| Ovarian cancer is the most frequent and lethal gynecological tumor with a five year survival of only30-40%, due to lack of proper method for early diagnosis and effective therapy. It is shown that ovarian surface epithelial cells (OSEC) are responsible for up to80%of the ovarian cancer and most of the ovarian cancers are chromosomal unstable with major chromosome numerical abnormalities and minor structure abnormalities. However, it is still not clear through which pathway aneuploid cells are generated and which proteins are involved. In this study, by establishing the spontaneously transformed cell line through isolation and continuous passage of primary MOSEC, to mimic repetitive ovulation process in vivo which would cause insult to OSEC, wound healing, subsequent neoplastic transformation and ultimately tumor formation, we examined the chromosome diversification during this process by cytogenetic methods and the mitotic behaviors of MOSEC were then tracked by live cell imaging to examine through which pathway chromosome instability occurred. We found that:(1) Primary MOSEC initially proliferated very slowly, but they started to proliferate faster after about five passages, and finally became transformed. Cytogenetic analysis using FACS, metaphase spread and FISH demonstrated that MOSEC were diploid at p6, with more than90%of the cells containing40chromosomes. The percentage of tetraploid cells increased significantly accompanied by the reduction in diploidy from p9to p19. By p26, the proportion of near-tetraploidy had increased, including both hypo-and hyper-tetraploid chromosomal constitution, with the former significantly higher than the latter. Almost all the cells became aneuploid at p36, mainly containing hyper-tetraploid cells.(2) Live cell imaging performed on MOSEC at passage8showed that tetraploid cells were resulted from cytokinesis failure of diploid cells and76.8%of them passed through bipolar mitosis. Binucleated cells showed significantly higher proportion of lagging chromosomes at anaphase or telophase than mononucleatd cells undergoing bipolar mitosis. Centrosome analyzing showed that21.9%of the cells at this time had extra centrosomes but formed bipolar spindle mainly by centrosome clustering. All these evidence suggested that higher rate of improper kinetochore and microtubule attachment contributed to chromosome missegreagation in tetraploid cells.(3) FISH performed on cytochalasin D induced binucleated cells showed that1.94%tetraploid cells had chromosome nondisjunction during bipolar mitosis. It is suggested that near-tetraploidy was mainly from this way.(4) RT-PCR results showed that the mRNA level of P-catenin was significantly increased during binucleation. Ectopic expression of P-catenin in293T cells increased occurance of binucleation and multinucleation, which was suppressed by Quercetin, an inhibitor of β-catenin.(5) MOSEC at late passage cells which were nearly all aneuploid could induce tumorigenesis in vivo. Chromosome loss occured in ascite cells as showed by FISH, which indicated that aneuploid cells were under selective pressure in vivo. In conclusion, high rate of nondisj unction during mitosis of tetraploid cells which result from cytokinesis failure of diploid cells could lead to aneuploid cells, and then near tetraploid ovarian tumor. And abnormal high expression of β-catenin contributes to cytokinesis failure and binucleation. This provides a novel explanation for ovarian tumor initiation and a theoretical basis for using β-catenin as biomarkers for early diagnosis of ovarian cancer. |
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