The Pathway And Mechanism Of Chromosome Instability In Colorectal Cancer Cell Lines

The persistent presence of chromosome instability (CIN) has been demonstrated in majority of human cancer, particularly in solid cancers. Aneuploidy, an alteration in chromosome number, and chromosome instability (CIN), a high rate of chromosome mis-segregation, which are hallmarks of vast majority of human solid tumors, including colorectal cancers have been thought to be a major driving force in the development of carcinogenesis.Cancer cell evolution has been accused for drug resistance and relapse of tumors, but the underlying mechanism remains largely unknown.Various and mutually repulsive mechanisms, including defects in genes ensuring the fidelity of chromosome replication and segregation, lagging and bridging chromosomes, multiple centrosomes, defects in kinetochore-microtubule attachment, and cytokinesis failure, have been proposed to be responsible for aneuploidy and/or CIN. From point of molecular view, CIN is found to result from mutations in genes that control mitosis, DNA damage repair, DNA replication, chromosome condension, centrosome structure and function, telomeres, hypomethylation and so on, but the mechanisms responsible for the acquisition and accumulation of these alterations are less well understood. However, whether divisions with such mitotic errors could produce aneuploid daughter cells spontaneously and what the fate is for the aneuploid progenies is remain to be tested.In the study, we select3colorectal cancer cell lines (HCT116, HT29and SW480).9single cell subclones were created and expanded through a defined number of passages, then their karyotype were examined by fluorescence in situ hybridization (FISH, with a panel of centromeric probes to measure chromosome number changes). At the same time, cytokinesis-block micronucleus assay (CBMN) was employed to detect the chromosomal loss and nondisjunction events. By using Long-term live cell real-time imaging(follow cell mitosis) watching chromosome segregation during cell division and determining chromosome complement of daughter cells of the divisions using fluorescence in situ hybridization (FISH) following long-term live cell imaging, we can calculate the ratio of aneuploidy exactly. By using technique of long-term live cell real-time imaging, FISH and immunofluorescence stain, we study the ongoing rates of numerical instability in selected cancer cell lines clone and investigate the consequences of these rates to karyotypic progression and analysis which is the major way in aneuploidy occurrence. The main results were summarized as followings:The different between CIN and MIN include:(1) Anaphase bridges was higher in CIN cell lines (SW480and HT29) than microsatellite instability (MIN) cell line HCT116.(2) Lagging chromosome, including chromosome that left behind the other at anaphase/telophase, was also high in CIN compared to MIN.(3) Chromosome nondisjunction, which generates discrete chromosomal copy number variations, was found in both MIN and CIN cell lines.(4) Micronucleus (MN) and nuclear blub (NB) were a common phenomenon in SW480cell line.(5) Multipolar division will cause daughter cells with high variably chromosome complements and only a few of special karyotype cells can clongenic.(6) Cell cycle difference:homogenous cells with different karyotype had distinct cell cycle time, which will lead to certain karyotype accumulating while others reducing in the total population.Whether and which is the major pathway to create CIN? Here, we demonstrate, by watching chromosome segregation during cell division and determining chromosome complement of daughter cells of the divisions using fluorescence in situ hybridization (FISH) following long-term live cell imaging, that most newly generated aneuploid cells (>83%) were generated from multipolar divisions and very few (-16.7%in non-CIN cells and-8.4%in CIN cells) from bipolar divisions with chromosme nondisjunction in single cell clones from CIN and non-CIN colorectal cancer cell lines. Nondisjunction Divisions with lagging chromosomes were2-5folders more in CIN than in non-CIN cells, but only one cell was detected to give birth of one daughter cell with chromosome content different from their mother cells. Mutipolar divisions could be taken by mononucleated and binucleated parental cells, and produced daughter cells containing various copies of the chromosomes analysed. However, the daughters containing the same chromosome number as cells in the mother clone or missing a copy from mother subclone were most prevalent. More interestingly, the daughter cells missing a copy for certain chromosomes were found to survive and grow into clones with shorter cell cycle duration over others.Karyotype evolution in different clones maybe developed during different time or with different rate. By comparing the data in our experiment, we provided the first systematic explaination for cancer cell evolution that rapid proliferation of daughter cells from multipolar mitosis promoted colonal evolution in colorectal cancer cells. CIN cell proliferation may depend on the tight endogenous control of these processes.