It is generally believed that divergence in cancer cell lines is the consequence of differences in culture conditions, which change the selective pressure and, thus, favor the selection of new genomic anomalies. If this situation is extended on a large number of cell passages it will lead to important differences between cellular stocks. The level of divergence can be directly related to that of genetic instability and breast cancer cell lines seem particularly prone to it. Evidence for this can be found in recent work by Davidson and colleagues  and Kytola and colleagues , who studied breast cancer cell lines using 24 color caryotyping or SKY. Seven cell lines were studied by both groups and, for 3/7, reported data presented extensive differences. Interestingly, MCF-7 cells were the most divergent in both studies adding further evidence to existing data on phenotypic or caryotypic variations in this cell line. MCF-7 cells of different origins are characterized by their variable chromosome numbers, which range from 55 to 90. Noticeably, some subsets present a bimodal distribution with a first peak at 70 chromosomes and a second one at 130 , indicating the coexistence of two cellular subpopulations, one of which had undergone endoreduplication.
Data presented here document that different MCF-7 variants underwent divergence at both the genomic and the RNA expression levels. Furthermore, they indicate that this can occur rapidly according to the MCF-7 variant considered. All the MCF-7 variants studied here showed extensive differences in their CGH profiles. These differences affected the number of regions of either losses or gains, which ranged from 28 in MCF-7-ATCC to 41 in MCF-7-MG, as well as the size of the regions involved. Remarkably, closely related sublines such as MCF-7-R and its 3 daughter clones MCF-7-R-D4, MCF-7-R-F3 and MCF-7-R-G1 presented variations in their CGH profiles as well. Daughter cells presented aberrations which were absent in the mother subline and, this was less expected, had lost anomalies present in the mother line. Furthermore, sister clones showed different sets of anomalies indicating that these cells bore the capacity to diverge over a limited number of cell generations, even kept in identical culture conditions. It is questionable whether this rapid upsurge of anomalies fits a linear progression model, where mutations are supposed to occur sequentially and be retained due to positive selection. We think more plausible that the differences shown by the 3 subclones be related to the oligoclonal nature of MCF-7-R parent cells. Anomalies found in the subclones in fact preexisted in MCF-7-R cells and were brought to light by cell cloning. In comparison MCF-7-MVLN and its two tamoxifene resistant derivatives MCF-7-MVLN-6ms7 and MCF-7-MVLN-6ms8 were less divergent. MCF-7-MVLN correspond to MCF-7 cells stably transfected with ERE-Luciferase construct and went through a gentamycin selection process. This could have lead to the loss of the preexisting genetic heterogeneity. We propose that MCF-7 cells contain an undetermined number of coexisting clones, out of which one (or several) possess stem clone potential and are responsible for the genetic oligoclonality.
The oligoclonal nature of MCF-7 cells can be related to aberrant or instable mitoses. Indeed, cells that have lost proper mitotic controls are prone to unequilibrated sister chromatid exchanges and tolerate the propagation of damaged chromosomes. As such they rapidly become aneuploid and tend to accumulate physical aberrations. Such anomalies have been reported in cellular models in which the anaphase checkpoint gene MAD2 was disabled [27–29], as well as in human tumors . Consequently instable mitoses will lead to rapid caryotypic changes. We, thus, verified the integrity of the M phase in MCF-7-R cells. MCF-7 cells did not show proper G2-M arrest when challenged with Nocodazole, a spindle inhibitor (data not shown). Our data are concordant with recently reported data by Yoon and coworkers  which showed that 7/9 breast cancer cell lines, among which MCF-7, presented important chromosome number variations.
The capacity to generate oligoclonality could be a strong selective advantage for cancer cells because it allows for rapid changes and as such confers an elevated genetic plasticity. Such tumor systems would evolve according to a nodal scheme (possibly through bursts) rather than following a linear selection model. Arguments in favor of a nodal evolution scheme stemmed from the phylogenetic analysis we have performed to reconstruct the history of MCF-7 sublines and identify diagnostic characters (CNAs). Because a number of analogies exist between evolution of species and that of tumor cells, classification methods developed for systematics have become increasingly employed to analyze genetic data in cancer. Approaches, based on hierarchical clustering or other distance-based models, have been applied to classify LOH  or CGH results [33, 34]. We chose the maximum parsimony approach in a cladistic framework because it is a character based classification method and, as such, was considered to be best adapted to meet our goals . We reconstructed the phylogeny of the MCF-7 clade and, interestingly, MCF-7-ATCC, which was the most divergent MCF-7 subline in our study, was positioned closest to the common ancestor. MCF-7-R came in second, positioned as the ancestor of all other MCF-7 sublines. Out of the total of 62 CNAs present in all the sublines tested, only 8 were selected as diagnostic of the MCF-7 clade. This means that this set of 8 events is shared by all the MCF-7 cells tested here and the original tumor possibly developed upon them. Thus, according to this phylogenetic tree MCF-7-ATCC and MCF-7-R, which bear respectively 28 and 34 CNAs, evolved from a common node. The robustness of these results was reinforced by bootstrap and Bremer analyses.
Given the extensive differences observed at the genomic level we were interested to check different MCF-7 sublines at the transcriptome level. Our RNA expression profiling results confirmed the divergent position of MCF-7-ATCC cells, which clustered with at some distance of other MCF-7 sublines. It, thus, appears from the expression profiling analysis, that MCF-7 sublines can show substantial differences at both the genomic and RNA expression levels and this strongly suggests that the genomic differences could translate into phenotypic differences of possibly equivalent importance. MCF-7 cells are the most commonly used model for hormone responsive breast cancer and there is generally little knowledge concerning the variant used. Our data indicate that this may bear some importance, given the level of genetic variability these cells show and the rapidity with which they evolve.