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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 9, 2005 as doi:10.1096/fj.04-2276fje. |
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* Departament de Biologia Cel·lular, Fisiologia i Immunologia and Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, Spain; and
IDIBELL-Institut de Recerca Oncològica, Hospital Duran i Reynals, L’Hospitalet, Barcelona, Spain
1 Correspondence: Laboratori de Citogenètica, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. E-mail: jordi.camps{at}uab.es
SPECIFIC AIMS
In the present study, we quantify the ongoing structural chromosome alterations in two archetypes of colorectal cancer cell lines: HCT116, and SW480 and its single subclones using the multicolor-FISH. The application of the cytokinesis-block micronucleus (CBMN) assay allowed a detailed measurement of numerical instability, to elucidate the origin of the aneuploidy and to predict the structural chromosome instability level by measuring the abnormal nuclear shape events by performing centromeric and pancentromeric FISH.
PRINCIPAL FINDINGS
1. Rates of structural chromosome instability
Several efforts have been assessed to characterize and quantify the ongoing appearance of structural chromosome instability. In the present study, multicolor-FISH and CGH were performed for both the parental cell lines, HCT116 and SW480, and its derivative subclones (S1, S2, S4, S2.3, and S2.4). To quantify the rate of structural instability of each cell line and its subclones, the maximum rate (maxr) of instability was calculated. This rate was assessed by the de novo nonclonal alterations divided by the number of cells analyzed. The parental cell lines did not display any de novo clonal chromosome rearrangements, but the subclones did show clonal alterations even though the rate was much lower than the nonclonal chromosome alterations. The SW480 subclones tended to display a maxr 3- to 5-fold higher than the parental cell line. De novo nonclonal alterations mostly appeared as unbalanced translocations and usually involved the whole arm of a chromosome with breakpoints near the centromere. Complex translocations were frequently observed. Our results suggested that the structural heterogeneity observed in subclones not only was generated by remodeling chromosome markers observed in the parental cell lines, but also involved normal chromosomes. Low differences were observed between CGH data of parental cell line and its subclones, suggesting that structural rearrangements were not fixed in the genome. Consequently, these imbalances will not appear represented in a large enough population to be detected by CGH.
To understand the generation of these chromosome rearrangements, we might propose a dynamic engine of structural chromosome instability that permits the emergence of new chromosomal alterations, preferentially unbalanced over balanced. In our case, since the rate of structural chromosome rearrangements is significantly lower in the parental SW480 cell line compared with its subclones, we suggest a mechanism that triggers a karyotypic destabilization when subclones are generated. The chromosome aberration spectrum could be stabilized through the in vitro passages in a similar way to what has been observed during the in vivo progression.
2. Distribution and quantification of aneuploidy events
Intracellular chromosome number variability was evaluated as the percent of nuclei with a number of centromere signals for chromosomes 4, 7, 15, and 17 different from the modal number according to the composite karyotype of each cell line and subclone. The differences in the numerical variability average among HCT116 and SW480 were highly significant (P<0.0001), but the numerical variabilities displayed by the sublones of the SW480 were in the same range as the parental cell line.
The CBMN assay allowed us to elucidate the origin of the aneuploidy events, either nondisjunction or chromosome loss. By using chromosome-specific centromeric probes, different patterns were observed. Nondisjunction was considered as a gain of one or more centromeric signals in one daughter cell together with the corresponding loss in the other daughter cell. On the other hand, chromosome loss was detected when a micronucleus showed at least one or more centromeric signals, whereas the main nucleus lacked one or more signals (Fig. 1
).
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The analysis of the binucleated cells confirmed the low level of aneuploidy displayed by the HCT116 cell line. The SW480 cell line and its subclones showed a 10-fold higher rate of numerical instability when compared with HCT116 (P<0.0001). Only chromosome 15 showed notable differences between nondisjunction and chromosome loss events. In this case, nondisjunction events were predominant over chromosome losses.
From the analysis of the aneuploidy rate displayed by each chromosome analyzed (4, 7, 15, and 17), we calculate the aneuploidy rate for the whole genome applying a correction factor of mn/8 to HCT116 and mn/14 to the rates of parental SW480 and each subclone, mn being the modal chromosome number, and, 8 and 14 the number of total centromeric signals observed. No differences were observed among parental SW480 cell line and its subclones (Table 1
). Nevertheless, differences in the aneuploidy rate for the chromosomes analyzed were detected and the most affected chromosome by an aneuploidy event was 15, followed by 7, 17, and 4.
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3. Abnormal nuclear shapes are a sensitive measure of chromosome rearrangements: application of the CBMN assay
The CBMN assay allowed us to score the abnormal nuclear shapes (ANS). This term includes micronuclei, nucleoplasmic bridges, and nuclear blebs (Table 2
), and it has been associated to mitotic instability. The low frequency of ANS observed in the HCT116 cell line confirmed once more the chromosomal stability of these cells. On the other hand, SW480 parental cell line and its subclones displayed a major number of abnormal nuclear shapes, especially nucleoplasmic bridges and micronuclei, whereas nuclear blebs were much less represented. Among subclones, S2 displayed, in general, the highest number of ANS. These data agree with the high rate of structural chromosome instability detected by M-FISH in this subclone. When analyzing the relative frequencies of micronuclei with 
-satellite signals for the chromosomes tested, we did not find differences between parental SW480 cell line and its subclones, except for subclone S1.
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To deeply study the ANS in the subclone S2, we hybridized with a pancentromeric probe. Results did not show differences between the frequency of micronuclei with centromere signal and micronuclei without it. Nevertheless, we noticed that most of the nuclear blebs (41 out of 43) showed centromeric signals, indicating that nuclear blebs could eliminate not only pools of fragmented DNA or gene amplifications, but also entire chromosomes, and could serve as a mechanism of chromosome loss.
CONCLUSIONS AND SIGNIFICANCE
Genomic instability is described as one of the most important features during carcinogenesis. To date, only microsatellite instability (MSI) has been well-characterized, and it has been related to mutations of the mismatch repair genes. On the other hand, chromosomal instability (CIN) refers to an increased rate of imbalances in chromosome number (aneuploidy) or acquisition of chromosome rearrangements. In most of the cases these two patterns of instability do not coexist. To understand the origin of the chromosome instability, we have performed an array of molecular cytogenetic techniques which allow us to easily detect a rate of chromosome rearrangements and aneuploidies.
Our results demonstrate the stable karyotype of the HCT116 cell line and the high structural instability in SW480 cell line. Moreover, we reveal in this study that the parental SW480 cell line shows approximately the same aneuploidy rate than its subclones, and that subclones are clearly characterized by the acquisition of multiple chromosome rearrangements as a tool to generate tumor cell heterogeneity. These data suggest that some tumor cells are submitted to a mechanism which might destabilize the karyotype. Two interpretations are possible for the observed higher structural chromosome heterogeneity in the subclones as compared with parental cells: that the instability is higher in the subclones, or that the instability is the same but the effect (heterogeneity) is restrained in parental cells due to competitivity. In either case, the biological implications are the same: clonal episodes appear to favor an increase of potential heterogeneity. Whether this structural instability might act in an in vivo situation is still to be determined.
FISH with chromosome-specific centromeric probes has been used on nuclei as a tool to measure cell-to-cell variations in chromosome number (heterogeneity). Using the CBMN assay in tumor cells, we precisely assess a rate of numerical instability, which allows the analysis of the nondisjunction and chromosome loss events. This study demonstrates that the CBMN assay, one of the most commonly methods used in mutagenesis, can provide a comprehensive measurement of chromosome breakages in tumor cells by means of the analysis of abnormal nuclear shapes.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2276fje;
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