency of Cre-mediated recombination for 33 different P-clones. In these clones, Aprt+ colonies were generated at frequencies between 1x10–2 and 1x10–6 per transfected cell (Supplementary Material, Table S1). The Aprt+ colonies were pooled from each P-clone to generate 83 independent recombinant pools (R-pools). We pooled the colonies for three reasons. First, every cell from a given P-clone is expected to generate the same translocation (or limited number of translocations depending on the number of plasmid cassette insertions) following any Cre-mediated event, and assaying multiple clones from 83 different P-clones would be prohibitive. Second, if DRT/DMC occurs in a stochastic manner and could occur at any translocation breakpoint, pooling the colonies would allow us to detect chromosomes with DRT/DMC in every pool at similar frequencies. Third, chromosomes that display DRT/DMC are unstable (8
) and pooling the Aprt+ colonies would allow for fewer generations prior to karyotypic analysis.
One critical aspect in the analysis of chromosomes with DMC is that a 2–3-h pre-treatment of the cultures with colcemid prior to mitotic harvest interferes with our ability to detect the under-condensed phenotype (8). Therefore, to determine whether any of the R-pools contained chromosomes with DMC, each R-pool was harvested for mitotic cells in the absence of colcemid. Mitotic spreads from each R-pool were analyzed for the presence of chromosomes with DMC. We scored a cell as DMC-positive if it contained one or more chromosomes with at least two of the following characteristics: (1) at least twice as long as any other chromosome within the same spread, (2) less than half as wide as any other chromosome within the same spread and/or (3) contained a bend of greater than 180° as previously described (8,9). Note that the parental HTD114 cells contain three 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-pools displayed a relatively low frequency of DMC, ranging from 0 to 4% (Table 1). In contrast, 13 pools contained chromosomes with DMC in greater than 5% of mitotic cells, ranging from 6 to 50%. This observation indicated that DMC did not occur in all R-pools with similar frequencies, suggesting that only certain Cre-mediated chromosome rearrangements resulted in DMC. Examples of mitotic spreads containing chromosomes with DMC from this initial screen are shown in Figure 2.
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Because there is a low but detectable frequency of DMC in the parental HTD114 cells, each engineered translocation had to be 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-clones and their respective R-pools that contained DMC in >6% of the mitotic spreads, as well as on four P-clones and their respective R-pools that showed a low frequency of DMC. This initial karyotypic analysis was carried out in the presence of colcemid to facilitate the characterization of the chromosomes involved. Initially, we used fluorescence in situ hybridization (FISH), with the Aprt plasmid cassettes as probes, followed by R-banding to identify the sites of insertion. We next used whole chromosome paints (WCPs) to confirm the insertion sites in the P-clones and to visualize the Cre-dependent translocations in the R-pools. Finally, the chromosome translocations present in the R-pools were confirmed using G-banding. An example of this analysis is shown for P175 and its Aprt+ pool R175 in Figure 3. P175 contains plasmid cassette insertions in 6q14–15 and 10q11.2 (Fig. 3A and B). As expected, R175 contains a new translocation involving chromosomes 6 and 10 (Fig. 3C–E). The breakpoints for this new translocation are at the plasmid cassette insertion sites and generated a balanced translocation, t(6;10)(q14–15;q11.2). A schematic view of the plasmid cassette insertions and the t(6;10) is shown in Figure 3F. A summary of this karyotypic analysis for all 13 clones is shown in Table 1. In addition, we have characterized the plasmid cassette insertions in this set of P-lines and in their respective R-pools for copy number and for reconstitution of the Aprt cassettes using southern blot hybridizations (Supplementary Material, Fig. S2).
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One of the advantages of this approach is that the same translocations can be generated in multiple, independent events. Therefore, to determine if a particular translocation displayed DMC following independent Cre events, additional R-pools were generated and analyzed for DMC in mitotic spreads prepared in the absence of colcemid. We analyzed two independently derived pools of R175 (R175A and R175B) using FISH with chromosome 6 and 10 WCPs. Figure 4A–D shows examples of this analysis and indicated that chromosomes with DMC hybridized to both the chromosome 6 and 10 probes in both isolates of R175. Analysis of P268 and two Aprt-expressing pools, R268A and R268B, indicated that a balanced translocation involving the long arm of chromosome 15 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 independently derived pools of R175 and R268, and it occurred on the Cre-dependent t(6;10) and t(15;16), respectively. A similar analysis of R27, R186 and R276 showed that DMC also occurred on Cre-dependent balanced translocations (Table 1). In contrast, DMC was not detected on balanced translocations generated by Cre in two independent pools each of R38, R161, R244, R248 or R263, with over 200 metaphase spreads analyzed from each pool (Table 1, Supplementary Material, Fig. S4, data not shown). These results indicate that DMC was detected in independent isolates of certain Cre-dependent chromosome translocations and not in others, indicating that this abnormal chromosomal phenotype is not the consequence of a stochastic process that could occur at any translocation breakpoint.
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Only one or both derivative chromosomes display DMC
Because the DMC phenotype occurred on balanced translocations and every balanced translocation generates two derivative chromosomes, we next determined whether one or both derivative chromosomes displayed the DMC phenotype. To address this question, we analyzed mitotic spreads for chromosomes with DMC using FISH with chromosome-specific centromeric probes. Figure 4E and F shows two examples of this analysis for R175 and indicated that the chromosomes with DMC hybridized to the chromosome 10 centromeric probe and not to the chromosome 6 centromeric probe. In total, we detected 10 DMC chromosomes that hybridized to the chromosome 10 probe and no DMC chromosome that hybridized to the chromosome 6 probe. A similar analysis on R268 indicated that the chromosome 15 derivative, not the chromosome 16 derivative, displayed DMC (Supplementary Material, Fig. S3). Analysis of a third translocation with DMC, a t(3;16)(p13;p13.3) present in R27, also indicated that only one product, the 16 derivative, displayed DMC (data not shown). Although it would be impossible to prove that any given chromosome ‘never’ displays DMC, these observations suggest that only one of the products from these 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 cassette insertion, 3(q29), 11(p15) and 13(q14). However, the only new translocation detected in two independent R186 pools was a t(3;13)(q29;q14), indicating that the 11(p15) insertion site did not participate in the Cre events that generated the Aprt+ cells in the R186 pools. The reason that the 11p cassette has not been detected in a translocation in R186 is unknown, but it may contain a deleted or rearranged cassette that prevents reconstitution of a functional Aprt gene, or perhaps the frequency at which the 11p15 cassette can participate in a Cre event with the other cassette may simply be much lower than the frequency for the other insertion sites (Supplementary Material, Table S1). Regardless, using FISH with chromosome 3 and 13 centromeric probes, we detected DMC on both derivative chromosomes from t(3;13) (Fig. 5F and G). These observations indicate that either one or both of the products from certain balanced translocations display the DMC phenotype.
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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 with DMC also display DRT, we analyzed replication timing of the chromosomes with DMC using a BrdU incorporation assay combined with FISH using chromosome-specific centromeric probes. Figure 6A shows a schematic illustration of this replication timing assay. Analysis of mitotic spreads from R175B harvested for late replication indicated that the chromosomes with DMC hybridized to the chromosome 10 centromeric probe and incorporated BrdU along their length at a time when the fully condensed chromosomes did not show any detectable BrdU incorporation. An example of this analysis is shown in Figure 6B–D. This analysis indicates that the derivative chromosome 10 present in R175 displays both DRT and DMC.
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Plasmid insertions and small intra-chromosomal deletions are not sufficient to induce DRT/DMC
The results described earlier indicate that DRT/DMC occurs on balanced translocations induced by Cre-mediated recombination, and that one or both of the derivative chromosomes display the phenotype. These observations suggest that DRT/DMC occurs following inter-chromosomal exchanges at specific chromosome locations. However, it was also possible that simple plasmid cassette insertion or Cre-mediated recombination at these specific chromosome locations was responsible for inducing DRT/DMC. Therefore, to determine whether DRT/DMC was simply the result of plasmid cassette insertion, we analyzed mitotic spreads for DMC in P-clones that generated DMC at a high frequency in their R-pools. None of the P-clones assayed displayed DMC in greater than 4% of the spreads (Table 1). In addition, P175 and P27 were assayed for DMC combined with FISH with centromeric probes. This analysis indicated that P175 and P27 did not retain DMC chromosomes that hybridized to the chromosome 6 and 10, or 3 and 16 centromeric probes, respectively (Table 2). Therefore, simple plasmid cassette insertion at these chromosomal locations is not sufficient to induce DRT/DMC.
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Next, to determine whether DRT/DMC was simply the consequence of Cre-mediated recombination at these specific chromosomal locations, 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-chromosomal Cre events are markedly more efficient than inter-chromosomal Cre events (13
). Thus, the design of this set of experiments was to transiently transfect P-clones with a Cre-expression vector and isolate clones that experienced Cre-mediated intra-chromosomal deletion of the Neo and Hyg genes but did not generate ICTs. To aid in the isolation of Cre-transfected cells, we co-transfected a GFP-expression vector and isolated the GFP-positive cells by fluorescence-activated cell sorting (FACS). The GFP-positive cells, and presumably Cre-positive cells, were plated at clonal densities, allowed to form colonies in the absence of Aprt selection and isolated as individual clones. For this analysis, we chose two clones, P175 and P27, that generated DRT/DMC on Cre-dependent ICTs. Deletion of the Neo and Hyg genes occurred in >90% of these clones as assayed by southern blot hybridization (Supplementary Material, Fig. S2C), and the absence of the translocations was confirmed by southern blot hybridization for reconstitution of the Aprt gene and FISH with WCPs (data not shown). This set of experiments resulted in the generation of cell lines that experienced Cre-mediated intra-chromosomal deletion of the Neo and Hyg genes at the same chromosomal locations involved in the translocation events produced in R175 and R27. The clones with the Neo and Hyg genes removed were designated P175
NH and P27NH, respectively. Three independent P175NH clones were assayed for DMC after hybridization with chromosome 6 and 10 centromeric probes and two independent P27NH lines were assayed for DMC after hybridization with chromosome 3 and 16 centromeric probes. Chromosomes with DMC were not detected in any of the P175NH or P27NH clones with any of the centromeric probes (Table 2). Taken together, these observations indicate that insertion of the plasmid cassettes and Cre-mediated intra-chromosomal deletions at these specific chromosome locations is not sufficient to induce DRT/DMC and suggest that DRT/DMC is the result of inter-chromosomal exchanges at specific chromosomal locations. However, it is important to point out that this analysis does not rule out the possibility that other intra-chromosomal deletions or rearrangements could result in DRT/DMC.
DRT/DMC occurs on translocations
The results described earlier indicate that DRT/DMC can occur following Cre-mediated site-specific recombination, but only on specific translocation derivatives. To determine if a different mechanism for generating the translocations could produce DMC on one of these derivatives, we took advantage of the fact that our 5' and 3' cassettes have I-Sce1 sites in the second intron of the Aprt gene (Fig. 1). We transiently transfected P175 cells with an I-Sce1-expression vector and selected for Aprt-expressing cells. Because DSBs induced by I-Sce1 are subject to exonuclease activity prior to NHEJ and could potentially result in translocations with different breakpoints (12), we isolated Aprt+ clones, named the RS175 series. Karyotypic analysis of RS175-1 indicated that the expected balanced translocation, a t(6;10)(q12–13;q11.2), was generated (Fig. 7A–C). Although we have not sequenced the breakpoints in RS175-1, t(6;10) is cytogenetically identical to the translocation produced by Cre in the R175 pools. In addition, southern blot analysis indicated that the Aprt gene was reconstituted in the second intron, and the Neo and Hyg genes were still intact (Fig. 1; data not shown). Therefore, the translocation breakpoints in RS175-1 are located within the plasmid cassettes, indicating that the DSBs induced by I-Sce1 did not experience significant exonuclease digestion prior to the generation of the t(6;10) in this clone. To determine if DMC was occurring on the chromosome 10 derivative, as it is in R175, we analyzed mitotic spreads from RS175-1 using FISH with the chromosome 6 and 10 centromeric probes. An example of this analysis shown in Figure 7D indicated that the chromosome 10 derivative, not the chromosome 6 derivative, displayed DMC. A similar analysis on I-Sce1-induced translocations from P244 and P38, which did not generate DMC following Cre, showed undetectable levels of DMC 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 generate chromosomes with DRT/DMC.
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Chromosomes with DRT/DMC are unstable
We previously found that chromosomes with DRT/DMC participate in frequent secondary rearrangements and translocations (8
). During characterization of the R-pools described here, we also observed unexpected rearrangements involving the chromosomes with DRT/DMC (Fig. 5 and Supplementary Material, Fig. S3). To further characterize these secondary rearrangements and to compare the stability of DRT/DMC chromosomes to non-DRT/DMC chromosomes, clones containing Cre-induced translocations were isolated 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 different P-clones: three that generated DRT/DMC following Cre-mediated recombination (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 of the chromosomes prior to the generation of the Cre-induced translocations, the P-clones were transfected with an intact Aprt-expression vector and stable Aprt-expressing clones isolated; these clones were named the P175F, P186F, P268F and P244F series. Analysis of these Aprt-transfected clones would allow us to determine the stability of the chromosomes prior to creation of the translocations and to control for any variability in chromosome stability between different P-clones subjected to transfection, Aprt selection and clonal expansion in AA media. Because the cells constituting each clone were cultured through a similar number of generations (20–22) and were grown under identical conditions, the stability of the chromosomes could be directly compared both before and after the translocation events and between DRT/DMC and non-DRT/DMC containing clones. In addition, we analyzed a series of P268F clones that had experienced Cre transient transfection, but did not generate the translocation. These clones, designated P268NH, were generated by co-transfection with Cre and GFP-expression vectors, FACS for GFP-expressing cells, expansion in the absence of selection and screening for deletion of the Hyg gene by southern blot hybridization (data not shown). These clones were cultured through a similar number of generations as the other clones (20–22) and served as an additional control for non-specific affects of Cre transient transfection. Mitotic spreads from each clone were analyzed using FISH with WCPs that hybridized to the Cre-dependent translocations, and 100 mitotic spreads were scored for each clone with each WCP. This set of experiments allowed us to determine the frequency at which unexpected gross chromosomal rearrangements (GCRs: translocations, deletions, insertions and rearrangements) occurred on Cre-dependent translocations, with or without DRT/DMC.
These cell lines could clearly be segregated into two groups based on the frequency of new GCRs. All the control Aprt-transfected clones (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 frequencies of new GCRs involving the appropriate chromosomes. For example, we did not detect any GCRs involving chromosomes 15 and 16 in the P268F or P268NH clones, and we did not detect any GCRs involving chromosomes 13 and 16 in the P244F clones (Table 3). In addition, we detected only one GCR involving chromosome 13 in one cell from one R244F clone (Table 3 and Supplementary Material, Fig. S5). In contrast, the DRT/DMC containing clones showed dramatically higher frequencies of GCRs involving the DRT/DMC chromosomes. For example, every R268F clone contained GCRs that hybridized to the chromosome 15 and/or 16 WCPs, and cells with GCRs were also 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 many of these GCRs represented translocations to other, and presumably non-DRT/DMC chromosomes (Fig. 8). Interestingly, and consistent with our observation that only the chromosome 15 derivative displayed DRT/DMC in the R268 pools (Supplementary Material, Fig. S3), the chromosome 16 derivative was still present and 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. In addition, to determine whether any of the Cre-treated clones had integrated the Cre-expression vector, which could potentially contribute to new GCRs via Cre-mediated recombination between endogenous or cryptic loxP sites (14,15), we carried out southern blot hybridizations on genomic DNA extracted from all of the Cre-treated clones shown in Table 3. This analysis indicated that none of these clones retained the Cre-expression vector (data not shown).
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Finally, using a fluctuation analysis (16
), as modified by Lin et al. (17) to allow for estimating high mutation rates, we estimate that the rate at which cells acquired new GCRs involving the DRT/DMC chromosomes was 7x10–1 (or one new GCR every 14 cell divisions) for the t(6;10) in R175, 1.6x10–1 (or one new GCR every six cell divisions) for the t(3;13) in R186 and 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 likely to be an underestimate of the rate for GCR formation involving these translocations, as many of the R-clones retained GCRs in a large fraction of the cells, ranging from 2 to 100% of the cells (Table 3). In contrast, we estimate that the rate at which cells acquired new GCRs involving the appropriate non-rearranged chromosomes (i.e. in P175F, P186F, P244F and P268F clones), non-DRT/DMC translocations (i.e. the t(13;16) in R244F) and in 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 an 30–80-fold increase in the rate at which new GCRs occur on the affected chromosomes. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Chromosomes with DRT/DMC were detected in five of seven tumor cell lines and five of 13 primary tumor samples, suggesting that chromosomes with DRT/DMC are common in tumor cells in vitro and in vivo (8
). In addition, following exposure to IR, chromosomes with DRT/DMC were detected in as many as 25% of surviving cells, indicating that DRT/DMC is also quite common in cells exposed to IR (9). Furthermore, IR-induced chromosomes with DRT/DMC in primary cells, SV40 immortalized cells and tumor-derived cells at similar frequencies, indicating that the DRT/DMC phenotype is not restricted to cells with unregulated cell cycles or defective cell-cycle checkpoints (9). Here, we show that 10% of random chromosome translocations generated using a chromosome engineering strategy display the DRT/DMC phenotype. These results are consistent with our previous observations that 5% of chromosome translocations induced by IR display DRT/DMC (9). In addition, we found that two distinct methods for generating the same translocations, site-specific homologous recombination or NHEJ, give rise to DRT/DMC, but only on certain derivative chromosomes. Moreover, the majority of chromosome translocations do not display DRT/DMC, indicating that DRT/DMC is not the result of a stochastic process that could occur at any translocation breakpoint. Taken together, our data suggest that 5–10% of all chromosome translocations induced by different mechanisms, site-specific recombination, NHEJ or exposure to IR display DRT/DMC.
Genetic instability
Genetic instability can occur at distinct levels. In most cancers, the instability occurs at the chromosome level, resulting in gains or losses of whole chromosomes (4). This type of instability is a dominant trait and is independent of p53 mutations (18). Another common form of genetic instability found in cancer cells is 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 of intact chromosomes as chromosome instability (CIN) (4) and to the process that results in the generation of frequent translocations and rearrangements as translocation instability (TIN). Unfortunately, the molecular mechanisms responsible for either CIN or TIN in tumor cells are still poorly understood (4,19).
Another example of genetic instability found in mammalian cells occurs at a delayed time following exposure to IR (20,21). This delayed or persistent chromosomal instability occurs in vitro and in vivo and is characterized by the appearance of new chromosome translocations and rearrangements in subsequent generations after the initial exposure to IR (22). Interestingly, many aspects of this delayed instability cannot be explained by simple mutational inactivation of trans-acting factors. For example, sub-cloning experiments of irradiated cells indicate that this type of genomic instability is unevenly transmitted to sibling sub-clones and that the chromosomal rearrangements that occur within the unstable clones are non-randomly distributed throughout the karyotype (23,24). So, alternative epigenetic and/or cis-acting mechanisms have been proposed to explain this poorly understood process (reviewed in 25,26).
Given our limited understanding of genomic instability in mammalian cell systems, it is currently not known whether the delayed chromosomal instability observed in irradiated cells and the TIN associated with cancer cells are caused by similar or distinct mechanisms. However, we previously showed that chromosomes with DRT/DMC are common in tumor cells in vitro and in vivo (8). In addition, we found that chromosomes with DRT/DMC were present in as many as 25% of cells exposed to IR (9). In this study, we found that chromosomes with DRT/DMC have a 30–80-fold increase in the rate at which new GCRs occur on the affected chromosomes. Therefore, because chromosomes with DRT/DMC are common in tumor cells and in cells exposed to IR, we propose that chromosomes with DRT/DMC represent a common source of the genomic instability observed in cancer cells (i.e. TIN) and in cells exposed to IR (i.e. delayed chromosomal instability). In addition, because the DRT/DMC phenotype occurs only on certain derivative chromosomes, our data provide support for previous models 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 in the DRT/DMC phenotype? This is an intriguing question given that the translocation breakpoints that induced DRT/DMC in this study occurred within the plasmid cassette sequences and not within the chromosomal DNA. In addition, it appears that inter-chromosomal exchanges are required for the phenotype, as plasmid cassette insertions and small Cre-mediated intra-chromosomal deletions at the same chromosome locations did not result in DRT/DMC. Furthermore, on certain balanced translocations, only one of the derivative chromosomes displayed the phenotype. Taken together, these observations indicate that the DRT/DMC phenotype is regulated by a cis-acting mechanism that occurs following specific chromosomal exchange. However, it is important to point out that intra-chromosomal rearrangements, involving chromosomal DNA instead of plasmid DNA, may be capable of generating DRT/DMC. Regardless, we are considering two possibilities to explain how specific chromosomal exchanges can give rise to the DRT/DMC phenotype. First, it is possible that these chromosomal exchanges result in deletion or mutation of a cis element that normally establishes early replication timing for the entire chromosome. Loss of this element would then result in delayed replication of the entire chromosome. Second, it is possible that these specific chromosomal exchanges generate dominant interfering elements that act in cis to delay normal chromosome replication timing by some unknown mechanism. Although we cannot distinguish between these possibilities at the present time, the chromosome engineering strategy described here, combined with ‘mixing and matching’ of loxP-tagged chromosomes and directing loxP sites to specific chromosome locations, should provide for a system in which the molecular mechanisms responsible for the DRT/DMC phenotype can be elucidated.
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MATERIALS AND METHODS |
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Cell culture
HTD114 cells are a human APRT null cell line derived from the HT1080 fibrosarcoma cell line (11
). This cell line was grown in DMEM (Gibco) supplemented with 10% bovine calf serum (Hyclone). HTD114 P-line derivatives were grown as above with the addition of 500 µg/ml Geneticin (Gibco) and 200 µg/ml Hygromycin B (Calbiochem). HTD114 R-line derivatives were grown in DMEM supplemented with 10% dialyzed fetal bovine serum, 10 µg/ml azaserine (Sigma) and 10 µg/ml adenine (Sigma) to facilitate selection for Aprt-expressing cells. Cells were grown in a humidified incubator at 37°C in a 5% carbon dioxide atmosphere.
Mitotic harvests
Cells were exposed to 10 ng/ml of colcemid (Sigma) for 1 h. Trypsinized cells were centrifuged at 1000 rpm (300 g) for 10 min in a swinging bucket rotor. The cell pellet was resuspended in 75 mM potassium chloride for 15 min at 37°C, recentrifuged at 1000 rpm for 10 min and fixed in 3 : 1 methanol : acetic acid. Fixed cells were added drop-wise to microscope slides to make mitotic chromosome spreads using standard methods (27).
Fluorescence in situ hybridization
Slides with mitotic spreads were baked at 85°C for 20 min and then treated with 0.1 mg/ml RNase for 1 h at 37°C. After RNase treatment, the slides were washed in 2x SSC (1x SSC is 150 mM NaCl and 15 mM sodium citrate) with three changes for 3 min each and dehydrated in 70, 90 and 100% ethanol for 3 min each. The chromosomes were denatured in 70% formamide in 2x SSC at 70°C for 3 min and WCPs were used according to the manufacturer's recommendations (American Laboratory Technologies and Vysis). Detection of digoxigenin-dUTP probes used a three-step incubation of slides with sheep FITC-conjugated anti-digoxigenin antibodies (Roche) followed by rabbit FITC-conjugated anti-sheep antibodies (Roche) followed by goat FITC-conjugated anti-rabbit antibodies (Jackson Laboratories). Slides were stained with DAPI (12.5 µg/ml) or propidium iodide (0.3 µg/ml), cover slipped and viewed under UV fluorescence with FITC filters (Zeiss).
Centromeric probes
Mitotic chromosome spreads were prepared as described in the previous section. Slides were treated with RNase at 100 µg/ml for 1 h at 37°C and washed in 2x SSC and dehydrated in 70, 90 and 100% ethanol. Chromosomes were denatured at 75°C for 3 min in 70% formamaide/2x SSC, followed by dehydration in ice cold 70, 90 and 100% ethanol. Probe cocktails (Vysis) were denatured at 75°C for 10 min and pre-hybridized at 37°C for 30 min. Probes were applied to slides and incubated overnight at 37°C. Post-hybridization washes consisted of three 3-min rinses in 50% formamide/2x SSC, three 3-min rinses in 2x SSC and finally three 3-min rinses in PN buffer (0.1 M Na2HPO4+0.0 M NaH2PO4, pH 8.0, +2.5% Nonidet NP-40), all at 45°C. Slides were then counterstained with either propidium iodide (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 previously described (8). Asynchronously growing R175B cells were exposed to 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. The cells were treated with 75 mM KCl for 15 min at 37°C, fixed in 3 : 1 methanol : acetic acid and dropped on wet slides. The chromosomes were denatured in 70% formamide in 2x SSC (1x SSC is 150 mM NaCl and 15 mM sodium citrate) at 70°C for 3 min. Incorporated BrdU was detected using a FITC-labeled anti-BrdU antibody (Becton Dickinson). Slides were stained with propidium iodide (0.3 µg/ml), cover slipped and viewed under UV fluorescence (Zeiss).
Chromosome engineering
Three micrograms of the 5'-AP-Neo plasmid was linearized at a unique NotI site, electroporated (300 V, 950 µF in PBS; Bio-Rad) into HTD114 cells and grown under 500 µg/ml G418 (Geneticin, Gibco) selection for 10–14 days. G418-resistant (NeoR) colonies were then pooled and expanded. Three micrograms of 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/ml G418 and 200 µg/ml Hygromycin B selection for 10–14 days. 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 fluorescent protein (GFP) expression plasmid (pCSGFP) and either 3 µg of a Cre recombinase expression plasmid (pBS185, Gibco) or an empty vector control (pBluescript SK, Stratagene). Average transfection efficiencies were between 5 and 10%, as determined by GFP expression. Cre-transfected cells were grown for 14–21 days in 10 µg/ml azaserine and 10 µg/ml adenine (AA selection). The resulting Aprt+ colonies were pooled to generate 83 independent pools and expanded. A preliminary description of this system was described previously (9). Alternatively, individual Aprt+ clones were picked and expanded to approximately 4x106 cells and analyzed for the presence of ‘new’ rearrangements using FISH with WCPs.
Deletion of neomycin and hygromycin genes
P27 and P175 were co-transfected (Lipofectamine, Gibco) with 3 µg of a Cre-expression plasmid (pBS185) and 1 µg of a GFP-expression plasmid (pcsGFP). After 24 h, the cells were trypsinized, washed once in Hank's balanced salt solution (Gibco) and 1x106 cells were subjected to FACS with gating set to collect GFP-expressing cells. Typical transfection efficiencies before FACS, based on the number of GFP-positive cells, were between 15 and 25%. After FACS, the collected cell fractions were suspended in 1 ml of fresh Hank's balanced salt solution and 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 dishes containing DMEM supplemented with 10% fetal bovine serum. Individual colonies were picked, expanded and challenged to grow in media containing 500 µg G418 or 200 µg Hygromycin B. Clones that failed to grow in both G418 and Hygromycin B were expanded and subjected to southern analysis to confirm loss of the Neo and Hyg markers. The absence of translocations was confirmed using FISH with appropriate WCPs.
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SUPPLEMENTARY MATERIAL |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Supplementary Material is available at HMG Online.
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ACKNOWLEDGEMENTS |
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The authors wish to thank Dr M. Turker, Dr E. Epner, Dr D. Pankratz, Dr D. Stauffer, and Dr B. Chang for critically reading the manuscript. This work was supported by NIH grants CA97021 and CA104693 (M.J.T). Funding to pay the Open Access publication charges for this article was provided by NIH grant CA97021.
Conflict of Interest statement. None declared.
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