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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 3, 2002 as doi:10.1096/fj.02-0425fje. |
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* Institut de Recerca Oncològica, Hospital Duran i Reynals, L’Hospitalet, Barcelona, Spain;
Departament de Biologia Cellular, Fisiologia i Immunologia, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain; and
Institut Català d’Oncologia, Hospital Duran i Reynals, L’Hospitalet, Barcelona, Spain
2Correspondence: Institut de Recerca Oncològica, Hospital Duran i Reynals, Granvia km 2,7, L’Hospitalet, 08907 Barcelona, Spain. E-mail: mpeinado{at}iro.es
SPECIFIC AIMS
To characterize chromosomal instability in cancer cells, we analyzed genetic clonal divergence in three colon cancer cell lines (LoVo, HCT116, and SW480) regarded as archetypes in cancer research. A dynamic setting using comprehensive analyses at chromosomal level (G banding cytogenetics, comparative genomic hybridization, and centromeric fluorescence in situ hybridization, or FISH) was designed to allow the calculation of mutation rates.
PRINCIPAL FINDINGS
1 Cytogenetic characterization of cell lines
LoVo and HCT116 are diploid cells with a modal number of 49 and 46 chromosomes, respectively. SW480 are aneuploid cells showing a very complex karyotype. Its modal chromosome number was 57 with several chromosome markers in all metaphases. Due to the heterogeneity observed among metaphases, no major subpopulations were identified. All karyotypes agreed with previous reports and the ATCC catalog.
2 Rates of chromosomal instability in cell clones
The appraisal of chromosomal instability as a mutation rate is feasible only in dynamic settings. We have only considered the de novo mutations occurring in a defined interval of time (number of cell divisions here). Conservative (minimum rate, minr) and less strict (maximum rate, maxr) estimates of chromosomal instability were calculated. The maximum rate only takes into account nonclonal alterations and assumes they took place in the last generation (maxr=number of nonclonal alterations/number of cells). This assessment is an indicator of genetic instability irrespective of the viability of the cells harboring the novel genetic alterations that may or may not be transmitted to further generations. The minr assumes the accumulation of clonal and nonclonal alterations through generations with the same viability (minr=number of alterations/number of cells/number of generations).
Regarding numerical alterations, all three cell lines showed low rates (Table 1
). On average, SW480 clones showed a 
fivefold increase in the minr (0.005±0.001) compared with the HCT116 and LoVo clones (0.001±0.002) (Mann-Whitney test, U=25, P=0.036). These differences were not observed in the maxr. At the structural level, cell lines displayed different rates of instability. LoVo clones showed a stable karyotype. In HCT116 cells, a certain degree of heterogeneity was observed in all clones, with 7–27% of the metaphases displaying novel chromosomal abnormalities, most of them unbalanced and nonclonal. The rates of chromosomal alterations in the clones of these two cell lines were of the same order for structural and numerical alterations (Table 1)
. All clones of SW480 showed novel structural rearrangements in all metaphases analyzed, resulting in rates 100-fold higher than those found in LoVo and 10- to 50-fold more than HCT116 cells (Table 1)
. Differences in the minr and maxr of the three cell lines were statistically significant (Krustal-Wallis nonparametric ANOVA test, minr KW=10.08 P<0.0001). Structural alterations largely surpassed numerical alterations in SW480 cells (8-fold in parental cells and 20- to 70-fold in their clones) (minr paired t test, P=0.002). Altogether, our observations suggest that the relative contribution of structural chromosomal instability to cell heterogeneity may be of special relevance in colorectal cancer cells.
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3 Prevalence of structural over numerical instability
CGH analysis confirmed the karyotypes obtained by G banding in HCT116 and LoVo cells. SW480 cells displayed a very complex pattern that affected most chromosome profiles. In contrast to the striking heterogeneity observed in G banding analyses, CGH revealed a consistent profile for all clones. As a whole, these results imply that chromosomal reorganizations resulting in new chromosome markers in derivative clones do not involve substantial gains or losses of DNA.
The low prevalence of numerical alterations was confirmed by centromeric FISH of interphase cells using probes for chromosomes 3, 7, and 15. Numerical chromosome instability in this assay is revealed as heterogeneity with regard to the modal chromosome number of each clone (Table 2
). Parental cells presented modal chromosome numbers in agreement with the G banding and CGH data. Since the cells were grown in chamber slides and processed in situ, it was possible to determine whether the changes observed in chromosome number corresponded to independent mutational events or were clonal and propagated. In such a case, they were considered only once because they represented a single mutational event. All clones maintained the modal chromosome number of parental cells, except for chromosome 7 in one of the clones of SW480 cells (tetrasomy in parental cells and trisomy in the clone). In concordance, most of the remaining variations in chromosome number consisted of losses, with infrequent gains that affected principally chromosome 3 (Table 2)
. As a whole, the adjusted mutation rate was 0.002 ± 0.002 per chromosome and per generation in LoVo and HCT116 cells vs. 0.005 ± 0.003 in SW480 cells (Mann-Whitney test, U=86.5, two-tailed P=0.027).
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CONCLUSIONS AND SIGNIFICANCE
Our results reveal the existence of a type of instability characterized by multiple translocation events with a low variability in chromosome number. Structural instability is extremely high in SW480 (average minr=0.118), low in HCT116 (average minr=0.004), and absent in LoVo cells (average minr <0.001). We also show that aneuploid instability, understood as a high rate of gains and losses of whole chromosomes, occurs at low rate in the two near-diploid cell lines and is 2.5- to 5-fold higher in the aneuploid cell line SW480.
To understand our estimates of chromosomal instability and how close they are to genuine rates, it is necessary to emphasize some considerations we made when analyzing the data. Actual numerical alterations appear in the form of aneuploidy or polyploidy. In assessing numerical changes (by G banding and FISH analyses), we did not take into account polyploid endoreduplications because it is a well-characterized process driven by different mechanisms to aneuploidy. Clonal populations displayed a striking homogeneity in chromosome number, suggesting the clonal nature of most changes. Therefore, the heterogeneity in the chromosome number distribution is largely explained by sparse numerical mutations that are propagated by clonal expansion of stable cells.
Part of the confusion in the interpretation of chromosomal alterations arises from the biased reading of molecular data: aneuploidies and polyploidies are usually mixed and unbalanced rearrangements are frequently interpreted as numerical changes, precluding the identification of the underlying cause of the so-called chromosomal instability. The complex CGH profiles of most chromosomes in SW480 cells are suggestive of multiple rearrangements, and other studies have also noted the intricacy of the patterns of allelic loss and retention in different chromosomes in human colorectal cancer.
Chromosome instability can actually be rated only in dynamic settings such as the one described here. This type of assessment is precluded by undefined elapsed time and unknown generation number in in vivo studies of human cancer. Moreover, mutation rate is likely to be different according to the environmental conditions, which obviously are quite different in in vivo and in vitro settings. In consequence, our results can not be directly extrapolated to obtain estimates of chromosomal instability in primary tumors in vivo. Despite these limitations, our findings may help to interpret data generated in the genetic analysis of biopsies from cancer patients.
In summary, genetic instability is a landmark in colorectal cancer, but there is no straightforward approach to its analysis. The use of single methodologies and the entanglement in the interpretation of genetic alterations detected in molecular approaches have confounded the characterization of chromosomal instability. A realistic picture of the ongoing genetic processes taking place in tumor cell evolution can be obtained only in dynamic settings like the one reported here. Concomitant application of multiple and comprehensive methodologies is indispensable. We demonstrate that the nature of the chromosomal instability in the SW480 cell line is in essence structural.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0425fje; to cite this article, use FASEB J. (December 3, 2002) 10.1096/fj.02-0425fje 
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