Abstract
Abstract Breast cancer is the most common form of cancer and the second-leading cause of cancer-related deaths in women in the United States. While the causative factors and tumor characteristics are very heterogeneous between cases, Cyclin D1 has been shown to be expressed in approximately 50% of all breast cancers. While it may not be generally considered a canonical partner, Cyclin D1 has also been shown to form active complexes with Cdk2 and transient expression of Cyclin D1 causes irreparable chromosomal instability, the gain or loss of whole chromosomes, in a Cdk2 dependent manner. To study the role of Cyclin D1/Cdk2 complexes in this chromosomal instability, we are utilizing a Cyclin D1/Cdk2 (D1K2) fusion protein, which exhibits constitutive protein kinase activity. Mice with mammary expression of D1K2 under control of the MMTV promoter generate spontaneous tumors that exhibit aneuploidy and centrosome amplification. Karyotype analysis shows that the tumors consist of distinct populations clustered around 2N and 4N DNA content, suggesting mechanisms capable of doubling the DNA content of the cell in addition to causing small stochastic changes, likely through centrosome amplification. Live cell microscopy of a cell line derived from one such tumor shows the occurrence of failed cytokinesis. The cleavage furrow forms and the cells begin to pull apart but appear to fail abcission and merge back together to form a binucleate cell. The resultant cell is then tetraploid with an abnormal number of centrosomes. Following the cell further, it later reenters mitosis and completes division, providing a glimpse of what could be the underlying cause of the centrosome amplification and chromosomal instability seen in these cells. We have also engineered the MCF10A cell line, a normal human breast epithelial cell line, to express the D1K2 protein. Rb and NPM are hyperphosphorylated in these cells, providing potential mechanisms for loss of cell-cycle control and centrosome amplification, respectively. When treated with 1 μM paclitaxel for 72 hours, the cells expressing D1K2 show a distinct population with an 8N DNA content when analyzed by propidium iodide staining and flow cytometry, indicative of either endoreduplication or failed cytokinesis, whereas cells expressing only a hygromycin resistance vector do not. This effect is dependent on the kinase activity of the D1K2 as both cells expressing a kinase dead mutant of D1K2 and D1K2 cells in the presence of the specific Cdk2 inhibitor CVT313 yield results similar to the control cells. This implicates the constitutive Cdk2 activation in altering the spindle assembly checkpoint and/or the G1/S tetraploidy checkpoint since cells would need to both slip through the paclitaxel induced mitotic arrest and reenter S phase to attain an 8N ploidy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3923. doi:10.1158/1538-7445.AM2011-3923
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call